ollshment of in vitro syshodhganga.inflibnet.ac.in/bitstream/10603/337/10/10_chapter 2.pdf ·...

ollshment of In vitro Sy

2.1 Introduction

Scientists have, over the years, outgrown their initial fascination with the basic aspects of in vitro techniques such as expression of totipotency, dedifferentiation and redifferentiation of cultured plant cells, tissues and organs. The focus has now shifted to more applied aspects of tissue culture and the technique of culturing is now given the consideration only as a first step in achieving much deeper goals. Three major applied aspects of tissue culture are currently the in vogue. They are listed below:

I. Classic micropropagation as an alternative means of plant vegetative propagation. Asexual reproduction through tissue culture benefits i . two ways; large-scale multiplication and production of true-to-type clones of ornamentals and horticultural crops (Bhojwani and Razdan, 1983).

2. Genetic transformation - a more advanced technique - for breeding programmes of crop plants and qualitative and quantitative improvement of yield (Van Stint Jan et al., 1996).

3. As an alternative for the production of a number of high-value secondary metabolites (Lipsky, 1992; McDonald et al., 1995; Xu et al., 1998). Micropropagation and standardization of protocols for the establishment and maintenance of regenerants of rare, endangered and also high product-yielding species of medicinal plants is recognized as one of the five thrust areas of biotechnological approach for exploiting plant-derived secondary metabolites (Rao and Ravishankar, 2002).

Despite the existence of a plethora of literature serving as guidelines for exploring diverse aspects of the establishment of cultures, in vitro technique and the like, the actual effort has to begin from the basics with a unique assembly of skill, planning, large-scale experimentation for the initiation, establishment

68 Part I1 - Establishment of in vitro systems

and maintenance of cultures since the in vitro behaviour varies from species to species. Nutritional requirement of tissues from different parts of the same plant may have different requirements for satisfactory growth (Murashige and Skoog, 1962). When starting a new system using a particular tissue, it is essential to work out a medium that would fulfil the specific requirements of that tissue (Bhojwani and Razdan, 1983).

Against this background, in continuation of activities described in Part I, in vitro techniques were designed and developed for the two high-solasodine- yielding plants of S. hilobaturn and S. wendlandii, with the ultimate aim of increasing its content by in vitro manipulation. The attempt was to define basic culture conditions for callogenesis, suspension cultures, root cultures and whole plant regeneration from leaf explants in the two selected species of Solanum.

Cultivated species of Solanum are seasonal plants and hence it is difficult to get plant material throughout the year for drug manufacture. Micropropagated plants could be a source of continuous supply of planting material. Thus, in vitro cultures of Solanum are of great importance with respect to the secondary metabolites and micropropagation aspects in the current scenario. The cultivation of S. viarum at present is labour-intensive because of the thorny nature of the plants. Low disease resistance of the plants is also a serious problem (Krishnan, 1995).

S. trilobatum is an important medicinal herb (Emmanuel et al., 2000) and also has a high content of solasodine. This plant has come to receive increasing attention in recent years for the afore-said two reasons. S. wendlandii has not so far been investigated to any extent in in uitro conditions. An efficient plant regeneration system for these two plants would alsobe necessary for the development of genetic techniques in connection with solasodine.

2.1.1 Aims and objectives

The following aims and objectives are applicable to both the species of Solanurn studied here.

I. To develop protocol for maximum callus production in a simple defined media.

Introduction 69

2. To study the effect of auxins on callogenesis and the trends therein.

3. To initiate cell suspension cultures and establish their growth curve in a pre-determined PGR regime.

4. To establish root cultures from in vitro adventitious roots. 5. To regenerate whole plants from leaf explants and also for raising

regenerants for assessment of solasodine content. 6. To establish protocol for clonal propagation and study its in vitro aspects.

