CHEMICAL MANIPULATION OF SEED VIABILITY, PLANT...

CHEMICAL MANIPULATION OF SEED VIABILITY,

PLANT GROWTH, METABOLISM, PRODUCTIVITY

AND PHYTOCHEMICAL ANALYSIS OF

ABRUS PRECATORIUS L. AND DOLICHOS BIFLORUS L.

THESIS SUBMITTED FOR

THE DEGREE OF DOCTOR OF PHILOSOPHY IN BOTANY

OF

THE UNIVERSITY OF BURDWAN

BY

SUBHOJIT OJHA, M. Sc. (BOTANY)

DEPARTMENT OF BOTANY

THE UNIVERSITY OF BURDWAN

BURDWAN-713 104

WEST BENGAL

INDIA

SEPTEMBER, 2013

Dedicated To

My

Late Grand Parents

ACKNOWLEDGEMENTS

I wish to express my deep regards and sincere gratitude to my respected

teacher and supervisor Dr. Aloke Bhattacharjee, Professor of Botany, The University

of Burdwan for his valuable guidance and supervision, constant encouragement and

co-operation throughout the entire period of investigation and also during preparation

of the thesis.

My sincere thanks also go Professor Smriti Kumar Sarkar, Hon’ble Vice

Chancellor, The University of Burdwan (B.U.) and to Dr. Abhijit Bandyopadhyay,

Head of the Department of Botany, B.U. for their kind permission to conduct my

research and providing necessary laboratory facilities.

I would acknowledge the University Grants Commission (UGC) for financial

support in the form of a Project Fellow under CAS (Phase I) Programme of the

Department of Botany at Burdwan University.

Sincere thanks are due to Professor Kajal Gupta and Professor Radhanath

Mukhopadhyay, Department of Botany, The University of Burdwan for their constant

encouragement and support towards carrying out the research work.

I am grateful to all the faculty members, office staff members of Department

of Botany, The University of Burdwan, for rendering me their necessary help and

kind cooperation.

I am also grateful to Dr. Srikanta Chakraborty, University Science and

Instrumentation Centre (USIC), The University of Burdwan for his valuable help to

take photographs of Scanning Electron Microscopy of my research material.

I would like to acknowledge the valuable help rendered by Professor A. K.

Ghosh, Chairman, Life Science Division and to Dr. Shanti Mohan Mondal,

Superintendent In-charge, Central Research Facility Section of Indian Institute of

Technology (IIT), Kharagpur, Paschim Medinipore, W. B. for their help to carry out

the work on Phytochemical analysis using HPLC and MALDI-TOF MS.

I am very much grateful to Professor Subrata Laskar, Department of

Chemistry, The University of Burdwan for his valuable help for undertaking

Phytochemical analysis part of my research work.

I record my gratitude to Mr. Kaushik Sarkar, Technical Assistant, Gr.-II,

Department of Botany for his technical help particularly in terms of photographic

work both in laboratory and in research field.

I can never forget the help I received from my fellow researchers Mrs. Irani

Biswas Sur, Mr. Hemanta Kumar Ghosh, Mr. Avisek Dey, Mr. Joydeep Mazumdar,

Mr. Subhabrata Ghosh, Mr. Arnab Jash, Ms. Priyanka Charaborty, Mr Samir Halder,

Ms Moitreyee Kundu, Mr. Arijit Ghosh, Mrs Sutapa Pal, Mr. Jayanta Sikdar, Mr.

Surajit Roy, Dr. Debjyoti Das, Mr. Alokmoy Basu, Mrs. Shilpa Dutta Basu, Mr.

Tausif Ahmed, Ms. Mousumi Das who were always ready to spare their time to help

me as and when required.

Thanks are also due to my ERS Hostel mates of Burdwan University specially

Dr. Tirthankar Mandal, Dr. Krishnendu Bhattacharyya, Mr. Milan Mandal, Mr.

Sumitava Khan, Mr. Udipta Ranjan Chatterjee, Mr. Koushik Das, Mr. Subhasis Roy,

Mr. Somnath Choubey, Mr. Suman Roy for their constant appreciations and

encouragement.

Finally, I would like to express my immense depth of gratitude to my

grandfather Late Rasbehari Ojha, grandmother Late Genubala Ojha, my father

Mihirlal Ojha, mother Baruni Ojha and my elder brother Dr. Suprakash Ojha, my

elder sisters Mrs. Sudipta Ojha Layek and Mrs. Sudeshna Ojha Layek who had

always been a source of inspiration to me. I am indebted to all my relatives and well

wishers for their constant encouragements during the period of my research work.

…………………………………………..

Date: (Subhojit Ojha)

CONTENTS

SUBJECTS PAGE

A. SYMBOLS AND ABBREVIATIONS USED 1

B. INTRODUCTION 4

C. OBJECTIVES 8

E. REVIEW OF THE LITERATURE 9

F. MATERIALS AND METHODS 43

G. PHOTOGRAPHS 57

H. RESULTS, TABLES AND FIGURES 68

I. DISCUSSION 120

J. SUMMARY AND CONCLUSION 127

K. FUTURE VISION 135

L. REFERENCES 136

M. LIST OF PUBLICATIONS 155

N. PHOTOCOPIES OF PUBLISHED PAPERS

AND ABSTRACTS

158

1

SYMBOLS AND ABBREVIATIONS USED IN THE TEXT

SYMBOLS AND

ABBREVIATIONS FULL FORM OF THE SYMBOLS AND

ABBREVIATIONS

AR = Analytical reagent

BDH-AR = British Drug House-analytical reagent

BSA = Bovine serum albumin

CCC = Chloro choline chloride

ºC = Degree centigrade

cm = Centimeter(s)

DNA = Deoxyribo nucleic acid

ΔOD = Delta optical density

e.g. = For example

et al. = And associates

FeCl3 = Ferric chloride

fr. wt. = Fresh weight

g = Gravity

g = Gram(s)

GA3 = Gibberellic acid

h = Hour

HCl = Hydrochloric acid

HClO4 = Perchloric acid

HgCl2 = Mercuric chloride

HPLC = High performance liquid chromatography

H2O = Water

H2O2 = Hydrogen peroxide

H2SO4 = Sulphuric acid

IAA = Indole 3-acetic acid

i.e. = That is

ISTA = International Seed Testing Association

L. = Linnaeus

LSD = Least significant difference

μ = Micron

2

SYMBOLS AND


ABBREVIATIONS

M = Molar

MALDI-TOF MS = Matrix Assisted Laser desorption/ionisation

Time-Of-Flight Mass Spectrometry

μg = Microgram

mg = Milligram(s)

MgSO4 = Magnesium sulphate

min = Minute(s) (time unit)

ml = Millilitre(s)

mM = Millimolar

MnCl2 = Manganese chloride

MnO2 = Manganese dioxide

(N) = Normal

NaDK = Sodium dikegulac

Na2HPO4 = Disodium monohydrogen phosphate

NaH2PO4 = Monosodium dihydrogen phosphate

NaOH = Sodium hydroxide

NC = Not calculated

nm = Nanometer

Nos. = Numbers

NS = Not significant

OD = Optical density

p. = Page

PGR(s) = Plant growth regulator(s)

pp. = Pages

%

%T

=

=

Percentage

Transmittance

RH = Relative humidity

RNA = Ribonucleic acid

sec. = Second (time unit)

sp. = Species

Syn. = Synonym

3

SYMBOLS AND


ABBREVIATIONS

t = Time

T50 = Time required for 50% germination

TCA = Trichloroacetic acid

TTC = 2, 3, 5 - Triphenyl tetrazolium chloride

Tv = Total volume

v

v/v

viz.

=

=

=

Volume

Volume/volume

Namely

W. B. = West Bengal (a state of India)

wt = Weight

w/v = Weight/volume

Introduction

4

INTRODUCTION

Medicinal plants play a pivotal role on the health care problems of a large

number of people in developing and underdeveloped countries particularly to almost

all tribal groups in India and other third World countries. The traditional knowledge

on ethnomedicinal plants dates back to prehistoric times and uncivilized people

started getting remediation of their ailments by using some unique medicinal plants on

trial and error basis. A group of intelligent people from among the ethnic communities

learned the art of health care system not only using some selected plants as a whole

but by using some selected parts of the plants. They also identified the appropriate

collection time of specific medicinal plants in different seasons as well as collection

of plants in particular developmental phases for having optimum bioremediary action.

However, right from the beginning of the usage of medicinal plants as crude extracts

in Vedic age it has been well established that some medicinal plants can exert

dramatic effect for alleviation of a wide range of ailments in human beings (Jain,

1968; 1981; Jain and Mudgal, 1999; Fox et al. 2008; Sur et al. 2012). Different health

care systems were established and these include: Unani, Ayurveda, Siddhha,

Homoeopathy, Naturopathy etc. However, in recent times the allelopathic system of

medicine is, in fact, the advanced one which deals with scientific approaches for

dealing the ailments in a perfect manner and quality improvement is often done by

biotechnological approaches (Khan and Khanum, 1999).

India, being one of the important megabiodiversity countries in the World is

bestored with huge varieties of medicinal plants for curing numerous diseases. And

such plants are the hidden treasure for our country for preparation of traditional

medicines as well as formulation of drugs employing advanced technology. In

Atharba Veda, Charak Sanhita and other allied Indian literature, attempts were made

to systematically record the use of medicinal plants along with different ingredients to

get rid of some common as well as dreadful diseases by perfect application of various

preparations and drug formulations from various medicinal plants (used singly or in

combination with some other plants).

In the back drop of this ancient literature and further contribution of

knowledge on medicinal plants by the present day researchers, attempts are being

Introduction

5

made to enrich the herbal health care systems by scientific methods and to improve

the productivity of medicinal plants by biotechnological approaches (Das et al., 1999;

Sharma et al., 1999; Sharief et al., 1999; Rao and Raju, 1999; Chand et al., 1999;

Bhalla et al., 1999). In fact, scientists all over the world are engaged in work on

medicinal plants with special emphasis on identifying the appropriate bioactive

compounds which play crucial roles for remediation of various ailments (Khan and

Khanum, 1999; Keshri and Mukhopadhyay, 2012).

However, the present investigation is aimed at the identification of the

specific problems of two medicinal plants Abrus precatorius L. and Dolichos biflorus

L. and to design experiments for possible alleviation of the problems for augmented

productivity of seeds which are the main reservoirs of bioactive compounds. Both the

experimental species have already been established as potent medicinal plants for

curing various health related problems particularly in India and neighboring countries.

The major problem of Abrus is its poor seed germinability under ambient

climatic conditions prevailing in India. Abrus seeds are often considered as stubborn

type in respect of germination and these need optimum scarification for germinability

under laboratory condition (Ojha and Bhattacharjee, 2012 Qadir et al. 2012). In fact,

poor germination performance is mainly due to the factors like hard coated seeds,

imperviousness of gaseous exchange, hindrance for entry of water and moisture,

excess amount of inhibitors, waxy nature of seeds as well as some intrinsic factors

including allelochemicals (Bewley and Black, 1982; 1994; Ojha et al., 2013b). Thus,

this particular species requires special attention for overcoming the physical and

chemical barriers for allowing normal germination. Reports exist in the literature that

properties like high germinability, enhanced vigour and viability, lower T50 values,

quicker field emergence of seeds give rise to healthy seedlings and potential plant

establishment (Bhattacharjee and Choudhuri, 1986; Chhetri et al., 1993; Bhattacharjee

et al., 2009; Chakraborty et al., 2013). All these beneficial features finally lead to

enhanced productivity in terms of seeds. Both Abrus and Dolichos seeds are the major

store house of a number of potent bioactive compounds responsible for rendering

medicinal properties. Moreover, plant type modification or production of ideotypic

Abrus plant by some manipulative technique is often considered to be a prerequisite

for production of flowers and pods in flashes due to the short statured plant habit with

Introduction

6

multiple branches. Again, subnormal or impaired source-sink relationship of many

cereal and pulse crop is also considered to be a major stumbling block for successful

filling of the seeds with photosynthates from the contributory leaves. This results in

production of partially filled smaller seeds and thus seed productivity is reduced.

Hence, keeping in mind these problems i.e. poor seed germinability, undesired

plant type as well as poor source-sink relationship for impaired productivity, an

attempt was made to overcome such problems of Abrus by employing manipulation

technique with two growth promoters (IAA and GA3) and two growth retardants

(CCC and Na-DK). Scarification agent H2SO4 is reported to overcome some physical

barriers of seeds and stimulatory agent like GA3 can trigger seed germination (Bewley

and Black, 1982; Ojha and Bhattacharjee, 2012). Again ideotypic plant production

can be achieved by employing growth retarding chemicals like CCC and NaDK

(Bhattacharjee, 1984). Source and sink strength are also reported to be enhanced by

judicious chemical manipulation with growth retardants and growth promoters

(Bhattacharjee et al., 1984; Kanp, 2007).

In case of Dolichos biflorus there exists some problems which are related to

impairment of productivity. These include: poor storability of seeds under ambient

environmental condition, rapid loss of seed vigour and viability under storage, poor

seedling and plant establishment from low vigour of seeds. These consequently result

in poor plant health due to reduced growth, metabolism, substandard yield attributes

etc. Keeping in mind such problems of Dolichos species attempts were made to

improve storability, vigour and viability of seeds by chemical manipulative technique.

To obtain expeditious results, accelerated ageing technique was employed and this

technique accurately justifies the vigour status of seeds under storage (Heydecker,

1972). Seed vigour and viability status was again evaluated through a number of

reliable physiological and biochemical parameters like percentage germination, time

required for 50% germination of seeds (T50), speed of germination, analysis of some

growth and biochemical parameters and finally critical analysis of yield attributes of

chemically hardened or invigourated and normal seeds. In view of the specific

problems of these two seed species, attempts were taken to get rid of such problems to

achieve my prime target i.e. enhancement of productivity.

Introduction

7

To get an insight into the status of a few phytochemicals present in the seeds of

the two experimental species, an attempt was also made to analyse the samples by

using IR Spectra, HPLC and MALDI-TOF MS. In fact, this phytochemical analysis is

the beginning of a comprehensive programme where critical assessment on the role of

bioactive compounds will be pinpointed for exerting the medicinal property of the

experimental plants.

Objectives

8

OBJECTIVES

The prime objective of this investigation was to undertake a comprehensive

field and laboratory work using a minor pulse cum medicinal crop Dolichos biflorus

L. and a wildly available medicinal plant Abrus precatorius L. growing in West

Bengal. However, the two experimental plants have different pinpointed problems. In

case of Abrus, the main problem is its stubborn germination behaviour and low

productivity mainly due to undesired source-sink relationship. On the other hand, in

case of Dolichos the main problem is its poor seed storability under ambient climatic

condition because seeds tend to become nonviable due to quicker deterioration under

storage. Again, plant type modification in terms of multiple branched plant with

bushy habit is required for higher yield attributes and productivity. Keeping in mind

these vital problems the following objectives were set for possible alleviation of these

problems with a view to augmenting seed productivity:

1. To evaluate the relative efficacy of different concentrations of sulphuric acid

for optimization of scarification of Abrus seeds.

2. To determine the effect of scarification followed by hormonal treatment on

further enhancement of germination behaviour of Abrus seeds over

scarification technique.

3. Production of desired plant type of both Abrus and Dolichos by chemical

manipulation employing IAA, GA3, NaDK and CCC.

4. To analyse the chemical-induced manipulation of the vigour and viability

status of Dolichos seeds under natural and accelerated aging condition.

5. To probe the influence of the chemical manipulants on alteration of yield

attributes leading to enhanced productivity.

6. To analyse the existence of some phytochemicals in seeds of both Abrus and

Dolichos by employing Spectrophotometer, IR-Spectra, HPLC, MALDI-TOF

MS.

Review of the Literature

9

REVIEW OF THE LITERATURE

This investigation was undertaken as a part of a comprehensive work on

medicinal plants which is being continued in our department focusing on alleviation

of the specific problems of a number of selected medicinal plants available in our area

with the prime objective to improve productivity. This review includes a brief preface

on medicinal plants with special reference to Indian scenario and the aspects dealt in

this investigation.

India has a glorious past in respect of the science of life like Ayurveda, which

not only deals with medicines but enables us to live a healthy and happy life. It is an

established fact that man learnt the art of healing with the help of medicinal plants. In

our country a large number of people, residing particularly in rural belt, still depend

on medicinal plant-related health care system to get rid of various kinds of ailments.

In fact, our age-old ethnic knowledge of medicinal plants is gradually diminishing at

an alarming rate due to adoption of modern system of medicine and neglecting the

traditional medicine. Thus, it is urgently required to collate indigenous knowledge of

herbal medicine from tribal communities and other sources, conserve medicinal plants

and to undertake a comprehensive research for a thorough understanding of different

modern aspects of medicinal plants. A huge volume of work is now available in

literature on diverse areas of medicinal plants (Kirtikar and Basu, 1933; 1935; 1991;

Chopra et al., 1969; Vedavathy et al. 1997; Deora and Singh, 2002; Chakrabarti,

2003; Chakraborty et al., 2003; Paria, 2005; Evans, 2005; Chakraborty and

Bhattacharjee, 2006; Fox et al., 2008).

Keeping in mind the problem and immense prospects of medicinal plants, an

investigation was undertaken pertaining to chemical manipulation of seed vigour and

viability alongwith growth, metabolism, productivity and phytochemical analysis of

two important medicinal plants, Indian Liquorice (Abrus precatorius L.) and Horse

Gram (Dolichos biflorus L.). However, major thrust was given on chemical-induced

augmentation of productivity of seeds and also analysis of some phytochemicals

present within the seeds. In fact, reports available in literature that some chemical

manipulative agents are used as efficient tools for enhancement of productivity of


10

many crop plants (Bhattacharjee, 1984; Kanp, 2007; Dolui, 2008) including medicinal

plants by chemical and biotechnological approaches (Khan and Khanum, 1999).

The present review deals with the various aspects of the two experimental

medicinal plants Abrus precatorius and Dolichos biflorus as well as the research work

done on seed viability, modulation of growth, metabolism, productivity and

phytochemical analysis. These are captioned as follows:

I. About the experimental plants

II. About the chemicals used in this investigation

III. Medicinal values of the experimental plants

IV. Vigour, viability, longevity and germination of seeds

V. Modulation of plant growth, metabolism and productivity

VI. Phytochemistry of the experimental plants

I. ABOUT THE EXPERIMENTAL PLANTS:

Abrus precatorius:

Systematic Position:

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Fabales

Family: Fabaceae

Subfamily: Faboideae

Genus: Abrus

Species: A. precatorius


11

The plant Abrus precatorius L. is characterised by high-climbing, twining, or

trailing woody vine with slender herbaceous branches. Leaves alternate, petioled,

even-pinnately compound with 5-13 pairs of leaflets, oval to oblong, margins entire.

Flowers shaped like pea flowers, white to pink or reddish, small, in short stalked

dense clusters at leaf axils. Fruit a short, oblong pod. Pods turgid, oblong, appressed

hairy, with a sharp deflexed beak, silky-textured, generally 3 to 6 seeded, seeds

elliptic to sub-globose, smooth, glossy, shining red with black blotch around the

hilum. The seeds were traditionally used to weigh jewellery in India. The measure

ratti is equal to the weight of one seed.

Habitat and distribution:

It is used medically in china, Indo china, Islands, West Indies, Guina, Brazil,

udan, South Africa, Madascar and India. Plant found all throughout the plains of

India, from Himalaya down to Southern India and Ceylon (Nadkarni, 1999).

Phenology:

Flowers in winter; fruits ripen in summer (Kirtikar and Basu, 2005).

Propogation: By seeds (Kirtikar and Basu, 2005).

Parts Used:

The roots, leaves and seeds of the plant are used medicinally (Nadkarni, 1999,

Daniel, 2006).

Dolichos biflorus:

Systematic Position:

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Fabales

Family: Fabaceae

Genus: Dolichos

Species: D. biflorus


12

The plant Dolichos biflorus L. (Syn. Vigna unguiculata sub. sp. unguiculata;

Macrotyloma uniflorus Lamp Verdc.) under the family Fabaceae is named variously

in different Indian languages ( Sanskrit - Kulattha; Hindi - Kulthi ; Tamil - Kollu ;

Malayalam - Muthira ; Kannada - Huruli ; Bengali - Kulthikalai; Punjabi - Kulthi). It

is a popular pulse crop of South India and parallels with gram of Northern India. It is

a good human food with 22 per cent protein and have some medicinal value for

patients suffering from urinary trouble. It also provides good green fodder for cattle

and horses. The grains are also given boiled or lightly parched as a part of concentrate

ration to milch animals and also to working animals (Thakur, 1975; Sundararaj and

Thulasidas, 1993; Kar, 2003).

It is a hardy drought resistant annual grain legume. It derives its name from

the fact that it is fed to horses. The bhusa of the crop after removal of the pods is used

as a cattle feed. It is distributed throughout the tropics and is cultivated in Malaya,

Mauritius, Sierra Leone, Transval and West Indies and grows wild in the Eucalyptus

forests of Queensland. It has been brought under cultivation in India since prehistoric

times and according to Vavilov India is its centre of origin.

It is the most extensively grown pulse crop of Peninsular India and is

cultivated up to 5000 feet elevation. The southern states are the major Dolichos

growing states in India. It is also grown in Bihar, Jharkhand, Madhya Pradesh and

Uttar Pradesh states of India.

Botany of the horse gram:

Terrestrial suberect herb, tap root system with nodules, basal part erect,

terminal part twining; stem solid, semiwoody, pubescent with prominent nodes and

internodes; leaves alternate, pinnately trifoliate, stipulate, stipules free lateral, small,

stipels subulate; leaflets ovate to lanceolate, multicostate reticulate, pubescent, entire,

acute, lamina unequal considering midvein; flowers axillary (1-3 per axil), bracteate,

bracteolate (bracteole minute), short pedicillate, zygomorphic, bisexual, complete,

hypogynous, medium size, yellowish white; sepals-5, gamosepalous, lobes very short,

obtuse, two upper connate in an entire very short lip, persistent; petals 5, sub-equal,

papilionaceous ; standard orbicular, auriculate at the base, wings falcate, adnate to the


13

keel, keel much incurved, beaked; stamens 10, diadelphous (9+1), vexillary, 1 stamen

free, others connate in a sheath, anthers 2 celled basifixed, uniform, dehiscence

longitudinal; carpel 1, superior subsessile ovary, elongated, one chambered with many

ovules in the chamber, marginal placentation; style-thickened upwards and beared

longitudinally down the front, stigma penicillate; fruit - a flat linear recurved pod,

continuous within; seed-thick or flattened, hilum short with slender funicle, elongated

and covered by thickened subpersistent apex of funicle.

Cultivation:

The sowing time of horse gram ranges from the month of August to October.

The land is prepared by ploughing and harrowing. It is sown either broadcast or in the

lines 20-25 cm apart. The usual seed-rate is 45 kg/ha. Although no manuring is in

practice in rural areas, application of 10 kg N and 40 kg P2O5 / ha may be quite

useful for the crop.

Interculturing

Where the crop is sown in rows interculturing should be practised. At places slight

grazing is supposed to be helpful. Horse gram is sown in many places with a mixed

crop of niger, which is grown in rows about 90 - 180 cm apart simultaneously with it.

