IN VITRO DIRECT AND INDIRECT

ORGANOGENESIS AND PLANT REGENERATION OF KENAF (Hibiscus cannabinus L) VAR. KB6

ZETI ERMIENA SURYA BT MAT HUSSIN

MASTER OF SCIENCE

2016

In Vitro Direct And Indirect Organogenesis And Plant

Regeneration Of Kenaf (Hibiscus cannabinus L) Var. KB6

by

Zeti Ermiena Surya Bt Mat Hussin

A thesis submitted in fulfilment of the requirements for the degree of

Master of Science

Faculty of Agro Based Industry

UNIVERSITI MALAYSIA KELANTAN

2016

THESIS DECLARATION

I hereby certify that the work embodied in this thesis is the result of the original

research and has not been submitted for a higher degree to any other University or

Institution.

OPEN ACCESS I agree that my thesis is to be made immediately

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approved by the Post Graduate Committee.

CONFIDENTIAL (Contains confidential information under the Official

Secret Act 1972)*

RESTRICTED (Contains restricted information as specified by the

organization where research was done)*

I acknowledge that Universiti Malaysia Kelantan reserves the right as follows.

1. The thesis is the property of University Malaysia Kelantan.

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SIGNATURE SIGNATURE OF SUPERVISOR

I/C/PASSPORT NO. NAME OF SUPERVISOR

Date: Date:

ACKNOWLEDGEMENTS

In the name of Allah Taala, The Most Gracious, with God’s help and His

amazing grace, I’m finally completed this dissertation after going through the four

years of challenging and stressful period. Nevertheless, this period is so meaningful

because it gives me an opportunity to step foot into the realm of true research work. I

would like to take this opportunity to express my appreciation to their efforts and

kindness.

First and foremost, I thank my chairperson supervisory committee, Dr. Dwi

Susanto for his invaluable guidance, suggestions, and help throughout the course of

this study. I also wish to express my deep sense of gratitude and sincere thanks to Dr

Mohammed Arifullah and Dr. Fatimah Kayat who kindly provided me with their

knowledge, guidance, and advice in carrying out this study as well as completion of

this thesis. They also struggled to setup new laboratory for tissue culture in Jeli

Kampus to give the best tissue culture laboratory to us.

I also would like to million thank to all staff members at tissue culture

laboratory, especially to En. Suhaimi from Faculty of Agro Based Industry, Universiti

Malaysia Kelantan, which is person I considered as a good helper and very supportive.

I also want to thank my labmates, Mr. Ahmed, Husna, Ilfah Husna and Vikram, I owe

a special word of thanks for their ready help and timely courtesy. Not forgetten my

“Jeli Penthouse group” which act as a spirits around me and also their endless care,

help and moral support given to me, I’d really appreciate our memorable moments.

Last but not least, I would like to express my deepest gratitude to my beloved

family especially to my parents, my lovely husband Mohd Hamka and my dear uncle

Dato’ Pahamin A. Rajab for their unstinting love, endless encouragement, concern,

patience and sacrifices which had helped me in undertaking and completing this study.

I could not have asked for better without them, My study would not have been

possible. ALHAMDULILLAH!