The ultimate aim of the study presented here in Part II was to obtain a reliable set of culture conditions suitable for each type of system to generate sufficient study material for the next stage of experiments, which is presented in Part 111. Tissue culture itself is academically interesting to the researcher and a

sizeable share of the effort was spent in analyzing the response of cultures and the factors affecting it, although at a superficial level. To add a word of caution, the possibilities of this part should not be overestimated since delving deep into the mysteries of the interplay of growth regulators vis-a-vis plant cells and the expression/non-expression of totipotency were beyond the scope of this study. 1

2.2. Review of Literature

a. 2. I Callogenesis

When explant is placed in a suitable nutrient medium it undergoes cells division in random planes with loss of organized structures, resulting in callus. When subcultured regularly on agar media, callus cultures are found to exhibit an S-

shaped or sigmoid pattern of growth during each passage (Rao, 1997). Thus callus forms a coherent but unorganized amorphous mass of tissue (George and Sherrington, 1984). That it was possible to culture plant callus was discovered in the period 1935-40. In the years between 1945 and 1985~20-25 percent df all reported cultures had been in the callus phase (George et al., 1987). Callus is the easiest to establish in culture especially for broad-leafed dicots (George and Sherrington, 1984). A scrutiny of available literature on the subject reveals that most of the solasodine-related work was done in the callus phase.

2,4-D is the most preferred auxin for callogenesis in Solanurn species (refer Table 25). It was selected as the auxin producing maximum callus either alone or in combination with cytokinin. 2,4-D alone was sufficient to produce callus in S. xanthocarpum (Heble et al., 1971)~ S. khasianum (Uddin and Chaturvedi, 1979), S. eleagnifolium (Khanna et al., 1977;' Nigra et al., 1987);

S. laciniatum (Hosoda andyatazawa, 1g79), S. auiculare (Galanes et al., 1984,

S. plataniJolium (Jaggi et al., 1988), S. tuberosum (Keller et al., 1996) and S. nigrum (Shahzad et al., 1999). There are innumerable reports where 2,4-D was used in combination with a cytokinin for callogenesis especially with Kn

(Chaturvedi et al., 1979; Chandler and Dodds, 1983a; Ehmke and Eilert 1986; Mak and Doran 1993; Suardi et al., 1994; Aburjai et al., 1996; Muhlenbeck et al., 1996; Villarreal et al., 1997; Kittipongpatana et al., 1998; Ikenaga et al., 2000; Bhalsing et al., 2000; C u ~ n o et al., 2001).

Review of Literature 71

NAA was used to induce callus in S. chacoense (Zacharius and Osman, 1977), S. surattense (Barnabas and David, 1986), S. sarrachoides (Banerjee and Ahuja, igg3), S. trilobatum (Krishnamul-thy et al., 1996), S. paludosum (El Badaoui et al., 1996) S. laciniatum (Syahrani et al., 2000) and S. platanifolium (Jaggi and Singh, 2000). IAA was also employed for callus formation but only in a few species such as S. khasianum (Uddin and Chatruvedi, 1979), S. nigrum (Bhatt et al., 1983), S. surattense (Malpathak and David, 1994) and S. aculeatissimum (Manjula and Nair, 2002). Callus studies in S. surattense helped to understand the underlying mechanisms of morphogenesis changes in

the activities of key enzymes, and metabolic constituents during differentiation (Swarnkar et al., 1986)

2.2.2 Cell suspension cultures

Suspension cultures of plant cells began during the 1950s (George et al., 1987). Cell suspensions are initiated by transfer of callus pieces into flasks containing liquid medium that are then placed on a gyratory shaker to provide aeration to the cells. Cells in suspension can exhibit much higher rates of cell djvision than callus (Rao, 1997). - .In culture, cell clumps consisting of active centres of cell division are required for active growing suspensions to divide and give rise to new cells. Most of the free cells especially during the lag phase are predestined to enlarge, senesce and die in the primary suspension culture after a long time in that mode of culture (Wang and Nguyen, 1990). The duration of the lag phase would largely depend on the growth phase of the stock culture at the time of subculture and the size of the inoculum (Bhojwani and Razdan, 1983). Usually low auxin levels are used for suspensions employed in studies of secondary metabolites. Suspension cultures offer the advantage offaster growth and more direct control of media factors than callus cultures (George and Sherrington,

1984).

Almost all the reports on cell suspensions of Solanurn species were carried out for recording solasodine content in free cells and media. Suspension cultures are established also with the aim of obtaining free cells for immobilization (Jirku et al., 1981; Subramani et al., 1989; Malpathak and David, 1992) and cell line selection (George and Sherrington, 1984). In S. tuberosum cell suspension was established for studying genetic variability (Pij nacker et al., 1986) glycoalkaloid

72 Part 11 - Establishment of in vitro systems

toxicity in potato tuber slices (Osman et aL, 1980). In connection with solasodine, cell suspensions have been established in eight species. The most commonly employed growth regulators for the suspensions were 2,4-D (0.1 - 2 mg/l ) and Kn (0.1 - 1 mg/l) (Chandler and Dodds, 1983a; Ehmke and Eilert, 1986; Subramani et al., 1989; Mak and Doran, 1993 and Quadri and Giulietti, 1993). ,

Occasionally other growth regulators such as IAA, NAA as auxins and BAP as cytokinin were also employed (Manjula and Nair, 2002; Kittipongpatana et a[.,

1998; Malpathak and David, 1992,1994).

