Review

of Literature

Chapter-2

REVIEW OF LITERATURE

The initial wave in the history of plant tissue culture was made by Henri-Louis

Duhumel du Monceau in 1756, who during his pioneering studies on wound

healing in plants, observed callus formation on the decorticated elm plants

(Gautheret 1985). However, the theoretical framework and experimental basis of

modern plant tissue culture is derived from the concepts of cellular totipotency

(which is the ability of a single cell to divide and produce a whole plant). The

science of plant tissue culture takes its roots from path breaking research in botany

like the discovery of cell and propounding of cell theory. The concept of

totipotency is inherent in the cell theory of Schleiden (1838) and Schwann (1839),

which recognized cell as the primary unit -elementary part- of all living

organisms. In 1838, Matthias Jakob Schleiden, a botanist, suggested that every

structural elements of plant is composed of cells or their products. In the following

year, Theodar Schwann, a zoologist, made similar conclusions and elaborated for

animals. They independently proposed “Cell theory” that holds the fact that the

cell is the unit of structure and function in an organism and therefore, capable of

autonomy and even demonstrated the potential for totipotency. The idea was

tested by several researchers and interesting observations were made by Votching

(1878) suggesting ‘polarity’ as a characteristic feature guiding the development of

plant fragments. In his classical experiments on stem cuttings, he observed that

upper portion of a piece of stem always forms buds and the basal region callus or

roots. Further the grafting experiments which he undertook among species of

Opuntia, Salix, Beta and other trees, demonstrated that the behavior of a tissue is

not altered by contact with other tissue because of the dependence of

morphogenetic capacity on hereditary internal factors is very strict.

No sustained or organized attempts were made to list the validity of the

concept of totipotency that is inherent in the cell theory until the beginning of

twentieth century. The Austro-German plant physiologist, Gottlieb Haberlandt in

15

1902, was the first to try to obtain experimental evidence of totipotency by

culturing plant cells in nutrient solutions in the hope of regenerating whole plants.

He cultured isolated chloroplasts containing differentiated cells from leaves of

Lamium purpureum, cells from petioles of Eicchornia crassipes, glandular hairs of

Pulmonaria and Urtica, and stamen hairs of Tradescantia on Knop’s (1865) salt

solution in hanging drop cultures. The cells grew in size but failed to divide and

eventually lost to infection. He failed in his experiments but based on his

experiences he made some bold predictions. He advocated the use of embryo sac

fluids (coenocytic liquid endosperm, such as coconut milk that was later widely

and successfully used in tissue culture studies) for inducing cell divisions in

vegetative cells, and pointed out the possibility of cultivating artificial embryos

from vegetative cells in nutritive solutions. It is for these reasons Haberlandt is

rightfully regarded as Father of Plant Tissue Culture. He thus clearly established

the concept of totipotency and further indicated that the technique of cultivating

isolated plant cells in nutrient solutions permits the investigations of important

problems forming a new experimental approach (Haberlandt 1902).

In 1904, Hanning successfully cultured embryos of several crucifers by

excising them at a near mature stage and grew them to maturity on mineral salts

and sugar solutions. A new approach to tissue culture technology was conceived

simultaneously in 1922 independently by Kotte in Germany, a student of

Haberlandt and Robbins in USA, in growing isolated root tips. They postulated

that a true in vitro culture could be made easier by using meristematic cells such as

those operate in root tip or bud. However, an important breakthrough for

continuously growing isolated tomato root tips cultures was made by Phillip

Rodney White in 1934 a. In 1932, he found Knop’s nutrient solutions as well as

the formulation used by Robbins (1922) to be unsatisfactory. This prompted an

effort made by many individuals to develop nutrient solutions that could

adequately support the growth of isolated plant tissues. White developed a new

nutrient solution- The White’s medium (White 1943, 1963), where he included

16

glycine, nicotinic acid, pyridoxine and thiamine. It was the most widely used

nutrient solutions for plant tissue cultures until the 1960s.

In 1928, Frits Went at Leiden in The Netherlands successfully discovered a

naturally occurring auxin, indole-3-acetic acid (IAA). The development of

improved nutrient solutions, use of the newly discovered plant growth regulator,

indole-3-acetic acid (IAA), informed choice of plant material and appreciation of

the importance of aseptic cultures, led to the sustained growth of root tips and

carrot root and tobacco stem tissues for indefinite period of time (Gautheret 1934,

1939, 1985; Nobecourt 1939; White 1939). This important milestone was achieved

by Roger Gautheret and Pierre Nobecourt in France, and Philip R. White in the

United States, within weeks of each other. Gautheret was engaged in

experimentation with excised root tips and the cultivation of cambial tissues

removed under aseptic conditions from Salix capraea, Acer pseudoplanatus and

Populus nigra and other trees on agar solidified Knop’s solution containing

glucose and cysteine hydrochloride and recorded that they proliferated for a few

months. In 1939, White reported the establishment of similar cultures from tumor

tissue of the hybrid Nicotiana glauca × N. langsdorfii. These were the first plant

tissue cultures in strict sense of prolonged cultures of unorganized materials. The

methods and media now used are in principle modifications of those established

by these three pioneers. Philip R. White, Roger J. Gautheret and Pierre Nobecourt

can be credited with providing a significant impetus to the field with the

publications of their authorative handbooks.

Skoog and Tsui (1948) showed that addition of adenine and high levels of

phosphate allowed non-meristematic pith tissues to be cultured and to produce

shoots and roots but only in the presence of vascular tissues. This led to the

addition of a variety of plant extracts, including coconut milk to nutrient medium

in an attempt to replace the effect of vascular tissues and to identify the active

factors responsible for their stimulatory effect (Van Overbeek et al. 1941). Further

studies were conducted on nucleic acid i.e., the addition of DNA to the medium

which greatly enhanced the cell division activity in cultured pith tissues (Vasil

17

2008). These findings eventually led to the isolation of kinetin from aged

autoclaved herring sperm DNA by Carlos Miller, then a postdoc in Skoog’s

laboratory (Miller et al. 1955). Kinetin, which was active in inducing cell divisions

at astonishingly great dilutions (as low as 1 part per billion) in the presence of

auxins, was soon identified as N6-furfurylaminopurine and synthesized (Miller et

al. 1956).

Soon after the discovery of cytokinins, Skoog and Miller (1957) made

another significant contribution by demonstrating the hormonal (auxin-cytokinin)

regulation of morphogenesis in plants, which allowed the controlled formation of

shoots and roots in callus tissues and is rightly considered an important milestone

in understanding plant morphogenesis, micropropagation and regeneration of

plants from cultured tissues. A relative high level of auxin to kinetin favored

rooting while the reverse led to the shoot formation and intermediate levels to

callus or wound parenchyma tissue. In addition to the unipolar shoot buds and

roots, the formation of bipolar somatic embryos was first reported in carrot

independently by Reinert (1958, 1959) and Steward et al. (1958).

In 1960s, Toshio Murashige, a graduate student in the laboratory of Folke

Skoog, at the University of Wisconsin, was trying to obtain optimum and

predictable growth of cultured tobacco pith tissues which Skoog needed for

performing reliable bioassays of cytokinin activity. He found that the addition of

an aqueous extract of tobacco leaves to White’s medium resulted in greater than

fourfold increase in growth. Based on their observations, they together formulated

a new and completely defined nutrient solution, the Murashige and Skoog’s (1962)

or MS medium which includes concentrations of some salts 25 times higher than

that of Knop’s solution. In particular, the level of NO3- and NH4

+ were very high

and the array of micronutrients was increased. It also includes chelated iron in

order to make it more stable and available during the life of the cultures, myo-

inositol and a mixture of four vitamins. It is the most widely used formulation for

culture of plant tissues and the publications describing the MS medium remains

one of the most highly cited publications in plant biology.

18

Ball in 1946, successfully produced plantlets by culturing shoot tips with a

couple of leaf primodia of Lupinus and Tropaeolum. Early studies by White

(1934b) showed that cultured root tips were free from viruses. This was confirmed

when virus free Dahlias and Orchids plants were obtained from infected plants by

culturing their shoot tips (Morel and Martin 1952; Morel 1960).

Direct and unequivocal evidence of the totipotency of plant cells was

finally provided by Vasil and Hildebrandt (1965 a and b), who regenerated whole

plants from a single cell of a hybrid tobacco grown in a fresh nutrient solution in a

microculture chamber, thus demonstrating the totipotency of plant cells.