Zeti Ermiena Surya Bt Mat Hussin

TABLE OF CONTENTS

PAGE

THESIS DECLARATION i

ACKNOWLEDGMENT ii

TABLE OF CONTENTS iii

LIST OF TABLES vi

LIST OF FIGURES ix

LIST OF ABBREVIATIONS xii

ABSTRACT xiv

ABSTRAK xv

CHAPTER 1 INTRODUCTION

1.1 Uses of kenaf (Hibiscus cannabinus L) 1

1.2 Objectives of the study 5

CHAPTER 2 LITERATURE REVIEW

2.1 Plant tissue culture 6

2.2 Micropropagation 7

2.3 Kenaf 10

2.3.1 Plant describtion 12

2.3.1.1 Stem (Stalk) 12

2.3.1.2 Leaf 13

2.3.1.3 Inflorescence 15

2.3.1.4 Fruit & seed 16

2.4 Kenaf variety KB6 18

2.5 A brief review on in vitro studies of kenaf 19

CHAPTER 3 MATERIALS AND METHODS

3.1 Materials 22

3.1.1 Plant collection 22

3.1.2 Chemical, glassware and equipment list 23

3.2 In vitro propagation method 24

3.2.1 Washing and storage of glassware 24

3.2.2 Preparation & composition of nutrient media 24

3.2.3 Preparing the sterile transfer hood 27

3.2.4 Culture condition and subculture 27

3.3 Experimental protocol for kenaf (Hibiscus cannabinus L) 28

3.3.1 Direct shoot organogenesis 28

3.3.2 Indirect shoot organogenesis 30

3.3.3 Acclimitization procedure 32

3.4 Statistical analysis 32

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Shoot tip culture 34

4.1.1 Direct shoot organogenesis from shoot tip explant 34

4.1.2 Indirect shoot organogenesis from shoot tip explant 39

4.2 Node culture 46

4.2.1 Direct shoot organogenesis from node explant 46

4.2.2 Indirect shoot organogenesis from node explant 50

4.3 Leaf culture 56

4.3.1 Direct shoot organogenesis from leaf explant 56

4.3.2 Indirect shoot organogenesis from leaf explant 60

4.4 Petiole culture 67

4.4.1 Direct shoot organogenesis from petiole explant 67

4.4.2 Indirect shoot organogenesis from petiole explant 68

4.5 Acclimitization 76

4.5.1 The effect of humidity 76

CHAPTER 5 CONCLUSION

5.1 Summary & Conclusion 79

5.2 Recomendation for future research 81

5.3 Schematic sketch of micropropagation protocol for kenaf var. KB6 82

REFERENCES 83

APPENDIX 92

Appendix A-Table A.1 92

Appendix B-Figure B.1 93

-Figure B.2 94

NO.

LIST OF TABLES

PAGE

2.1 Previous study on kenaf propagate through tissue culture 21

technique

3.1 Distribution of plant growth regulator (PGR) BAP and IAA 29

(mg/1) in MS medium for direct organogenesis shoot induction

3.2 Distribution of PGR NAA and IAA (mg/1) MS medium for root 30

induduction

3.3 Distribution of PGR 2,4-D and Kinetin (mg/l) in MS medium to induce

callus in indirect organogenesis

31

3.4 Distribution of PGR BAP and IAA (mg/l) in MS medium for shoot 31

induction from callus

4.1 The percentage multiple shoot, average number of shoot 35

induction average of shoot length and average number of leaves

for shoot tip culture using MS medium with different

concentration of BAP and IAA (mg/l)

4.2 Percentage and average of root induction, root length, root number 37

from regenerated shoot using NAA (mg/l) verses IAA (mg/l)

4.3 The percentage pf explants forming callus and cullusing morphology 41

from shoot tip induction of callus using MS medium different

concentration of KN (mg/1) and different concentration of

2,4-D (mg/1)

4.4 Frequency of callus generate into multiple shoot, average of shoot

induction per explants and average of shoot length from shoot tip

42

callus using MS medium with different concentration of BAP (mg/l)

and IAA (mg/l)

4.5 Rooting induction of regenerated shoot from shoot tip callus on 43

MS medium with various concentration of NAA and IAA (mg/1)

4.6 The percentage of multiple shoot, average number of shoot induction 47

average of shoot length and average number of leaves for node

cultureusing MS medium with different concentration of BAP (mg/l)

and different concentration of IAA (mg/l)

4.7 Percentage and average of root induction, root length, root 48

number from node culture regenerated shoots using MS medium and

different level of NAA (mg/l) and MS medium and different level

of IAA (mg/l)

4.8 The percentage of explants forming callus and callusing morphology 51

from node induction of callus using MS medium different

concentration of KN (mg/l) and different concentration of

2,4-D (mg/l)