2.2.2.1 Callus cell aggregates

In Solanurn species, formation of compact bead-like aggregates consisting of parenchymatous cells was reported in S. dulcamara (Ehmke and Eilert, 1986)

and S. aviculare (Tsoulpha and Doran, 1991). Aggregates have also been reported in cultures in connection with solasodine production but studies of alkaloid synthesis in this mode of biocatalyst were limited to a few species. They were Tagetes patula (Hulst et al., 1989), Taxus (Ellis et al., 1996) and Rhodiola

/

sachalinensis (Xu et al., 1998; Xu et al., 1999; Wu et al., 2003). Self- immobilization or aggregation of cells has not received as much attention as gel- or foam- entrapment even though the same advantages apply (Tsoulpha and Doran, 1991). Moreover, the tendency for aggregation in some cases eliminate the need for artificial immobilization supports and may be exploited for large- scale phytochemical screening (Xu et al., 1gg8), Liquid cultures of CCA may have the same advantages as the artificially immobilized cell cultures, which include lower liquid viscosity, better mixing and improved secondary metabolite synthesis (Xu et al., 1998).

2.2.3 Root cultures

Root cultures are normally established from root tips excised from primary or lateral roots of plants or their in vitro counterparts (George and Sherrington, 1984). The root tips develop as organ cultures having the morphology and cellular characteristics of seedling roots. Cultured roots show only primary root anatomy or very limited secondary vascular tissue (Street, i973a).


Work on root cultures were started in 1922 independently by Robbins and Kotte. It was followed by pioneering work by White in 1934. During 1939- 50, extensive work on root culture was carried out by Street and his students to understand the role of vitamins in plant growth and shoot-root relationship (Bhojwani and Razdan, 1983). In the earlier attempts root cultures, were employed exclusively for the investigation of basic mineral nutrition of plants (Street, 1g73a). Untransformed root cultures have assumed importance in connection with secondary metabolite production only in the last two decades. Root cultures were sidelined also because they do not feature in the micropropagation techniques. According to Street (1973a), successful initiation and continuous culturing of root cultures not only depends on the species and plant source but also on the culture conditions employed. Torrey (1976) found the cultural parameters such as sucrose concentration, light quality and intensity and interactions with growth regulators as being critical for root growth. George and Sherrington (1984) reported nitrate/ammonium concentration and growth regulators especially auxins to be most crucial for root cultures.

Culture of untransforrned roots has been rare, if the number gf published reports were an indication. Root cultures were successfully established in S. melongena for studying the effect of polyamines on growth by Sharma et al. 1997. In S. eleagnifolium, root cultures were not successful (Nigra et al., 1990).

2.2.4 Regeneration in Solanurn species

Only those reports of studies pertaining to non-tuberous species of Solanum conducted in connection with solasodine production or basic micropropagation are reviewed here.

Direct regeneration of adventitious shoots was reported in S. sarrachoides (Bane j e e et al., 1985), S. commersonii (Iapichino et al., ~ g g ~ ) , S. nigrum (Shahzad et al., 1999, S. trilobatum (Emmanuel et al., ~ O O O ) , S. surattense (Pawar et al., 2002) and S. xanthocarpum (Seetharam et al., 2003). In S. commersonii, twelve shoots were produced in presence of 1 or 2 mg/l IAA and 5 mg/l zeatin. Direct buds were formed from leaves (25 shoots) and nodes (23 shoots) of S, xanthocarpum were produced in 2 mg/l BAP + 1 mg/l kinetin and 4 mg/l BAP and I mg/l Kn respectively. In S. nigrum, both direct and indirect caulogenesis from leaf explants were produced with 2,4-D as auxin and 2 mg/l


Kn. A protocol for multiple shoot induction from S. surattense from leaf and shoot tip explants was reported as yielding 210-215 adventitious shoots in 25- 30 days on culture in MS medium fortified with equal concentrations (15 1M) of BAP.