Pests and diseases

Horse gram is attacked by hairy caterpillar and grass hoppers. The crop is also

susceptible to root-rot, bean rust, anthracnose and die back.

Harvesting and threshing

The plants are harvested by pulling out the whole plants, and are carted to threshing

floor, stacked for a week, put out to dry and threshing is done by conventional or

mechanical process.

Yield

Under the existing conditions of cultivation the crop yield is 6-7 q / ha

Anthesis and pollination:

The flowers are borne in axillary fasciles. Dehiscence of the anthers takes

place from 4 p.m. onwards. The dehiscence is by longitudinal sutures. The pollen


14

grains are massed together and they do not shed; as the stigma is on the same level at

the time of dehiscence it gets fully covered with the pollen. The individual pollen

grains are very big. The standard opens during night and continues for twenty-four

hours. It then fades in colour and folds in the same bud position and so it is often

difficult to distinguish these from the unopened buds. Self pollination is the rule in

horse gram.

Varieties:

There are two distinct varieties which can be made out from the colour of the

seed. The common variety is the one with the buff seed coat (brownish colour) and

the other has a black seed coat. The black seeded type is generally shorter in duration

and low in vigour.

The varieties grown in difference states of India are –

Bihar – BR 5 : Flower colour pale yellow; seed deep black; average yield 850 kg/ha.

BR 10 : Flower colour pale yellow ; seed deep black ; flowering in 48 days;

average yield 921 kg/ha, generally grown in Santhal Pargana.

Kulthi - 7/4/6-3: Another promising variety.

Tamil Nadu – Culture No. 35, isolated at Coimbatore, has given significantly higher

yield than the existing selection No. D. B. 7.

Improvement:

The improvement work on Kulthi was initiated from 1941 onwards in

different states of India under the Pulse Improvement Scheme of I.C.A.R. Although

there is lack of proper breeding material, the following objectives are desired to

induce suitable characters in the newly evolved varieties:

i) High yield, ii) Good cooking quality, iii) High protein value, iv) Drought tolerance,

v) Early maturity and vi) Resistant to diseases.


15

Variations in plant characters:

The stem and petioles are mostly purple pigmented. Flowers are always purple

pigmented whereas the vegetative parts are pigmented or not. There is a purple eye on

the standard, and a light purple wash on the wings, keels and the style. The pods are

either erect or drooping from the leaf axils. The seed colour is brown or black. In the

same plant brown and chocolate coloured seeds are formed due to the differential

environment during the ripening process. In the black seeded type, black-mottled and

black-patchy seeds are also met with. Haploid chromosome number is 12.

Ecological aspects:

The horse gram is a hardy plant and thrives in areas of low rainfall and requires

little or no manuring. In regions of high rainfall it is sown after rainy season is over.

It is considered as a crop suited for poor soils and invariably cultivated as a rainfed

crop in dry lands. It is grown on deep red loams, black cotton soils and clayey soils.

As a matter of fact it is grown on a wide range of soils. Alkaline soils are not

supposed to be suitable for this crop. A new land which is to be brought under

cropping often has horse gram grown on it in the beginning. Once established with the

rains, the crop makes satisfactory growth even with dew when enough soil moisture is

not available (Mishra, 2006).


16

II. ABOUT THE CHEMICALS USED IN THIS INVESTIGATION:

i) IAA (Indole -3-acetic acid):

IAA (Formula C10H9NO2 & Molecular weight 175.18)

IAA is a plant hormone that is a naturally occurring and it promotes growth and

rooting of plants. Chemically IAA is a carboxylic acid in which the carboxyl group is

attached through a methylene group to the C-3 position of an indole ring. As indole

ring present in its structural make up IAA is called a typical indole auxin which

perform a wide range of functions in plant as well as in microbial system. Details are

not mentioned about this phytohormone as a comprehensive literature is available on

this plant growth promoter.

ii) GA3 (Gibberellic acid):

GA3 (Formula C19H22O6 & Molecular weight 346.38)

Gibberellic acid is a simple gibberellin, a pentacyclic diterpene acid promoting

growth and elongation of cells in plants. As many as 140 gibberellin species are

available in various groups of plants. Among these some are C19 type and some are

C20 type on the basis of the numbers of carbon found in their structural make-up.

Since its discovery from the fungus Gibberella fujikuroi and its subsequent

http://en.wikipedia.org/wiki/Carboxylic_acid

http://en.wikipedia.org/wiki/Carboxyl_group

http://en.wikipedia.org/wiki/Methylene_group

http://en.wikipedia.org/wiki/Indole

http://en.wikipedia.org/wiki/Gibberellin

http://en.wikipedia.org/wiki/Polycyclic_compound

http://en.wikipedia.org/wiki/Diterpene


17

characterization, a huge volume of literatures are available on this potent plant growth

promoter. Hence, details of this phytohormone needs little or no introduction to the

plant physiologists.

iii) NaDK (Sodium di-kegulac):

NaDK (Formula C12H17NaO7 & Molecular weight 296.25)

Dikegulac sodium (Sodium 2,3:4-6-di-0-isopropylidene-α-Lxylo- 2-

hexalofuranosate) or ATRINAL (trade name) has been established as a potent plant

growth retardant (Bocion et al., 1975). A considerable volume of work on different

aspects of growth, development, metabolism, productivity etc. of a large number of

plant species has been accumulated (Bocion et al., 1975; Arzee et al., 1977; Zilkah

and Gresel, 1980; Purohit, 1980; Bhattacharjee et al., 1984, Chhetri et al., 1993;

Kanp, 2007; Bhar, 2011). Particularly due to its chemical pinching property, the

chemical evokes attention of agrihorticulturists. NaDK produced as an intermediate

product in the commercial synthesis of L ascorbic acid, is a sugar hormone which is

monosaccharide in nature and of the different salts, sodium-linking to it was found to

be most effective with respect to exhibiting the hormonal activity. Arzee et al. (1977)

demonstrated that it works counter to auxins or to gibberellins but it is neither an

antiauxin or an antigibbrellin in true sense. The chemical possesses extremely low

bee, fish and mammalian toxicity; it is not irritate to eyes and skin (Bocion et al.,

1975). Physically it is white, odourless, solid with a m. p. 300ºC and does not exhibit

photosensitivity. Na-Dikegulac is stable in aqueous solutions at pH 7 and above,

highly soluble in water, methanol and ethanol and less soluble in chloroform, acetone,

cyclohexane and hexane.


18

iv) CCC (Chlorocholine chloride):

CCC (Formula C5H13Cl2N & Molecular weight 158.06)

A group of quaternary ammonium compounds was added to the list of growth

retarding chemicals (Tolbert, 1960). These were analogues of the extremely important

natural base choline. Its trivial name was chlorocholine chloride, abbreviated to CCC.

This chemical was proved to be effective for retardation of plant growth and

metabolism on much wider variety of plant species.


19

III. MEDICINAL VALUES OF THE EXPERIMENTAL PLANTS:

1. Abrus precatorius

GENERAL USES:

i) Leaves, roots and seeds Abrus precatorius are used for medicinal purposes.

Decoction of leaves is taken orally for cough and flu (Nadkarni and Nadkarni, 1954;

Chopra et al. 1956).

ii) Grinded roots of Abrus precatorius are taken with pure clarified butter thrice a day

for four days to cure cough. Dry seeds of Abrus precatorius are powdered and taken

one teaspoonful once a day for two days to cure worm infection (Kirtikar and Basu,

1956).

iii) Root is chewed as a snake bite remedy (Watt and Breyer-Brandwijk, 1962).

iv) Hot water extract of fresh root is administered orally as an anti-malarial and anti-

convulsant (Adesina, 1982).

v) Decoction of dried root of Abrus is taken orally to treat bronchitis and hepatitis

(Chukuo et al., 1995).

vi) Abrus seeds have also the potential of good insecticide and antimicrobial activity

(Saxena and Vyas, 1986).

vii) Seeds are considered as abortifacient, anodyne, antimicrobial, diuretic, emetic,

expectorant, emollient, febrifuge, hemostat, laxative, purgative, refrigerant, sedative,

vermifuge, antidote and used in various ailments to cure headache, conjunctivitis,

convulsion, cough, diarrhoea, fever, gastritis, gonorrhea, jaundice, malaria, night-

blindness, ophthalmia, rheumatism, diabetes and chronic nephritis. Dried Abrus seeds

are taken orally as an aphrodisiac (Chopra, 1933; Nath and Sethi, 1992).

viii) Abrus seeds are also taken for tuberculosis and painful swellings (Arseculeratne

et al., 1985).


20

TRADITIONAL USES:

Leaves:

Leaves of Abrus are used as aphrodisiac, useful in eye diseases, cures

leucoderma, itching, skin diseases and wounds, and also cure fevers, stomatitis, head

complaints, asthama, thirst, tuberculous glands and caries of teeth. Extracted juice

from the fresh leaves, mixed with some blend oil, applied externally, seems to relieve

local pain (Kirtikar and Basu, 2005). Mixture of leaf powder with sugar given in case

of leucoderma and menorrhagia (Chadha, 2004). The leaves of Abrus precatorius also

used as diuretic, diarhhoea, gastritis, heart diseases, kidney diseases, insomnia, Cancer

and CNS sedative (Rose and Lori, 2003).

Root:

Abrus root is often considered as emetic and alexiteric. The watery extract is

useful in relieving obstinate coughs. Roots are taken for sore throat and rheumatism

(Kirtikar and Basu, 2005). The root also used as diuretic, diarhhoea, gastritis, heart

diseases, kidney diseases, insomnia, Cancer and CNS sedative (Rose and Lori, 2003).

The roots also possess usefulness in gonorrhoea and jaundice and other infection

(Daniel, 2006).

Seed:

Internally, Abrus the seeds are described as poisonous and useful in affections

of the nervous system, and externally, in skin diseases, ulcers, affections of the hair.

The seeds reduced to a paste are recommended to be applied locally in sciatica,

stiffness of the shoulder joint, paralysis, and other nervous diseases. In alopesia a

paste of the seed is recommended to be rubbed on the bare scale. The seeds are used

as purgative. Taken internally by women, the seed disturbs the uterine functions and

prevents conception. Reduced to a paste they are used for contusion and inflammation

(Kirtikar and Basu, 2005). The root also used as diuretic, diarhhoea, gastritis, heart

diseases, kidney diseases, insomnia, Cancer and CNS sedative (Rose and Lori, 2003).


21

USES OF ABRUS IN SOME TRIBAL GROUPS ARE AS FOLLOWS:

Directions of use by SANTAL: (i) grind the roots, make small pills, encase the

pills in molasses and eat the same to treat night-blindedness; (ii) make a plaster by

grinding the roots of white-fruited variety and apply the plaster on the painful part of

inflammated sections of the gum.

MUNDA: Root-paste in gonorrhoea.

ORAON: dried root-powder as mild purgative.

AGNI PURANA: (i) husks of A. precatorius along with the same of Vitis vinifera and

the decoction of Polyalthia longifolia and Moringa pterigosperma, as well as fruits of

Terminalia belerica, Terminalia chebula and Emblica officinalis destroys all intestinal

worms.

SOME OTHER REPORTED ACTIVITIES OF ABRUS ARE AS FOLLOWS:

Neuromuscular Effects:

Some neuromuscular effects of the crude extracts of the leaves of Abrus

precatorius were found (Wambebe and Amosun, 1984).

Renal Protective activity:

Renal protective activities of the seed extract of Abrus precatorius was found

in rats (Ae et al., 2009).

Nephroprotective activity:

Nephroprotective study of aqueous extract of aerial parts of Abrus

preacatorius shows that Abrus has best recovery effect and can be used for the

prevention or treatment of renal disorders (Sohn et al., 2009a; 2009b).

Immunostimulatory properties.

In vitro immunostimulatory effect of Abrus lectins was found (Maiti et. al.,

2009).

Anti-inflammatory activity:

Two triterpenoid, saponins 1 and 2 isolated from Abrus precatorius plant and

their acetate derivatives, 3 and 4 shows anti-inflammatory activity (Anam, 2001)


22

Antiepileptic activity:

Information from the literature shows that Abrus precatorius L. can be used by

people for the treatment of epilepsy (Moshi et al., 2005).

Antimalarial activity:

An isoflavanquinone, abruquinone, was isolated from the extract of aerial

parts of Abrus exhibited antimalrial acitivity (Limmatvapirat et al., 2004).

Antiplasmodial activity and cytotoxicity in the assessment of antimalarial activity was

further done by Menan et al., 2006.

Anthelmintic activity:

Aqueous extract of stem and root of Abrus precatorius shows anthelmintic

activity (Molgaard et al., 2001).

Antithrombin effect:

Methylene chloride and methanol fraction of Abrus shows in vitro

antithrombin activity (Chistokhodova et al. 2002).

Antifertility effect:

Antiferitility activity of Abrus prectorius root as well as seed extracts was

found and seeds are used as an antifertility agent or contraceptive (Agarwal, et al.,

1970; Jahan et al., 2009).

Sperm antimotility effect:

Sperm antimotility properties of a seed extract of Abrus precatorius was found

(Ratnasooriya et al., 1991; Anand et al., 2010; Gupta et al., 2013).

2. Dolichos biflorus

TRADITIONAL ETHNIC USES:

SANTAL: (i) plant: dysuria, sores, tumours; (ii) leaf: in burns; (iii) seed: in adenitis,

fistula ani, intercostal neuralgia, pleurisy, pneumonia, prolapsus ani; MUNOA :

aqueous extract of seed: to women after childbirth; IRULA, KOTA, TOOA (Nilgiri) :

seed: in menstrual complaints; RURAL FOLKS: Aqueous extract of seed: in urinary

troubles and kidney stone.


23

CHARAKA SAMHITA: seed: useful in piles, hiccup, abdominal lump, bronchial

asthma, in causing and regulating perspiration; SUSHRUTA SAMHITA . seed powder:

useful in stopping excessive perspiration;BAGBHATTA: seed: useful in

spermatocalcali (Shukrashman); CHAKRADATTA : decoction of seed: beneficial in

urticaria; RAJANIGHANTU : beneficial in piles, colic, epistasis, flatulence,

ophthalmia, ulcer.

AYURVEDA: decoction of seed: useful in leucorrhoea, menstrual troubles, bleeding

during pregnancy, colic caused by wind, piles, rheumatism, heamorrhagic disease,

intestinal worms; seed powder: antidiaphoretic; seed (in combination with milk):

work as anthelmintic, soup prepared from seeds is beneficial in enlarged liver and

spleen.

SIDDHA: seed: used in preparing a medicine named Kollu (Botanical survey of

India).

SOME MEDICINAL VALUES OF DOLICHOS ARE AS FOLLOWS:

i) The aqueous extracts of Dolichos biflorus L. seeds are used to cure urinary

troubles, acid peptic disorder (gastritis), constipation, sun-burn, kidney stone,

female diseases (leucorrhoea, menstrual troubles, bleeding during pregnancy,

post partum excessive discharges), colic caused by wind, piles, rheumatism,

hemorrhagic disease, intestinal worms etc. (Laskar et al., 1998; Mishra, 2006;

Pati and Bhattacharjee, 2013).

ii) As per “Charak Samhita”, the seed are useful for the cure of piles, hiccup,

abdominal lump, bronchial asthma, in causing and regulating perspiration. It is

also mentioned in the “Sushruta Samhita” that the seed powder is useful in

stopping excessive perspiration. It contains essential amino acids having great

nutritional value.

iii) The seeds are anthelmintic, astringent, diaphoretic, diuretic, emmenagogue,

expectorant, febrifuge, ophthalmic and tonic. They are useful in tumours,

scrofula, bronchitis, heart diseases, nephrolithiasis, urolithiasis, worms,

splenomegaly, cough, asthma, strangury, hiccough, ophthalmopathy, fever,

urticaria and rheumatoid arthritis (Mishra, 2006).


24

iv) If the water in which horse gram had been soaked for the whole night (and is

mashed in the same water in the morning) is taken daily, taken twice then it

cures "stones". Drinking semi liquid solution of horse gram powder cures

flatulation.

v) Seeds are used to cure stomach pain, the pain of dry piles.


25

IV. VIGOUR, VIABILITY, LONGEVITY AND GERMINATION OF SEEDS:

In the history from ancient times, human beings appreciated the truth of this

quotation from the Bible “Care with seeds, joy with the harvest”. So people from

the prehistoric age understood the necessity of preserving seeds for better plant

growth and crop yield.

„Lives crop up from the grains, and the grains evolve out of the plants which

are even produced by way of sprinkling water from time immemorial‟. This practical

knowledge is also theoretically marked in the first half of sloka no. 14 of adhyaya no.

3 of Srimadbhagavad Gita: “annad bhavanti bhutani parjanyad annasambhavah”.

Which is again pronounced in Indian Sanskrit Literature: “Yatha vijam tathankurah”

i.e. like seed like shoot. So the very motto of yielding seeds for the sake of vitality

reigns over the human civilization forever. Thus, our ancient literature laid special

emphasis on seeds – the basic input in agriculture for higher productivity of crops.

Seed vigour is simply defined as relative potential of seeds and seed viability

can be defined as the ability of seeds to germinate under favourable environmental

situation. However, seed vigour and viability are two interrelated phenomena and

generally loss of vigour precedes loss of viability (Anderson and Baker, 1983;

Karivartharaju, 1990; Basu, 1994). The vigour of seeds has been interpreted and

defined in a number of ways. An accepted definition of vigour is „the sum total of

those properties of the seed which determine the level of activity and performance of

the seed or seed lots during germination and seedling emergence‟ (Chin, 1988).

Despite the fact that the concept is still vague and requires the indexing of many

components, vigour is well appreciated by researchers and those involved in the seed

trade. Vigour measurements by tetrazolium test and assessment of quality seeds by a

number of reliable physiological and biochemical parameters (Tekrony, 2001;

Phartyal et al., 2003; Verma et al., 2003) can be used for predicting the potential

performance in the field. Vigourous seeds will produce more uniform, vigourous

stands of plants resulting finally in higher yield per hectare. Thus, the degree or level

of viability of a seed lot can be considered as "seed vigour" or in other words vigour is

the potential status of viable seeds.


26

Seed viability is the capacity of a seed to germinate under favourable

conditions provided that any dormancy in the seed is broken before testing

germinability. In a standard germination test of a seed lot in which individual seeds of

the lot may be either alive or dead, the germination percentage would give a

quantitative measure of viability. However, two seed lots of the same cultivar with

identical germination percentage may differ in their average rates of the germination

and seedling growth, tolerance to stress conditions, field emergence and often their

subsequent performance in terms of biological and agricultural yield. The question of

seed vigour assumes significance under such situations. But seed vigour, though

understood in essence, it is difficult to define and still more difficult to quantify.

Following the underlying principles in the definitions proposed by Perry (1978) and

McDonald (1980), we may look upon vigour as the quality of the seed responsible for

rapid and uniform germination, extended storability, good field emergence and ability

to perform well over a wide range of edaphoclimatic conditions. The storability factor

is indeed a very important aspect of seed vigour and in the absence of data on field

performance, may serve as a very useful index of vigour.

Because of empirical nature of the vigour concept, quantification of the vigour

has been difficult. Stress tests like the cold test of maize would distinguish between

high and low vigour seed and indicate their relative planting values under specific

situation. Conductivity of seed leachate, activities of certain enzymes and controlled

ageing test would also do the same but still fail to quantify vigour of a seed lot as is

available for its viability level.

An excellent vigour test was made by Basu and his collaborators (Basu et al.,

1990; Sur and Basu, 1990; Basu, 1994) in which high vigour jute (Corchorus olitorius

L.) seed (bioassay material) was germinated along with wheat (Triticum aestivum L.)

seeds of different vigour levels (stock materials) in separate air tight containers: Each

and every seed lot or even a single seed differ in vigour level from other lots of seeds.

Seeds of different sizes, colour, position, variety and maturity show differences in

vigour and are affected by various factors which may be genetic, physiological,

morphological, mechanical and microbial. Faulty harvesting, processing and

transportation can seriously affect seed vigour, differences in vigour as affected by


27

various factors are not often detected by ordinary germination test. In fact, specific

vigour tests are needed to determine field stand potential.

1. Test for viability:

The various methods used to determine seed viability other than the seed

germination test.

a. Assays for viability:

Barton (1961) discussed several methods for testing seed viability. One of the

earliest attempts to assess viability of fresh seeds was the “cutting test”. Seeds were

cut in to half to determine if they were empty or full. This test, of course, revealed

nothing about deterioration in the embryo. The “excised embryo technique” was more

a means of circumventing the problem of dormancy by removing a hard or

impermeable seed coat and thus allowing germination to proceed. “Plasmolysis”, or

the shrinking of the protoplasm from the cell wall in the presence of solutions such as

saturated sugar or 2 M potassium nitrate (KNO3), has not been used extensively

owing perhaps to the skill required in preparation and in the microscopic

determination of germination values. Enzyme assays, staining techniques,

conductivity tests and X-ray analysis are all commonly used in attempting to assay

viability.

b. Staining techniques:

Since the 1940s, numerous staining techniques have been developed to

measure seed viability. Several organic dyes including indigo-carmine, acid violet,

neutral red, methylene blue, Bismark Brown, Cresol red, orange G, Congo red and

malachite green have been used (Barton, 1961).

The use of tetrazolium to assay seed viability is second only to the standard

germination test. Originally developed by Kuhn and Jerchel (1941), the technique was

quickly adopted and perfected by Lakon (1949). Little more need be said on the utility

of this seed assay method. The use of the X-ray contrast method of viability testing

was developed by Simak (1957). It has been used almost exclusively for tree seeds.

Seeds are soaked in a heavy metal solution [usually barium chloride (BaCl2)] then X-


28

rayed with a soft X-ray. Seeds which are damaged or have lost their membrane

integrity take up the salt and thus, show a shadow on the radiograph.

c. Conductivity measurement:

Use of conductivity measurements of seed leachates to determine seed

viability is best of the fundamental work of Osterhout (1922) who established the

relationship between cell death and release of electrolytes. Using this technique, Fick

and Hibbard (1925) studied the relationship between seed viability and electrical

conductivity and were able to obtain a correlation between these two variables.

As with the tetrazolium test, conductivity measurement has been widely used

as a vigour test (Association of Official Seed Analysts, 1983; 1984).

d. Enzyme tests:

Many enzymes have been studied in an attempt to correlate seed viability or

vigour with the amount of enzyme activity (Barton, 1961; Macleod, 1952;

Woodstock, 1973). Catalase has been a popular enzyme to assay (Baldwin, 1935).

Other enzymes which have been assayed in seeds included diastase (White,

1909), peroxidase (Mc Hargue, 1920), phenolase (Davies, 1931), malic

dehydrogenase (Throneberry and Smith, 1955), alcohol dehydrogenase (Throneberry

and Smith, 1955), cytochrome oxidase (Throneberry and Smith, 1955) and glutamic

acid decarboxylase (Linko, 1961).

2. Seed storage:

Maintenance of high seed germination and vigour after harvest until planting

is of utmost importance in a seed storage programme. Good seed storage facility is,

therefore, a basic requirement in seed programme.