4.9 Frequency of callus generate into multiple shoot, average of 52

shoot induction per explants and average of shoot length from

node callus using MS medium with different concentration of

BAP (mg/l) and different concentration of IAA (mg/l)

4.10 Rooting induction of regenarated shoot for node callus using 54

MS medium with different level of NAA (mg/l) and MS

medium and different level of IAA (mg/l)

4.11 Frequency of shoot induction, percentage of root form, average 57

number of root per explants, average of root length in induction of

shoot stage from leaves culture using MS medium with different

concentration of BAP and IAA (mg/l)

4.12 The frequency explants forming callus and callus morphology, 62

in indirect organogenesis from leaves culture using MS medium

with different concentration of KN and 2, 4-D (mg/l)

4.13 The frequency of callus generate into multiple shoot (%), average 63

of shoot induction per explants and average of shoot length

from leaves culture callus using MS medium with different

concentration of BAP and IAA (mg/l)

4.14 Rooting induction of regenerated shoot for leaves callus using 64

MS medium with different level of NAA (mg/l) and MS medium

and different level of IAA (mg/l)

4.15 Percentage of explants forming callus and callus morphology 70

from petiole culture induction of callus using MS medium

with different concentration of KN and 2, 4-D (mg/1)

4.16 The frequency of callus generate into multiple shoot (%) average

of shoot induction per explants and average of shoot length (cm)

from petiole culture callus using MS medium with different

concentration of BAP and IAA (mg/l)

73

4.17 Rooting induction of regenerated shoot for petiole callus using 71

MS medium with different level of NAA (mg/l) and MS medium

and different level of IAA (mg/l)

4.18 Different high of plantlets, the percentage of explants survive

and the different number of explants leaves for acclimitization

process from effect of humidity

74

LIST OF FIGURES

NO.

2.1a

Leaves of kenaf was picked fresh from farm

PAGE

14

2.1b Canabis sativa or Marijuana leaf identical to kenaf's

leaf (Source from Wikipedia)

14

2.2a Flower of the kenaf freshly picked from farm 16

2.2b Center of the flower, red in colour 16

2.2c Pollen grain under the microscope (source from Ahmed et al, 2014) 16

2.3a The wedges-shaped seed of kenaf, seed sample collected from

MARDI Telong

17

3.1 Preparation of stock solutions and volumes taken from the

stocks to prepare 1 liter of MS medium. Gamborg and shyluk (1981),

Gamborg (1982), 1991) and Dodds and Roberts (1982) in

(Aurifullah, 2006)

26

4.1a Single initiation of shoot tip explants on MS medium on 1st

day of inoculation, rapid observation was done on first 10 days

to observe the contamination

38

4.1b Single shoot initiation of shoot tip explants on MS medium with

0.05 mg/l BAP and 0.01 mg/l IAA after 2 weeks

38

4.1c Shoot proliferation on MS medium with 0.05 mg/l BAP and

0.01 mg/l IAA after 3 week

38

4.1d Multiple shoot production MS medium with 0.05 mg/l BAP 38

and 0.01 mg/l IAA after 5 weeks

4.1e Induction and elongation of multiple shoot on MS medium

with 0.05 mg/l BAP and 0.01 mg/l IAA after 6 weeks

38

4.1f Elongation of single plantlet and rooting of in vitro raised

shoot on MS basal medium on 8 weeks

38

4.2a Shootip explants were inoculated in MS medium with KN

and 2, 4-D on first day

45

4.2b Callus induction for shoootip explants in MS medium

with 0.2 mg/l 2,4-D after 10 days

45

4.2c Shoot induction from callus in MS medium with 0.1 mg/l BAP 45

and 0.03 mg/l IAA after 20 days

4.2d Shoot elongation and multiplication on MS medium with 0.1 mg/l BAP 45 and 0.03 mg/l IAA after 5 weeks

4.2e Single shoot elongation & rooting in MS basal medium on 45

2nd week in rooting medium

4.3a Direct organogenesis of node explants in MS medium with 0.1 mg/l 49 BAP after 6 days