Indirect organogenesis in Solanum species was reported in S. laciniatum (Conner, 1987; Lemmonier e t al., 1989). S. khasianum (Bhalsing and Maheshwari, 1997), S. nigrum (Shahzad et al., 1999) and S. aculeatissimum (Manjula and Nair, 2002).

In S. nigrum, a protocol for direct organogenesis was developed where MS medium supplied with 0.5 mg/l N U and 2 mg/l BAP produced shoots on the twentieth day. For S. aculeatissimum, 0.5 mg/l IAA and 4 mg/l Kn was useful for producing 38 shoots in culture. For S. laciniatum, in two separate studies, BAP was the preferred cytokinin but as auxin, NAA was effective in one (Conner, 1987) and IAA in the other (Lemmonier et al., 1989). Shoot bud formation in dark took place in the presence of BAP 3 mg/l in S. khasianum (Bhalsing and Maheshwari, 1997). I

2.2.4.1 Rooting

Often, for rooting of shoots that were induced in full strength MS medium, the salt concentration was reduced to half or a quarter (Bhojwani and Razdan, 1983). Rooting of shoots in reduced salt concentration was reported in S. tn'lobatum (Emmanuel et al., ~ O O O ) , S. cornmersonii (Iapichino et al., 1991) and S. sarrachoides (Bane j e e et al., 1985). Rooting in full strength MS was also reported, though rare, in S. aculeatissimum (Manjula and Nair, 2002) and S. khasianum (Bhalsing and Maheshwari, 1997).

Adventitious shoots developed in cultures in presence of a cytokinin often lack roots. To obtain full plants, the shoot must be transferred to rooting medium containing either IBA or NAA (Bhojwani and Razdan, 1983). This requirement of auxin is dispensable for Solanum species as observed for S. comrnersonii (Iapichino et al., 1991), S. trilobatum (Emmanuel et al., ~ O O O ) , S. carolinense, S. dulcamara, S. laciniatum, S. ly copersicoides, S. melongena, S. pennellii, S. nigrum and S. surattense (George et al., 1987), which rooted in hormone-free medium.


Solanum species that require an auxin were also found. S. aculeatissimum produced six roots per shoot in IBA (I mg/l) (Manjula and Nair, 2002) and S. khasianum in NAA (2 mg/l) (Bhalsing and Maheswari, 1997). S. sarrachoides required both ZAA (2 mg/l) and NAA (0.1 mg/l) for producing roots (Banejee et al., 1985).

2.2.5 Somatic embryogenesis

Somatic embryogenesis is defined as a non-sexual developmental process, which produces a bipolar embryo from the somatic tissues (Evans et al., 1981). The potential of somatic embryogenesis has been realized in vitro propagation, synthetic seed production, rejuvenation, plant transformation and germplasm conservation and the single-cell-origin of regenerants (Becher et al., 1992; Murthy and Saxena, 1998; Gupta et al., 2001).

Direct somatic embryogenesis offers definite advantages over classical breeding procedure for plant improvement and clonal propagation (Denchev et al., 1991) and is also associated with greater genetic and cytological uniformity (Maheshwaren and Williams, 1984). The multiplication rate of somatic embryos can be faster than adventitious shoots and a separate step of rooting of shoots is not necessary since normal embryos have a shoot and root apex (Ammirato,

1983). The hypothesis put forward by Sharp et al. (1982) that direct embryogenesis occurs in vitro when cells within the explant were predisposed to become embryo even before culture and that nutrient media and other in vitro conditions serve only to enhance the process is getting more and more credibility.

However, experience of many workers suggests somatic emblyogenesis to be an infrequent and irregular phenomenon in cell or tissue cultures, so that

its hormonal control was difficult to ascertain (George and Sherrington, 1984). Avariety of somatic embryos that develop in culture are reported to vary greatly in their developmental patterns (Padmanabhan et ol., 1998) and their ability to grow into plantlets (Kerns et al., 1986, Merkle et al., 1990) chiefly due to non- shoot forming variants.