In the temperate countries year round chilling or cool atmospheric temperature

helps to store the seeds under ambient condition for longer period. But in tropical and

subtropical countries like India seeds of many crops (specially vegetables, flowers,

forest plants etc.) lose viability quickly due to high atmospheric temperature and

humidity. Suitable temperature and moisture content conditions are to be manipulated


29

during storage of seed to minimize loss of viability. Indigenous facilities available to

the common farmers need modification or substitution to sustain the essential quality

standards of seed up to planting. Seed is stored for:

(i) “Short term” for about 6 months from one harvest to next sowing,

(ii) “Medium term” for 18 to 36 months to overcome crop failure, natural

hazards, fluctuations in price and market demand, and

(iii) “Long term” for 5 to 50 years to preserve germplasm and breeder stock.

Various factors which affect seed viability and germinability in storage should be

avoided by carefully attending to the following aspects:

(i) Seed should be stored in dry and cool place.

(ii) Before storing, seeds should be dried to safe moisture limit.

(iii) Only high quality seed should be stored, i.e. thoroughly and properly

processed seed with high germination and vigour.

(iv) Proper sanitation should be maintained in seed stores.

(v) Seeds should be properly protected in storage against diseases, insect pests

and rodents.

Factors affecting seed longevity in storage: The maintenance of vigour and

germinability of seed in storage depends on the following factors:

(i) Genetic effects: Genes influence seed storage life. Long storability of seed is

dominant over short storability. Good storability in male or female parent of pearl

millet as dominant trait was transmitted to hybrid seeds. The influence of female

parent appeared to be predominant in storage indicating the role of cytoplasm in

controlling storability. Germplasm specificity for storability of variety/kind of the

seed is remarkable. Some kinds are naturally short lived and have very little shelf life,

e.g. onion, soybean, groundnut; while cotton and wheat seeds can be stored for


30

medium period; other seeds e.g. rice, beans etc. are long lived and can be stored for

longer period.

(ii) Seed quality: Vigorous, healthy and undamaged seed with high

germinability can be stored for long period. Storability or shelf life depends on the

extent of severity of damage due to weathering, mechanical injury etc., even when the

seed lot has good germination.

(iii) Provenance: Seed samples originating in different places show difference

in storability. Red clover seeds grown in Canada required 4 years compared to 3 years

required by seeds grown in England and New Zealand to deteriorate to 80%

germination when stored under ambient condition of Cambridge, U.K.

(iv) Moisture content: The amount of moisture in the seeds is probably the

most important factor influencing storability. The deterioration rate increases as the

seed moisture content increases through moisture regain during storage.

Seeds with more than 13% moisture content deteriorate very rapidly due to

mold growth. Very low moisture (below 4%) may also damage seeds due to extreme

desiccation, or cause hard seededness in some kinds. The safe moisture content varies

depending on storage length, type of storage structure, kind/variety of seed and type

of packaging material used. For cereals, in ordinary storage conditions for 1 to 1.5

years, seed moisture content of 10% appears quite right. However, for storage in

sealed container, drying up to 5 to 8% moisture content may be necessary depending

on the kind/variety.

(v) Relative humidity and temperature: Seeds attain a specific and

characteristic moisture content when subjected to a given level of atmospheric

humidity. This is known as Moisture Equilibrium Content (MEC). MEC for a

particular kind of seed at a given RH tends to increase as temperature decreases and

deterioration progresses. This is possible as the seeds are highly hygroscopic

(Yanping et al., 2000).

Relative humidity (RH) is a measure of water vapour in air relative to the

amount that air can hold at saturation at a given temperature. With the increase in air


31

temperature the water holding capacity of air also increases. If the absolute weight of

moisture remains constant, RH decreases on heating, the reverse will be true if the air

is cooled.

Roberts (1972) developed a formula to describe the relationship between

temperature, seed moisture and period of viability. From those relationships it was

possible to construct a seed viability Nomograph for different crops. These are helpful

in predicting the retention of seed viability in a defined storage environment for a

particular period or to determine combinations of temperature and moisture content

which will ensure the retention of a desired level of seed viability for a specified

period.

(vi) Effect of gas during storage: Regardless of the kind of seeds or the

atmosphere in the sealed container, only adequately dried seeds (about 5% moisture)

are able to retain their germination well during long periods of storage. The added

advantage, if any, of any gas other than air is not realized except during very long

period of storage for 40 to 50 years. Oxygen is generally harmful for seed storage at

seed moisture content of about 10% but it is beneficial for storing if seed moisture

content is sufficiently high (more than 25%) for repair mechanisms to operate. Heat

injury to kidney bean embryos decreased in reduced oxygen pressure and that the

application of cystein overcome the injury to some extent. Starch phosphate is very

effective in prolonging the viability of onion and okra seeds and alpha tocopherol had

some beneficial effect on onion seeds.

(vii) Storage conditions:

(a) Very high moisture content, extreme desiccation of seed, very high RH or high

temperature of storage atmosphere impairs storability.

(b) Bacteria and fungi prevalent in storage are completely inactive below 62% RH

and have very little activity below 75% RH, while above 75% RH the growth of fungi

in seed often shows as exponential relationship with increase of RH. The storage

bacteria require at least 90% RH for activity and growth. Certain organisms can grow

at temperature as low as 8oC and others at temperatures as high as 80

oC. Since high

temperatures rapidly decrease seed viability, the practical method of controlling


32

microfloral activity is by deep freezing. There is no satisfactory chemical control

method.

(c) Insects and mites are inactive at seed moisture content below 8%. But if grains are

already infected, increased activity may appear up to about 15% moisture content.

The important storage insects become more active around 28o to 38

oC. The

temperature below 22o to 17

oC are unsuitable for insect activity. By

fumigation/contact insecticide application insects and mites can be controlled to some

extent. But often chemicals have adverse effect on seed viability or vigour and some

of these are dangerous to handle.

(viii) Storage loss in transit: Adequate precautions should be taken by proper

packaging and careful handling so that the rate of deterioration is very low during

transit.

(ix) Seed structure: The presence or absence of glumes (lemma and palea) in

grasses influence their life-span. Husk, chaff or both have inhibitory effect on the

growth of mold and thereby help to increase the life span of cereal seeds during

storage. Oats and timothy grass seeds had a longer life span when stored with glumes

intact compared to machine hulled caryopses.

Types of storage for different end use: Types of storages related to end use and

period of storage are as follows:

(a) Foundation, stock and enforcement seed: Foundation and stock seeds

may be stored for several years to minimize genetic drift caused by multiplication in

field. The Enforcement seed samples should be kept for a year or more with high

germination percentage. These specifications call for much better seed storage

conditions. Storage area requirement is small as the seed quantities to be stored are

not large. To maintain high germinability for 3 to 5 years, the storage area should

have 25% RH at 30oC or less or 45% RH at 20

oC or less. This is possible by making

the room moisture proof by using a dehumidifier and arrangement for ambient to

subambient temperature control. Alternately, small seed lots may be stored in

moisture proof metal boxes or polyethylene bags (7 mm thick) maintained at proper

RH by a desiccant (e.g. silica gel). Since these containers will be opened from time to


33

time, the desiccant will have to be redried at intervals. If necessary, refrigeration may

be used to maintain very low temperature.

(b) Commercial seeds: About 75 to 80% of the seed produced are stored for

about 6 to 8 months from harvest for the next planting time of the crop. The

requirement for storage are relatively simple if the seeds are harvested and stored

during winter months. When storage is required during rainy season, the storage

conditions and containers should be such that seeds should be well protected against

moisture regain. The general preconditions are that the:

(i) seeds must be clean, free from trash which may otherwise harbour insects

and micro-organisms and prevent free circulation of air,

(ii) seeds should be undamaged to minimize decline in germination and

vigour,

(iii) must be dried to less than 14% moisture content for starchy and less than

11% for oily seeds,

(iv) storage containers should not be steel bins which when exposed to the sun

in a hot climate would damage the seed viability, and

(v) adequate pest control measure should be taken.

(c) Carry over seeds: Generally, remaining 20 to 25% of seeds are carried over

in storage through one growing season for the next sowing time. Thus the storage

period is about 16 to 20 months. In dry and cool areas, seeds to be stored can maintain

high germinability even in ambient condition. However, in warm humid areas seed of

cotton, soybean, onion, may flower and tree species deteriorate rapidly and the

storage requirements for such area and for these types of crop are:

(i) the store house should be made as cool as possible by constructing false

ceiling with ventilation between ceiling and roof and by insulation of thick

stone or brick wall and ceiling. Ventilating fans are also useful,

(ii) seeds should be dry and should not be allowed to come in contact with

damp floor. Storage in steel bins with tight fitting lids or in a moisture


34

proof bag will only be useful if the seed moisture content is low enough

for sealed storage.

(d) Germplasm seeds: Germplasm seeds for the gene bank are to be kept for

very long periods. Long term storage requires the coldest temperature (5o to –10

oC)

with 20% to 25% RH. The stored samples should be dried to proper moisture level.

Safe storage: A good seed store should have one door but no window. The

entrance should be about 90 cm above the ground. There should be rat-proof lip

around the building at about 90 cm height extending out to 20 cm. Such construction

prevents entrance of rats through the walls unless there are cracks in the foundation.

The godown must be rain-proof, relatively moisture/vapour proof and insect proof.

There should be no cracks in wall or on floor. The seed bags should not be kept

directly on the floor, but on wooden pallets and should be at least 50 cm away from

the walls. An exhaust fan may be fixed for ventilation when outside temperature is

lower than the seed store, but RH of the outside air should also be kept in view while

planning to ventilate the seed store. If ventilation is used carefully, it can reduced both

temperature and seed moisture. Bags should be stacked only on pallets with at least 10

cm open space for air movement and minimum 20 to 30 cm open space between

stacks and wall. Germination percentage and health of stored seed should be checked

regularly. Fertilizers, feeds, fuels etc., should not be stored with seed. Stored should

be kept clean, free from insects and pests by fumigation, seed treatment, space spray,

poison baits and other appropriate methods.

The thumb rule proposed by Harrington (1972) is useful:

(i) for every decrease of 5.6oC (10

oF) in storage temperature (between 0

o to

50oC) the life of seed doubles and

(ii) for every decrease of 1% in seed moisture content (between 5 to 14%)

the life of the seed doubles.

3. Seed germination:

Seed retains total biological potentiality of the parent plant at a threshold level

of biological function indicated by minimal respiration rate. The cells of seed


35

maintain living condition at the lowest level of moisture content. Such a seed contains

the inactive quiescent or dormant embryo. The activation of this embryo to its

morphological differentiation and polar growth at the radicle and plumule end is

known as germination. Germination is the beginning of growth and development of

the dormant embryo. It involves various changes till the embryo develops into a

seedling.

The development process of germination starts when the dry seed regains

moisture. With the intake of water (imbibition) the cells of the seed turn turgid and

physiologically active. These two phases can be defined physiologically as the steps

for resumption of active metabolism in all the seed parts including the embryo. This is

followed by the third phase i.e. triggering of the embryonic cells for growth and

differentiation. Cell growth starts with the activation of maturation promotion factor

(MPF) through the regulated function of cell division cycle genes.

The germination of seeds is an external manifestation of the process of

conversion and utilization respectively of the metabolites and enzymes in the

endosperm/storage tissue for cell multiplication and formation of new structures in the

embryo. The formation of the new structures takes place as a result of protein, lipid

and carbohydrate metabolism, which is controlled by genes. The accumulation of

energy in the form of ATP and other energy rich compounds is made possible by

oxidative phosphorylation in mitochondria. Therefore, a study of physiological

activities associated with structural changes in nuclei, mitochondria and ribosomes of

cells of embryo could provide important information not only for understanding the

processes taking place at the time of seed germination; but also defining those

morphological deviations which are observed in plants developing from seeds

subjected to different kind of treatments, immature embryos or embryos injured

through biotic or abiotic stresses.

Hence study of several external factors of the seed environment and internal

factors which control the regime of physiological-genetical functions for seed

germination is imperative.


36

Factors influencing germination:

External factors

(a) Water

(b) Temperature

(c) Light

Internal factors:

(a) Food and some phytohormones

(b) Resting period

(c) Viability

Agronomic and other factors:

(a) Preharvest seed production, seed harvesting and storage

(b) Special treatments

(c) Germination and ecology

(d) Germination and salinity

Changes during germination:

Both viable and nonviable dry seeds absorb water by imbibition. The imbibition

process involves.

(i) diffusion of water by dry cell wall of the seed coat; and

(ii) osmotic intake by the living cells.

During imbibition the following events occur sequentially:

(i) rapid water uptake by biocolloids in dry seeds;

(ii) reactivation of pre-existing macro-molecules and organelles; and

(iii) acceleration of respiration rate resulting ATP formation which provides

energy for synthesis of substrates.


37

Dry seeds must absorb water in fair proportion before the beginning of germination.

Water requirement on the basis of dry weight of seed is 40% in maize and 70% in

beans. This is followed by changes at the tissue and cell level.

(a) Changes at morphological level: The transformation of the quiescent embryo

into a seedling after imbibition of water follows a sequence of morphological

events.

(b) Changes at physiological level: Water uptake triggers physiological activity by

increasing the rate of respiration and reverting the cell infrastructure to activated

state, activating the lipo-protein membrane to promote translocation of enzymes

capable of degrading the stored carbohydrate, protein and lipid molecules. This

initiates the primary steps of cyto-differentiation and influences the activation of

silenced genes. The growth and differentiation at the target site of quiescent

embryo begins which is expressed as the development of embryo during

germination through the sequestered activity of the genes which remained

predominantly repressed in the genome before germination. Increased activity of

protoplasm of seed cells is supplemented by increase in respiration rate

providing energy for the synthesis and mobilization of early enzymes involved

in digestion of stored food to functional substrates at target sites. The

metabolism in germinating seeds is amphobolic. It is catabolic due to

degradation of reserve compounds to substrates for utilization in germination

process and early growth of seedling and anabolic due to production of

structural and functional proteins for the biogenesis of various organelles and

synthesis of new cells and tissues. During germination major catabolic functions

are located in storage tissue, i.e. the endosperm or the cotyledon; whereas true

anabolic activity takes place in embryo. During seed formation the metabolites

were translocated to the reserve tissues and converted into complex molecules as

reserve food.

(c) Changes at cell layer: Young cells of embryo rapidly developing to maturity

contain mitochondria with many cristae, dictyosomes with abundant vesicles

and ribosomes frequently in polysomial aggregates in Secale cereale at one or

two weeks after anthesis. In the dry mature embryo in seed the mitochondria


38

have few cristae and few dictyosomes and compact polysomes. Lipid bodies

remain distributed throughout the activated cytoplasm but in dry state these are

packed against the plasmalemma. During germination, the ultra-structural

changes in cell take place after water imbibition and uptake by the embryonic

cells. The rough compact and concentric layers of endoplasmic reticulum

proliferate to form many surrounding circlets preceeded by the increase in

respiration as well as number of mitochondria and number of cristae per

mitochondria. Autoradiographic studies revealed that protein synthesis increased

prior to initiation of the early cell divisions for embryo development. Electron

microscopic studies showed that membranes were scarce in mitochondria of

dormant seeds, but are well developed in germinated seed.

The aleurone layer was responsible for digestion of galactomannan for

carbohydrate mobilization during germination of Trigonella foenumgraecum,

Trifolium incarnatum and Medicago sativa. During the period of active

digestion of galactomannan the aleurone grain contents disappear and the cells

become vacuolated. This indicated that enzymes responsible for degradation of

galactomannan were translocated to storage cells at early stages of germination.

In fact, residual pool on enzymes in dry seeds remained in active state as found

in heat killed seeds. The lypo-protein membrane control translocation from

storage cell to the target. As the germination process proceeds with the

triggering and mobilization of stored molecules, the protein, carbohydrate and

lipid contents are progressively depleted in the storage cells with the

concomitant progress in development and growth of embryo.

(d) Changes at biochemical level: Germination is indicated by sequential reactive

changes taking place in the cells of seeds. Oxidative and hydrolytic functions are

triggered through activation of synthesis and mobilization of enzymes

concerned. Gibberellic acid have been found to play a vital role in activation and

initiation of the function these enzymes. This is followed by enzymatic

degradation of stored reserves. The pattern or sequence of events controlling the

hydrolysis of these reserves may vary from species to species (Bewley and

Black, 1994).


39

Phytic acid is the main phosphate reserve in many seeds. Phytase hydrolyses

the phytin to release phosphates which activate the protoplasm. Starch as amylose or

amylopectin is degraded generally to glucose, α- and β-maltose, and dextrin by α-

amylase and β-amylase. Lipase degrades the lipids to glycerol and fatty acids. The

fatty acids are rapidly degraded and converted to carbohydrate; while glycerol is

utilized directly as substrate for respiration or translocated to cotyledons and

embryonic axis. Degradation of protein reserves by protease liberates amino acids.

The amino acid pool thus formed is translocated to embryo axis for synthesis of

proteins. The cells of bean cotyledons are filled with starch and proteins, those of

soybean with oil and proteins. The germs (embryo, including scutellum or the single

cotyledon) of wheat and maize contains much oil and is rich in protein, but the

endosperm is largely starchy. The stored food in date palm and carrot seeds is

hemicellulose. Organic and inorganic phosphors present during germination are also

important for the transfer of energy for growth.

Whether a seed is albuminous or exalbuminous, the pattern of mobilization of

simple metabolites derived through degradation of food reserves are quite different in

different species of angiospermous seeds. The differences indicate the possible

genetic control of the amphibolic physiological functions. Reserve food mobilization

during germination of cereal seed is a regulated function initiated by the residual

hormone reserved in the seeds. The pattern of mobilization, therefore, is a sequential

pathway of utilization of stored carbohydrates and proteins. In case of non-cereal

seeds, however, the pattern of mobilization of food stored in endosperm or cotyledon

may be different. Experimental results obtained from detached cotyledon or

endosperm of non-cereal seeds have the drawback of incorporating some injury to

storage tissues, unlike that of cereals. This may give rise to deviations from the

interpretation derived from whole seed. It is evident that availability of oxygen is the

most important factor in initiating food mobilization during germination. However,

the picture will be clear to some extent by examining specific cases of the pattern of

food mobilization in respect of protein rich legume seeds and lipid rich oil yielding

seeds (Das, 2008).


40

V. MODULATION OF PLANT GROWTH, METABOLISM AND PLANT

PRODUCTIVITY:

Role of plant growth regulators on modulation of plant growth, metabolism as

well as productivity have been well documented since the discovery of various plant

growth regulators (Cathey, 1964, 1975; Abou-Zeid et al., 1978; Ben-Gad et al., 1979;

Bhattacharjee et al., 1986; Lama, 2000; Bhar, 2011). It is needless to mention that

growth promoters like IAA, GA3, kinetin etc. can potentially enhance plant growth as

well as metabolism (Taiz and Zeiger, 2010; Hopkins and Hüner, 2009) and growth

retardants like CCC; 2, 4-DNC; morphactins; alar; MH etc. induce suppression of

stem elongation with formation of bushy habit of plants (Cathey and Stuart, 1961;

Humphries, 1963; Weaver, 1972; Bhattacharjee et al., 1986). From the analysis of the

sensitivity of the wide range of plant species towards retardants, it seems that dicot

species are more responsive than monocot ones (Krishnamoorthy, 1981). To ensure

effective growth modulation different methods of PGR application have been

practiced viz. foliar application, soil drench application, seed soaking or even by

injection. (Sterrett, 1979; Kanp, 2007). However, each treatment has its merits and

demerits. Literature also revealed that the time of application with respect to specific

growth stages of plants plays a vital role on PGR-induced modification of growth and

metabolism (Maurer, 1976; Kamp and Nightingle, 1979; Bhattacharjee, 1984; Pati,

2007). Guardia et al. (1974) reported that height reduction was resulted in sunflower

by application of CCC, SADH and ethrel. Again, inverse effect of gibberellin and

AMO 1618 on growth and some enzyme activities have been reported (Halevy, 1962).

Effect of NaDK on the alteration of plant growth, metabolism and productivity has

been studied by a number of workers ( Bocion et al., 1975; Heild et al., 1978; Orson

and Kofranek, 1978; Bhattacharjee and Gupta, 1984a; 1984b; Kanp, 2007).

Hormonal regulation of productivity by manipulation of source and sink systems as

well as by deferral of senescence have been well documented in the literature (Knypl,

1967; Warieng and Patrick, 1975; Bhattacharjee, 1984; Dolui, 2008, Bhar, 2011).

Thus, it is quite apparent from the literature that plant growth regulators play a

pivotal role on desired modulation of growth, metabolism as well as productivity.


41

VI. PHYTOCHEMISTRY OF THE EXPERIMENTAL PLANTS:

Abrus precatorius:

With regard to the phytochemistry of the plant, following chemical constituents

are reported to be present in different parts of Abrus precatorius:

LEAVES:

The leaves which are sweet in taste contain up to 10% Glycyrrhizin, triterpene

glycosides pinitol and alkaloids such as abrine, choline and precatorine. Other

compounds of the leaves are tritepenes abrusgenic acid, abruslactone A and methyl

abrusgenate and flavonoids vitexin, liquirtiginin-7-mono- and diglycosides and

toxifolin-3-glucosides (Ghosal and Dutta, 1971; Daniel, 2006; Solanki and Zaveri,

2012).

ROOTS:

Root contains glycyrrhizin and alkaloids like abrasine and precasine besides

abrine and related bases.

SEEDS:

The seeds yield alkaloids, steroids, flavonoids, and anthocyanins. The

alkaloids of the seeds are abrine, hypaphorine, choline and precatorine. The oil

content of seed is only 2.5%, which is rich in oleic acid and linoleic acids.

Stigmasterol, abricin, and cholesterol are the steroids present. The colour of the seed

is due to glycosides of abranin, pelargonidin, and delphinidin. Lectines are the chief

constituents of the seeds, the principal ones being abrin. Lectins are both toxic (abrin)

and non toxic (Abrus agglutinin). Abrins are denoted by abrin a, b, c and d and consist

of one large polypeptide chain and short polypeptide chain joined by disulphide bond

(Lin et al., 1971; Herrmann and Behnke, 1981; Daniel, 2006, Prathyusha et al. 2010;

Anand et al., 2010).


42

Dolichos biflorus:

Chemical constituents of Dolichos seeds are reported to be as follows:

SEEDS:

In Dolichos, seeds are the major storehouse of the bioactive compounds.

These are Urease, strepogenin, β-sitosterol, genistein, 2-hydroxygenistein,

dalbergioidin, kievitone, phaseollidin, isoferreirin, coumesterol, psoralidin, phyto-

haemagglutinins, β-N-acetyl glucosaminidase, α & β-galactosidases, α -mannosides,

β-glucosides, 5-hydroxy-7,3'4'-trimethoxy-&methyl isoflavone-5-neohesperidoside,

D-glucose, D-galactose, L-rhamnose, D-arabinose and L-ascorbic acid and amino

acids viz., glycine, alanine, cysteine, serine and aspartic acid (Mishra, 2006).

LEAVES:

Leaves contain genistein, 2'-hydroxygenistein, dalbergioidin, kievitone,

phaseollidin, coumesterol, psoralidin, lectin like glycoprotein (leaves and stem);

dolichin A and dolichin B (Mishra, 2006).