4.3b Shoot proliferation & petiole fall down from node explants 49 in MS medium with 0.5 mg/l BAP and 0.05 mg/l IAA after 14 days

4.3c Multiplication of shoot in MS medium with 0.5 mg/l BAP 49 and 0.05 mg/l IAA after 5 weeks

4.3d Elongation of single plantlet and rooting of in vitro raised shoot 49 on MS medium basal on 8 weeks

4.4a Indirect organogenesis of node explants in MS medium 55 with 0.2 mg/l 2, 4-D on 1st 7 days start to swollen at the bottom

4.4b Callus induction from nodal explants on MS medium 55

with 0.2 mg/l 2,4-D after 12 days

4.4c Shoot induction from pieces of callus on MS medium 55 with 0.1 mg/l BAP after 3 weeks

4.4d Shoot induction from nodal callus on MS medium 55

with 0.1 mg/l BAP after 5 weeks

4.5a Direct organogenesis of leaves on MS medium with BAP and IAA in first week

59

4.5b Root form from leaves direct organogenesis on MS medium 59

with 0.05 mg/l BAP and 0.01 mg/l IAA after 9 days

4.5c Abudant of root from leaves culture on MS medium 0.1 mg/l BAP 59

with 0.05 mg/l IAA after 4th weeks

4.6a Indirect organogenesis of leaves on MS medium

with KN and 2, 4-D first week of inoculation

66

4.6b Callus induction of leaves indirect organogenesis on MS medium 66

with 0.2 mg/l 2, 4 D after 14 days

4.6c Callus induction of leaves indirect organogenesis on MS medium 66

with 0.2 mg/l 2, 4 D after 25 days

4.6d Shoot induction from callus in MS Medium with 0.3 mg/l BAP 66

and 0.03 mg/l IAA after 20 days

4.7a Indirect organogenesis of petiole in MS medium 75

with KN and 2, 4-D on 1st day of inoculation

4.7b Callus induction after 15 days on MS medium with 0.2 mg/l 2,4-D 75

4.7c Callus induction after 10 days on MS medium 1.0 mg/l KN 75 with 0.1 mg/l 2,4-D

4.7d Initiation of multiple shoot from callus after 25 days on MS medium 75 with 0.1 mg/l BAP and 0.02 mg/l IAA

4.7e Elongation and rooting of multiple shoot after 47 days on MS medium 75 with 0.1 mg/l BAP and 0.02mg/l IAA

4.8a Combination of sand, coco pit and vermiculite with 3:2:2 ratios 78

4.8b Plantlets were covered with the hole plastic containers on 78 1

st day of acclimatization

4.8c The plantlet survive after 9 days of acclimatization under shaded roof 78

4.8d Field plant of kenaf plantlet was successfully tested after 15 days in 78

shaded roof. The plantlets were 40 days

LIST OF ABBREVIATIONS

µm Micron

μg Microgram

μm Micrometer

µl Microlitre

% Percent

°C Degree Celsius

2,4-D 2,4-Dichloro-phenoxy acetic acid

BAP Benzyl-6-aminopurine

C2H5OH Ethyl Alcohol

cm Centimeter

Fig. Figure

g Gram

h Hour

HgCl2 Mercuric Chloride

IAA Indole-3-acetic acid

KIN Kinetin

KNO3 Potassium Nitrate

m Meter

M Molarity

min Minute

ml Millilitre

mg Miligram

mg/l Miligram/litre

mm Millimetre

MS Murashige and Skoog’s Medium

n Sample size

NAA Naphthalene-3-acetic acid

PGR Plant growth regulator/s

s Second

Sp Species

Direct and indirect organogenesis and plant regeneration of an industrial plant

kenaf (Hibiscus cannabinus L) var. KB6

ABSTRACT

As an important fiber crop, many potential applications of kenaf are being

identified and developed in 21 century, especially in developed countries such as