Technique of in vitro pollination and fertilization was pioneered by Kanta

et al. (1962) using Papaver somniferum. Guha and Maheswari (1964, 1966)

obtained haploid plants of Datura innoxia from the cultured anthers. This

discovery received significant attention since plant recovered from double haploid

cells are homozygous and express all recessive genes thus making them ideal for

pure breeding lines. Next breakthrough in tissue culture came with the isolation of

protoplasts first demonstrated by Prof. Edward C. Cocking in 1960 using cell wall

degrading enzymes. In 1968, Takabe et al. for the first time demonstrated the

totipotency of protoplasts and successfully obtained tobacco plants from

mesophyll protoplasts. This was followed by regeneration of first interspecific

hybrid plants (Nicotiana glauca × N. langsdorfii) by Carlson et al. 1972.

2.1. Micropropagation

It is a technique of tissue culture in which plants are multiplied in an

enormous number under aseptic condition and controlled environment.

Micropropagation allows the production of large numbers of identical plants from

small pieces of stock plant in a relatively short period of time. In most of these

cases, the original piece of tissue is taken either from the leaf, shoot-tip, axillary

bud, stem or root tissue. In some cases, the original explant is not destroyed in the

process, a factor of considerable importance to the owner of a rare or endangered

or unusual plant. This methodology is a true means of accelerated asexual

19

propagation and the plants produced by these techniques respond similarly to any

own-rooted plant (Akin-Idowu et al. 2009). It has been estimated that axillary bud

proliferation approach typically results in an average of ten-fold increase in shoot

numbers per monthly culture passage (Chawla 2003).

Murashige (1974) introduced the basic micropropagation plan involving

three major stages. Stage I: Establishment of axenic cultures. Stage II:

Proliferation and multiplication of shoots from the established explants. Stage III:

Rooting of in vitro regenerated shoots. A stage IV is also included in some cases

where establishment of plantlets in soil is done i.e., the acclimatization stage. This

is one of the most important and crucial step in micropropagation process. It

involves slowly weaning of the plantlets from a high humidity, low light and warm

environment to what would be considered as a normal growth conditions (low

humidity, high temperature and ample of microbes) for the species in question.

Sometimes, a Stage 0 is also added as an additional stage which involves selection

and maintenance of stock plants for culture initiation by Debergh and Maene

(1981). Numerous factors are reported to influence the success of in vitro

propagation of different medicinal plants as reviewed by Murashige (1974),

Hussey (1980), Ammirato (1983), Hu and Wang (1983), Bhagyalakshmi and

Singh (1988), Shorts and Roberts (1991), Rout et al. (2000a) and Akin-Idowu et

al. (2009). Recently, tissue culture gained an unbeatable recognition in plant

species leading to the commercial application. A number of plant species have

been investigated around the globe, out of which review of some important

medicinal plants has been discussed in this chapter.

2.1.1. Direct plant regeneration

Clonal propagation of plants in vitro was started as a result of significant

observations made by Morel (1960) during the culture of virus free orchid,

Cymbidium. The discovery of cytokinin and tissue culture media (MS 1962)

provided further impetus to meristem tip culture technique and recently proved as

a commercially viable technology for mass propagation of numerous crop plants.

Direct plant regeneration by enhanced axillary bud proliferation offers several

20

advantages over indirect organogenesis. It assures the production of identical

plants with similar characteristics features of the parent tissue while indirectly

obtained plants do have some genetic abnormalities or instability. Consequently,

clonal propagation played an important role in the development of a worldwide

industry that produces more than 250 million plants (Kane 2000). In vitro

approaches have been proved handy in establishing plants that are genetically

uniform and enriched in selected characters (Alcaraz and Alya-Rocha 1982).

2.1.1.1. Explant type

The selection of suitable explant for the initiation of a cloned culture is

largely dependent on the method to be adopted for in vitro culture. Rapid shoot

regeneration has been achieved with a wide range of species with initial explants

being taken from normal aerial shoots of field grown medicinal plants including

Asclepias curassavica (Pramnaik and Datta 1986), Ocimum spp. (Pattnaik and

Chand 1996), Ocimum basilicum (Sahoo et al. 1997; Siddique and Anis 2007 b

and c, 2008), Jasminium officinale (Bhattacharya and Bhattacharya 1997), Alpinia

galanga (Borthakur et al. 1999), Murraya koenigii (Babu et al. 2000), Cunila

galioides (Fracaro and Echeverrigaray 2001), Myrica esculenta (Bhatt and Dhar

2004), Rauvolfia tetraphylla (Faisal et al. 2005), Gloriosa superba (Hassan and

Roy 2005), Vitex negundo (Ahmad and Anis 2007) and Holarrhena antidysentrica

(Mallikarjuna and Rajendurdu 2007). However, propagules derived from aseptic

seedlings or from established cultures are successfully used for tissue culture

purposes. These propagules are favorable as they rule out contamination

associated with ex vitro derived explants and also obviate the dependency on field

material. The use of aseptic explants are widely utilized in propagation of many

medicinal and ornamental plants viz., Melissa officinalis (Tavares et al. 1996),

Glycine max (Kaneda et al. 1997), Liquidambar styraciflua (Kim et al. 1997),

Vicia faba (Khalafalla and Hattori 1999), Sesbania drumondii (Cheepala et al.

2004), Lathyrus sativus (Barik et al. 2004), Mucuna pruriens (Faisal et al. 2006 a

and b), Artemesia vulgaris (Sujatha and Kumari 2007), Cassia angustifolia

(Siddique and Anis 2007 a), Andrographis paniculata (Purkayastha et al. 2008),

21

Cardiospermum halicacabum (Jahan and Anis 2009) and Tuberaria major

(Goncalves et al. 2010).

A variety of explants are available for micropropagation purposes but the

most productive and responsive are those with actively growing meristems (Elliot

1970; Davies 1980; Sivakumar and Krishnamurthy 2004; Avani et al. 2006;

Prakash and Staden 2007). The most important technique in micropropagation is

meristem proliferation, wherein shoot meristems i.e., shoot tips or apical

meristems or axillary buds are cultured to regenerate multiple shoots without any

intervening callus phase. Nodal segments possessing an axillary bud is one of the

most commonly, rapidly and widely used explant which showed better

performance in the event of multiple shoot production in a large number of plant

species like Cephalis ipecacuanha (Jha and Jha 1989), Mucuna pruriens

(Chattopadhyay et al. 1995; Faisal et al. 2006 a), Orthosiphon spiralis

(Elangomathavan et al. 2003), Psoralea corylifolia (Jeyakumar and Jayabalan

2002; Anis and Faisal 2005), Holostemma ada-kodien (Martin 2002), Annona

squamosa (Amin et al. 2002), Morus alba (Anis et al. 2003), Rauvolfia tetraphylla

(Faisal et al. 2005), Bambusa vulgaris (Ndiyae et al. 2006), Cassia angustifolia

(Siddique and Anis 2007 a), Cardiospermum halicacabum (Jahan and Anis 2009),

Oroxylum indicum (Gokhale and Bansal 2009), Solanum nigrum (Sundari et al.

2010) and Ceropegia attenuata (Chavan et al. 2011).

Another popular explant widely utilized for multiple shoot production is the

shoot tip explant. Multiplication of plants via shoot tips harboring apical meristem

results in the development of genetically homogenous, healthy and vigorous plants

free from viral and fungal infections. The method was successfully applied in

Digitalis thapsi (Herrera et al. 1990), Gossypium meristem (Saeed et al. 1997),

Saussurea lappa (Johanson et al. 1997), Acacia catechu (Kaur and Kant 2000),

Lippia alba (Gupta et al. 2001), Phyllanthus amarus (Ghanti et al. 2004),

Basilicum polystachyon (Amutha et al. 2008) and Chrysanthemum morifolium

(Waseem et al. 2009).

22

Actively growing materials especially meristems were more responsive

than dormant buds. However, there are cases where successful regeneration was

carried by using diverse types of explants apart from nodal and shoot tips. These

includes cotyledonary nodes as used in Melissa officinalis (Tavares et al. 1996),

Gossypium hirsutum (Rauf et al. 2004), Pterocarpus marsupium (Anis et al.

2005), Capsicum annuum (Siddique and Anis 2006), Nyctanthes arbor-tristis

(Siddique et al. 2006), Aegle marmelos (Nayak et al. 2007), Crotolaria verrucosa

(Hussain et al. 2008), leaf explants of Centella asiatica (Banerjee et al. 1999),

inflorescence of Ocimum sanctum (Singh and Sehgal 1999) and Carthamus

tinctorius (Sujatha and Kumar 2007). Mao et al. (1995) observed that nature and

condition of explant of Cleodendron colebrookianum had a significant influence

on the multiplication rate.