Materials and Methods

43

MATERIALS AND METHODS

Experiments of the present investigation were carried out with the viable and

healthy seeds two medicinal plants Abrus precatorius L. which was collected from

wild sources and Dolichos biflorus L. which was procured from the local market of

Burdwan. In this investigation, the chemical manipulative agents like indole acetic

acid (IAA), gibberellic acid (GA3), sodium dikegulac (NaDK) and chlorocholine

chloride (CCC) were selected after an initial screening experiment.

Keeping mind the ethnomedicinal value of both Abrus and Dolichos species,

an attempt was made aiming at the augmented productivity of the experimental plants.

However, emphasis was given on the four plant growth regulator (PGR)-induced

manipulation of seed viability, plant growth, metabolism and productivity of the

plants as well as analysis of phytochemicals present in the seeds of experimental

plants.

The methodologies used in the present investigation are given as follows:

EXPERIMENT NO. I

Scarification, germination behaviour and TTC stainability of Abrus seeds:

(Tables 1-3)

In Abrus seeds, scarification was done by immersing healthy seeds at different

concentrations of H2SO4 (0, 5, 10, 20, 25 and 30 percent) for different duration (10,

20, 30 and 40 min) prior to surface sterilization of the seed samples using 0.1% HgCl2

for 90 seconds (Ojha and Bhattacharjee, 2012). Percentage germination of seeds was

recorded after 48, 96, 144, 192 and 240 hours of seed soaking in distilled water.

(Table 1)

After optimization of germination in 25% H2SO4 for 30 minutes, Abrus seeds

were presoaked with IAA, GA3, NaDK and CCC or distilled water for 24 hours.

Percentage germination and T50 values of seeds were recorded after 10 days of seed

soaking in distilled water.

Percentage seed germination: To analyse the percentage germination, the individual

seed lots in four groups of 100 seeds i.e. 400 seeds of each treatment were transferred


44

to separate Petri dishes containing filter paper moistened with 10 ml distilled water.

Germination data were recorded after 10 days of seed soaking following the

International Rules for Seed Testing (ISTA, 1976). (Table 2)

T50 values (time in h required for 50% germination) of germination: The time for

50% germination of seeds (T50) was determined following the method described by

Coolbear et al. (1984). (Table 2)

AGEING TREATMENT (NATURAL AGEING):

Abrus seed samples were allowed to undergo natural ageing under ambient

climatic conditions in the laboratory for 36 months. Data on germination behaviour,

field emergence capacity, TTC stainability and biochemical changes were recorded up

to 36 months at 9-months intervals of seed storage. Thus, altogether five observations

were made at 0, 9, 18, 27 and 36 months after seed storage. After natural ageing

treatment of Abrus seeds following experiments were undertaken (Tables 3-11):

Seed germinability: Method for recording seed germinability in terms of percentage

germination was already mentioned as per ISTA, 1976. (Table 3)

Field emergence capacity: For determining field emergence capacity, seeds were

sown in the research field of Burdwan University, containing well prepared soil

mixed with farmyard manure. After 20 days of sowing emergence of plumule above

the soil was considered as field emergence index of germinating seeds. This method

was followed after Halder (1981). (Table 3)

T50 values (time in h required for 50% germination) of germination: Method for

determining T50 values have already been mentioned. This was done as per the

methods of Coolbear et al. (1984). (Table 4)

TTC stainability: For analysing TTC-stainability, Abrus seeds (100 Nos.) of each

treatment were allowed to imbibe 0.5% TTC (2, 3, 5-triphenyl tetrazolium chloride)

solution (w/v) in Petri dishes for 24 h in dark condition. The percentage TTC-stained

(red coloured) seeds were calculated from the total number of seeds of each treatment.

This method was adapted essentially after Halder (1981). (Table 4)


45

Free amino acids (from seed leachate): Free amino acid levels from the seed

leachates of each treatment and each ageing period were analysed after immersing 10

g seed sample of Abrus in 100 ml distilled water for 24 h. From the leachate stock,

free amino acid level was quantified following the method of Moore and Stein (1948)

modified by Bhattacharjee (1984). Test tubes containing 1 ml seed leachate and 3 ml

of 0.1% ninhydrin solution (in 80% ethanol) were kept in a water bath for 15 min with

glass marbles at the top of the test tubes. When the reaction mixture turned to violet

colour, the test tubes were taken out, cooled and volume was made up to 4 ml with

80% ethanol. The absorbance of the solution was measured at 580 nm by UV-VIS

spectrophotometer. The quantitative estimation was made by comparing the optical

density (O.D.) values from a standard curve prepared from glycine. (Table 5)

Soluble carbohydrates (from seed leachate): Sampling procedure of soluble

carbohydrates from seed leachates was the same as done in case of leachable amino

acids, and from the same leachate stock, soluble carbohydrate level was determined

following the method of McCready et al. (1950) with slight modification. For

quantitative measurement of sugar, 1 ml of the seed leachate from each treatment and

each ageing period was taken in a test tube after necessary dilution and to it added 3

ml freshly prepared, precooled, 0.2% anthrone reagent (200 mg anthrone dissolved in

100 ml concentrated H2SO4). After 30 min, the intensity of green colour in terms of

O.D. was measured by UV-VIS spectrophotometer at 610 nm. Actual quantity was

evaluated from a previously prepared standard curve with glucose. (Table 5)

Soluble carbohydrates (from seed kernels): For analysis of soluble carbohydrate

levels from seed kernels of Abrus, 100 mg seed kernel of each sample was thoroughly

homogenized in a mortar with pestle using 5 ml 80% boiling ethanol. After

centrifugation at 6000 g for 10 min the filtrate was taken as the source of soluble

carbohydrates. One ml sample was taken from the stock solution and quantitative

analysis was done using 0.2% anthrone reagent as mentioned earlier. (Table 6)

Insoluble carbohydrates (from seed kernels): For the analysis of insoluble

carbohydrates, the same residue after centrifugation of the sample (vide soluble

carbohydrate extraction from seed kernel) was digested with 5 ml 25% H2SO4 (v/v) at

80°C in a water bath for 30 min. The extracted material was taken as a source of


46

insoluble carbohydrates. For quantitative measurement 1 ml of extracted sample was

taken in test tubes after necessary dilution and insoluble carbohydrates level was

determined with 0.2% anthrone reagent as per the method described earlier. (Table 6)

DNA and RNA (from seed kernels): Extraction of nucleic acids (DNA and RNA)

was done from 100 mg seed kernel as per the method described by Cherry (1962).

Estimation of both DNA and RNA were analysed from a common stock where the

sample was finally extracted with 5% perchloric acid. An outline of extraction

procedure of nucleic acids (DNA and RNA) is as follows:

Plant tissue (100 mg)

↓

Homogenized in 5 ml chilled methanol

↓

Centrifuged at 5000 g for 15 min

↓

Supernatant Residue

Methanol-soluble phosphate Extracted with 5 ml 0.2 M perchloric acid

(discarded) ↓


↓

Supernatant Residue

Acid-soluble phosphate Extracted with 5 ml ethanol

(discarded) ↓


↓

Supernatant Residue

Ethanol-soluble phosphate Extracted with 5 ml ethanol:ether (2:1) at 50oC for 30 min

(discarded) ↓


↓

Supernatant Residue

Lipid-soluble phosphate Extracted with 5 ml 5% perchloric acid at 70oC for 40 min

(discarded) (refrigeration overnight aids in clarification)

↓


↓

Supernatant Residue

Taken for nucleic acids source (both DNA & RNA) (discarded)


47

Estimation of DNA: one ml of the nucleic acid extract in a test tube was mixed with

5 ml freshly prepared diphenyl amine reagent (100 ml glacial acetic acid (BDH, AR)

+ 2.7 ml conc. H2SO4 + 1 g AR grade diphenyl amine). The mixture was boiled in a

water bath for 30 min with a glass marble at the top of the test tube. After cooling in

running tap water, intensity of blue colour was measured spectrophotometrically at

610 nm. DNA content was quantified from the O.D. values of a standard curve

prepared with herring sperm DNA. (Table 7)

Estimation of RNA: Three ml each of seed kernel extract (in 5% perchloric acid) in

separate test tubes was treated with an equal volume of freshly prepared orcinol

reagent (1 g AR grade orcinol dissolved in 100 ml concentrated HCl containing 0.1%

FeCl3, 6H2O), and boiled in a water bath for 20 min with glass marbles at the top of

the test tubes. The mixture was then cooled, and the intensity of the blue green colour

was measured at 700 nm in the spectrophotometer according to the method of

Markham (1955) modified by Choudhuri and Chatterjee (1970). The blank used

contained a mixture of 3 ml distilled water and 3 ml orcinol reagent which was treated

in an identical manner. RNA level was calculated from a standard curve prepared with

yeast RNA. (Table 7)

Protein (from seed kernels): Samples for protein were taken from Abrus seed

kernels of each treatment and each ageing period. Seed kernels (100 mg) were

homogenized in a mortar with 80% ethanol and centrifuged at 6000 g for 10 min. To

make the pellet free from phenol, it was washed successively with 10% (w/v) cold

trichloroacetic acid (twice), ethanol (once), ethyl alcohol: chloroform (3:1, v/v, once),

and finally with solvent ether as per the method of Kar and Mishra (1976). The pellet

was then evaporated to dryness. The protein was solubilised by treating 0.5 (N) NaOH

at 80°C for 1 h. A definite volume (4 ml) was made with the extraction medium. It

was then estimated by reacting the protein solution with Folin phenol reagent and

measuring the OD values at 650 nm according to the method of Lowry et al. (1951).

Quantitative determination was made by comparing the OD values with a standard

curve previously prepared using bovine serum albumin (BSA, Fractin-V, Sigma

Chemical CO., USA). (Table 8)


48

Dehydrogenase activity (from seed kernels): To analyse dehydrogenase (total)

activity 1 g Abrus seeds of each treatment were immersed in 0.5% TTC solution in

test tubes and incubated for 12 h in dark. The hydrogen atoms released by the total

dehydrogenase enzymes which are involved in the respiration process of living

tissues, reduce tetrazolium to red coloured formazan (Moore, 1973). The TTC-stained

1 g embryonal axes of the seeds of each treatment was extracted with 5 ml of 2-

methoxyethanol for 24 h and OD values of the solutions were recorded at 520 nm.

The activity of total dehydrogenases of intact seeds was analysed by the reaction of

tetrazolium chloride according to the method of Rudrapal and Basu (1979). (Table 9)

Catalase activity (from seed kernels): Five hundred mg seed kernel of each

treatment were homogenized with 8 ml of chilled 0.1 M phosphate

(Na2HPO4/NaH2PO4) buffer (pH 6.5). The homogenate was centrifuged at 3000 g for

15 min followed by 10,000 g for 20 min in cold condition. The volume of the

supernatant was made up to 10 ml with the same buffer, and this was used as crude

enzyme source. The enzyme activity was determined following the method of Snell

and Snell (1971) modified by Biswas and Choudhuri (1978). The reaction mixture for

catalase consisted of 1 ml of the above extract and 2 ml 0.05 M H2O2, incubated

together at 370C for 2 min. The reaction was stopped by adding 2 ml 0.1% titanium

sulphate in 25% H2SO4 (v/v), and the mixture was centrifuged at 6000 g for 15 min.

The intensity of the golden yellow colour was measured at 420 nm. The blank was

prepared by inactivating (heat killed) enzyme with the addition of titanium sulphate

prior to H2O2 addition. (Table 9)

Peroxidase activity (from seed kernels): For assay of peroxidase 500 mg Abrus seed

kernel were homogenized in cold 10 ml 0.05 M sodium phosphate buffer (pH 6.5).

The homogenate was centrifuged at 10,000 g for 15 min. The filtrate was used as

enzyme source.

Activity of this enzyme was assayed following the method of Kar and Mishra

(1976) with slide modifications. Five ml of the assay mixture containing 1 ml 300

mM of sodium phosphate buffer (pH 6.8), 2 ml 50µM H2O2, 1 ml 50µM catechol and

1 ml of crude enzyme extract were incubated for 30 min. After incubation at 25°C for

5 min, the reaction was stopped with addition of 1 ml of 10% H2SO4. Then


49

purpurogallin (Golden yellow colour) was read at 430 nm in UV-VIS

spectrophotometer. (Table 10)

Amylase activity (from seed kernels): It was estimated following the method

described by Khan and Faust (1967) with necessary modification. Seed kernel (500

mg) of each sample was homogenized with 10 ml of 0.1 M phosphate buffer (pH 6.5).

The homogenate was centrifuged at 5000 g for 15 min. The supernatant was taken as

the crude source of the enzyme. One ml of the enzyme solution was mixed with an

equal volume of 0.1% starch solution in 0.1 N sodium acetate buffer, pH 5.0 and

incubated at 370C for 10 min. The reaction was stopped with 3 ml iodine-HCl solution

(600 mg KI and 60 mg I2 in 100 ml of 0.05 N HCl). The blank was prepared after

inactivating the enzyme with 3 ml iodine-HCl solution prior to addition of starch. The

intensity of blue colour was measured at 620 nm. (Table 10)

IAA-oxidase activity (from Abrus seed kernels): Extraction of this enzyme was

made from 100 mg seed kernel crushed with 12 ml of cold 0.2 M sodium phosphate

buffer (pH 6.1). The homogenate was centrifuged at 10,000 g for 15 min. The

supernatant was taken as the crude source of the enzyme. The activity of IAA-oxidase

was assayed following the method of Gordon and Weber (1951) as modified by

Ramadas et al. (1968). The reaction mixture contained 1 ml 1 mM 2, 4-

dichlorophenol, 1 ml 1 mM MnCl2, 6 ml of 0.03 M sodium citrate buffer (pH 4.5) and

1 ml of enzyme extract. This was incubated for 1 h at 350C temp. and then the

reaction was stopped by pouring 1 ml of 20% HClO4 to the reaction mixture. One ml

of the assay mixture was reacted with 3 ml of Salkowski reagent (50 ml 35% HClO4 +

1 ml 0.5 N FeCl3), and the reading was taken at 525 nm in a spectrophotometer.

(Table 11)

Protease activity (from seed kernels): Extraction procedure of this enzyme was the

same as that of catalase except that the pH of the buffer solution used was 6.5.

Protease activity was measured by incubating the reaction mixture consisting of 1 ml

enzyme extract, 0.1 ml 0.1 M MgSO4, 7H2O and 1 ml BSA [(0.5 mg/ml dissolved in

distilled water)] for 1 h at 370C followed by adding 1 ml 50% trichloroacetic acid

(TCA) and subsequent analysis of residual protein by Folin-phenol reagent (Lowry et.


50

al., 1951). This assay procedure was followed as per the modified method of Biswas

and Choudhuri (1978). (Table 11)

In each case of enzyme assay, value at zero time was taken as blank, and the

activity of each enzyme was expressed as [( A×Tv)/(t × v) × g wt. of tissue], where

A is the OD value of blank OD minus sample OD, Tv is the total volume of the

filtrate, t is the time (hour) of incubation with the substrate and v is the volume of

filtrate taken for incubation (Fick and Qualset, 1975).

EXPERIMENT NO. II

Chemical manipulation of germinability, TTC stainability and metabolism of

Dolichos seeds stored under both accelerated and natural ageing condition

(Tables 12-20)

AGEING TREATMENTS:

i) Accelerated ageing:

Accelerated ageing treatment was given only on Dolichos seeds along with

parallel natural ageing. Five seed lots (200 g each) pretreated with IAA, GA3, NaDK

and CCC or distilled water were taken in separate porous cloth bags and thus stored in

separate desiccator in which an environment of 99.5% relative humidity (RH) was

artificially imposed by keeping 250 ml 1.57% H2SO4 within it (Maity et al., 2000;

Das et al., 2003; Chakrabarti, 2003; Kanp 2007). This experimental set-up was kept at

32+20C allowing the seeds to experience forced ageing treatment for 60 days and

H2SO4 was changed at 7-day intervals to restore the desired RH within the desiccator

throughout the experimental period of 60 days.

From the seed lots of each sample some physiological and biochemical

analyses were made after 0, 20, 40 and 60 days of accelerated ageing treatment.

ii) Natural ageing:

This experiment was carried out under natural environmental conditions. This

experiment was performed using freshly harvested Dolichos seeds. Seed samples

were allowed to experience natural ageing under ambient storage situation in Plant


51

Physiology and Biochemistry Laboratory at Department of Botany, Burdwan

University.

Method of surface sterilization of seeds and pretreatment procedure using the

test chemicals were the same as mentioned earlier. Subsequently all the seed lots (200

g for each treatment) were taken in separate porous cloth bags and thus stored in

separate desiccator. This experimental set up was kept in laboratory under ambient

environmental situation.

The experimental set up was kept for 24 months. Seeds were taken out from

desiccators and some physiological and biochemical analyses were made after 0, 8, 16

and 24 months of natural ageing. In this experiment, water-soaked seeds served as the

‘Control’ set.

After accelerated and natural ageing treatment of Dolichos seeds, following

experiments were undertaken:

Seed germinability and field emergence capacity: To analyse the seed germination

percentage of Dolichos seeds stored under both the ageing treatments was done as

mentioned earlier following the method ISTA, 1976. (Table 12)

In case of field emergence capacity study, method was followed as before

(Halder, 1981). (Table 12)

T50 values of germination and TTC stainability: Method for determining T50 values

have already been mentioned. This was done as per the methods of Coolbear et. al.

(1984). (Table 13)

For analysing TTC-stainability, Dolichos seeds (100 Nos.) of each treatment

were allowed to imbibe 0.5% TTC (2, 3, 5-triphenyl tetrazolium chloride) solution

(w/v) in Petri dishes for 24 h in dark condition. The percentage TTC-stained (red

coloured) seeds were calculated from the total number of seeds of each treatment.

This method was adapted essentially after Halder (1981). (Table 13)

Free amino acids and soluble carbohydrates (from Dolichos seed leachate): Free

amino acid levels from the seed leachates of each treatment and each ageing period


52

were analysed following the method of Moore and Stein (1948) modified by

Bhattacharjee (1984) have already been mentioned earlier. (Table 14)

Leaching of soluble carbohydrate levels from Dolichos seed was determined

following the method of McCready et al. (1950) with slight modification. (Table 14)

Soluble and insoluble carbohydrates (from seed kernels): Soluble carbohydrate

levels from seed kernels of Dolichos, was determined as per the method of McCready

et al. (1950) with slight modification. (Table 15)

Determination of insoluble carbohydrates from Dolichos seed kernels was

done following the method described earlier as in case of Abrus. (Table 15)

Protein and nucleic acid (DNA and RNA) contents from seed kernels: For

analysis of protein as well as nucleic acid contents from Dolichos seed kernels in both

the natural and accelerated ageing treatments was same as mentioned before. (Tables

16, 17)

Enzyme assay from seed kernels of Dolichos was done following the methods

as previously described in case of Abrus. (Tables 18, 19 and 20)

EXPERIMENT NO. III and IV

i) Chemical modulation of phenophases of Abrus and Dolichos:

In this experiment, the changes of the inception of different phenological

phases/ onset of the major events in the life cycle of Abrus (Table 21) and Dolichos

(Table 24) plants were done.

ii) Analysis of some growth variables of Abrus and Dolichos:

Root and shoot length of Abrus:

Optimally scarified (25% H2SO4 for 30 minutes) Abrus seeds were presoaked

with the aqueous solution of the chemicals or distilled water for 6h and then sun dried.

The seeds were then allowed to germinate in the experimental field for establishment

of plants. Root and shoot lengths of plants, raised from the treated seeds, were

recorded from 30 and 60 days old uniformly grown plants. (Table 22)


53

Root and shoot length of Dolichos:

Seeds were presoaked with the aqueous solution of the chemicals or distilled

water for 6h and then sun dried. The seed samples were then separately allowed to

experience accelerated ageing treatment (99.5% RH, 32±2°C temperature) in

desiccators for 45 days. The seeds were then allowed to germinate in the experimental

field for establishment of plants. Root and shoot lengths of plants, raised from the

treated seeds, were recorded from 30 and 60 days old uniformly grown Dolichos

plants. (Table 25)

Fresh and dry weight of Abrus:




of plants. Fresh and dry weights were analysed by weighing five uniformly growing

plants and weighing the same plants after oven drying (80OC for 10 days)

respectively. (Table 23)

Fresh and dry weight of Dolichos:





field for establishment of plants. Fresh and dry weights were analysed by weighing

five uniformly growing plants and weighing the same plants after oven drying (80OC

for 10 days) respectively. (Table 26)

STATISTICAL ANALYSES

All the data in this investigation were statistically analysed at the treatment

and replication levels (Panse and Sukhatme, 1967). In tables LSD (least significant

difference) values (at 5% level) were incorporated. In figures SEM (standard error of

means) values were presented.


54

EXPERIMENT NO. V

Methods for yield attributes of Abrus:

Optimally scarified seeds were presoaked with the aqueous solution of the

chemicals or distilled water for 6h and then sun dried. Pretreated and sundried seeds

were then sown in the experimental field for plant establishment. Foliar treatment was

given on the 50 and 100 days old Abrus plants which were raised from the pretreated

seeds. Yield attributes data viz. pod number per plant (Fig. 1), pod volume (Fig. 2),

seed number per pod (Fig. 3) and 1000 seed weight (Fig. 4) were recorded from five

uniformly growing plants of each treatment.

EXPERIMENT NO. VI

Methods for yield attributes of Dolichos:

In case of Dolichos, seeds were presoaked with the aqueous solution of the

chemicals or distilled water for 6h and then sun dried. Pretreated and sundried seeds

were then sown in the experimental field for plant establishment. Foliar treatment was

given on the 20 and 40 days old Dolichos plants which were raised from the

pretreated seeds. Yield attributes data viz. pod number per plant (Fig. 5), pod volume

(Fig. 6), seed number per pod (Fig. 7) and 1000 seed weight (Fig. 8) were recorded

from uniformly growing plants of each treatment.

EXPERIMENT NO. VII

Qualitative phytochemical analysis of Abrus and Dolichos seeds:

A preliminary phytochemical analysis of aqueous and methanolic extracts of

untreated Abrus and Dolichos seeds was done using specific reagents as described by

Sofowara (1993) and Harborne (1973). Methods are as follows:

Test for alkaloids (Using Mayer’s reagent): Dried seed extract was heated on a

boiling water bath with 2% HCl. After cooling, the mixture was filtered and treated

with a few drops of Mayer‘s reagent. A white yellowish precipitate or formation of

turbidity indicates the presence of alkaloids. Alternatively, to the solution of extract


55

Dragendorff‘s reagent was added and formation of orange-red precipitate or turbidity

confirms presence of alkaloids.

Test for terpenoids (Salkowski test): About 2 g of extract was boiled in 25ml of

dehydrated alcohol for 15 minutes and after cooling 5ml of this alcoholic extract was

mixed in 2ml of chloroform and 3ml of conc. H2SO4 was carefully added to it to form

a layer. A reddish brown coloration of the interface was formed to show positive

results for the presence of terpenoids. In case of triterpenoids the color is reddish pink.