America, Japan, and France and Malaysia as well. The present study report a protocol

for the efficient in vitro propagation of kenaf (H.cannabinus L) to initiate multiple

shoot from mother plant part (shoot, petiole, node and leaf) through direct and indirect

organogenesis using MS medium + BAP + IAA for direct organogenesis and MS

medium + KN+2,4-D for indirect organogenesis to get callus and after 8 weeks, the

calli were put in the MS medium + BAP +IAA for shoot induction. The highest

number of shoots produced from node explants part via direct organogenesis

(16.33/explants) in MS medium + 0.5 mg/l BAP +0.05 mg/l IAA. The highest

percentage explants forming callus and callus generate into shoot also from node

explants part which was induced 75% callus from explants and 73.33% of callus turn

into shoot in MS medium + 0.1 mg/l BAP. Several subcultures were drived in order to

enhance the multiplication rate. The treatments have their significant different with

others. The shoots then were transferred to the root induction medium. Shoots showed

the vigorous roots in the MS basal medium. The in vitro rooted plantlets were

acclimatized in sand+ coco pit + vermiculite with ratio 3:2:2 and were covered with

hole container for 0-15 days to test the effect of humidity to plantlets. The 9th-15

th days

explants covered with container showed 100% of survive. Survive well rooted

plantlets were transferred to the field. Plants grew well into maturity without any

remarkable morphological variations within the treatments.

Regenerasi Tumbuhan Secara Langsung & Secara Tidak Langsung Tumbuhan

Industri, Kenaf (Hibiscus cannabinus L) var. KB6

ABSTRAK

Sebagai salah satu tanaman yang penting, banyak aplikasi potensi kenaf sedang

dikenalpasti dan dibangunkan di abad 21, terutama di negara-negara maju

membangun seperti Amerika, Jepun, dan Perancis dan juga Malaysia. Laporan kajian

ini adalah untuk mengkaji protokol yang terbaik dalam pembiakan in vitro kenaf

(H.cannabinus L) dengan menggunakan eksplan dari pelbagai bahagian pada induk

kenaf variasi KB6 (pucuk, daun, nod dan daun nod) melalui organogenesis langsung

dan tidak langsung dengan menggunakan MS medium + BAP + IAA untuk

organogenesis langsung dan MS sederhana + KN + 2,4-D untuk organogenesis tidak

langsung untuk mendapatkan kalus dan selepas 8 minggu, kalus telah dimasukkan ke

dalam medium MS + BAP + IAA untuk mendapatkan pucuk. Bilangan tertinggi pucuk

dihasilkan dari nod eksplan bahagian melalui organogenesis langsung (16.33 /

eksplan) dalam MS medium + 0.5 mg / l BAP 0,05 mg / l IAA. Peratusan tertinggi

eksplan yang membentuk kalus dan kalus berpotensi mengeluarkan pucuk juga dari

nod eksplan sebahagian yang disebabkan 75% kalus daripada eksplan dan 73.33%

daripada kalus bertukar menjadi pucuk di MS medium + 0.1 mg / l BAP. Beberapa

subkultur telah dilakukan bagi meningkatkan kadar pengeluaran pucuk. Setiap rawatan

mempunyai perbezaan yang signifikan dengan rawatan lain. Pucuk kemudian

dipindahkan ke medium induksi akar. Pucuk mengeluarkan akar yang baik dalam

medium asas MS. In vitro anak pokok berakar telah dipindahkan ke medium pasir +

coco pit + vermikulit dengan nisbah 3: 2: 2 dan ditutup dengan bekas berlubang untuk

0-15 hari untuk menguji kesan kelembapan ke atas anak pokok. Anak pokok yang

ditutup dengan bekas selama 9-15 hari menunjukkan 100% daripada bermandiri. Anak

pokok yang berakar dipindahkan ke tanah lapang di luar kawasan rumah hijau. Anak

pokok tumbuh dengan baik matang tanpa apa-apa perubahan morfologi yang luar biasa

dalam rawatan.