76 Part I/ - Establishment of in vitro svstems

Published reports of embryogenesis in Solanum species were also rarely encountered during the review of literature, especially in the non-tuberous species. Embryogenesis was reported in S. carolinense and S. melongena (George et al., 1987). In another study, positive correlation between the spatial distribution of polyamines and the embryogenic capacity of the explant of ,

S. melongena was reported b y Yadav and Rajam (1997).

2.2.5.1 TDZ and somatic embryogenesis

Thidiazuron or TDZ is a phenylurea derivative, which has been recognized as the most biologically active of all adenine type of cytokinins (Mok et al., 1982). TDZ is a common commercially used defoliant of cotton (Huetteman and Preece, 1993; Magioli et al., 1998). Huetteman and Preece (1993) published a thorough review of the works on TDZ till 1993. According to that review and many other reports that were done in later years, TDZ was ideal for axillary bud multiplication, adventitious shoot formation and somatic embryogenesis of woody plant species.

/

The action of TDZ is in producing high cytokinin-like activity in in vitro cultivated cells (Wang et al., 1986; Fiola et al., 1990; Saxena et al., 1992). The mechanism of TDZ action is partly related to the inhibition of cytokinin degradation by cytokinin oxidase resulting in increased levels of endogenous cytokinin (Hare and van Staden, 1994).

2.2.5.2 Maturation and gemination of somatic embryos

ABA is the most commonly used growth regulator, on account of its active role in the promotion of somatic embryo development. Low concentrations of ABA stimulate somatic embryo initiation and embryo growth (George and Sherrington, 1984). Moreover, ABA prevents precocious germination of somatic embryos and is used for embryo maturation in many plant species (Emons et al., 1993; Dong et al., 1997; ~ucreux et al., 1998). ABAplayed a major role in

advanced embryo development in Pimpinella anisurn after 2,4-D mediated induction (Bela et al., 1999). Somatic embryo maturation in Prunus avium (Reidiboyrn et al., 1998) and Dendrocalamus strictus (Sumathi et al., 2003)


have also been reported.

ABA acts by antagonizing the endogenous cytokinins thereby regulating them and permitting the formation of embryos (Yarnaguchi and Nakajima, 1972; George and Sherrington, 1984).

G& although occasionally reported to be promotive to maturation of somatic embryos (Bhojwani and Razdan, 1983) is generally considered as an inhibitor of somatic embryo formation (George and Sherrington, 1984). The results obtained in Geranium suggested that bath exogenously supplied as well as endogenous GAS play a role, albeit a negative one, during somatic embryogenesis (Hutchinson et al., 1997). Exogenous application of GA3 has already been reported to inhibit development of a,4-D-induced embryo in carrot (Fujimura and Komamine, 1975) and anise cultures (Noma et al., 1982).

There were also reports of stimulation of embryo maturation by GA3 application in Eryngium foetidurn (Ignacimuthu et al., 1999) and Panax ginseng (Chang and Hsing, 1980). The action ofGA3 is by modification of tbe activity of

specific enzymes or by promoting the availability of endogenous auxins (George and Sherrington, 198~) .

2.3 Materials and Methods

2.3.1 Materials

The materials used for conducting experiments in Part I1 of the present study are described below.

2.3.1.1 Chemicals

The chemicals used to prepare the stock solutions were of LR or equivalent grade.

Plant growth regulators (PGRs) used in the study comprised auxins such as

2,4-dichloropheno~yacetic acid (n,4-D), I-Naphthaleneacetic acid (NAA), Indole- 3-acetic acid (IAA), Indole-3-butyric acid (IBA) and cytokinins such as Benzylaminopurine (BAP), 6-furfurylaminopurine (Kn), 2-isopentenyl aminopurine (2iP), Thidiazuron (TDZ). Gibberellic acid (GA3) and Abscissic acid (ABA) were procured from Sigma, USA or HiMedia Laboratories Pvt. Ltd., Mumbai.

Stock solutions of macro nutrients OX), micro nutrients (loox) and PGRs (0.5 mg/ml) were prepared in bulk and stored in amber coloured glass bottles at q°C.

2.3.1.2 Glassware

Beakers, measuring jars, round bottom flasks, conical flasks, pipettes, petri dishes, watch glass and standard flasks, all of Borosil or equivalent grade, were used in the preparation of media and for other related purposes such as drying and weighing.

Solid cultures were initiated in round bottom test tubes (25 x 150 mm) and plugged with cotton bungs. Bottles (250 ml) with polypropylene lids and

Materials and Methods 79

sealed with stretch film were used for subculture or for mass cultures and root cultures. For acclimatization during hardening, one-litre bottles were used as micro chambers. Rooting of the adventitious shoots was achieved by placing the shoots on paper bridges in contact with liquid media in culture tubes.