Test for phenols (Ferric Chloride test): Test extract was treated with 4 drops of

alcoholic FeCl3 solution. Formation of bluish black colour indicates the presence of

phenol.

Test for tannins: Small amount of extract was boiled in 20 ml of water in a test tube

for few minutes and then filtered. 0.5 ml of filtrate was diluted with 1 ml of water and

1-2 drops of 0.1% aqueous FeCl3 solution was added and observed for brownish

green, blue black or green black coloration. Blue black color was observed for gallic

tannins and green black color for catecholic tannins (Iyengar, 1995).

Test for anthraquinones: A little powder is shaken with 10ml of FeCl3 solution,

mixed with 5ml of 10% HCl and heated on a water bath for 10 minutes. After

filtration and cooling, the filtrate is extracted with 10ml CCl4. The organic layer is

separated, washed with 5ml of water and shaken with 5ml of dilute ammonia

solution. A cherry red precipitate is obtained. It confirms the presence of

anthraquinone derivatives.

Test for flavonoids: A small amount of extract was heated with 10 ml of ethyl

acetate in a water bath for 3 min. The extract was filtered and 4ml of each filtrate

were shaken with 1 ml of dilute ammonia solution. An yellow colouration was

observed indicating a positive test for flavonoids (Harborne, 1973).

Test for saponins (Foam test): A small amount of extract was taken in a test tube

and shaken vigorously with small amount of aqueous sodium carbonate solution. A

stable honey comb like foam indicated the presence of saponins.


56

Test for glycosides: Glycosides are compounds which upon hydrolysis give rise to

one or more sugars (glycones) and a compound which is not a sugar (aglycones). In

case of plant extracts, 5ml of extract was treated with 2ml of glacial acetic acid

containing one drop of ferric chloride solution. This was underlayed with 1ml of

concentrated sulphuric acid. A brown ring at the interface indicates deoxysugar

characteristics of cardenolides. A violet ring may appear below the brown ring in the

sulphuric acid layer, while a greenish ring may form in the acetic acid layer (Keller-

Killani test).

EXPERIMENT NO. VIII

Phytochemical analysis by using IR-Spectra, HPLC and MALDI-TOF MS study

of Abrus and Dolichos seeds:

IR-spectra (KBr discs, 4000-400 cm-1

) of untreated Abrus and Dolichos seed

powder were recorded by using a Perkin-Elmer FTIR model RX1 spectrometer

(Tables 27, 28 and Figures 9, 10).

HPLC analysis of the methanolic extracts of untreated seeds of Abrus (Fig. 11)

and Dolichos (Fig. 14) was done by using Agilent 1100 series HPLC (with the flow

rate 1ml/min, 80:20 Methanol: Water, C18 column, Signal 280nm).

Finally, Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight Mass

Spectrometry (MALDI-TOF MS) analysis of the eluted samples of HPLC

chromatogram of both Abrus and Dolichos was done by using Voyager- DE Pro

MALDI TOF MS instrument for detection of the compounds (Figs. 12, 13 in case of

Abrus precatorius and Figs. 15, 16 in Dolichos biflorus).

57

PLATE 1

Fully matured seeds of Abrus precatorius after harvest

Top view of H2SO4 -scarified Abrus seed under SEM Close-up view of hilar point of scarified seed

58

PLATE 2

Unscarified Abrus seed under SEM Scarified Abrus seed showing cracked region

Unscarified Abrus seed at a portion of hilum Scarified Abrus seed at the strophiolar region

59

PLATE 3

Scarified Abrus seed without GA3 treatment Scarified followed by GA3 treated germinating seed

Abrus sapling raised from scarified Abrus plant showing initiation of floral bud

followed by GA3 treated seed

60

PLATE 4

Inflorescence of Abrus plant Early pod initiation from flowers

Developing pods in clusters Splitting of pods showing attached seeds

61

PLATE 5

Pods in clusters in untreated plants Pods in dense clusters in NaDK treated plants

Fully mature seeds of NaDK treated plants Worker holding a pod at departmental plot

62

PLATE 6

Fully desiccated seeds still in pods (above); fully desiccated seeds separated from pods (below)

63

PLATE 7

Freshly harvested seeds of Dolichos biflorus

Soaked seeds prior to inception of germination Germinating seeds showing radicles

64

PLATE 8

A few on field germinating seeds (left); Field emergence of seeds (right)

Seedling emergence with a pair of leaves Worker holding trailing plant upright

65

PLATE 9

Close up view of a mature Dolichos plant

Axillary flowers (right) and development of tender pods from leaf axil (left)

66

PLATE 10

Plant with tender pods of untreated plant (above) and that of NaDK treated plant (below) of same

age.

67

PLATE 11

Drying pods of NaDK treated senescing plants (above) and single enlarged pod from the same

plant (below).

Results

68

RESULTS OF EXPERIMENT NO. I


Abrus seeds (Tables 1-11).

Table 1: A screening experiment was done for optimization of scarification technique

using appropriate percentage of H2SO4 and exact duration of treatment with the

scarifying agents for maximum germinability. Percentage germination of Abrus seeds

was recorded using different concentrations (0, 5, 10, 15, 20, 25, and 30 percent) of

H2SO4 for different durations (10, 20, 30 and 40 minutes). Data were recorded at 48 h

intervals up to 240h. Results showed that maximum germination was found at 25%

H2SO4 treatment for 30 minutes and maximum germination percentage was recorded

after 240 hours of seed soaking.

Table 2: In table 2 the most potent plant growth regulator (PGR) and its most

effective concentration for seed germinability as well as T50 hours (time in hour

required for 50% germination) was shown using the optimally scarified Abrus seeds.

Results clearly revealed that in Abrus, scarification followed by GA3 (500 μg ml-1

)

treatment exerted the best response for stimulating seed germination. Percentage

germination was significantly less than control in both the retardant treatments i.e.

CCC and NaDK. However, both the growth promoters IAA and GA3 enhanced seed

germination in comparison to control samples. T50 values were found to be

significantly less in GA3 (500 µg ml-1

) in comparison to control and other treatments

(1AA, NaDK and CCC), and 50% seed germination was not at all achieved in all such

treatments irrespective of their concentrations.

Table 3: Seed germinability and field emergence capacity of the scarified followed by

PGR-treated Abrus seeds were shown in table 3. Results showed that significant

reduction of percentage germination with concomitant decrease of field emergence

capacity were found in seeds which underwent ageing treatment, and the later

observations (27 and 36 month of ageing) were found to be much more remarkable.

Results

69

Table 4: All the PGR treatments except GA3 caused nonattainment of 50%

germination irrespective of the various durations (0, 9, 18, 27 and 36 months) of

natural ageing. However, in GA3 treatment at least 50% germination occurred

although the T50 values were recorded to be higher and these increased with the

increase of ageing period. TTC stainability of Abrus seeds started decreasing with the

progressive increase of natural ageing. However, all the PGRs, tended to increase

TTC stainability over control values. The magnitude of TTC staining percentage was

found to be maximum in GA3-treated seed samples.

Table 5: Both amino acid and soluble carbohydrate levels increased in Abrus seed

leachates at all the treatments as well as in control samples with the advancement of

ageing months. However, extent of leaching was found to be significantly less in all

the treatments, and slowing down of leaching of the soluble substances was much less

in NaDK-treated seed samples.

Table 6: Data on the PGR-induced changes of soluble and insoluble carbohydrates in

Abrus seed kernels were found to be distinctly reverse of each other. In fact, insoluble

carbohydrate showed a steady state decrease with the increase of ageing months with

concomitant increase of the soluble carbohydrate in Abrus seeds.

Table 7: In this table nucleic acid contents (both DNA and RNA) showed a

progressive decreasing trend in seed samples which experienced natural ageing for 36

months in all the treatments. The ageing-induced reduction of both the nucleic acid

levels was found to be partially alleviated in all the PGR treatments. The alleviatory

effect of the treatments was found to be most promising in NaDK-treated seed

samples.

Table 8: As regards the changes of protein content an identical trend, as found in case

of nucleic acids (table 7), was recorded in the experimental plant growth regulators.

Here also, NaDK-induced changes of proteins were found to be most significant.

Table 9: The activities of dehydrogenase and catalase were found to decrease with

natural ageing for 0 to 36 months. The PGRs averted the alarming loss of the

activities of the beneficial enzymes over control samples. The plant growth retardants

Results

70

NaDK and CCC were promising over the growth promoters 1AA and GA3 in this

regard.

Table 10: The changes of the activities of peroxidase and amylase were found to be

diametrically opposite. Data clearly showed that peroxidase activities decreased with

the progress of seed ageing while amylase activities started increasing. In case of

peroxidase, the magnitude of changes were maximum in NaDK-treated seed samples

and in case of amylase, GA3- induced increase of the activities was most significant.

Table 11: As regards the changes of IAA oxidase and protease activities almost an

identical trend was recorded regardless of the treatments and ageing period. The

ageing- induced gradual enhancement of the deleterious enzymes was potentially

alleviated by the growth retarding chemicals NaDK and CCC.

Tables

71

TABLES OF EXPERIMENT NO. I

Table 1: Influence of acid scarification with different concentrations of H2SO4 for

different duration on percentage germination of Abrus precatorius seeds.

Seeds were pretreated with different concentrations (0-30%) of H2SO4 for

different durations (10-40 min) on percentage germination of Abrus seeds recorded at

48h intervals up to 240h.

H2SO4

(Percent) Treatment

duration

(min)

Percentage germination after hours of seed soaking

48 96 144 192 240

0

10

20

30

40

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

5

10 0.0 0.0 0.0 0.0 0.0

20 0.0 0.0 0.0 0.0 0.0

30 0.0 0.0 0.0 0.0 0.0

40 0.0 0.0 0.0 5.0 6.5

10

10 0.0 0.0 0.0 0.0 10.0

20 0.0 0.0 10.0 14.2 20.0

30 0.0 0.0 10.5 16.0 20.9

40 0.0 0.0 10.6 20.0 22.8

15

10 0.0 0.0 0.0 10.0 20.0

20 0.0 0.0 10.0 16.0 30.9

30 0.0 0.0 10.5 18.4 32.0

40 0.0 0.0 11.5 22.0 33.5

20

10 0.0 0.0 0.0 10.5 20.5

20 0.0 0.0 12.4 15.5 32.2

30 0.0 0.0 15.2 22.2 35.0

40 0.0 0.0 16.4 31.5 35.0

25

10 0.0 0.0 0.0 15.5 25.2

20 0.0 0.0 15.6 20.5 30.5

30 0.0 10.5 20.9 30.8 45.9

40 0.0 0.0 15.2 25.8 35.5

30

10 0.0 0.0 0.0 10.0 15.5

20 0.0 0.0 10.0 12.5 25.2

30 0.0 0.0 15.0 20.5 30.5

40 0.0 0.0 15.0 22.3 30.0

LSD (P=0.05) - - NC 1.20 1.26 1.33

(NC= Not calculated)

Tables

72

Table 2: Influence of seed pretreatment with the plant growth promoters (IAA

and GA3) and the plant growth retardants (NaDK and CCC) on percentage

germination (%) and T50 values (h) of Abrus precatorius seeds.

Abrus seeds were presoaked with the chemicals or distilled water for 24h after

scarifying them with 25% H2SO4 for 30 minutes and data were recorded after 10 days

of seed soaking in distilled water.

Treatments

(µg/ml)

Abrus precatorius

Percentage germination (%) T50 values (h)

Control 0

40.26

NA

IAA 100

500

43.00

NA

45.70

NA

GA3 100

500

50.33

241.6

66.50

193.5

NaDK 100

500

36.00

33.76

NA

NA

CCC 100

500

37.25

35.52

NA

NA

LSD (P=0.05) 3.70

17.20

(NA= Nonattainment of 50% germination)

Tables

73

Table 3. Influence of seed pretreatment with IAA, GA3, NaDK and CCC (500 μg

ml-1

each) on germinability (%) and field emergence capacity (%) of Abrus

precatorius seeds stored under natural ageing condition for 36 months.


with the aqueous solution of the chemicals or distilled water for 6h and then sun

dried. The seed samples were then separately allowed to experience natural ageing (in

laboratory condition) in a desiccator. Data were recorded after 0, 9, 18, 27 and 36

months of natural ageing of seeds.

Treatments

Seed germinability (%)

Natural ageing (months)

0 9 18 27 36

Control 40.5 40.0 40.0 32.4 20.1

IAA 46.8 45.0 45.0 38.6 30.4

GA3 68.6 68.0 68.0 58.8 52.5

NaDK 32.5 32.0 32.0 25.5 24.0

CCC 36.0 36.0 35.0 32.6 32.0

LSD (P=0.05) 3.20 3.15 3.08 2.35 1.98

Treatments

Field emergence capacity (%)


0 9 18 27 36

Control 38.6 38.0 38.2 30.0 17.5

IAA 45.0 43.0 43.0 35.6 27.0

GA3 68.0 66.5 66.5 55.4 50.0

NaDK 30.5 30.0 30.0 23.0 23.0

CCC 35.0 35.0 35.0 30.4 30.0

LSD (P=0.05) 2.85 2.90 2.90 2.10 1.66

Tables

74


ml-1

each) on time (hours) to 50% germination (T50) and TTC stainability (%) of

Abrus precatorius seeds stored under natural ageing condition for 36 months.

Treatments are same as in table 3. Data were recorded after 0, 9, 18, 27 and 36


Treatments

Time (hours) to 50% germination (T50)


0 9 18 27 36

Control NA NA NA NA NA

IAA NA NA NA NA NA

GA3 190.5 202.6 202.6 227.4 235.0

NaDK NA NA NA NA NA

CCC NA NA NA NA NA

LSD (P=0.05) NC NC NC NC NC

Treatments

TTC stainability (%)


0 9 18 27 36

Control 42.5 42.0 42.0 34.8 22.6

IAA 50.0 47.0 47.0 40.2 32.5

GA3 72.8 70.0 70.0 60.5 55.7

NaDK 35.7 35.0 35.0 32.5 28.2

CCC 40.2 38.0 38.0 35.6 34.0

LSD (P=0.05) 2.85 2.90 2.90 2.10 1.66

NA: Nonattainment of 50% germination, NC: Not calculated.

Tables

75


ml-1

each) on leaching of free amino acids and soluble carbohydrates (mg/g/10

ml) from Abrus precatorius seeds stored under natural ageing condition for 36

months.



Treatments

Amino acids (mg/g/10 ml)


0 9 18 27 36

Control 0.67 0.95 1.12 1.76 3.10

IAA 0.66 1.10 1.36 2.58 2.86

GA3 0.64 1.25 1.68 2.35 2.72

NaDK 0.64 0.84 1.05 1.55 1.80

CCC 0.66 0.90 1.05 1.61 2.10

LSD (P=0.05) NS 0.08 0.10 0.15 0.17

Treatments

Soluble carbohydrates (mg/g/10 ml)


0 9 18 27 36

Control 1.77 2.85 3.24 4.00 6.52

IAA 1.74 3.52 3.81 4.44 7.27

GA3 1.75 3.68 4.50 4.93 7.88

NaDK 1.72 2.00 2.25 2.84 3.90

CCC 1.75 2.16 2.42 3.13 4.26

LSD (P=0.05) NS 1.97 2.12 2.68 3.85

NS: Not significant.

Tables

76

Table 6. Influence of seed pretreatment with IAA, GA3, NaDK and CCC

(500 μg ml-1

each) on the changes of soluble and insoluble carbohydrate contents

(mg/g fr. wt.) in the kernels of Abrus precatorius seeds stored under natural

ageing condition for 36 months.

Treatments are same as in table 3. Data were recorded after 0, 9, 18, 27 and

36 months of natural ageing of seeds.

Treatments

Soluble carbohydrates (mg/g fr. wt.)


0 9 18 27 36

Control 55.6 62.4 68.2 77.5 85.0

IAA 56.0 68.2 76.3 87.6 92.5

GA3 57.3 76.4 82.8 90.2 95.8

NaDK 56.5 48.1 40.2 36.0 32.4

CCC 57.0 51.4 45.8 41.6 37.1

LSD (P=0.05) NS 4.31 3.80 3.45 3.01

Treatments

Insoluble carbohydrates (mg/g fr. wt.)


0 9 18 27 36

Control 186.5 165.2 144.6 131.8 115.5

IAA 184.8 158.2 131.2 115.3 106.2

GA3 185.0 153.4 136.6 121.5 97.0

NaDK 188.2 186.5 168.3 155.2 137.1

CCC 187.5 173.3 161.2 146.7 123.5

LSD (P=0.05) NS 12.01 10.98 10.00 9.56


Tables

77


ml-1

each) on the changes of DNA and RNA contents (µg/g fr. wt.) in the kernels

of Abrus precatorius seeds stored under natural ageing condition for 36 months.



Treatments

DNA (µg/g fr. wt.)


0 9 18 27 36

Control 92.5 73.6 61.0 53.2 41.8

IAA 91.5 78.0 70.5 59.0 48.2

GA3 92.0 76.5 66.8 57.5 45.1

NaDK 90.0 85.2 78.5 65.3 55.0

CCC 91.3 82.6 76.0 60.5 50.2

LSD (P=0.05) NS 7.26 6.05 5.10 3.98

Treatments

RNA (µg/g fr. wt.)


0 9 18 27 36

Control 210.6 185.5 154.0 131.7 115.3

IAA 212.5 189.7 161.3 135.2 122.0

GA3 211.8 193.6 166.8 141.2 127.6

NaDK 210.5 205.4 192.3 178.2 167.5

CCC 211.4 200.3 186.5 173.8 161.0

LSD (P=0.05) NS 18.37 15.05 12.96 11.05


Tables

78


ml-1

each) on the changes of protein contents (mg/g fr. wt.) in the kernels of

Abrus precatorius seeds stored under natural ageing condition for 36 months.



Treatments

Protein (mg/g fr. wt.)


0 9 18 27 36

Control 215.6 190.2 156.3 137.2 122.0

IAA 215.5 178.6 144.0 128.3 115.2

GA3 217.0 167.8 135.6 112.5 101.6

NaDK 216.5 202.0 172.5 150.5 135.2

CCC 217.6 195.6 160.3 130.6 127.5

LSD (P=0.05) NS 13.50 12.98 9.05 9.85


Tables

79


ml-1

each) on the changes of dehydrogenase (ΔOD/g wet wt./5ml) and catalase

(unit/h/g fr. wt.) activities in the kernels of Abrus precatorius seeds stored under

natural ageing condition for 36 months.



Treatments

Dehydrogenase (ΔOD/g wet wt./5ml)


0 9 18 27 36

Control 0.77 0.60 0.55 0.41 0.31

IAA 0.77 0.62 0.56 0.44 0.35

GA3 0.78 0.63 0.57 0.47 0.39

NaDK 0.76 0.69 0.61 0.54 0.46

CCC 0.76 0.65 0.58 0.49 0.42

LSD (P=0.05) NS 0.05 0.05 0.04 0.03

Treatments

Catalase (unit/h/g fr. wt.)


0 9 18 27 36

Control 68.2 60.7 54.0 49.6 41.3

IAA 67.5 61.5 55.4 51.1 45.8

GA3 68.4 62.0 56.1 52.4 47.6

NaDK 69.1 65.8 61.4 56.3 50.5

CCC 69.4 63.7 58.8 54.6 52.0

LSD (P=0.05) NS NS 4.30 3.80 4.01


Tables

80

Table 10. Influence of seed pretreatment with IAA, GA3, NaDK and CCC (500

μg ml-1

each) on the changes of peroxidase (unit/h/g fr. wt.) and amylase

activities (unit/h/g fr. wt.) in the kernels of Abrus precatorius seeds stored under




Treatments

Peroxidase (unit/h/g fr. wt.)


0 9 18 27 36

Control 96.4 75.2 66.0 57.5 40.4

IAA 95.8 78.3 69.1 58.4 51.2

GA3 96.0 81.5 73.2 62.7 56.0

NaDK 95.2 86.4 81.4 68.5 60.7

CCC 95.5 82.7 78.6 66.0 57.8

LSD (P=0.05) NS 6.42 6.00 5.66 4.88

Treatments

Amylase (unit/h/g fr. wt.)


0 9 18 27 36

Control 48.6 72.5 92.1 106.3 121.5

IAA 48.0 77.3 95.0 111.5 133.8

GA3 48.8 88.2 107.2 124.8 145.6

NaDK 48.0 61.8 78.6 87.3 100.0

CCC 48.2 66.2 83.5 96.7 108.2

LSD (P=0.05) NS 6.05 7.74 8.52 9.88


Tables

81


μg ml-1

each) on the changes of IAA-oxidase (unit/h/g fr. wt.) and protease

activities (unit/h/g fr. wt.) in the kernels of Abrus precatorius seeds stored under




Treatments

IAA-oxidase (unit/h/g fr. wt.)


0 9 18 27 36

Control 2.10 2.56 3.28 3.87 4.23

IAA 2.04 2.27 2.70 3.24 3.48

GA3 1.96 2.38 2.95 3.40 3.67

NaDK 2.00 2.13 2.43 2.85 3.10

CCC 2.02 2.20 2.56 3.08 3.28

LSD (P=0.05) NS 0.20 0.20 0.26 0.30

Treatments

Protease (unit/h/g fr. wt.)


0 9 18 27 36

Control 2.55 3.78 4.33 5.62 6.40

IAA 2.51 2.96 3.65 4.10 4.96

GA3 2.48 3.12 3.94 4.66 5.85

NaDK 2.60 2.75 3.27 3.55 5.10

CCC 2.58 2.88 3.42 3.84 5.23

LSD (P=0.05) NS 0.25 0.30 0.35 0.47


Results

82

RESULTS OF EXPERIMENT NO. II


Dolichos seeds (Tables 12-20).

Table 12: Percentage germination and field emergence capacity of Dolichos seeds,

which experienced natural as well as accelerated ageing, were shown in this table.

Results revealed that both germinability and field emergence capacity were declined

with the progress of ageing period and the magnitude of the reduction was found to be

very rapid under accelerated ageing condition. Both the growth retardants (NaDK and

CCC) averted the rapid loss of seed germinability as well as field emergence capacity

but NaDK was found to be most responsive under accelerated ageing condition and

GA3 was most responsive under natural ageing condition.

Table 13: Results on PGR- and ageing-induced changes of T50 values and TTC

stainability were represented in this table. Three (Control, IAA and GA3) seed

samples, failed to attain 50% germination in case of accelerated ageing for 60 days,

and nonattainment of 50% germination under natural ageing for 24 months was

recorded in control, NaDK- and CCC-treated seed samples. TTC stainability

proportionately decreased with the advancement of both accelerated and natural

ageing and maximum reduction occurs in control samples. The retardants and

promoters showed ageing-induced reduction TTC stainability, when compared with

control samples.

Table 14: Leaching of free amino acids and soluble carbohydrates occurred with the

progress of both accelerated and natural ageing duration. NaDK and CCC potentially

checked profuse leaching of the soluble substance particularly at later observation

periods of both the ageing processes.