CHAPTER 1

INTRODUCTION

1.1 Uses of kenaf (Hibiscus cannabinus L)

Kenaf (Hibiscus cannabinus L.) is widely known as an important family of

Hibiscus for modern needs. The reason behind it is apparently because it produces high

biomass, and also able to adapt well with local environment. Kenaf has been adopted in

many countries ever since, including Malaysia. This unique plant is still new in Malaysia

and has been cultivated generally in the eastern part of the country, especially Kelantan.

Initially, kenaf is to replace tobacco industry since it is no longer supported by the

government. Far in Africa, kenaf is used in anaemic therapy (Charles et al., 2002).

This plant can rise up to 60-120cm height as young as 2-3 months. U.S.

Department of Agriculture reported that kenaf can produce up to 10 tons of dry fiber per

acre per year. That is 3 to 5 times greater than the production of Southern pine trees, in

which it would take 7 to 40 years before it can be harvested (McLean et al., 1992). As

known in the botanic field, kenaf is classified in various types in which each of them can

perform at their best in certain locations, conditions and other variables. These groups of

kenaf are known to produce different flowering schedule. Some may be harvested earlier

than the other and some later. The good part of it is the flowering can last up to 3 to 4

weeks or more, with each individual bloom can last only for one whole day (Charles et

al., 2002).

Kenaf’s flowers would eventually bloom at the end of growing season. The

process takes place when the flower drops off and leave a seed pod behind. The seeds,

however, will not get matured in many parts of U.S. Although some of the plant’s

varieties are known to bloom early, its biomass production is still not tangible enough to

provide adequate amount of fiber economically. This is because of their African origin

require an additional 60-90 days of frost free condition for the seeds to germinate.

Because of this particular, kenaf is not suitable for massive plantation across the country

like a normal weed. It also presents some interesting challenges for developers to ensure

a consistent supply of seed for next year’s crop (McLean et al., 1992).

The stem of the plant consists of two recognizable types of fiber. The first layer of

fiber is called ‘bast’. It is made up of roughly 40% of the stem’s total dry weight. The

refined ‘bast’ fiber measures at 2.6mm and is similar to the fiber commonly used to make

paper. The core is the second layer. It is made up of 60% of the stem’s total dry weight.

Its refined fiber measures at approximately 6mm and is comparable to hardwood tree

fiber which is used in wide range of paper products (Rowell and Cook, 1998).

Harvesting kenaf is not difficult as it can be processed in a common mechanical

fiber separator. It is very similar to a cotton gin. While the two fibers are separated

through the mechanical separator, they are allowed to be processed independently, in

which it provides raw materials for a wide range of products, such as paper, particle

board, bioremediation aids and animal bedding (McLean et al., 1992).

According to Duke 1983, kenaf was propagated by seed, but seed availability is

limited (Sullivan, 2003). Seed production strategies are affected by the cultivar, location-

especially latitude, and cultural practices. The first issue to address is the cultivar

photosensitivity, whether the cultivar is an ultra-early, an early to medium, or a late

maturing cultivar (Webber III & Bledsoe, 2002). Researchers and seed producers have

reported that the strategy for maximizing harvestable kenaf seed is very different than the

production approach in maximizing kenaf fiber yields (Mullens, 1998; Webber III &

Bledsoe, 2002). Kenaf seed has high oil content (Mohamed et al., 1995) and seed

viability decreases over time when stored at higher relative humidity and higher

temperatures (Webber III & Bledsoe, 2002). Propagation by seed is not recommended

due to the heterogeneous nature of the seedlings owing to its cross pollination. Thus, any

kenaf variety will decrease in purity, leading to the increment of seed yields and

reduction of fiber yields (Ramesh, 2016).

The application of plant tissue culture as a micropropagation technique has

become an important biotechnological tool which offers additional advantages such as

the rapid multiplication rate (Edwin et al, 2008). Therefore, in vitro propagation methods

would be important to propagate this crop for high and stable cellulosic fiber production,

as large numbers of genetically identical plants and in a relatively short time for the

industrial plantation. Axillary buds are widely used for micropropagation as they have

entire rudimentary vegetative shoot and can be induced to develop into plants easily

which are similar to the parental type (Zapata et al., 1999; Srivatanakul et al., 2000).