Suspension cultures were established and maintained in 250 ml Erlenmeyer flasks (Borosil) plugged with cotton bungs. Glassware were washed thoroughly in soap solution and finally rinsed in distilled water after each use.

2.3.1.3 Accessories

Blade holders, sterile blades, long forceps, spatula, sieves (2 mm mesh size), spoons, droppers, scissors, pipettes - all made of either glass, stainless steel or plastic were used while working in the laminar air flow for doing inoculation, subculture, harvest, sample collection, etc.

2.3.1.4 Instruments I

Several electrical electronic instruments and apparatus were employed at the different stages of medium preparation and establishment of cultures. They are listed below.

I. Autoclave - Lab Agencies 2. Bacterial filtration unit - Tarsons

3. Cellulose nitrate membrane filters (0.45 pm) - Whatman 4. Centrifuge - Rotek

5. Digital pH meter "pH Scan I" - Eutech Instruments 6. Electronic balance - Sartorius

7. Hot air oven - Kumar Industries 8. Hot plate 9. Laminar horizontal airflow chamber - Yorko Scientific Industries 10. Orbital shaker - Labline 11. Refrigerator - Allwyn 12. Vacuum pump - Labline 13. Water distillation unit - Magnum

80 Pad / I - Establishment of in vitro systems

2.3.1.5 Media

The nutrient composition suggested by Murashige and Skoog (MS) (Table 4) in 1962 was used as the basal medium throughout the study. Required quantities of stock solutions were pipetted out and the final volume made up in a clean .

and dry standard flask after dissolving sucrose and meso-inositol. The pH of the medium was adjusted between 5.7 and 5.8 using o.iN NaOH or o.iN HCI. For the preparation of solid media, 0.8 percent (w/v) agar was gelled with the medium after adjusting the pH. When the agar was completely dissolved in hot medium, it was dispensed into culture vessels, plugged with cotton bungs or closed with caps.

Table 4. Composition of MS medium (Murashige and Skoog, 1962)

Stock Nutrient mgll

Na, . EDTA I I I

FeS04. 7H20

MnS04. 4H20

ZnSO, .7H,O

H3B03 IV K f

Na2Mo0,. 2H20

CoCI2 . 6H20

C U S O ~ . 5H20

Vitamins

Glycine

Nicotinic acid

Pyridoxine - HCI

Thiamine - HCI

Meso-inositol

Sucrose

Agar

pH


2.3 .I. 6 Sterilization of media, glassware, accessories

The media and entire set of accessories used during inoculation were sterilized by autoclaving at 121°C at 15 lbs. for 20 minutes. Containers and accessories were wrapped in autoclavable plastic covers. Glassware after final rinse in distilled water were placed in hot-air oven for dry sterilization at I O O O C for 2 hours. The practice of autoclaving glassware with contaminated cultures before washing in order to reduce sources of contaminants in the working area was strictly followed. Thennolabile chemicals such as GA3 and ABA in required quantities were added to a small quantity of the medium and passed through membrane filter inside the sterile atmosphere of the laminar air flow chamber. This was immediately added to the already autoclaved bulk of the medium, and dispensed into culture vessels. In the media with agar, the chemical was added before the agar was set.

2.3.1.7 Source of explant

S. trilobatum plants procured from wild areas in Tamil Nadu and S. wendlandii grown in the botanical garden of the Sacred Heart College, Thevarq and home gardens nearby were collected and grown in the home premises of the research scholar. Healthy,' juvenile leaves of S. trilobatum (2-3 cm long) and S. wendlandii (5-8 cm long) were excised from the 3" to the 5" nodes of the mother plants. The young stem - split longitudinally into two - was also used in regeneration studies.

2.3.1.8 Surface sterilization of explant

The explants were initially washed in a strong jet of water under the tap. They were then coarsely trimmed and immersed in tap water along with a few drops of labolene (1 drop/~oo ml) for lo minutes followed by the same treatment in sterile distilled water for another lo mintues. The explants were then soaked in 0.1 percent HgCl, (w/v) mixed with a few drops of labolene for 1-22 minutes. These were then washed four or five times with sterile distilled water to remove all traces of the surface sterilant. I n vitro derived explants, being sterile, did not require any surface sterilization.