Table 15: A differential result was recorded in case of the changes of soluble and

insoluble carbohydrate levels with the ageing process. Data clearly revealed that

soluble carbohydrate started increasing with the progression of both natural and

accelerated ageing process but NaDK and CCC arrested the speed of leaching. On the

Results

83

other hand, insoluble carbohydrate contents showed decreasing trend irrespective of

the samples analysed.

Table 16: Alarming fall of protein level was found in seeds which experienced

accelerated ageing treatment but the PGRs, particularly retardants, significantly

slowed down the ageing-induced reduction of protein. In natural ageing also the

retardants NaDK and CCC arrested the rapid decrease of protein level and the effect

was found to be more remarkable at later observation periods.

Table 17: Results also revealed that the PGRs more or less checked the loss of both

the nucleic acid levels, particularly RNA in kernels of Dolichos seeds under both

accelerated and natural ageing condition. However, the reduction of the nucleic acid

was found to be faster in forced ageing treatment.

Table 18: Activities of the enzymes dehydrogenase and catalase gradually decreased

with the progress of seed ageing process in all the treated and control seed samples.

However, the PGRs particularly NaDK and CCC efficiently reduced the magnitude of

reduction of the enzyme activities and the effect was found to be more significant at

later periods of observation. Results of the changes recorded at zero days after ageing

(both natural and accelerated) were insignificant.

Table 19: This table clearly shows that the ageing-induced changes of the activities of

the enzymes peroxidase and amylase are differential. In fact, peroxidase activities

were diminished with the ageing duration (natural as well as accelerated) and amylase

activities were increased with the ageing process. All the seed pretreating agents

arrested the decrease of peroxidase and increase of amylase, and the NaDK was found

to be most responsive in this regard.

Table 20: Data on the PGR-induced as well as the ageing-induced changes of the

activities of IAA oxidase and protease showed that activities of the enzymes started

increasing with the ageing periods. But the plant growth regulators slowed down the

speed of increase of the enzyme activities at least up to 60 days of accelerated ageing

and 24 months of natural ageing period. Ageing-induced alarming increase of the

enzyme activities was significantly arrested and among the PGRs, NaDK was found

to be most efficient.

Tables

84

TABLES OF EXPERIMENT NO. II


μg ml-1

each) on germinability (%) and field emergence capacity (%) of Dolichos

biflorus seeds stored under accelerated ageing condition for 60 days and natural

ageing condition for 24 months.



experience accelerated ageing treatment (99.5% RH, 32±2°C temperature) in a

desiccator and natural ageing (in laboratory condition) in a separate desiccator. Data

were recorded after 0, 20, 40 and 60 days of accelerated ageing and 0, 8, 16 and 24


Treatments

Seed germinability (%) Field emergence capacity (%)

Accelerated ageing (days)

0 20 40 60 0 20 40 60

Control 100 78.5 48.6 20.0 85.5 73.0 44.0 12.8

IAA 100 85.0 73.8 45.8 96.0 82.0 66.5 35.2

GA3 100 88.2 78.5 49.5 100 83.0 69.0 37.4

NaDK 100 93.7 85.3 55.6 95.2 83.1 76.0 47.2

CCC 100 90.8 82.6 50.0 94.2 81.2 75.1 44.4

LSD (P=0.05) NC 7.02 4.67 1.98 NS 7.10 4.14 1.10

Treatments Natural ageing (months)

0 8 16 24 0 8 16 24

Control 100 70.6 48.2 27.5 100 66.1 44.1 12.5

IAA 100 81.5 67.6 51.0 100 78.5 60.6 35.2

GA3 100 87.4 74.6 60.7 100 85.8 68.5 40.1

NaDK 100 54.8 55.5 52.5 100 49.9 51.7 30.1

CCC 100 60.6 53.2 45.6 100 50.8 52.1 32.7

LSD (P=0.05) NC 5.21 4.75 3.68 NC 4.28 4.06 1.16

NC: Not calculated; NS: Not significant.

Tables

85


(500 μg ml-1

each) on time (hours) to 50% germination (T50) and TTC

stainability (%) of Dolichos biflorus seeds stored under accelerated ageing

condition for 60 days and natural ageing condition for 24 months.

Treatments are same as in table 12. Data were recorded after 0, 20, 40 and 60

days of accelerated ageing and 0, 8, 16 and 24 months of natural ageing of seeds.

Treatments

T50 values (hours) TTC-stainability (%)


0 20 40 60 0 20 40 60

Control 32.0 82.5 135.0 NA 100 78.6 51.5 24.5

IAA 31.5 65.0 123.8 NA 100 86.0 75.5 48.3

GA3 32.6 60.2 100.5 NA 100 88.8 80.1 51.4

NaDK 32.1 50.7 105.3 125.5 100 94.5 87.2 58.2

CCC 32.0 53.8 110.6 135.5 100 92.5 85.1 55.4

LSD (P=0.05) NS 4.85 8.67 NS NC 7.70 5.08 2.23


0 8 16 24 0 8 16 24

Control 32.0 112.5 145.0 NA 100 88.1 74.1 52.5

IAA 31.5 95.0 113.8 162.4 100 90.5 70.6 65.2

GA3 32.6 88.2 128.5 150.5 100 91.8 78.5 62.1

NaDK 32.1 130.7 145.3 NA 100 95.9 85.7 70.1

CCC 32.0 123.8 138.6 NA 100 93.4 80.8 68.7

LSD (P=0.05) NS 8.21 10.58 NS NC 8.54 6.88 5.05

NA: Nonattainment of 50% germination; NC: Not calculated;


Tables

86


μg ml-1

each) on leaching of free amino acids (mg/g/10 ml) and soluble

carbohydrates (mg/g/10 ml) from Dolichos biflorus seeds stored under

accelerated ageing condition for 60 days and natural ageing condition for 24

months.



Treatments

Amino acids Soluble carbohydrates

(mg/g/10 ml) (mg/g/10 ml)


0 20 40 60 0 20 40 60

Control 0.88 2.85 3.92 5.98 2.12 5.23 7.65 9.59

IAA 0.89 2.15 3.01 3.34 2.15 5.70 7.96 10.20

GA3 0.87 2.48 3.29 3.77 2.13 6.18 8.23 11.56

NaDK 0.88 1.53 2.62 3.03 2.12 4.52 5.27 6.28

CCC 0.87 1.72 2.96 3.82 2.13 4.68 5.45 6.52

LSD (P=0.05) NS 0.15 0.29 0.30 NS 0.44 0.51 0.58

Treatments


0 8 16 24 0 8 16 24

Control 0.88 1.85 2.92 3.98 2.12 4.25 6.68 8.50

IAA 0.89 1.77 2.01 2.34 2.15 4.78 6.86 9.20

GA3 0.87 1.98 2.29 2.77 2.13 5.37 7.56 9.77

NaDK 0.88 0.98 1.31 1.88 2.12 2.82 3.90 4.63

CCC 0.87 1.12 1.58 2.12 2.13 3.01 4.25 5.22

LSD (P=0.05) NS 0.08 0.12 0.18 NS 0.29 0.37 0.45


Tables

87


(500 μg ml-1

each) on the changes of soluble and insoluble carbohydrate contents

(mg/g fr. wt.) in the kernels of Dolichos biflorus seeds stored under accelerated

ageing condition for 60 days and natural ageing condition for 24 months.



Treatments

Soluble carbohydrates Insoluble carbohydrates

(mg/g fr. wt.) (mg/g fr. wt.)


0 20 40 60 0 20 40 60

Control 58.1 73.4 88.0 98.2 208.5 188.9 102.5 66.8

IAA 57.4 69.1 78.6 90.2 210.2 166.5 115.2 86.3

GA3 57.8 71.2 84.8 95.3 209.4 155.4 95.2 60.5

NaDK 58.5 64.0 75.1 79.2 209.8 196.5 132.6 93.5

CCC 58.0 68.8 78.6 81.5 209.0 190.2 125.7 98.8

LSD (P=0.05) NS 3.88 3.95 3.22 NS 12.6 9.2 5.0


0 8 16 24 0 8 16 24

Control 58.1 65.2 73.6 83.1 210.5 186.8 132.5 110.6

IAA 58.5 68.1 76.4 87.0 209.0 187.0 155.6 122.3

GA3 58.0 70.0 82.5 93.3 208.6 188.6 162.2 132.7

NaDK 58.2 62.0 66.1 72.2 209.8 200.5 182.3 158.8

CCC 58.1 64.2 67.6 78.0 210.0 196.4 175.2 146.1

LSD (P=0.05) NS 4.52 3.88 3.02 NS 12.5 12.1 8.5


Tables

88


(500 μg ml-1

each) on the changes of protein contents (mg/g fr. wt.) in the kernels

of Dolichos biflorus seeds stored under accelerated ageing condition for 60 days

and natural ageing condition for 24 months.



Treatments

Protein

(mg/g fr. wt.)

Accelerated ageing (days) Natural ageing (months)

0 20 40 60 0 8 16 24

Control 295.4 172.6 96.5 62.8 294.5 233.4 178.7 138.6

IAA 296.0 180.7 125.9 70.2 296.4 244.1 185.2 140.0

GA3 296.8 176.2 112.5 66.7 295.0 236.3 178.5 146.7

NaDK 295.7 188.5 158.2 75.7 293.2 276.8 245.7 196.5

CCC 297.6 190.8 175.8 86.4 294.0 263.4 232.2 174.2

LSD (P=0.05) NS 16.8 9.41 5.8 NS 22.5 16.7 8.5


Tables

89


μg ml-1

each) on the changes of DNA and RNA contents (µg/g fr. wt.) in the

kernels of Dolichos biflorus seeds stored under accelerated ageing condition for

60 days and natural ageing condition for 24 months.



Treatments

DNA RNA

(µg/g fr. wt.) (µg/g fr. wt.)


0 20 40 60 0 20 40 60

Control 85.6 58.3 47.6 33.4 202.4 148.5 98.3 74.0

IAA 83.6 60.1 50.0 37.3 199.0 156.3 135.7 98.3

GA3 85.0 64.2 55.6 40.5 200.5 147.3 125.2 87.2

NaDK 86.2 76.3 65.4 58.2 198.3 168.5 148.2 115.7

CCC 87.0 73.2 58.3 49.3 201.5 161.1 137.2 98.4

LSD (P=0.05) NS 5.5 4.2 2.9 NS 13.8 9.6 6.8


0 8 16 24 0 8 16 24

Control 86.2 76.3 66.5 50.2 200.5 175.7 155.7 142.6

IAA 85.3 79.6 70.2 56.8 202.3 176.5 158.2 148.4

GA3 87.1 78.3 68.4 55.0 201.2 178.5 166.2 151.2

NaDK 86.8 82.5 76.0 61.2 199.3 187.2 176.3 166.1

CCC 86.0 80.0 74.3 60.2 203.1 182.2 171.0 160.3

LSD (P=0.05) NS 3.5 6.4 4.9 NS 10.8 14.9 14.1


Tables

90


μg ml-1

each) on the changes of dehydrogenase (ΔOD/g wet wt./5ml) and catalase

(unit/h/g fr. wt.) activity in the kernels of Dolichos biflorus seeds stored under


months.



Treatments

Dehydrogenase Catalase

(ΔOD/g wet wt./5ml) (unit/h/g fr. wt.)


0 20 40 60 0 20 40 60

Control 0.58 0.35 0.22 0.21 65.2 50.2 37.3 24.6

IAA 0.60 0.42 0.27 0.23 64.6 53.6 39.2 34.5

GA3 0.58 0.45 0.30 0.25 64.8 55.2 41.3 36.7

NaDK 0.59 0.52 0.37 0.28 65.1 60.5 53.2 46.5

CCC 0.59 0.48 0.34 0.31 63.8 58.6 48.3 41.2

LSD (P=0.05) NS 0.03 0.02 0.02 NS 5.00 3.60 2.38


0 8 16 24 0 8 16 24

Control 0.56 0.38 0.25 0.24 65.0 46.6 33.9 27.1

IAA 0.58 0.39 0.31 0.26 66.1 55.7 42.5 39.0

GA3 0.57 0.42 0.35 0.27 64.7 58.4 46.1 41.5

NaDK 0.57 0.54 0.41 0.34 65.5 61.7 56.5 51.2

CCC 0.56 0.51 0.37 0.30 64.0 59.2 49.1 50.3

LSD (P=0.05) NS 0.03 0.02 0.02 NS 4.56 3.20 2.70


Tables

91


μg ml-1

each) on the changes of peroxidase (unit/h/g fr. wt.) and amylase

activities (unit/h/g fr. wt.) in the kernels of Dolichos biflorus seeds stored under


months.



Treatments

Peroxidase Amylase

(unit/h/g fr. wt.) (unit/h/g fr. wt.)


0 20 40 60 0 20 40 60

Control 92.4 68.5 45.6 28.7 55.6 80.5 108.6 115.8

IAA 93.8 68.7 51.6 38.2 55.8 117.4 133.6 151.3

GA3 95.2 71.2 56.3 33.7 56.5 122.2 145.7 161.5

NaDK 94.0 86.8 73.3 65.5 55.8 79.5 87.2 98.5

CCC 93.5 81.7 67.8 60.3 56.0 81.2 93.5 102.4

LSD (P=0.05) NS 6.78 4.50 2.77 NS 7.85 8.52 9.78


0 8 16 24 0 8 16 24

Control 93.5 71.0 56.2 43.5 55.0 86.5 112.8 128.4

IAA 94.2 71.6 58.3 48.2 56.1 112.7 133.5 145.6

GA3 95.8 74.4 60.5 55.2 54.7 133.4 148.1 167.5

NaDK 94.0 88.1 76.2 70.4 55.5 80.0 88.3 96.8

CCC 94.5 83.6 69.1 63.3 55.2 83.2 92.1 101.3

LSD (P=0.05) NS 7.03 5.02 4.28 NS 7.83 8.56 9.45


Tables

92


μg ml-1

each) on the changes of IAA-oxidase (unit/h/g fr. wt.) and protease

activities (unit/h/g fr. wt.) in the kernels of Dolichos biflorus seeds stored under


months.



Treatments

IAA-oxidase Protease

(unit/h/g fr. wt.) (unit/h/g fr. wt.)


0 20 40 60 0 20 40 60

Control 2.03 3.78 4.65 5.23 2.46 4.25 6.88 7.95

IAA 1.86 2.83 3.24 4.15 2.00 3.18 3.91 4.88

GA3 1.91 2.71 3.13 3.98 2.13 3.24 4.00 5.60

NaDK 1.72 2.52 2.83 3.15 1.96 2.91 3.62 4.90

CCC 1.65 2.64 2.88 3.34 1.89 2.96 3.71 5.12

LSD (P=0.05) 0.15 0.24 0.27 0.30 0.18 0.28 0.35 0.48


0 8 16 24 0 8 16 24

Control 2.03 2.87 3.44 4.15 2.46 3.98 5.65 6.23

IAA 1.86 2.13 2.66 3.61 2.00 2.95 3.74 4.76

GA3 1.91 2.17 2.75 3.80 2.13 3.02 3.87 5.10

NaDK 1.72 1.97 2.28 2.96 1.96 2.63 3.65 4.82

CCC 1.65 2.01 2.37 3.08 1.89 2.70 3.78 4.95

LSD (P=0.05) 0.15 0.19 0.21 0.28 0.18 0.27 0.35 0.47

Results

93

RESULTS OF EXPERIMENT NO. III

Chemical modulation of phenophases, and some growth variables of Abrus

(Tables 21-23).

Table 21: Data on the inception of selected developmental stages of Abrus

precatorius plants have been incorporated in this table. Results showed that the

growth promoters IAA and GA3 tended to advance all the developmental stages of the

plant right from the radicle emergence to harvest. On the other hand, the growth

retardants NaDK and CCC deferred the onset of the stages and, GA3 and NaDK was

found to be most effective in manipulating the advancement and deferment,

respectively.

Table 22: Results showed that the PGRs modulated both root and shoot length of 30

and 60 days old Abrus precatorius plants. Here the growth retardants tended to reduce

both the root and shoot lengths in comparison to control samples.

Table 23: As regards the PGR-induced changes of fresh and dry weight of Abrus

seeds, all the said pretreating agents increased both the fresh and dry weight of Abrus

plants, but the magnitude of increment was found be highest in case of NaDK.

Tables

94

TABLES OF EXPERIMENT NO. III


μg ml-1

each) on changes of time (days) required for inception of the important

events in the life cycle of Abrus precatorius.

Seeds were scarified with 25% H2SO4 for 30 minutes and then treated with the

chemicals for 24h. Data were recorded from various developmental stages of the

plants, raised from the treated seeds. Average data of five uniformly growing plants

were put in the table.

Treatments

Events

Control IAA GA3 NaDK CCC

Days required for inception of events

Seed sowing

0 0 0 0 0

Radicle emergence

3.8 3.4 3.2 5.0 5.0

Field emergence

15.3 12.6 10.2 18.2 17.6

Leaf emergence

33.5 28.6 27.0 37.5 35.4

Branch initiation

45.3 42.8 40.1 49.8 47.5

Bud initiation

126.2 120.0 116.6 136.4 132.8

Flower initiation

155.0 148.2 144.8 164.2 160.6

Pod initiation

171.8 165.4 160.4 184.6 178.2

Pod maturation

188.2 175.2 170.6 195.6 191.8

Pod ripening

200.2 189.8 185.4 210.8 206.4

Senescence

225.6 216.8 210.8 236.8 230.4

Harvest

242.4

230.6 226.4 260.0 252.2

Tables

95


μg ml-1

each) on changes of root length and shoot length of Abrus plants.




of plants. Root and shoot lengths of plants, raised from the treated seeds, were

recorded from 30 and 60 days old uniformly grown plants.

Treatment

Root length (cm) Shoot length (cm)

Plant age (days)

30 60 30 60

Control 4.90 9.18 27.80 57.33

IAA 5.00 10.44 32.66 60.25

GA3 5.50 11.78 35.00 64.58

NaDK 4.82 8.78 23.87 51.40

CCC 4.85 8.96 25.30 53.74

LSD(P=0.05) 0.11 0.74 2.05 3.86

Tables

96


μg ml-1

each) on changes of fresh weight and dry weight of Abrus plants.

Treatments and recording of data are same as table 22.

Treatment

Fresh weight (g) Dry weight (g)

Plant age (days)

30 60 30 60

Control 37.90 89.18 7.80 17.33

IAA 41.50 100.44 9.50 20.25

GA3 48.50 102.78 10.77 24.58

NaDK 39.86 118.00 8.67 28.40

CCC 38.12 110.96 8.20 26.00

LSD(P=0.05) 2.56 7.66

0.65 1.45

Results

97

RESULTS OF EXPERIMENT NO. IV

Chemical modulation of phenophases, and some growth variables of Dolichos

(Tables 24-26).

Table 24: In this table, major phenological events of Dolichos biflorus were shown as

done in case of Abrus precatorius. Results revealed that life cycle of this monocarpic

medicinal pulse crop is completed at around 132 days, and the experimental PGRs

modulated the inception of all the events to some extent. Here also, the promoters

caused earliness of the events and the retardants slowed down the onset of the events,

thereby caused late appearance of the phenological phases.

Table 25: In case of Dolichos the PGR-induced regulation of root and shoot length

were recorded from the seed samples which were allowed to experience accelerated

ageing treatment for 0 and 45 days. Here, the growth retardants tended to decrease the

root and shoot length of plants raised from nonaged seeds, but the parameters were

found to increase in plants which were established from acceleratedly aged seeds for

45 days. However, the growth promoters increased both root and shoot length

irrespective of the plants, raised from aged and nonaged seeds. Again, a drastic

reduction of both roots and shoot length regardless of the samples, was recorded in

the plants, raised from the forcefully aged seeds for 45 days.

Table 26: As to the PGR-induced changes of fresh and dry weight of Dolichos seeds,

an increased trend was recorded in almost all the treatments when data were recorded

from plants which were developed from acceleratedly aged seeds, and here also both

fresh and dry weights were drastically reduced in samples due to accelerated ageing

treatment.

Tables

98

TABLES OF EXPERIMENT NO. IV


μg ml-1

each) on changes of time (days) required for inception of the important

events in the life cycle of Dolichos biflorus.

Seeds were treated with the chemicals for 24h. Data were recorded from

various developmental stages of the plants, raised from the treated seeds. Average

data of five uniformly growing plants were put in the table.

Treatments

Events

Control IAA GA3 NaDK CCC

Days required for inception of events

Seed sowing

0 0 0 0 0

Radicle emergence

1.0 0.8 0.6 2.0 2.0

Field emergence

5.1 4.5 4.0 6.1 6.3

Leaf emergence

8.2 7.7 7.1 9.0 9.5

Branch initiation

24.2 23.7 23.0 25.4 26.3

Bud initiation

46.3 45.0 44.2 49.0 47.4

Flower initiation

55.1 53.7 52.8 61.7 58.6

Pod initiation

64.3 62.4 61.0 69.1 67.8

Pod maturation

83.5 79.3 77.6 90.5 86.6

Pod ripening

95.5 91.2 88.7 107.5 103.2

Senescence

115.8 110.6 108.8 129 125

Harvest

132.4 124.5 122.1 145

140

Tables

99


μg ml-1

each) followed by accelerated ageing treatment for 45 days on changes of

root length and shoot length of Dolichos biflorus plants.





field for establishment of plants. Root and shoot lengths of plants, raised from the

treated seeds, were recorded from 30 and 60 days old uniformly grown plants.

Treatments

Root length (cm) Shoot length (cm)

Plant age (days)

30 60 30 60


0 45 0 45 0 45 0 45

Control 10.38 4.23 15.18 3.29 47.91 22.90 77.86 17.33

IAA 12.24 4.57 16.44 3.72 52.55 23.10 83.66 18.25

GA3 12.80 4.83 17.16 4.10 54.28 24.23 86.12 19.60

NaDK 8.96 6.92 13.70 5.10 44.00 25.60 73.29 22.55

CCC 10.00 6.09 14.39 4.80 45.63 24.00 75.67 20.51

LSD(P=0.05) 0.75 0.33 1.10 0.28 3.78 0.95 6.85 1.66

Tables

100


μg ml-1

each) followed by accelerated ageing treatment for 45 days on changes

of fresh weight and dry weight of Dolichos biflorus plants.

Treatments and recording of data are same as table 25.

Treatments

Fresh weight (g) Dry weight (g)

Plant age (days)

30 60 30 60


0 45 0 45 0 45 0 45

Control 52.0 19.5 298.9 90.0 9.7 3.0 62.4 20.9

IAA 56.2 19.6 315.4 95.8 10.2 3.4 67.5 22.5

GA3 58.0 21.0 324.6 105.2 11.2 3.8 68.8 22.8

NaDK 48.0 23.6 285.7 147.9 10.6 4.5 59.3 25.0

CCC 50.2 20.6 290.6 138.3 12.0 4.2 56.1 23.2

LSD(P=0.05) 3.85 1.16 22.5 8.44 0.66 0.22 4.06 1.45

Results

101

RESULTS OF EXPERIMENT NO. V

Chemical manipulation of yield attributes of Abrus (Figures 1-4).

Data revealed that pod number per individual plant (fig. 1) was increased in

samples which were developed from NaDK-treated seeds, and this increase was noted

to be still higher when seed treatment with NaDK (500 µg/ml) was followed by a

foliar treatment with the same PGR (100 µg/ml) on 50 and 100 days old Abrus plants.