The development of tissue culture and regeneration techniques are the most

important procedures that must be established in order to improve kenaf by genetic

transformation procedure (Banks et al., 1993). There are few reports on the in vitro

propagation of the Hibiscus genus, with kenaf being the most studied species in terms of

organogenesis regeneration. According to McLean et al. (1992), organogenesis of kenaf

via callus culture was not reproducible and resulted in very low regeneration efficiency

and the induction of heritable mutation. Preliminary research on shoot regeneration of

kenaf callus used the shoot apex still unsuccessful though direct shoot regeneration

without a callus phase has been achieved (Zapata et al., 1999; Srivatanakul et al., 2000),

while other authors reported the formation of multiple shoot from young shoot and

cotyledons (Khatun et al., 2003; Herath et al., 2004). A protocol for plant regeneration

from callus using segments of hypocotyls and cotyledons has been established for the H.

siryacus (Jenderek & Olney, 2001). The establishment methods for sterilization from

mother plant also have not been proposed by any researcher due to high contamination in

culture. Using seeds as a means of plant propagation is simple and can be produced by

farmers, but disadvantage are the progeny may be genetically different from the parents

and some plant have the dormancy period. So, in vitro propagation is the one of the

alternative techniques to solve these problems and can be used for mass propagation of

seedlings. That’s why this experiment was proposed.

1.2 Objectives of the present study

Therefore, the aims of this research were to:

(a) To analyses the effect of different plant growth regulator on direct and

indirect organogenesis of kenaf var. KB6

(b) To develop a protocol for kenaf var KB6 (Hibiscus cannabinus L) in vitro

regeneration using different explants like shoot tips, nodes, leaves and

petioles

CHAPTER 2

LITERATURE REVIEW

2.1 Plant tissue culture

Plant tissue culture has gone through a long developmental history to achieve its

current state. The history and milestones of tissue culture development has been

elaborated in many reviews (Gamborg, 2002; Thorpe, 2007; Vasil, 2008). Plant tissue

culture is an essential component of Plant Biotechnology. Plant cell and tissue culture has

already contributed significantly to crop improvement and has great potential for future

(Mehbooba et al., 2011). Research efforts in plant cell and tissue culture have increased

dramatically worldwide in recent years including efforts in developing nations. Plant cell

and tissue culture is defined as the capability to regenerate and propagate plants from

single cells, tissue and organs under sterile and controlled environmental conditions

(Murashige, 1974).

According to George & Debergh (2009), tissue culture techniques are now being

widely applied for improvement of field crop, forest, and horticulture and plantation crop

for increased agricultural and forestry production. Today tissue culture technology is

being exploited mainly for large scale production or micropropagation of elite planting

material with desirable characteristics. This technology has now been commercialized

globally and has contributed significantly towards the enhanced production of high

quality planting material. Recently emphasis has been on genetic transformation,

especially for (1) increased production of secondary metabolite for example, Bourgaud et

al., 2001, was studied about secondary metabolite from hairy roots callus and other organ

culture of Plumbago indica L. The work on in vitro production of secondary metabolites

also has been reviewed by (Ramachandra & Ravishankar, 2002). Many researchers have

reported the production of secondary metabolites by cell cultures where the synthesis and

yield of compounds approached or exceeded the levels found in natural plant resources

(Eran & David, 2000; Nakashima et al., 1997), (2) production of alkaloids

pharmaceutics, nematocidal compounds, and also some novel compounds not found in

the whole plants, regeneration of plant resistant to herbicides, disease, and pests (Leslie &

Johan, 2000) (3) scale up of cultures in bioreactors, Jacqueline & John (1999), was build

a research about hairy root culture by setup the bioreactor culture, (4) plants with

different morphological traits and (5) transgenic crops for production of recombinant

vaccines and anti-microbial antibodies (Peter & Stoger, 2011). These developments have

far-reaching implications in the improvement of medicinal plants as well (Bajaj, 1990).