2.1.1.2. Media type

The selection and development of a culture medium is vital to the success

of tissue culture programmes. No single medium will support the growth of all

cells, and changes in the medium are often necessary for different types of growth

responses from a single explant. A literature search will be useful for the selection

of a suitable medium. The outmost and the most promising media was formulated

by Murashige and Skoog in 1962 which was frequently applied in

micropropagation of many medicinal plants including Paulownia fortuneii

(Venkateswarlu et al. 2001), Ceropegia candelabrum (Beena et al. 2003), Acacia

catechu (Kaur and Kant 2000), Rauvolfia tetraphylla (Faisal et al. 2005), Glycine

max (Shan et al. 2005), Cassia angustifolia (Siddique and Anis 2007 d) and

Albizzia lebbeck (Perveen et al. 2011).

The composition, type and strength of basal medium also played an

important role in shoot multiplication. Full strength of MS medium was found

favourable for multishoot production in Tylophora indica (Faisal et al. 2003),

Ludwigia repens (Ozturk et al. 2004), Kalanchoe blossfeldiana (Sanikhani et al.

2006), Holarrhena antidysentrica (Mallikarjuna and Rajendrudu 2007), Rehmania

glutinosa (Park et al. 2009) and Cardiospermum halicacabum (Jahan and Anis

23

2009) and Nyctanthes arbor-tristis (Jahan et al. 2011 a). However, at times

reduction in MS salts to either half or one-third or to one-fourth enhances the

capacity of shoot proliferation. Koroch et al. (1997) reported higher rates of shoot

multiplication in Hedeoma multiflorum on half-strength MS medium than on its

full strength. Similar observations were made by Faisal et al. (2006 a and b) in

Mucuna pruriens and Aktar et al. (2008) on Dendrobium whilst Jang et al. (2003)

observed superiority of one-third and one-quarter strength MS medium over full

strength MS in Dionea muscipula.

Other than MS medium, there are a variety of basal medium formulations

for in vitro organogenesis. Superiority of B5 (Gamborg’s et al. 1968) over L2

(Philips and Collins 1979) and MS (1962) medium for the production of maximum

shoots in Glycine max has been established by Kaneda et al. (1997). Bhatt and

Dhar (2004) established higher efficiency of Woody Plant Medium (WPM, Lloyd

and Mc Cown 1980) over other types of medium like B5 and MS and their strength

tried for shoot proliferation in Myrica esculenta. They observed that neither MS

nor B5 gave satisfactory response even when the salt concentration is reduced to

half and shoot response was severely inhibited. Similarly, a modification of WPM

was successfully applied by Krogstrup et al. (2005) for micropropagation of

Dorstenia gigas while Normah et al. (1995) reported unsuitability of WPM

medium for shoot multiplication on Mangosteen and found full strength MS

medium as the best composition for optimum multiplication. Similarly, five

different media types i.e., MS, B5, NN (Nitsch and Nitsch 1969), N6 (Chu et al.

1975) and QL (Quoirin and Lepoivre 1977) were compared by Fracaro and

Echeverrigaray (2001) in Cunila galioides and reported highest shoot proliferation

in MS and NN media. Tetsumura et al. (2008) evaluated different basal medium

for four highbush blueberry cultivars and found a combination of MS and WPM in

equal parts produced best shoot growth.

2.1.1.3. pH

pH determines many important aspects of the structure and activity of

biological macromolecules. The pH range from 5.0 to 6.0 of the nutrient medium

24

has been reported to be suitable for in vitro growth of explant. The standard

procedure for pH adjustment in tissue culture media is to correct the pH of the

medium with an acid or a base prior to autoclaving (Skirvin et al. 1986). The pH

affects nutrient uptake as well as enzymatic and hormonal activities in plants

(Bhatia and Ashwath 2005). The detrimental effects of pH are generally related to

an imbalance in nutrient uptake rather than to direct cell damage (Huda et al.

2009).

Dougall (1980) reviewed the literature associated with in vitro pH changes

and believed that the cause of such changes is best explained in terms of

ammonium (NH4+) and nitrate (NO3

-) uptake from the medium. In addition, the

status of a solidifying agent is readily affected by a medium pH. Bhatia and

Ashwath (2005) reported that a high pH above 6.0 produces a very hard medium

and a pH lower than 5.0 does not sufficiently solidify the medium.

Parliman et al. (1982) tested the effect of different medium pH (3.5, 4.5, 5.5

and 6.5) in Dionea muscipula and found that the optimum pH for shoot

proliferation and elongation was 5.5 and it was severely inhibited in more acidic

medium. A similar study was conducted by Faisal et al. (2006 a and b) where a

wide range of pH was tested for shoot induction and found maximum

multiplication rate at 5.8 pH. Similar findings were reported currently by Siddique

and Anis (2007 a) in Cassia angustifolia and Jahan and Anis (2009) in

Cardiospermum halicacabum.

2.1.1.4. Plant Growth Regulators (PGRs)

Plant growth regulators are the organic compounds, either natural or

synthetic which regulates the plant growth and development. These are the signal

molecules produced in extremely low concentrations and are active at the site

remote from where they are produced. The influence of plant growth regulators

and their interaction on micropropagation of different plant species have been

discussed by Mor and Zieslin (1987), Rout et al. (1989, 2000a), Skirvin et al.

(1990) and Rout and Das (1997 a and b).

25

Khan et al. (2006) reported the use of hormone free MS medium for

achieving maximum results from shoot tip explants of Kalanchoe tomentosa. They

suggested that plant growth regulator had a very little role in the in vitro

development of K. tomentosa since very high rates of multiplication were obtained

in MS medium than with plant growth regulators. However, Taha et al. (2001)

reported that good results could be obtained in full strength MS medium but the

addition of plant growth regulators becomes essential to improve the response.

Since, the main function of cytokinin is the release of axillary bud from the

phenomenon of apical dominance. The requirement of growth hormone as a

supplement for obtaining optimal response for sprouting and further shoot

development is well documented in a number of species.

Cytokinins and auxins are the most widely used plant growth regulators in

plant tissue culture and usually used either singly or in various combinations.

Cytokinins comprise a separate class of growth regulator particularly stimulates

protein synthesis and participates in cell cycle. These compounds overcome apical

dominance and release lateral buds from dominancy, and thus help in axillary and

adventitious shoot formation. The use of cytokinin at a critical level has been

shown to be most important in multiplication of many important medicinal plant

species (Jha and Jha 1989; Tavares et al. 1996; Pattnaik and Chand 1996;

Borthakur et al. 1999; Fracaro and Echeverrigaray 2001; Faisal et al. 2006 a and b;

Sanikhani et al. 2006; Siddique and Anis 2007 a and b; Hussain et al. 2008;

Makara et al. 2010).

6-Benzyladenine or BA is one of the most frequently used cytokinin in

plant tissue cultures. Benjamin et al. (1987) reported that BA at high concentration

(1-5 ppm) stimulates the development of axillary meristems and shoot tips of

Atropa belladona. Bhattacharya and Bhattacharya (1997) developed an in vitro

culture protocol for Jasminium officinale using BA at elevated level (17.76 µM) in

MS medium. Similarly, Purkayastha et al. (2008) obtained maximum number of

shoots at 10 µM BA. A considerable higher range of BA (22.2 µM) was used by

Koroch et al. (1997) for micropropagation of Hedeoma multiflorum. However,

26

lower concentrations of BA (2.22 µM) was applied with full strength MS medium

by Elangomathvan et al. (2003) to obtain multiple shoots in Orthosiphon spiralis.

Nayak et al. (2007) investigated the effect of BA at low concentrations on shoot

proliferation in Aegle marmelos and found optimal response in MS medium

supplemented with 6.6 µM BA. Gokhale and Bansal (2009) obtained multiple

shoots directly from apical and axillary buds of Oroxylum indicum in MS medium

amended with 4.43 µM BA. The beneficial role of BA on shoot multiplication has

been reported in a number of cases including Ocimum spp. (Ahuja et al. 1982),

Melissa officinalis (Tavares et al. 1996), Cunila galioides (Fracaro and

Echeverrigaray 2001), Lathyrus sativus (Barik et al. 2004), Phyllanthus amarus

(Ghanti et al. 2004), Pterocarpus marsupium (Anis et al. 2005), Bambusa vulgaris

(Ndiaye et al. 2006), Mucuna pruriens (Faisal et al. 2006 a and b), Rosa hybrida

(Azadi et al. 2007), Ruta graveolens (Bohidhar et al. 2008), Moringa oleifera

(Marfori 2010) and Chlorophytum borivilliens (Sharan et al. 2010).