This pattern of increase in seed followed by foliar treatment was recorded in all the

chemical agents, and the best response was rendered by NaDK. The PGRs also

modulated the pod volume per plant (fig. 2), and here also NaDK showed the most

covetable result. In all the treated samples, the dual treatment of the chemicals (in

seed soaking mode and foliar mode) were found to be more encouraging than single

treatment given by seed soaking mode only. However, as to the chemical-induced

modulation of seed number per pod (fig. 3) the changes were found to be least

significant or insignificant, although the retardants were found to show a little bit

increasing trend. Changes on the 1000-seed weight (fig. 4) by the PGRs were found to

be promising and here also NaDK (seed followed by foliar treatment) exerted the best

response.

Figures

102

FIGURES OF EXPERIMENT NO. V

Fig. 1: Influence of seed pretreatment with IAA, GA3, NaDK and CCC (500 μg

ml-1

each) and seed pretreatment followed by foliar treatment with the same

chemicals (100 μg ml-1

each) on the changes of pod number per plant of Abrus

precatorius.

Seeds were presoaked with the aqueous solution of the chemicals or distilled water for

6h and then sun dried. Pretreated and sundried seeds were then sown in the

experimental field for plant establishment. Foliar treatment was given on the 50 and

100 days old Abrus plants which were raised from the pretreated seeds. Data were

recorded from five uniformly growing plants of each treatment.

The symbol indicates seed pretreatment only, and the symbol indicates seed

pretreatment followed by foliar treatment.

Each bar is mean value of 3 replicates and the vertical lines on the bar represent the

standard errors of the mean.

Figures

103


ml-1



each) on the changes of pod volume (cm3) of Abrus

precatorius.

Treatments and recording of data are the same as in fig. 1.





Figures

104


ml-1



each) on the changes of seed number per pod of Abrus

precatorius.

Seeds pretreatment and recording of data are the same as in fig. 1.





Figures

105


ml-1



each) on the changes of 1000 seed weight (g) of Abrus

precatorius.

Seeds pretreatment and recording of data are the same as in fig. 1.





Results

106

RESULTS OF EXPERIMENT NO. VI

Chemical manipulation of yield attributes of Dolichos (Figures 5-8).

Data on the results of the chemical-induced changes of pod number per plant

(fig. 5) clearly showed that the PGRs particularly NaDK and CCC increased the pod

number per plant regardless of plants which were raised from seed treatment only or

seed treatment followed by foliar treatment. However, the dual treatment i.e. seed-

followed by foliar treatment was found to be more effective for increasing pod

number. Almost an identical change was recorded when the results of pod volume of

the treated samples was considered. Here also the effects of PGRs particularly NaDK

and CCC, and the dual treatments were found more pronounced (fig. 6). As regards

the chemical-induced changes on seed number per pod (fig. 7) the regulatory action of

the PGRs was found to be less prominent than the other yield attributes. However, a

little bit increasing trend was still apparent in the chemical treated samples. Figure 8

clearly revealed that 1000-seed weight was significantly increased regardless of the

treatments either at seed level or at seed followed by foliar treatment level. Here,

NaDK most efficiently triggered to increase 1000-seed weight of Dolichos when

treatment was given by two different modes i.e. seed treatment followed by foliar

treatment.

Figures

107

FIGURES OF EXPERIMENT NO. VI


ml-1



each) on the changes of pod number per plant of Dolichos

biflorus.

Seeds were presoaked with the aqueous solution of the chemicals or distilled water for

6h and then sun dried. Pretreated and sundried seeds were then sown in the

experimental field for plant establishment. Foliar treatment was given on the 20 and

40 days old Dolichos plants which were raised from the pretreated seeds. Data were

recorded from uniformly growing plants of each treatment.





Figures

108


ml-1



each) on the changes of pod volume (cm3) of Dolichos

biflorus.






Figures

109


ml-1



each) on the changes of seed number per pod of Dolichos

biflorus.






Figures

110


ml-1



each) on the changes of 1000 seed weight (g) of Dolichos

biflorus.






Results

111

RESULTS OF EXPERIMENT NO. VII

Phytochemical analysis (qualitative) of Abrus and Dolichos seeds.

A preliminary qualitative phytochemical analysis of aqueous and methanolic

extracts of Abrus and Dolichos seeds was done to have an overall idea on the

existence or non existence of some secondary metabolites like alkaloids, terpenoids,

phenols, tannins, anthraquinones, flavonoids, saponins and glycosides (vide chart 1).

In tabular representation the presence of the test secondary metabolites has been

indicated by a plus (+) sign, and the minus (-) sign indicates that the specific

secondary metabolites have not been detected by the analytical methods adopted in

this investigation.

Chart

112

CHART OF EXPERIMENT NO. VII

Chart 1: Tabular representation of qualitative phytochemical analysis of aqueous and methanolic extracts of Abrus and Dolichos seeds

Extracts Alkaloids Terpenoids Phenols Tannins Anthraquinones Flavonoids Saponins Glycosides

Abrus

(aqueous) - + + - - - + -

Abrus

(Alcoholic) - - + + - - - +

Dolichos

(aqueous) + - + + + - + -

Dolichos

(alcoholic) + + + - - - - -

The symbol ‘+’ and ‘–’ signifies the detected or non-detected of the compounds, respectively.

Results

113

RESULTS OF EXPERIMENT NO. VIII

Phytochemical analysis by using IR-Spectra, HPLC and MALDI-TOF MS study

of Abrus and Dolichos seeds.

Analysis of the functional groups of a few phytochemicals of Abrus and

Dolichos seed samples was done by Infra-Red (IR) Spectral data and the results were

represented in tables 27 and 28 and also in corresponding figures 9 and 10

respectively. Concomitantly, HPLC analysis of the methanolic extract of Abrus seed

was depicted in figure 11. Further, fraction 1 and fraction 2 of the HPLC

chromatogram of Abrus were experienced MALDI-TOF MS analysis and data

showed that the fraction 1 contains genistein and fraction 2 contains

homoisoflavonoids. Again, results of HPLC analysis of methanolic extract of

Dolichos seed was represented in figure 14 where fraction 1 showed the compound

dimer-gallate and fraction 4 showed the compound 3, 4-di-o-caffeoylquinic acid after

MALDI-TOF MS study.

Tables & Figures

114

TABLES AND FIGURES OF EXPERIMENT NO. VIII

The Infrared Spectrum of seed of Abrus precatorius was recorded in KBr disc

in 4400-400 cm-1

. Some selected frequencies of this seed are set in table 27 and the

typical spectral pattern is shown in fig. 9.

Table 27 : Infrared spectral data (in cm-1

)

Sample Frequency (in cm-1

) Inference

Abrus

precatorius

3517.88 N-H stretching of secondary amide

3310.35 O-H stretching, may be -COOH or -OH group

2913.90 C-H stretching in CH2 group

1654.00 Probable C=O stretching in secondary amide

1555.33 N-H bonding

1074.93 C-O stretching

1241.68 Probable sulphate group

4400.0 4000 3000 2000 1500 1000 400.0

-0.40

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.00

cm-1

%T

3310.35

1654.00

1555.33

1404.46

1241.68

1074.93

705.70

2913.90

3517.88

Fig. 9: IR Spectrum of seeds of Abrus precatorius

Conclusion: It is a mixture of compounds and aliphatic in nature. Some compounds

may be cyclic ones.

Tables & Figures

115

The Infrared Spectrum of seed of Dolichos biflorus was recorded in KBr disc

in 4400-400 cm-1

. Some selected frequencies of this seed are set in table 28 and the

typical spectral pattern is shown in fig. 10.

Table 28 : Infrared spectral data (in cm-1

)

Sample Frequency (in cm-1

) Inference

Dolichos

biflorus

3517.88 N-H stretching of secondary amide

3164.17 O-H stretching of aliphatic -OH group or

-COOH group

2913.90 C-H stretching in CH2 group

1651.36 C=O stretching in secondary amide

1154.34 C-O stretching in an ester

1027.29 C-O stretching in an ester

1551.36 N-H bonding of secondary amide

1241.68 Probable sulphate group

4400.0 4000 3000 2000 1500 1000 400.0

-0.20

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.59

cm-1

%T

3164.171655.95

3517.88

2913.90

1551.36

1404.46

1241.68

1027.29

1154.34

578.66

701.73

852.60

Fig. 10: IR Spectrum of seeds of Dolichos biflorus

Conclusion: Mixture of cyclic aliphatic compounds. Some compounds may show

steroidal composition.

Tables & Figures

116

HPLC and MALDI-TOF MS analysis of Abrus:

min0 2 4 6 8

mAU

0

200

400

600

800

1000

1200

1400

DAD1 A, Sig=280,4 Ref=off (22121077.D)

Fig. 11: HPLC chromatogram of methanolic extract Abrus precatorius seeds.

0 200 400 600 800 1000

Mass (m/z)

0

1.0E+4

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sity

Stitched PSD[BP = 332.0, 10014]

332.0

162.3

445.2

333.4

354.9

499.6

Fig. 12: MALDI-TOF MS analysis of fraction 1 (HPLC) and determination of

Genistein from alcoholic extract Abrus precatorius seeds.

Tables & Figures

117

0 200 400 600 800 1000

Mass (m/z)

0

1.1E+4

0

10

20

30

40

50

60

70

80

90

100%

In

ten

sity


108.5

86.9

85.0

305.6

302.7

234.0

278.8

304.2189.2235.5

276.0173.888.7 280.2

328.9 741.6261.8581.5105.1 307.1237.4193.7

145.9301.253.5

106.7 180.4 771.6263.5 320.355.8 728.0125.2 222.1163.9 266.1 326.079.9 368.940.1736.2394.5 487.1 631.9445.6 577.2

532.2 785.2

805.6 871.8


Homoisoflavonoids from alcoholic extract Abrus precatorius seeds.

Tables & Figures

118

HPLC and MALDI-TOF MS analysis of Dolichos:

Fig. 14: HPLC chromatogram of methanolic extract of Dolichos seeds.

0 200 400 600 800 1000

Mass (m/z)

0

1.2E+4

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sity


44.2

238.6

97.949.618.9

263.4332.246.1

320.2265.1 729.3210.0 287.5248.7126.2 174.0

326.396.2 135.8 560.5367.842.3

735.6397.5

492.0 566.9436.8 755.1505.3 604.7553.0 769.7687.2 726.7

794.7


Dimer-gallate from alcoholic extract Dolichos biflorus seeds

Tables & Figures

119

0 200 400 600 800 1000

Mass (m/z)

0

6231.4

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sity


66.8499.8

96.0

445.4

115.6

481.9192.3

248.1

501.124.8

124.4 332.1 517.5

249.7 466.3534.9

446.7552.1

766.0


3, 4-Di-O-caffeoylquinic acid from alcoholic extract of

Dolichos biflorus seeds.

Discussion

120

DISCUSSION

Plants have been the traditional source of raw materials and finished medicinal

products since the dawn of civilization. A rich heritage of knowledge on preventive

and curative medicine was even available in ancient scholastic work included in the

Atharva Veda, Charaka, Sushruta etc. An estimate suggests that about 13000 plant

species are known to have World wise use as drugs. Twenty five million plant species

are known for medicinal uses, phytochemical test have been performed in about 5000

species. Nearly 1100 species are extensively exploited as Ayurvedic (80%), Unani

(46%) and allopathic medicines (33%) (Das et al., 1999; Evans, 2005; Das and

Pandey, 2007). It is reported that 41% prescriptions in USA and 50% in Europe

contains constituents from natural products and the trend of using natural product is

increasing keeping in mind the least or no side effect and safe use of the natural

products due to their non hazardous property. However, unlike agricultural crops,

large scale cultivation, scientific management, appropriate processing as well as

desired productivity of most of the medicinal plants have not been done.

Thus, the present investigation is an attempt to obviate the specific problems

of two medicinally important, locally available plants i.e. Abrus precatorius L. and

Dolichos biflorus L. In fact, chemical manipulative technique is adopted which is

reported to play a vital role for overcoming some undesired features of the plants

which are the route cause for impairing productivity (Khan and Khanum, 1999; Bhar,

2011; Das et al., 1999; Pati and Bhattacharjee, 2012; 2013). After a thorough survey

work, pinpointed problems of the experimental plants Abrus and Dolichos were

identified. A venture was undertaken for overcoming the seed germinability problem

of Abrus along with ideotypic plant production in terms of profusely branched plant

with higher plant potential by employing some selected growth promoters (IAA and

GA3) and growth retardants (NaDK and CCC). These, in fact, positively influence

yield attributes which, in turn, augment productivity. In case of Dolichos species, the

seeds are of low vigour and cannot retain standard viability for a considerably long

period (Mishra, 2006). Hence, often sub-normal and week seedlings and plants are

raised from such low vigour seeds. Again, there are also reports that due to

unbalanced source-sink relationship, the productivity of Dolichos is often impaired

(Chakrabarti, 2003; Mishra, 2006).

Discussion

121

In view of some negative factors and pinpointed stumbling blocks for

impaired productivity the experiments of the present investigation are designed with

the main objective for ameliorating the negative features using some selected

hormonal agents which are reported to modulate plant type, potentiate plant health,

maintain coveted source-sink relationship and thus enhance productivity

(Bhattacharjee, 1984; Bhar, 2011).

Results, of this investigation showed that, by optimization of acid scarification

technique, the vital problem of the germinability of Abrus seeds could be partially

overcome (Table 1) and germinability can further be triggered by application of GA3

on optimally scarified seeds using H2SO4 (Table 2). In fact, seed germination of

Abrus is considered to be stubborn in nature mainly because of hard and waxy seed

coat, imperviousness of moisture or water through the seed coat and very strong

membrane integrity (Paria, 2005; Ojha et al., 2010; Ojha and Bhattacharjee, 2012).

Reports available in the literature that dormancy of hard coated seeds can be

overcome by scarification technique of different types as well as by making way for

uptake of water by optimized cracking of seeds particularly in and around hilar region

(Khan and Khanun, 1999; Copeland and McDonald, 1995; 2001). Again, GA3 plays a

pivotal role for enhancing germination of seeds mainly by regulating the promoter-

inhibitor balance in seed kernels and stimulating the activity of -amylase enzyme

(Bhattacharjee, 1984; Kanp, 2007). In this investigation, scarification-induced

improvement of Abrus seed germinability and even still higher germinability of Abrus

seeds by GA3 treatment prior to scarifying the seeds with H2SO4, clearly indicates that

the optimized scarification resulted in weakening of hard seed coat to allow entry of

water. And remarkably higher increase of germinability in GA3 treatment over the

scarified seed sample indicates that possibly GA3 could balance the promoter and

inhibitor level in Abrus seed as well as stimulated the activity α-amylase enzyme in

the seed kernels. In fact, this result is on conformity with some reported observations

(Rai, 2000; Das, 2008; Ghosh et al., 2012). Higher germination behaviour by GA3

treatment is also corroborated with a reliable index T50 values, and this value is

inversely proportional to percentage seed germination. Thus, in this experiment the

enhanced seed germination percentage associated with the lower T50 values in GA3-

treated seed samples clearly indicates the potentiality of the manipulating agent on

improving the germinability status of Abrus seeds.

Discussion

122

Results of this investigation further revealed that scarification of Abrus seeds

followed by PGR treatment enhanced field emergence capacity with concomitant

increase of seed germinability (Table 3), increased TTC stainability and at least

attained 50% germination (Table 4), arrested the alarming increase of amino acids and

soluble carbohydrates in seed leachates (Table 5). This was associated with

enhancement of insoluble carbohydrates along with reduction of soluble

carbohydrates in NaDK and CCC treatments (Table 6), enhancement of nucleic acid

levels (Table 7) and protein contents in NaDK treatment (Table 8). Concomitantly,

enzyme activities like dehydrogenase and catalase were increased in all PGR

treatments (Table 9). In peroxidase and amylase activities in seed kernels of Abrus, an

opposite trend was found (Table 10), whereas IAA oxidase and protease activities

were decreased in comparison to control values (Table 11).

Enhanced field emergence capacity in Abrus seeds was resulted due to

enhanced germinability because these two parameters are directly correlated. As

mentioned earlier that higher germination capacity was resulted due to removing or

alleviating the hard seed coat-induced barrier along with promoter-inhibitor induced

hindering factor. But NaDK- and CCC-induced inhibitory effect on germination

particularly at initial observations might be due to the inhibitory effects of the two

growth retarding chemicals NaDK and CCC. However, at the final observation period

of 36 months of ageing the retardant-induced germinability was found higher than the

control samples. In fact, there are reports that the retardants usually render an initial

inhibitory effect on many physiological processes including seed germination but the

effect cannot persist for long. Because they can harden seeds and check the seeds

against quicker deterioration under adverse storage and thus show increased

germinability over untreated samples (Bhattacharjee and Gupta, 1985; Bhattacharjee

and Choudhuri, 1986; Bhattacharjee et al. 1999; Pati, 2006; Kanp, 2007). However,

all the PGR-induced enhanced field emergence capacity was very clear from this

study. This can be explained by the role of promoters on enhancement of seed

potential as well as α-amylase stimulating property of GA3 which caused higher seed

germinability with corresponding increase of field emergence capacity. In fact,

hardening effect of the PGRs possibly enhanced seed potential by decreasing the

ageing-induced seed deterioration and this was reflected in comparatively higher

germinability as well as field emergence capacity in the NaDK- and CCC-treated seed

Discussion

123

samples. Reports on both retardant- and promoter-induced enhanced performance on

germination behaviour, field emergence capacity as well as seedling and plant

performance in the field are well documented (Bewley and Black, 1982; Copeland

and McDonald, 2001; Bhattacharjee, 1984; Das, 2008).

Failing to attain 50% seed germination in all the Abrus seed samples except

GA3 treatment (Table 4) might be due to slow speed of germination along with the

inherent stubborn nature of seed germination in spite of overcoming the problem by

scarification and hormonal management to considerable extent. GA3-induced

enhanced α-amylase activity (Table 10) possibly maintained the germination speed

and thus could show T50 values although the values are much higher.

Rapid increase of soluble carbohydrates both in seed leachates (Table 5) and

in seed kennels (Table 6) of Abrus with ageing duration might be due to deterioration

of seeds and damaging of membranes which made the seeds leaky and thus finally

soluble carbohydrate levels were increased. In fact, ageing-induced deterioration of

seeds with concomitant derangement of seed membrane is well reported (Rai, 2000;

Bhattacharjee 1984a, b; Maity et al., 2000; Mishra, 2006; Copland and McDonald,

2001).

Again, comparatively higher level of soluble carbohydrate in NaDK- and

CCC-treated seed samples may be explained by less break down of insoluble

carbohydrate due to lower α-amylase activity and maintaining membrane integrity of

seeds. There are reports in the literature that some vital macromolecules are reduced

with enhancement of seed deterioration process and thus normal functional life of

seeds/plant organs is impaired. In plant senescence, drastic reduction of beneficial

macromolecules occurs with concomitant increase of some catabolic substances

including enzymes (Bhattacharjee, 1984; Bhattacharjee and Gupta, 1985; Pati, 2007).

In this investigation, ageing-induced reduction of nucleic acids (Table 7) and protein

(Table 8) in Abrus seeds might be due to the onset and progression of senescence,

commonly called seed senescence. However, in this study all the PGRs, particularly

the retardants, deferred seed senescence thereby arrested the rapid ageing induced loss

of the nucleic acid and protein levels.

All the PGRs averted the quicker ageing-induced loss of dehydrogenase and

catalase (Table 9) as well as peroxidase (Table 10) activities in Abrus seed kernels in

Discussion

124

comparison to control samples. In fact, beneficial and scavenger enzymes can

ameliorate harmful effects by reducing the toxic substances produced during

metabolic process. The positive influence of the PGRs on the regulation of the

activities of enzymes indicate that the agents could at last slow down the deterioration

of seeds, thereby increasing higher germinability, field emergence capacity etc. as

found in this investigation. Such reports also exist in the literature (Kanp, 2007;

Bhattacharjee et al., 2009; Ojha et al., 2012).

On the other hand, the chemical induced reduction of the activities of IAA-

oxidase and protease (Table 11) as well as that of the activities of amylase (Table 10)

in NaDK- and CCC-treated seed samples of Abrus might be due the suppressive

action of the chemical agents on the catabolic enzyme activities. This effect of the

PGRs is definitely beneficial for maintaining the anabolic activities even in the ageing

environment.

Keeping in mind the problem of seed viability and comparatively shorter life

span of seeds an attempt was made to overcome, at least to a considerable extent, this

inherent low vigour status of Dolichos biflorus seeds by employing the same chemical

manipulating agents i.e., IAA, GA3, NaDK and CCC. To evaluate the vigour and

viability status of the seeds accelerated ageing technique was employed with a parallel

assessment of the seed viability by allowing the seeds to experience natural ageing

under ambient environmental condition. Accelerated ageing test, as imposed by high

temperature and high relative humidity provides a powerful tool for studying the

processes of seed deterioration over a very short period, since such forced ageing

treatment augments the harmful biochemical processes associated with natural seed

deterioration (Heydecker, 1972). And the health status can be quickly and accurately

determined. In fact, accelerated aging process mimics the natural ageing one, and in

case of former the speed is exceedingly high than the later. (Heydecker, 1972; ISTA,

1996; Das, 2006).

Previous observation of Bhattacharjee (1984), that some PGRs shows delaying

effect on plant senescence, have prompted the present investigator to analyse the

efficacy of two growth retardants (NaDK and CCC) as well as two growth promoters

(IAA and GA3) on reducing seed senescence/deterioration under both accelerated and

natural ageing condition.

Discussion

125

The experiment on the influence of the test PGRs on the modulation of seed

germination behaviour of Dolichos biflorus shows that seed pretreatment with the

PGRs comparatively increased seed germinability as well as field emergence capacity

in comparison to the control samples irrespective of both accelerated and natural

ageing condition (Table 12). Seed germinability and rate of germination are

considered to be important visible criteria for evaluation of potential status of seeds

under storage (Anderson, 1970; Halder et al., 1983; Bhattacharjee et al., 1999; Pati

and Bhattacharjee, 2012). In this study, the chemical-induced higher germination and

field emergence percent indicated the ameliorating effect of the chemical

manipulants, particularly growth retardants, on maintaining comparatively higher

vigour status of seeds even under forceful accelerated as well as natural ageing

condition.

The efficacy of the PGRs on maintaining the storage potential of Dolichos

seeds can also be substantiated from the data of T50 values as well as TTC stainability

of the seeds (Table 13). Results clearly showed that three seed samples (Control, IAA

and GA3) failed to attain T50 values when stored in 60 days accelerated ageing and 24

months natural ageing condition, but in NaDK- and CCC-treated seed samples, at

least T50 values were shown in spite of accelerated ageing condition and this is

indicative of the fact that NaDK potentially hardened the Dolichos seeds under

storage.