2.2 Micropropagation

Micropropagation is a rapid multiplication of a selected plant using in vitro

culture techniques and it’s developed in past 35 years (Aneesha, 2015). In vitro clonal

propagation provides true to type plants of a selected genotype using in vitro culture

techniques (Thorpe, 2007). Micropropagation is also used to promote germplasm storage

for maintenance of disease-free stock in controlled environmental condition and in long

term via cryopreservation (Nukari et al, 2009). Micropropagation also gives the rapid

production of high quality, disease-free and uniform planting material (Ahloowalia et al,

2004). The plants can be multiplied under a controlled environment, anywhere,

irrespective of the season and weather, on a year-round basis (Bourgaud et al., 2001).

Production of high quality and healthy planting material of ornamentals, and forest and

fruit trees, propagated from vegetative parts, has created new opportunities in global

trading for producers, farmers, and nursery owners, and for rural employment

(Ahloowalia et al, 2004). One of the most exciting and important aspects of in vitro cell

and tissue culture is the capability to generate and propagate plants from cultured cells

and tissues. The simplest type of in vitro plant propagation is the stimulation of axillary

bud development (Edwin et al., 2008). This technique exploits the normal ontogenetic

route for branch development by lateral (axillary) meristems. The axillary buds are

treated with hormones to break dormancy and produce shoot branches (Zhu et al, 2015).

The shoots are then separated and rooted to produce plants. Alternatively, the shoots are

used as propagules for further propagation (Aneesha, 2015).

Plant regeneration from cultured tissue can also be achieved by culturing tissue

section lacking a preformed meristem (adventitious origin) or from callus and cell

cultures (de novo origin) (De Filippis, 2014). Axillary buds are preformed meristems. In

contrast, adventitious regeneration events occur at unusual sites of a cultured tissue such

as the internode, leaf blade, cotyledon, or root elongation zone, where meristem do not

naturally occur (Edwin et al, 2008). According to Nukari et al. (2009), adventitious plant

regeneration often is dependent upon the presence of organized explants tissue. In

comparison, de novo (literally, “to arise new”) plant regeneration occurs from callus and

cell cultures in the absence of organized explants tissues.

Whether adventitious or de novo in origin, plant regeneration can occur by one of

two processes. Organogenesis is the formation of individual organs, such as shoots or

roots. Somatic embryogenesis is the formation of a bipolar structure containing both

shoot and root meristems, and developing in a manner similar to zygotic embryos (Sara et

al, 2002).

According to Edwin et al (2008), most plant species are capable of plant

regeneration by either organogenesis or somatic embryogenesis, but very few species are

capable of both. Some species are easy to regenerate from callus or cell cultures, while

others regenerate only by an adventitious process. The choice of plant species and the

goal of the research will determine the plant regeneration or propagation procedure

(Vasil, 2008). When available for the same species, the different regeneration approaches

may yield different propagation rates (Chu, 1992). Axillary bud proliferation and culture

of individual nodes are the techniques most widely used in commercial micropropagation

and which show the least variation among the propagated plants (Ngezahayo & Liu,

2014). In contrast, adventitious shoot organogenesis and regeneration of plants from

callus by organogenesis or by somatic embryogenesis show the most variation, as well as

higher propagation rates (Gest, 2004). Direct (adventitious) somatic embryogenesis and

repetitive embryogenesis show the best balance of high propagation rates with relatively

few off-types (Gamborg, 2002).

There are several defined steps in a typical micropropagation system (Murashige,

1974). The first step is the initiation of a sterile culture of the explant (Stage I). The

second step is the multiplication of shoots or other propagules from the explants (Stage

II). Adventitious shoot proliferation is the most frequently used multiplication technique

in micropropagation systems (Chu, 1992). The culture media and growth conditions used

in Stage II systems are optimized for maximum rates of multiplication. The third step is

the development of roots on the shoots to produce plantlets (Stage III). Specialized media