Kinetin (Kn) or 6-furfurylaminopurine, is another most important cytokinin

applied usually for large scale micropropagation of medicinal and ornamental

plant species was originally isolated by Miller et al. (1955). Lal et al. (1988)

achieved rapid shoot proliferation rate in Picrorhiza kurroa using kinetin at 1.0 –

5.0 mg/l. Pattnaik and Debata (1996) obtained highest shoot regeneration

frequency in Hemidesmus indicus on MS medium amended with kinetin.

Likewise, Amo-Marco and Ibanez (1998) reported rapid shoot proliferation rate in

a threatened statice, Limonium cavanillesii on MS medium fortified with high

concentration of Kn (2-5 mg/l), while, Borthakur et al. (2000) used low

concentrations of Kn (0.05 mg/l) for establishing an in vitro regenerative protocol

of two traditional medicinal plants, Eclipta alba and Eupatorium adenophorum via

single step nodal cuttings method on full strength MS medium. However,

moderate levels of Kn were successfully applied by Kamastaityte and Stanys

(2004) for obtaining maximum micropropagation frequency in Allium cepa. Bhatt

and Dhar (2004) studied various factors controlling micropropagation of Myrica

esculenta and inferred best results in Woody Plant Medium supplemented with 10

27

µM Kn. Superiority of Kn over other growth regulators for direct in vitro shoot

multiplication in various medicinal plants has been well documented by several

workers including Jha and Jha (1989) in Cephalis ipecacuanha, Herrera et al.

(1990) in Digitalis thapsi, Paek and Murthy (2002) in Fritillaria thunbergii, Jang

et al. (2003) in venus fly trap. The successful application of this growth regulator

for multiple shoot production in a number of medicinally important plant species

includes Alpinia galanga (Borthakur et al. 1999), Eurycoma longifolia (Hussein et

al. 2005), Tinospora cordifolia (Gururaj et al 2007), Ricinus communis

(Chaudhary and Sood 2008) and Rauvolfia tetraphylla (Harisaranraj et al. 2009).

2-isopentenyladenine (2-iP) reported to be the best cytokinin for shoot

multiplication in blueberry by Cohen (1980) and in garlic by Bhojwani (1980).

Chattopadhyay et al. (1995) achieved rapid micropropagation protocol for Mucuna

pruriens using 2-iP. Mills et al. (1997) mentioned that 2-iP at higher concentration

of 30.5 mgdm-3 was optimum for differentiating maximum number of shoots in

Simmondsia chinensis. However, Taha et al. (2001) reported that shoot bud

proliferation ability of date palm shoot tips was strongly enhanced by low

concentration of 2-iP (3mgdm-3). Similarly, Jakola et al. (2001) obtained best

results in Vaccinium myrtillus and V. vitis-idaea utilizing higher levels of 2-iP

(49.2 µM and 24.6 µM) on modified MS medium while lower concentrations

(12.3 µM or 24.6 µM) were recommended by Pereira (2006) for other species of

Vaccinium cylindraceum micropropagation on Zimmermann and Broome

medium. In case of Rhododendrons, Vejsadova (2008) found highest shoot

multiplication rate on MS medium amended with 2-iP. Similarly, Singh and

Gurung (2009) proved 2-iP as a most effective cytokinin than BA or Kn for

multiple shoot induction in Rhododendron maddeni.

Thidiazuron (TDZ), a substituted phenyl urea (N-phenyl1, 2, 3-thidiazol-5-

ylurea) is a potent bioregulant known to possess high cytokinin activity. TDZ has

been shown to possess a powerful ability to induce high rates of shoot

proliferation in a number of medicinally and ornamentally important plant species

starting from herbaceous to recalcitrant woody plants (Fiola et al. 1990; Malik and

28

Saxena 1992; Huetteman and Preece 1993; Kanayand et al. 1994; Faisal et al.

2005, Ahmad et al. 2006; Siddique and Anis 2007 a and d; Hussain et al. 2008;

Corredoira et al. 2008; Jahan and Anis 2009; Kumar and Reddy 2010). Atta-Alla

and Van Staden (1997) achieved highest number of proliferated shoots of Yucca

aloifolia on MS medium supplied with 4.54 µM TDZ. An efficient

micropropagation protocol for Psiadia arguata using TDZ (0.5-1.0 mg/l) has been

developed by Kodja et al. (1998). Khalafalla and Hattori (1999) used two

cytokinins (TDZ or BA) for shoot multiplication in Vicia faba. They reported that

inspite of single cytokinin used, a combination of cytokinins acts efficiently.

Mohammad-Yaseen (2002) obtained complete plantlets of Eryngium foetidum in

addition to flower formation on TDZ supplemented MS medium. Faisal et al.

(2005) studied the effect of thidiazuron on in vitro shoot proliferation of Rauvolfia

tetraphylla. They obtained best results by applying higher concentrations of

thidiazuron (5 µM) together with full MS medium. Similarly, higher levels were

used by Siddique and Anis (2007 b) for multiplying Cassia angustifolia (5.0 µM).

While, Ahmad et al. (2006) investigated the effect of TDZ (0.1-10 µM) at varying

levels from nodal explants of Capsicum annuum and found 1.0 µM TDZ as a best

concentration for maximum shoot multiplication rate. However, lower levels of

this potent cytokinin have been recommended by Huetteman and Preece (1993)

for obtaining maximum shoot proliferation results. The shoot forming capacity of

Crotolaria verrucosa from cotyledonary node explants was strongly influenced by

very low TDZ concentration i.e., 0.1 µM (Hussain et al. 2008). Similarly, Jahan

and Anis (2009) achieved rapid shoot multiplication efficiently in Cardiospermum

halicacabum by applying TDZ at a very low concentration i.e., 0.3 µM amended

in MS medium using nodal explants of aseptic seedlings.

In addition to the tremendous ability of TDZ for multiple shoot production,

there are problems of conversion of TDZ induced shoots to complete plantlets. It

results in the formation of fasciated, distorted and hyperhydric shoots. Also, the

shoots are clumped together to form a rosette of shoots which did not elongate

further (Preece and Imel 1991; Huetteman and Preece 1993; Faisal et al. 2005;

29

Ahmad et al. 2006). This problem can be overcome by transferring these

regenerated shoot clusters to hormone free medium to nullify the negative effects

of thidiazuron. Such technique was successfully applied by various workers

including Huetteman and Preece (1993), Kodja et al. (1998), Ahmad et al. (2006),

Siddique et al. (2006), Ozturk et al. (2004), Raghu et al. (2006), Jahan and Anis

(2009), Makara et al. (2010) and Jahan et al. (2011 b).

Thidiazuron is selected for micropropagation of a wide array of plant species because of its tremendous ability to stimulate higher rates of shoot proliferation. When compared to purine based cytokinin, TDZ is found to be active at lower concentrations. The aminopurine cyotkinins have similar effects at higher concentrations i.e., in between 1 µM to 10 µM. This range with TDZ results in excessive callus formation and cessation of shoot growth (Huetteman 1988; Hutchinson et al. 2004). The TDZ is more effective than adenine-based compounds for inducing axillary shoot formation in many woody species (Huetteman and Preece 1993; Kim et al. 1997; Thimmappaiah et al. 2002; Ahmad and Anis 2007; Jahan and Anis 2009; Sivanesan et al. 2011). Whilst, there are many cases, where a short duration exposure to higher concentrations of TDZ (0-100 µM) in liquid state found influential in stimulating maximum regeneration potential within the explant. Such preconditional effect has been assessed by Singh and Syamal (2001) in rose, Prathanturarug et al. (2005) in Curcuma longa, Thomas (2007) in Curculigo orchioides, Siddique and Anis (2007 b) in Ocimum basilicum and Jahan et al. (2011 b) in Nyctanthes arbor-tristis.

In most of the cases, either the phenylurea type or the adenine derivative types of cytokinins alone were found effective in differentiating multiple shoots from the given explants (Garland and Stoltz 1981). The utilization of TDZ as a sole growth regulator in plant tissue culture systems for induction of multiple shoots has been well documented in a number of plant species including Scilla natalensis (Mc Carten and Van Staden 1998), Rauvolfia tetraphylla (Faisal et al. 2005), sugarcane (Gill et al. 2006), Kalanchoe blossfeldiana (Sanikhani et al. 2006), Pterocarpus marsupium (Husain et al. 2007), Crotolaria verrucosa (Hussain et al. 2008) and Cardiospermum halicacabum (Jahan and Anis 2009). However, reports are available proving positive modification of shoot formation

30

31

from cultured explants when they were exposed to TDZ together with a low concentration of auxin as reported by Atta –Alla and Van Staden (1997), Ozturk et al. (2004), Siddique and Anis (2007 a), Yucesan et al. (2007) and Farhani et al. (2008).