Chemical manipulation of germinability, TTC-stainability and metabolism of

Dolichos seeds with reference to the beneficial of the PGRs particularly NaDK and

CCC, as found in case of Abrus seeds, can be substantiated from the results shown in

different tables (Tables 12-20). The hardening effect of the PGRs especially NaDK

and CCC on Dolichos biflorus can be conclusively established from both accelerated

ageing and natural ageing experiments. In fact, in accelerated ageing experiment, the

efficacy of PGRs was more accurate and much faster than natural ageing. However, in

Dolichos the results are almost identical with that of Abrus, and thus the discussion on

the results of Dolichos is almost similar. Like Abrus, in Dolichos also the PGRs

checked the rapid leaching of amino acids and soluble carbohydrates (Table 14),

decreased soluble carbohydrates and increased insoluble one (Table 15) in seed

kernel, increased protein (Table 16) and nucleic acid (Table 17) contents, increased

dehydrogenase and catalase (Table 18), increased peroxidase and decrease amylase

Discussion

126

(Table 19), decreased activity of IAA-oxidase and protease (Table 20). As the PGRs

exert the same pattern of regulation on the parameters mentioned both in Abrus and

Dolichos there is least doubt to infer that beneficial effect of the experimental

chemicals are enforced regardless of the plant materials.

The influence of the PGRs on the regulation of the phenophases and some

growth variables of Abrus and Dolichos are also found to be similar. Results showed

that the inception of the phenological phases were delayed by the retardants (NaDK

and CCC) and advanced by the promoters (IAA and GA3). The promoters also

increased root and shoot length along with enhanced fresh and dry weight in both the

species.

The most promising aspect of the PGRs was their influence on positive

regulation most of the major yield attributes of Abrus (Fig. 1- 4) and Dolichos (Fig. 5-

8). In fact, these yield attributes resulted in the productivity of the medicinal plants.

A preliminary experiment was undertaken using untreated seeds of Abrus and

Dolichos to get an insight into the phytochemistry of the plant species. The

compounds analysed were alkaloids, terpenoids, phenols, tannins, anthraquinones,

flavonoids, saponins and glycosides (chart 1) and analysis was done on qualitative

basis.

Phytochemical analysis of the untreated seeds of Abrus and Dolichos was also

done by using Infra-Red Spectra (Tables 27, 28 and Figs. 9, 10) shows aliphatic

compounds and some are may be cyclic or steroidal in nature.

Concomitantly, HPLC analysis of the methanolic extracts of untreated seeds

of Abrus (Fig. 11) and Dolichos (Fig. 14) showed that the fraction 1 of HPLC

chromatogram (of Abrus) contains the compound genistein (Fig. 12) and fraction 2

contains homoisoflavonoids (Fig. 13) whereas fraction 1 of HPLC chromatogram (in

case of Dolichos) showed the compound dimer-gallate (Fig. 15) and fraction 4

contains 3, 4-di-o-caffeoylquinic acid (Fig. 16) after MULDI-TOF MS study.

Thus, the PGRs seem to be promising for augmentation of seed productivity.

However, the challenge to current and future scientists is to device research strategies

and methodologies in various research fields to probe into the prospects of IAA, GA3,

NaDK and CCC.

Summary and Conclusion

127

SUMMARY AND CONCLUSION

This investigation was performed using two medicinally important plants

Abrus precatorius L. and Dolichos biflorus L. Both the plants are used

ethnomedicinally by a number of tribal groups of West Bengal and the first one i.e.

Abrus occurs wildly and the second one Dolichos is used as a minor pulse crop cum

medicinal plant. In both Abrus and Dolichos plant, seeds are the major store house of

a number of potent bioactive compounds responsible for rendering medicinal

properties. The major problem of Abrus is its poor seed germinability under ambient

climatic condition followed by impaired seedling as well as plant establishment. In

case of Dolichos biflorus there exists some problems i.e. poor storability of seeds

under ambient environmental condition, rapid loss of seed vigour and viability under

storage, subdued seedling and plant establishment. These consequently result in poor

plant health due to reduced growth, metabolism, substandard yield attributes etc.

Keeping in mind such problems of Abrus and Dolichos species attempts were made to

improve vigour and viability status of seeds by chemical manipulative technique. To

obtain expeditious results, accelerated ageing technique was employed in case of

Dolichos seeds. In view of the specific problems of these two seed species, chemical

manipulation technique was employed to overcome such problems to achieve my

prime target i.e. enhancement of productivity. Thus, to overcome the pinpointed

problems of the experimental medicinal plants, attempts were made to get rid of such

problems by the chemical manipulative agents like indole acetic acid (IAA),

gibberellic acid (GA3), sodium dikegulac (NaDK) and chlorocholine chloride (CCC).

A screening experiment was done for optimization of scarification technique

using appropriate percentage of H2SO4 and exact duration of treatment with the

scarifying agents for maximum germinability. Percentage germination of Abrus seeds

was recorded using different concentrations (0, 5, 10, 15, 20, 25, and 30 percent) of

H2SO4 for different durations (10, 20, 30 and 40 minutes). Data were recorded at 48 h

intervals up to 240h. Results showed that maximum germination was found at 25%

H2SO4 treatment for 30 minutes and maximum germination percentage was recorded

after 240 hours of seed soaking.


128

The most potent plant growth regulator (PGR) and its most effective

concentration for seed germinability as well as T50 hours (time in hour required for

50% germination) was shown using the optimally scarified Abrus seeds. Results

clearly revealed that in Abrus, scarification followed by GA3 (500 μg ml-1

) treatment

exerted the best response for stimulating seed germination. Percentage germination

was significantly less than control in both the retardant treatments i.e. CCC and

NaDK. However, both the growth promoters IAA and GA3 enhanced seed

germination in comparison to control samples. T50 values were found to be

significantly less in GA3 (500 µg ml-1

) in comparison to control and other treatments

(1AA, NaDK and CCC), and 50% seed germination was not at all achieved in all such

treatments irrespective of their concentrations.

Seed germinability and field emergence capacity of the scarified followed by

PGR-treated Abrus seeds showed that significant reduction of percentage germination

with concomitant decrease of field emergence capacity were found in seeds which

underwent ageing treatment, and the later observations (27 and 36 month of ageing)

were found to be much more remarkable.

All the PGR treatments except GA3 caused nonattainment of 50% germination

irrespective of the various durations (0, 9, 18, 27 and 36 months) of natural ageing.

However, in GA3 treatment at least 50% germination occurred although the T50 values

were recorded to be higher and these increased with the increase of ageing period.

TTC stainability of Abrus seeds started decreasing with the progressive increase of

natural ageing. However, all the PGRs, tended to increase TTC stainability over

control (distilled water) values. The magnitude of TTC staining percentage was found

to be maximum in GA3-treated seed samples.

Both amino acid and soluble carbohydrate levels increased in Abrus seed

leachates at all the treatments as well as in control samples with the advancement of

ageing months. However, extent of leaching was found to be significantly less in all

the treatments, and slowing down of leaching of the soluble substances was much less

in NaDK-treated seed samples.

Data on the PGR-induced changes of soluble and insoluble carbohydrates in

Abrus seed kernels were found to be distinctly reverse of each other. In fact, insoluble


129

carbohydrate showed a steady state decrease with the increase of ageing months with

concomitant increase of the soluble carbohydrate in Abrus seeds.

Nucleic acid contents (both DNA and RNA) showed a progressive decreasing

trend in seed samples which experienced natural ageing for 36 months in all the

treatments. The ageing-induced reduction of both the nucleic acid levels was found to

be partially alleviated in all the PGR treatments. The alleviatory effect of the

treatments was found to be most promising in NaDK-treated seed samples.

As regards the changes of protein content an identical trend, as found in case

of nucleic acids, was recorded in the experimental plant growth regulators. Here also,

NaDK-induced changes of proteins were found to be most significant.

The activities of dehydrogenase and catalase were found to decrease with

natural ageing for 0 to 36 months. The PGRs averted the alarming loss of the

activities of the beneficial enzymes over control samples. The plant growth retardants

NaDK and CCC were promising over the growth promoters 1AA and GA3 in this

regard.

The changes of the activities of peroxidase and amylase were found to be

diametrically opposite. Data clearly showed that peroxidase activities decreased with

the progress of seed ageing while amylase activities started increasing. In case of

peroxidase, the magnitude of changes were maximum in NaDK-treated seed samples

and in case of amylase, GA3- induced increase of the activities was most significant.

As regards the changes of IAA oxidase and protease activities almost an

identical trend was recorded regardless of the treatments and ageing period. The

ageing- induced gradual enhancement of the deleterious enzymes was potentially

alleviated by the growth retarding chemicals NaDK and CCC.

Percentage germination and field emergence capacity of Dolichos seeds were

declined with the progress of ageing period and the magnitude of the reduction was

found to be very rapid under accelerated ageing condition. Both the growth retardants

(NaDK and CCC) averted the rapid loss of seed germinability as well as field

emergence capacity but NaDK was found to be most responsive under accelerated

ageing condition and GA3 was most responsive under natural ageing condition.


130

Results on PGR- and ageing-induced changes of T50 values and TTC

stainability were analysed. Three (Control, IAA and GA3) seed samples, failed to

attain 50% germination in case of accelerated ageing for 60 days, and nonattainment

of 50% germination under natural ageing for 24 months was recorded in control,

NaDK- and CCC-treated seed samples. TTC stainability proportionately decreased

with the advancement of both accelerated and natural ageing and maximum reduction

occurs in control samples. The retardants and promoters showed ageing-induced

reduction TTC stainability, when compared with control samples.

Leaching of free amino acids and soluble carbohydrates occurred with the

progress of both accelerated and natural ageing duration. NaDK and CCC potentially

checked profuse leaching of the soluble substance particularly at later observation

periods of both the ageing processes.

A differential result was recorded in case of the changes of soluble and

insoluble carbohydrate levels with the ageing process. Data clearly revealed that

soluble carbohydrate started increasing with the progression of both natural and

accelerated ageing process but NaDK and CCC arrested the speed of leaching. On the

other hand, insoluble carbohydrate contents showed decreasing trend irrespective of

the samples analysed.

Alarming fall of protein level was found in seeds which experienced

accelerated ageing treatment but the PGRs, particularly retardants, significantly

slowed down the ageing-induced reduction of protein. In natural ageing also the

retardants NaDK and CCC arrested the rapid decrease of protein level and the effect

was found to be more remarkable at later observation periods.

Results also revealed that the PGRs more or less checked the loss of both the

nucleic acid levels, particularly RNA in kernels of Dolichos seeds under both

accelerated and natural ageing condition. However, the reduction of the nucleic acid

was found to be faster in forced ageing treatment.

Activities of the enzymes dehydrogenase and catalase gradually decreased

with the progress of seed ageing process in all the treated and control seed samples.

However, the PGRs particularly NaDK and CCC efficiently reduced the magnitude of

reduction of the enzyme activities and the effect was found to be more significant at


131

later periods of observation. Results of the changes recorded at zero days after ageing

(both natural and accelerated) were insignificant.

The ageing-induced changes of the activities of the enzymes peroxidase and

amylase are differential. In fact, peroxidase activities were diminished with the ageing

duration (natural as well as accelerated) and amylase activities were increased with

the ageing process. All the seed pretreating agents arrested the decrease of peroxidase

and increase of amylase, and the NaDK was found to be most responsive in this

regard.

Data on the PGR-induced as well as the ageing-induced changes of the

activities of IAA oxidase and protease showed that activities of the enzymes started

increasing with the ageing periods. But the plant growth regulators slowed down the

speed of increase of the enzyme activities at least up to 60 days of accelerated ageing

and 24 months of natural ageing period. Ageing-induced alarming increase of the

enzyme activities was significantly arrested and among the PGRs, NaDK was found

to be most efficient.

Data on the inception of selected developmental stages of Abrus precatorius

showed that the growth promoters IAA and GA3 tended to advance all the

developmental stages of the plant right from the radicle emergence to harvest. On the

other hand, the growth retardants NaDK and CCC deferred the onset of the stages

and, GA3 and NaDK was found to be most effective in manipulating the advancement

and deferment, respectively.

Results showed that the PGRs modulated both root and shoot length of 30 and

60 days old Abrus precatorius plants. Here the growth retardants tended to reduce

both the root and shoot lengths in comparison to control samples.

As regards the PGR-induced changes of fresh and dry weight of Abrus seeds,

all the said pretreating agents increased both the fresh and dry weight of Abrus plants,

but the magnitude of increment was found be highest in case of NaDK.

Major phenological events of Dolichos biflorus were shown as done in case of

Abrus precatorius. Results revealed that life cycle of this monocarpic medicinal pulse

crop is completed at around 132 days, and the experimental PGRs modulated the


132

inception of all the events to some extent. Here also, the promoters caused earliness of

the events and the retardants slowed down the onset of the events, thereby caused late

appearance of the phenological phases.

In case of Dolichos the PGR-induced regulation of root and shoot length were

recorded from the seed samples which were subsequently allowed to experience

accelerated ageing treatment for 0 and 45 days. Here, the growth retardants tended to

decrease the root and shoot length of plants raised from nonaged seeds, but the

parameters were found to increase in plants which were established from

acceleratedly aged seeds for 45 days. However, the growth promoters increased both

root and shoot length irrespective of the plants, raised from aged and nonaged seeds.

Again, a drastic reduction of both roots and shoot length regardless of the samples,

was recorded in the plants, raised from the forcefully aged seeds for 45 days.

As to the PGR-induced changes of fresh and dry weight of Dolichos seeds, an

increased trend was recorded in almost all the treatments when data were recorded

from plants which were developed from acceleratedly aged seeds, and here also both

fresh and dry weights were drastically reduced in samples due to accelerated ageing

treatment.

Data revealed that pod number per individual plant was increased in samples

which were developed from NaDK-treated seeds, and this increase was noted to be

still higher when seed treatment with NaDK (500 µg/ml) was followed by a foliar

treatment with the same PGR (100 µg/ml) on 50 and 100 days old Abrus plants. This

pattern of increase in seed followed by foliar treatment was recorded in all the

chemical agents, and the best response was rendered by NaDK. The PGRs also

modulated the pod volume per plant and here also NaDK showed the most covetable

result. In all the treated samples, the dual treatment of the chemicals (in seed soaking

mode and foliar mode) were found to be more encouraging than single treatment

given by seed soaking mode only. However, as to the chemical-induced modulation of

seed number per pod the changes were found to be least significant or insignificant,

although the retardants were found to show a little bit increasing trend. Changes on

the 1000-seed weight by the PGRs were found to be promising and here also NaDK

(seed followed by foliar treatment) exerted the best response.


133

Data on the results of the chemical-induced changes of pod number per plant

clearly showed that the PGRs particularly NaDK and CCC increased the pod number

per plant regardless of plants which were raised from seed treatment only or seed

treatment followed by foliar treatment. However, the dual treatment i.e. seed-followed

by foliar treatment was found to be more effective for increasing pod number. Almost

an identical change was recorded when the results of pod volume of the treated

samples was considered. Here also the effects of PGRs particularly NaDK and CCC,

and the dual treatments were found more pronounced. As regards the chemical-

induced changes on seed number per pod, the regulatory action of the PGRs was

found to be less prominent than the other yield attributes. However, a little bit

increasing trend was still apparent in the chemical treated samples. 1000-seed weight

was significantly increased regardless of the treatments either at seed level or at seed

followed by foliar treatment level. Here, NaDK most efficiently triggered to increase

1000-seed weight of Dolichos when treatment was given by two different modes i.e.

seed treatment followed by foliar treatment.

A preliminary qualitative phytochemical analysis of aqueous and methanolic

extracts of Abrus and Dolichos seeds was done to have an overall idea on the

existence or non existence of some secondary metabolites like alkaloids, terpenoids,

phenols, tannins, anthraquinones, flavonoids, saponins and glycosides.

Analysis of the functional groups of the phytochemicals of Abrus and

Dolichos seed samples was done by Infra-Red Spectra. Concomitantly, HPLC and

MALDI-TOF MS analysis of the untreated seed samples showed that the compounds

genistein and homoisoflavonoids are present in Abrus precatorius and dimer-gallate

and 3, 4-di-O-caffeoylquinic acid in Dolichos biflorus.

CONCLUSION:

From the comprehensive results of the experiments performed, it can be

concluded that the stubborn germinability of Abrus seeds can be potentially overcome

by the optimised scarification technique using 25% H2SO4 for 30 minutes. And the

scarification effect can further be enhanced by seed treatments with the experimental

plant growth regulators (PGRs) on the scarified seeds. Scarification followed by GA3

treatment exerted the best response. Viability problem of Dolichos seeds can be


134

potentially ameliorated using the PGRs and here NaDK exerted the best response.

Concomitantly, the metabolic status of the treated experimental seeds was found to be

much better than the control under the ageing process. The PGRs, particularly the

NaDK and CCC successfully deferred the inception of the major events of both the

plants and thus facilitated the growing seeds of such plants to receive photoassimilates

for a longer duration. And this finally led to increased seed weight.

Thus, the productivity was found to be enhanced by virtue of the positively

influenced yield attributes by the PGRs along with enhanced sink strength.

Phytochemical status in the mature seeds of the untreated plants was analysed by

using IR-spectra, HPLC, and MALDI-TOF MS, and this was done for further in depth

study of the PGRs on regulation of the productivity of phytochemicals. Thus, this

work seems to be encouraging because the vital problems as mentioned were obviated

to some extent and seed productivity was enhanced.

Further research on the PGR-induced regulation of phytochemicals with

special reference to some potent bioactive compounds and their biosynthetic pattern

are in progress.

135

FUTURE VISION:

The comprehensive work of this investigation using the medicinal plants Abrus

precatorius and Dolichos biflorus opened some new avenues of research. The

following directions of research have been identified for undertaking further work in

this area and for appropriate exploitation of the promising chemical agents along with

others:

In addition to the present seed pretreating agents a wide range of chemical

agents should be skillfully tested with respect to their manipulative potential.

Optimisation of the suitable concentration (s) of chemical agents, their specific

mode of treatment and ideal stage of application on the plants should be done

for obtaining most covetable results in respect of higher plant potential,

productivity and content of phytochemical constituents.

Attempts should also be made to evaluate the efficacy of combined doses of the

identified chemical agents for determining any type of added positive influence

with respect to synergistic or additive action of the PGRs.

The expected beneficial results, as might be obtained in laboratory and

experimental field study, should be standardized through repeated analyses and

then may be popularized through extension work at the village farmers’ level.

In a separate study, an attempt may be made to critically analyse source-sink

relationship as well as to understand the actual mechanism(s) of action of the

chemical agents for obtaining expected beneficial results. If the mechanism(s) of

action of chemical agents can be pinpointed, the efficacy of these chemical

agents could further be exploited in a befitting and judicious manner.

A detailed work on the regulatory action of the PGRs on the qualitative and

quantitative aspects of the major bioactive compounds should be undertaken.

Subsequently, disease amelioration effect of specific bioactive compounds

should be evaluated by clinical analysis using albino rat as experimental animal

system.

References

136

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RESEARCH PUBLICATIONS:

1. S. Ojha, P. Mondal and J. Konar. 2010. Effect of promoters and pretreatment

with chemical on the seed germination of Abrus Precatorius L. Plant

Archives, 10, 641-645.

2. S. Ojha and J. Konar. 2011. Influence of leaf leachate of Shorea robusta on

the growth and nitrogen fixing capacity of Hybrid Azolla. Environment and

Ecology, 29 (1A): 462-464.

3. S. Ojha, C. K. Pati and A. Bhattacharjee. 2012. Seed invigouration and plant

potentiation of two pulse crop cultivars under stressful storage Condition. J

Botan. Soc. Bengal. 66(1): 63-67.

4. S. Ojha and A. Bhattacharjee. 2012. Technique for augmented germinability

of a medicinally important stubborn seed species Abrus precatorius Linnaeus

[Fabaceae]. Diversity and Conservation of Plants and Traditional Knowledge.

485 - 489. Edited by: S. Panda & C. Ghosh. Published by: Bishen Singh

Mahendra Pal Singh, Dehra Dun (India).

5. S. Ojha and A. Bhattacharjee 2013. A simple biofriendly method for

invigouration of Indian mustard seeds. Proceedings of the National Seminar

(UGC Sponsored) on “Challenges of Biology in 21st Century” (ISBN: 978-

9380663-66-1) 65-71. Edited by: Bikas Krishna Sinha Published by:

Department of Botany, M.U.C Women’s College, Burdwan and Levant

Books, 27 C, Creek Row, Kolkata 700014.

6. P. Chakraborty, S. Ojha, R. Mukhopadhyay and A. Bhattachrajee. 2013.

Herbal manipulation of seed vigour under storage and its rapid evaluation by

accelerated ageing technique. J Botan. Soc. Bengal. 67 (1): 21-27.

7. S. Ojha, C. K. Pati and A. Bhattacharjee. 2013b. Evaluation of allelopathic

potential of an aromatic exotic tree, Melaleuca leucadendron L. African

Journal of Plant Science. Vol. 7.

8. S. Ojha, C. K. Pati and A. Bhattacharjee. 2013a. Chemical-induced storage

potentiation of seeds of a medicinal pulse crop horse-gram (Dolichos biflorus

L. cv br-5). Indian Agriculturist. (In press).

156

Papers (Abstracts) in International, National and State level

Conferences / Seminars / Symposia:

1. S. Ojha, M. B. Ray, A. Bhattacharjee and K. Gupta (2008). Effect of

phytotoxicity of leaf leachates, bark and leaf extract of Melaleuca

leucadendron L. on mung bean (Vigna radiata L.) seeds measured in terms of

seed germination and amylase activity. National Seminar on “Medicinal

Plants: Prospects and Aspects”. March 15-16, UGC-CAS, Department of

Botany, The University of Burdwan, Golapbag, Burdwan, West Bengal, India.

pp. 67-68.

2. A. Nayek, S. Ojha and A. Bhattacharjee (2010). Physiobiochemical

approaches for evaluation of allelopathic potential of Eucalyptus. UGC

Sponsored National Seminar on “The Structure and Function of Coastal

Vegetation and its Relevance to the Society”. March 17-18, Department of

Botany, Ramnagar College, Depal, Purba Medinipur, West Bengal, India.

p.- 42.

3. S. Ojha, A. Nayek, S. Banerjee, M. B. Ray and A. Bhattacharjee (2010).

Physiobiochemical evaluation of chemically regulated potential status of horse

gram (Dolichos biflorus L.) seeds. Golden Jubilee International Seminar on

“Researches in Zoology – Basic and Applied”. (ISBN: 978-81-907741-8-5)

March 17-19, Department of Zoology, The University of Burdwan, Golapbag,

Burdwan, West Bengal, India. p.- 129.

4. S. Ojha and A. Bhattacharjee (2011). Influence of selected phytohormones

and plant growth retardants on changes of seed germination behaviour and

correlative biochemical changes of Abrus precatorius L. seeds. International

Conference on “Plant Science in Post genomic Era (ICPSPGE)”. Feb. 17-19,

School of Life Sciences, Sambalpur University, Jyoti Vihar, Orissa and The

Society for Plant Physiology and Biochemistry, New Delhi, India. pp. 126-

127.

5. P. Chakraborty, S. Ojha and A. Bhattacharjee (2013). Ecofriendly technique

for seed invigouration by herbal manipulation. 100th

Indian Science Congress,

Centenary Session, Section of Plant Science, Kolkata, January 3-7. p.- 231.

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