Sanikhani et al. (2006) reported that adding auxin in combination with TDZ did not improve shoot regeneration while the maximum aptitude was obtained with 0.45 µM thidiazuron in Kalanchoe blossfeldiana on MS medium. Similarly, addition of auxins failed to influence the rate of shoot multiplication in Crotolaria verrucosa (Hussain et al. 2008). A number of reports are available on a wide range of plant which exhibits maximum micropropagation frequency when cytokinins are applied alone including reports of Borthakur et al. (1999) on Alpinia galanga, Fracaro and Echeverrigaray (2001) on Cunila galioides, Elangomathvan et al. (2003) on Orthosiphon spiralis, Anis et al. (2005) on Pterocarpus marsupium and Jahan and Anis (2009) on Cardiospermum halicacabum.

Borthakur et al. (2000) reported micropropagation of Eclipta alba and Eupatorium adenophorum on MS medium amended with Kinetin as a sole growth regulator. Fracaro and Echeverrigaray (2001) evaluated the effect of growth regulators on shoot proliferation of Cunila galioides via axillary buds and achieved best multiplication rate on MS medium with 8.8 µM BA. They further established that addition of auxin together with cytokinins significantly reduced the number of shoots. Similarly, BA as a sole growth regulating hormone proved effective in multiple shoot formation in Balanites aegyptiaca (Ndoye et al. 2003), Lathyrus sativus (Barik et al. 2004) and Bambusa vulgaris (Ndiaye et al. 2006). Growth promoting ability of Kinetin has been well established by various workers in inducing multiple shoots including reports of Borthakur et al. (1999) in Alpinia galanga, Hussein et al. (2005) in Eurycoma longifolia and Kamstatiye and Stanys (2004) in Allium cepa. Amo-Marco and Ibanez (1998) reported shoot induction and multiplication of Limonium cavanillesii by applying Kn at 2mg/l. Pereira (2006) obtained an efficient in vitro shoot propagation method for Vaccinium cylindraceum using 2-iP. Similarly, Vejsadova (2008) supplied 2-iP only in MS medium for multiplication of Rhododendrons.

The shoot multiplication ability of cytokinins alone has been well established on a wide range of medicinal and ornamental plant species (Table 3).

Table 3. In vitro regeneration of medicinal plants.

Plants name Explants Used References

Alpinia galanga Rhizome Buds Borthakur et al. (1999)

Andrographis paniculata NS Purkayastha et al. (2008)

Bambusa vulgaris NS Ndiaye et al. (2006)

Banana Suckers Makara et al. (2010)

Bixa orellana ST, NS Sharon and D’ Souza (2000)

Capsicum annuum NS Ahmad et al. (2006)

Cardiospermum halicacabum NS Jahan and Anis (2009)

32 Chlorophytum brivilliens ST Sharan et al. (2010)

Cunila galioides AB Fracaro and Echeverrigaray (2001)

Eclipta alba NS Gawde and Paratkar (2004)

Eclipta alba and Eupatorium adenophorum NS Borthakur et al. (2000)

Holarrhena antidysentrica NS Raha and Roy (2001)

Kalanchoe blossfeldiana L, I Sanikhani et al. (2006)

32

Abbreviations: AB = Axillary buds; CN = Cotyledonary node; Hyp = Hypocotyl; I = Internode; NS = Nodal segment; L =

Leaves; ST = Shoot tip

Orthosiphon spiralis NS Elangomathavan et al. (2003)

Oroxylum indicum AB, AM Gokhale and Bansal (2009)

Vitex negundo NS Ahmad and Anis (2007)

Melaleuca alternifolia AB de Oliveria et al. (2010)

Pisum sativum Hyp Massimo et al. (1996)

Vigna radiata CN, Hyp Amutha et al. (2006)

Tribulus terrestris NS Raghu et al. (2010)

Phyllanthus amarus ST Ghanti et al (2004)

Lippia alba ST Gupta et al. (2001)

Rauvolfia tetraphylla NS Faisal et al. (2005)

Rosa hybrida AB Azadi et al. (2007)

Lathyrus sativus CN Barik et al. (2004)

Pterocarpus marsupium CN Anis et al. (2005)

33

33

The influence of plant growth regulators and their interaction played an important

role in shoot proliferation in many plant species. Cytokinins was required in an

optimal quantity for shoot proliferation in many genotypes but inclusion of low

concentration of auxins alongwith optimal concentration of cytokinin triggers the

rate of shoot proliferation (Jha and Jha 1989; Tsay et al. 1989; Makunga et al.

2005; Faisal et al. 2006 a and b; Siddique et al. 2007; Perveen et al. 2011; Jahan et

al. 2011 a). The synergistic effect of auxin and cytokinin positively modifies the

frequency of shoot regeneration parameters and has been found efficient on a

range of medicinal plants viz., Annona squamosa (Amin et al. 2002), Myrica

esculenta (Bhatt and Dhar 2004), Morus alba (Anis et al. 2005), Mucuna pruriens

(Faisal et al. 2006 a and b) and Aegle marmelos (Nayak et al. 2007).

Production of multiple shoots was higher in Plantago ovata using Kn (4-6

µM) in combination with NAA (0.05 µM) as indicated by Barna and Wakhlu

(1988). Applying the same combination (Kn-NAA), Herrera et al. (1990)

formulated one step shoot multiplication and rooting protocol for Digitalis thapsi.

Pattnaik and Debata (1996) reported micropropagation of Hemidesmus indicus

through axillary bud culture by augmenting MS medium with 1.15 µM Kn and

0.054 µM NAA. Similarly, Saeed et al. (1997) obtained best shoot development

media for Gossypium meristem on MS medium amended with a combination of

0.46 mM Kn and 2.68 mM NAA. Also, higher degree of shoot multiplication and

proliferation rate was achieved in Myrica esculenta using Kinetin in conjunction

with NAA by Bhatt and Dhar (2004). Complete plantlets of Ricinus communis was

successfully raised by Chaudhary and Sood (2008) while applying kinetin in

combination with NAA in MS medium. Kn-NAA synergism and their triggering

effect on shoot bud induction and multiplication has established perfectly in

Cephailis ipecacuanha (Jha and Jha 1989), Gossypium hirsutum (Rauf et al.

2004), Cordia verbenacea (Lameira and Pinto 2006), Alpinia officinarum

(Selvakkumar et al. 2007) and Gardenia jasminoides (Duhoky and Rasheed 2010).

34

35

However, Lal et al. (1988) reported a marked increase in the shoot forming

capacity of shoot tips when Kn was added with IAA. Likewise, Thakur and

Srivastava (2006) developed highly efficient plant regeneration method in Populus

ciliata via leaf explants on MS medium supplemented with Kn-IAA combination

while Kn-IBA synergism found effective in Holostemma ada-kodien (Martin

2002) and Arnebia euchroma (Manjkhola et al. 2005).

BA in combination with NAA increases the shoot forming capacity of

nodal and cotyledonary nodes manifolds in Mucuna pruriens as reported by Faisal

et al. (2006 a and b). Similar observations were made by Anis et al. (2005) in

Psoralea corylifolia, Priyakumari and Sheela (2005) in Gladiolus grandiflorus and

Girijashankar (2011) in Acacia auriculiformis. Whereas, BA-IAA combinations

was effective in Ocimum basilicum (Sudhakaran and Sivasankari 2002), Aegle

marmelos (Nayak et al. 2007) and Chrysanthemum morifolium (Waseem et al.

2009). Iapichino and Airo (2008) reported adding 2-iP with IAA found suitable for

propagation in Metrosideros excelsa.

The synergistic effect of auxins and cytokinins on shoot multiplication

ability of various medicinal and ornamental plants has been well documented in

literature (Table 4).

Table 4. In vitro propagation of medicinal plants

Plants name Explants Used References

Acacia auriculiformis NS Girijashankar (2011)

Artemesia judaica Intact Seedling Liu et al. (2003)

Azadirachta indica NS Arya et al. (1995)

Balanites aegyptiaca NS Siddique and Anis (2009); Anis et al. (2010) Calendula officinalis Hyp, C, CN Cocu et al. (2004)

Cassia angustifolia NS Siddique and Anis (2007a)

36

Cephalis ipecacuanha NS Jha and Jha (1989)

Clitorea ternatea Root Shahzad et al. (2007)

Decalpis hamiltonii ST Gridhar et al. (2005)

Hagenia abyssinica Meristem Feyissa et al. (2005)

Hemidesmus indicus AB Pattnaik and Debata (1996)

Holostemma ada-kodien NS Martin (2002)

36

37

Leptadenia reticulata NS Arya et al. (2003)

Ludwigia repens AM Ozturk et al. (2004)

Mellisa officinalis CN Tavares et al. (1996)

Mucuna pruriens NS, CN Faisal et al. (2006 a and b)

Myrica esculenta NS Nandwani (1994); Bhatt and Dhar (2004)

Nyctanthes arbor-tristis CN Siddique et. al. (2006); NS Jahan et al. (2011 a and b) Ocimum basilicum NS Sahoo et al. (1997); Siddique et al. (2007 b and c, 2008) Rehmania glutinosa L Park et al. (2009)

Tylophora indica AB, NS Sharma and Chandel (1992);

Zingiber spectable AM Faria and Illg (1995) Abbreviations: AB = Axillary buds; AM = Apical meristem; C = Cotyledon; CN = Cotyledonary node; Hyp = Hypocotyl; I = Internode; L = Leaves; NS = Nodal segment; ST = Shoot tip

Faisal et al. (2007)

32

2.1.1.5. Subculturing

The rate of in vitro shoot multiplication depends greatly on subculturing of

proliferating shoot cultures. Apostolo et al. (2005) reported that in prolonged

cultures, the concentrations of nutrients in the medium is gradually exhausted and

at the same time, the relative humidity of culture vessel decreases leading to

drying of developed shoots, as the high humidity in culture vessel helps to

promote the rapid shoot growth. Since, it is desired to establish cultures that could

be continuously multiplied in order to produce regular output of desired number of

shoots for field transfer, a regular subculturing has been recommended in order to

maintain rejuvenility of tissues during the period of culture (Debnath 2004). Bajaj

et al. (1988) obtained around 2200 plantlets of Thymus vulgaris from a single

shoot grown in vitro in 5 months (four passages). Ajithkumar and Seeni (1998)

reported that repeated subculturing of nodes and leaf from shoot cultures of Aegle

marmelos helped to achieve continuous production of callus-free healthy shoots at

least upto five subculture cycles. Borthakur et al. (1999) established a mass

multiplication protocol for Alpinia galanga by subculturing the regenerated

explants to kinetin supplemented medium for more than one year. They obtained

an average of 1000 plantlets with four to five successive subculture cycles i.e.,

within 40-45 days. Prevalek-Kozlina (1997) obtained highest shoot multiplication

rate after three subculture passages in Fibigia triqueira while repeated

subculturing of shoot tips and single node explants of Cunila galioides for eight

months as recommended by Fracaro and Echeverrigaray (2001) enabled mass

multiplication of shoots without any evidence of decline. Barik et al. (2004)

established shoot cultures of Lathyrus sativus or grasspea via subculturing original

cotyledonary nodes on fresh medium after each harvest of newly formed shoots.

Similarly, Hussain et al. (2008) established proliferating shoot cultures of

Crotolaria verrucossa by repeated culturing the original explant after harvesting

the newly formed shoots. They also reported that repeated subculturing was said to

be one of the method to maintain juvenility. The shoot forming potential of

original explant was not declined even after five subcultures. Likewise, repeated

38

subculturing of nodes induced a high frequency shoot multiplication rate in

Tribulus terrestris by Raghu et al. (2010) and Andrographis neesiana by

Karuppusamy and Kalimuthu (2010).

In case of Vigna radiata, Amutha et al. (2006) reported that an initial

exposure to high exposure to higher concentrations of TDZ (0.9 µM) followed by

three successive transfer to low concentrations (0.09 µM) resulted in the

production of 104 shoots from cotyledon and 30 shoots from hypocotyl explants.

Subculture effects are known to differ from one species to another. Shirin et al.

(2000) reported a 13-fold increase in the multiplication rate of shoots of

Kaempferia galanga upon subculturing while a much higher rate of shoot

proliferation was reported in Leptadenia reticulata by Arya et al. (2003). Hassan

and Roy (2005) established a shoot multiplication protocol for Gloriosa superba

by repeated subculturing original explants (shoot tips and single nodes) to the

regenerative medium which helped to achieve continuous production of shoots

upto five to ten subculture cycles. Purkayastha et al. (2008) achieved break free

rapid production of multiple shoots from nodal explants of Andrographis

paniculata by subculturing the mother explant twicely on MS medium

supplemented with 10 µM BA while Bohidhar et al. (2008) observed an increase

in the number of shoots with an increase in number of subculture passages (third

passage) in Ruta graveolens. Similarly, in Simmondsia chinensis, around 10-15

shoots were produced by repeated subculturing upto three successive subcultures

(Singh et al. 2008). In the event of multiple shoot production, the rate of shoot

proliferation reached an optimal level beyond which a gradual decline in shoot

parameters was noticed as demonstrated in investigations made on Vitex negundo

(Sahoo and Chand 1998), Holorrahena antidysentrica (Raha and Roy 2001),

Feronia limonia (Hiregoudar et al. 2003), Oroxylum indicum (Gokhale and Bansal

2009) and Balanites aegyptiaca (Anis et al. 2010).

Siddique et al. (2007 a and d) studied the effect of subculture passages on

the number of shoots and shoot length when thidiazuron induced cultures were

transferred to TDZ free MS medium. They reported the highest shoot numbers and

39

shoot length on fourth subculture, which became stable during fifth passage and

beyond which gradual decline in multiplication rate was noticed. A similar pattern

of shoot amplification was obtained by Yousef et al. (2007) in Alstroemeria, Jahan

and Anis (2009) in Cardiospermum halicacabum and by Jahan et al. (2011 b) in

Nyctanthes arbor-tristis where the rate of multiplication and proliferation reached

a maximum on fourth passage and stabilized by fifth one. Makara et al. (2010)

also studied the carry-over effect of TDZ that enabled shoots to continue to

proliferate on hormone free media as the culture cycles increased. Accumulation

of TDZ to high levels resulted in suppression of shoot proliferation but on

exposing such tissues to a cytokinin-free medium in subsequent subcultures would

result in increased shoot proliferation and elongation.

2.2. Rooting

The success of a micropropagation protocol depends strongly on the rooting

efficiency of regenerated shoots and their subsequent acclimatization to the field

condition. De novo formation of root meristems involves complex changes in the

metabolism and it is evident that endogenous factors interact in developmental

shift leading to adventitious root formation both at biochemical and at molecular

levels (Caboni et al. 1997). The intricacies involved in adventitious rooting were

reviewed by Haissig (1974), George and Sherrington (1984), Gaspar et al. (1994)

and Rout et al. (2000 b). The in vitro regenerated shoots of various medicinal

plants rooted readily on growth regulator free MS basal medium (Cristina et al.

1990; Binh et al. 1990; Faisal and Anis 2003; Shan et al. 2005; Mallikarjuna and

Rajendrudu 2007; Jahan and Anis 2009). The ease with which microshoots roots

in vitro in the absence of exogenously supplied hormones was supported by the

fact that there may occur high endogenous auxin in these in vitro regenerated

shoots. Full strength MS medium found satisfactorily well for root induction in a

number of plant species but as the concentration of salts were reduced to half or to

much lower levels (⅓ or ¼), a striking increase in the rooting efficiency was

observed. Murashige (1979) supported the fact that relatively low salt

concentration in the medium is known to enhance rooting efficiency of

40

microshoots. In case of Yucca aloifolia (Atta-Alla and Van Staden 1997), Rotula

aquatica (Martin 2003), Lathyrus sativus (Barik et al. 2004), Potentilla potaninii

(He et al. 2006) and Mucuna pruriens (Faisal et al. 2006 a and b), half strength

MS medium was found superior to full strength MS medium for root development.

Jahan and Anis (2009) reported that rate of root multiplication amplifies when

concentration of MS salts were further reduced to one-third in Cardiospermum

halicacabum.

In some cases, rooting ability of shoots was not influenced by the strength

of basal medium as reported by Sharma and Chandel (1992), Anis and Faisal

(2005), Ahmed et al. (2006) and Rajeshwari and Paliwal (2006). Therefore, for

better rhizogenesis addition of low concentration of auxin is necessary. Depending

upon the genotypic requirement, there is a marked variation in the rooting

potential of different species and systemic trial are often needed to define the

conditions required for root induction. Different types of auxins (IBA, IAA or

NAA) were added to MS medium to promote adventitious root formation.

Efficiency of IBA over IAA or NAA on in vitro root induction has been described

in Holostemma ada-kodien (Martin 2002), Ocimum americanum (Pattnaik and

Chand 2006), Mucuna pruriens (Faisal et al. 2006 a), Alpinia officinarum

(Selvakkumar et al. 2007), Clitorea ternatea (Barik et al. 2007), Ruta graveolens

(Bohidhar et al. 2008), Andrograpis paniculata (Purkayastha et al. 2008) and

Oroxylum indicum (Gokhale and Bansal 2009). However, there are a number of

cases where IAA proved as successful root inducer with 83% frequency in

Hedeoma multiflorum (Koroch et al. 1997), 100% efficiency in Cichorium intybus

(Yucesan et al. 2007), 89% in Metrosideros excelsa (Iapichino and Airo 2008) and

90% in Cardiospermum halicacabum (Jahan and Anis 2009). Barik et al. (2004)

reported IAA better than IBA in Lathyrus sativus. IAA found to be the most

suitable auxin favorable for in vitro root induction in Scilla natalensis (Mc Carten

and Van Staden 1998), Limmonium cavanilesii (Amo-Marco and Ibanez 1998),

Helianthus annuus (Vesperinas 1998), Populus ciliata (Thakur and Srivastava

2006), Artemesia vulgaris (Sujatha and Kumari 2007) and Crotolaria verrucosa

41

(Hussain et al. 2008). Similarly, NAA was used for root induction by many

workers including Lal et al. (1988) in Picrorhiza kurroa, Podwyszynska et al.

(1998) in Alstromeia x hybrida “Juanita”, Turker et al. (2001) in Verbascum

thapsus and Mandal and Gupta (2001) in safflower and Martin (2003) in Rotula

aquatica. 100% rooting efficiency in excised shoots of Zingiber spectable was

obtained by using 5 µM IAA or NAA in liquid or gelrite gelled medium (Faria and

Illg 2004). Auxins played a major role in rooting process and their efficiency

depends on several factors such as the affinity for auxin receptor protein involved

in rooting, the concentration of free auxin that reaches target competent cells, the

amount of endogenous auxin and the metabolic stability (De Klerk et al. 1999).

However, in case of Nyctanthes arbor-tristis, Rout et al. (2007 and 2008) used a

combined treatment of IBA and IAA to achieve rooting. Various phenolic

compounds such as phloroglucinol, chlorogenic acid and salicylic acid facilitate

rooting in in vitro regenerated shoots of various leguminous plant species. The

promotive role of phloroglucinol in root induction has been reported in

Pterocarpus marsupium by Anis et al. (2005) and Husain et al. (2007).

A two step rooting procedure involving a short term exposure of

microshoots to high concentrations of auxins followed by their transfer to

hormone free medium stimulates root differentiation. The auxin IBA is being

widely used for such procedures as an effective plant growth substance. Agnihotri

and Nandi (2009) exposed shoots of Dendrocalamus hamiltonii in 100 µM IBA

for ten days and then transferred them to IBA free MS medium which helped them

to achieve 90% success. Kozomara et al. (2008) achieved 50% rooting efficiency

in microshoots of Chimonanthus praecox by pretreating them in 2.0 mg/l IBA for

7 days followed by their transfer to hormone free medium. Similar, two step

procedures using IBA treatment followed by transfer to auxin-free media has been

reported by Bag et al. (2000) in Thamnocalamus spathiflorus, Santos et al. (2003)

in Olea europea, Feyissa et al. (2005) in Hagenia abysscinica, Rajeswari and

Paliwal (2006) in Albizia odoratissima, Siddique and Anis (2007) in Cassia

42

angustifolia, Singh et al. (2008) in Simmondsia chinensis and Jahan et al. (2011 b)

in Nyctanthes arbor-tristis.

2.3. Synthetic seeds

Synthetic seed technology has received considerable attention as a

potentially cost effective clonal propagation system. Production of artificial seeds

has unraveled new vista in plant biotechnology. The technology is designed to

combine the advantages of clonal propagation with those of seed propagation and

storage. The synthetic seed or artificial seed is defined as encapsulated somatic

embryos that functionally mimic seeds and can develop into complete plantlets

under suitable in vitro conditions. Successful cases of synthetic seed production

and plantlet regeneration have been reported for cereals, vegetables, fruits,

medicinal and ornamental plants and conifers (Redenbaugh et al. 1991;

Redenbaugh 1993; Fowke et al. 1994; Piccioni and Standardi 1995; Janeiro et al.

1997; Castillo et al. 1998; Pattnaik and Chand 2000; Mandal et al. 2000; Mamiya

and Sakamato 2001; Ganapathi et al. 2001; Hao and Deng 2003; Faisal and Anis

2007; Singh et al. 2010). Earlier, synthetic seeds were referred only to somatic

embryos but in the recent past, other micropropagules like shoot buds, shoot tips,

organogenic or embryogenic calli or unipolar structures have been employed in

the production of synseeds. Thus, the concept of synthetic seeds has been set free

from its bonds to somatic embryogenesis and links the term not only to its use

(storage and sowing) and product formation (plantlet) but also to other techniques

of micropropagation like organogenesis and enhanced axillary bud proliferation

system. There are few reports available which encapsulates vegetative propagules

like axillary or apical buds which could be used for mass clonal propagation as

well as long term conservation of germplasm. The concept of synthetic seeds was

first proposed by Murashige (1977). The production of synthetic seeds was first

performed by Kitto and Janick (1982) which involves encapsulation of carrot

somatic embryos followed by their desiccation. Later in 1984, Redenbaugh et al.

developed a technique for hydrogel encapsulation of individual somatic embryos

of alfalfa in sodium alginate gel. During cold storage, encapsulated nodal

43

segments requires no transfer to fresh medium, thus reduces the cost of

maintaining germplasm cultures (West et al. 2006).

Antonietta et al. (1999) studied the effect of encapsulation of somatic

embryos in calcium alginate beads with different artificial endosperm (with

nutrients and with or without GA3) was compared to non-encapsulated and

reported that a synthetic endosperm should contain nutrients and carbon source for

germination and conversion. Ganapathi et al. (2001) suggested that ingredients in

the encapsulation matrix contribute like an artificial endosperm to the developing

encapsulated somatic embryos of banana. Sodium alginate encapsulated unipolar

propagules can be used in clonal propagation, conservation and exchange of plant

materials between laboratories (Maruyama et al. 1997). Gel encapsulation using

sodium alginate and calcium salt was a useful technique as a method for

encapsulation. This is a good combination because the ions are non-damaging

materials easy to use, have a low price and embryo to plant conversion occurs

successfully (Redenbaugh et al. 1991).

The synseeds can be stored for longer duration as encapsulated axillary

buds of Morus alba, M. australis and M. cathyara have been stored at 4 °C for 60

days while that of M. bombycis, M. latifolia and M. nigra remained viable upto 90

days (Pattnaik and Chand 2000). According to species, storage needs and the use

of the synthetic seeds, the explant encapsulated in alginate matrix, supplemented

with necessary ingredients which served as an artificial endosperm, thereby

providing nutrients to the encapsulated explants for regrowth (Bapat and Rao

1992; Nieves et al. 1998). Faisal and Anis (2007) reported that encapsulated nodal

buds of Tylophora indica successfully regenerated after different periods of

storage at 4 °C.

The synthetic seeds demonstrated high adventitious rooting capacity after

sowing (Bapat 1993) but in some cases rooting does not takes place. Faisal et al.

(2006 c) excised regenerated shoots of Rauvolfia tetraphylla and subjected them to

rooting. Chand and Singh (2004) treated nodal segments of Dalbergia sisso with

IBA for 10 days, prior to encapsulation to allow root formation.

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2.4. Acclimatization

Acclimatization is the adaptation of tissue cultured plants to new external

uncontrolled environment during which normal photosynthetic activity and water

relations have to be developed (Desjardins et al. 1995). However, the commercial

utilization of the micropropagation technology is often limited due to poor

survival rates of many plant species during acclimatization period (Kozai 1991;

Preece and Sutter 1991). Since, the plantlets grown under optimal in vitro

conditions (high humidity, low temperature, ample sugar or heterotrophic mode of

nutrition and aseptic conditions) acquire some cultured induced phenotype that

render them unfit to survive external harsh environmental conditions of

greenhouse. The main problem of ex vitro transfer is high rates of water loss from

the shoots of plantlets taken out from the culture vessel. Even, if the water

potential of the substrate (sand or soil with nutrient solution) is usually higher than

the water potential of the media with sucrose, the plantlets may quickly wilt. The

cause is unrestricted rate of transpiration due to the retardation in the development

of cuticle, epicuticular wax and functional stomatal apparatus (Pospisilova et al.

2007). These physiological and anatomical characteristics of micropropagated

plantlets necessitates that they should be gradually acclimatized to the

environment of greenhouse or field (Hazarika 2003). During acclimation, the

leaves formed in vitro cannot cope with the external environmental conditions and

replaced by newly formed photosynthetically active leaves.

Apart from in vitro rooting, ex vitro rooting is being a preferred approach as

rooting and acclimatization steps are combined into a single step in some

commercial laboratories. It reduces a considerable reduction in the production cost

by avoiding an in vitro step and also a reduction in labour and time from

laboratory to field conditions. The normal procedure employed for ex vitro rooting

is by dipping the cut ends in high auxin concentration followed by their plantation

in different potting mixtures. Davies and Hartman (1988) reported though auxin

promotes rooting, adventitious root formation is a synchronized developmental

process involving biochemical, physiological and histological events in the

45

induction, initiation, development and elongation of root primordial. Rooting extra

vitrum has been reported in Veronica (Stapfer et al. 1985), Liquidambar

styraciflua (Kim et al. 1997), Fraxinus pennsylvanica (Kim et al. 1998),

Camptotheca acuminta (Liu and Li 2001), Rotula aquatica (Martin 2003),

Nyctanthes arbor-tristis (Siddique et al. 2006) and Malus zumi (Xu et al. 2008). In

majority of cases, IBA was most effective in inducing roots ex vitro (Bhatia et al.

2002, Siddique et al. 2006 and Jahan et al. 2011) while IAA found preferable in

Hagenia abyssinica (Feyissa et al. 2007) and NAA in Siratia grosvenorii (Yan et

al. 2010). Like in vitro rooting, ex vitro is also affected by the nature and

concentration of auxin and the explant source (Yan et al. 2010). The plants raised

by ex vitro practice have better developed root system compared to the ones raised

after in vitro rooting. This technique is economical, less labor, chemical and

equipments demanding.

2.5. Physiological Studies

Plants grown under heterotrophic conditions in vitro have leaves with low

chlorophyll contents and low rates of photosynthesis which impedes growth

(Grout and Millam 1985). This is due to low light intensities, low carbondioxide

concentrations (Infante et al. 1989) and inhibition of photosynthesis by sugar

concentration in the medium (Sheen 1990, Lees et al. 1991 and Reuther 1991).

Nevertheless, after transfer to ex vitro conditions, most micropropagated plants

develop a functional photosynthetic apparatus, although the increase in light

intensity is not linearly translated in an increase in photosynthesis (Kozai 1991). In

in vitro plantlets, the response of photosynthesis to light is similar to that of shade

plants, characterized by low photosynthetic rates, low light compensation

saturation point (Chaves 1994). Many plants transferred from tissue culture may

show a reduced photosynthetic rate due to a sudden shortage of nutrients in

substrate. However, in vitro cultured plants adjust to ex vitro conditions when

switched from heterotrophic to autotrophic conditions (Donnelly and Vidaver

1984; Kozai 1991). The development of photoautotrophy in micropropagated

plants represents one of the main goals during the transfer from in vitro to

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greenhouse conditions. The leaves formed in vitro were unable to develop further

in ex vitro conditions and after a few weeks they were replaced by the newly

formed normal leaves possessing functional stomata similar to that of seed grown

plants (Diettrich et al. 1992).

The stabilization of water status is prerequisites of plantlet survival, but

their further growth requires sufficient photosynthetic rate under correspondingly

high irradiance (Pospisilova et al. 2009). An increase in the chlorophyll contents

(Chlorophyll a and b) after transplantation has been reported by Trillas et al.

(1995), Rival et al. (1997), Pospisilova et al. (1998), Van Huylenbroeck et al.

(2000) and Osorio et al. (2005). Such an increase in the photosynthetic ability

might be attributed to the improvement in chloroplast ultrastructure (Wetsztein

and Sommer 1982). Piqueras et al. (1998) reported that as acclimatization

proceeds, a significant increase in the activity of enzyme of sucrose metabolism in

the leaves was observed revealing the plant’s growing photosynthetic competence.

However, there are reports available where an initial abrupt decrease in

chlorophyll contents during the starting days followed by a continuous and

subsequent increase was noticed as in Ocimum basilicum (Siddique and Anis

2008) towards the final days of acclimatization. A similar pattern of

photosynthetic efficiency in micropropagated plants of neem was detected by

Lavanya et al. (2009). Faisal and Anis (2010) compared chlorophyll contents of ex

vitro formed leaves of Tylophora indica with that of in vitro ones during

acclimation period and found significant higher levels of pigments in the fully

hardened plantlets at 28 day of acclimation. Amancio et al. (1999) indicated that

high light regime during acclimatization has a direct influence on the transition to

in vitro characteristics and on final yield, without symptoms of light stress.

2.6. Biochemical Studies

Plant tissue culture has been viewed as a key technology for the production

of true to type plants. However, the commercial application of this technology

finds limitations when the plantlets are transplanted from in vitro to ex vitro

condition. Many of the regenerated plants cannot cope with the external conditions

47

due to the tissue culture induced abnormalities and needs a period of

acclimatization to correct them. Under in vitro conditions, plants are exposed to

low photosynthetic photon flux density (PPFD) and high humidity conditions.

Once transferred to greenhouse, plants experienced water stress because of higher

PPFD and low humiditaceous environment. A synergic action of high irradiance

and water stress reduces the capacity of a photosynthetic system to utilize incident

radiation and causes oxidative stress through the formation of Reactive Oxygen

Species (ROS) or Active Oxygen Species (AOS). These include Superoxide

radicals (O2.-), singlet oxygen (1O2), hydrogen peroxide (H2O2) and hydroxyl

radicals (OH.) which causes tissue injury (Foyer et al. 1994). These are highly

reactive species and can seriously disrupt normal metabolism through oxidative

damage to membrane lipids, protein pigments and nucleic acid and ultimately

results in cell death. To counter the hazardous effect of reactive oxygen species

under stress, plants have developed or have evolved a complex antioxidative

defense mechanism system which involves both enzymatic and non-enzymatic

metabolites antioxidant such as Superoxide dismutase (SOD), Catalase (CAT),

Ascorbate Peroxidase (APX) and Glutathione Reductase (GR) which are efficient

antioxidant enzymes while the non-enzymatics includes ascorbate (AsA), GSH,

GSSG and Vitamin E (Sairam et al. 1998 and Ahmed et al. 2002). In the

enzymatic reactive oxygen species scavenging pathways, Superoxide dismutase

converts superoxide radicals (O2.-) directly to hydrogen peroxide (H2O2).

Furthermore, the accumulation of hydrogen peroxide is restricted through the

action of catalase or by the action of ascorbate-glutathione cycle, where ascorbate

peroxidase (APX) reduces it to water. Finally, glutathione reductase catalyses the

NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced

glutathione (GSG) (Noctor et al. 2002). Changes in Catalase and Superoxide

dismutase activity have been reported earlier by Hertwig et al. (1992), Fangmeir et

al. (1994) and Sgherri and Navari-Izzo (1995). Van Huylenbroeck et al. (1998)

investigated the activity of Superoxide dismutase and Catalase at different time

period during acclimatization. They observed an increase in catalase activity with

48

49

its maximum value after four weeks of acclimatization in both Spathiphyllium and

Calathea and thereby decreased afterwards, while the total SOD activity

increased with plant growth and attained a maximum range in the 24th week of

acclimatization in both the plants. Chakraborty and Datta (2008) reported an

increase in the activity of all four enzymatic antioxidants viz., SOD, CAT, APX

and GR in Gerbera reaching its maximum value at the beginning of

acclimatization process and decreased thereafter. However, after 20 days again

these enzymes activities increased which may be due to the plantlets being

exposed to field conditions after 15 days of humidity chamber (80-90% RH) might

faced oxidative stress again. In Tylophora indica, Faisal and Anis (2010) noticed a

time dependent variation in the activities of antioxidant enzymes. Increase in SOD

activity has been noticed in the plantlets acclimatized at high light intensities.

Furthermore, the effect of high PPFD elicitated an increase in catalase activity

during the whole period of acclimatization. Likewise, photoexposure of the

plantlets with both photosynthetically active radiations elevated the level GR

against 0 days plantlet. Nonetheless, the APX activity increased with varying

periods of acclimatization.