82 Part I! - Establishment of in vifro svstems

2.3.1.9 Inoculation and incubation

All the operations requiring sterile conditions were done inside the laminar airflow chamber. The cabinet was sterilized by exposure to UV rays for 30 minutes prior to inoculation along with autoclaved media and accessories. The ,

laminar airflow was switched on 5 minutes prior to inoculation. The working area was thoroughly swabbed with ethanol. Autoclaved accessories used during inoculation were kept immersed in ethanol and frequently flamed immedietly before inoculation to ensure maximum sterility. Cultures were incubated in sterile air-conditioned room at 25 * 1°C. Cool white fluorescent light of 2000 lux intensity at 1618 hr photoperiod and 55 - 60 percent relative humidity were maintained. Dark chamber was used for raising root cultures.

2.3.2 Methods

For developing protocols for each culture system, experiments were designed based on the critical hormonal, nutritional, chemical and physical factors suitable to each type. Likewise, only appropriate parameters necessary for evaluation and selection of optimum conditions of growth were recorded.

2.3.2.1 Callus culture

Three auxins - 2,4-D, NAA and IAA - were used at 0.5, I, 2, 3, 4, and 5 mg/l

concentrations to induce callus from leaf explants of S. trilobatum and S. wendlandii. The establishment of growth curve and optimization of callogenesis were combined as a single experiment. The number of days taken for initiation of callus, ratio of the number of cultures producing callus to the total number of cultures, expressed as percentage of response, increase in the callus mass over a period of two months at intervals of 15 days were recorded. The colour, texture and nature of the callus were also observed. Increase in fresh weight (fresh wt.) was measured without sacrificing samples. The callus was transferred to a pre-weighed culture tube and again the weight of the tube with the callus was recorded. The weights were recorded immediately before and after inoculation. The difference in weights was taken as the fresh weight of the callus. All readings are means of lo replicates. The selection of the best medium was done based on FGI (Fresh Growth Index) afier 30 days and calculated using the following equation.


FGI - - ~ r e s h wt. of explant Fresh wt. after 30 days - Fresh wt. of explant

2.3.2.2 Cell suspension culture

Batch suspension cultures were established based on the guidelines recommended by Rao (1997) Mother suspensions from friable callus of both S. trilobatum and S. wendlandii were established by dispersing approximately 2 g of actively proliferating callus tissue - cut into small pieces - in 50 rnl of liquid MS basal media supplemented with 0.5 rng/l2,4-D and 0.5 mg/l Kn. For S. trilobatum, two types of calli were used to initiate suspensions. To select the medium suitable for optimum growth and cell division, full (MS) and half strength ( 0 . 5 ~ MS) salt concentrations and full (3%) and half (1.5%) strength sugar concentrations were employed.

Periodic determination of the viability of cells was done using 1 percent solution of Evan's blue (Franklin and Dixon, 1994). Under the microscope, the viable cells remained unstained while the dead cells absorbed the st& and were coloured blue. - .

For determination of growth curve, fine suspensions were employed. These fine suspensions consisted of single cells and small cell-aggregates (up to 25

cells per cluster) obtained from mother suspensions by sieving through 2 mm mesh sieve. Ten ml of this suspension was added as inoculurn into fresh 50 ml medium. Packed cell volume (PCV), fresh weight and dry weight (dry wt.) of these cell suspensions were the growth parameters recorded at 7-day intervals for 35 days (Franklin and Dixon, 1994 ). Three replicates were harvested every week. To determine the PCV, the entire contents of the flask were transferred to a pre-weighed graduated centrifuge tube. Tubes were spun at 300 g for 4 minutes until the supernatant was free of cells. The volume of the pellet after centrifugation is expressed as the fraction of the volume of the pellet to the total culture volume (ml/l). The weight of the centrifuge tube with the pellet was again noted. The difference was taken as the fresh weight. The cells were then dried in an oven at 60°C until no change in the dry weight was observed.

84 Part I / - Establishment of in vitro systems

2.3.2.3 Root culture

To establish root cultures, adventitious roots produced in selected media from leaves of both S. trilobatum and S. wendlandii were inoculated into liquid media. To determine the optimal strength of the medium which produced maximum ,

growth of roots, salt concentration at full (MS) and half strength (0.5~ MS) was used. Also the effect of ammonium ion concentration on root growth with half strength, quarter strength and complete absence was experimented. The effect of I mg/l IAA was also ascertained. To raise these cultures, 3-4 cm long roots were transferred to 50 ml liquid medium.

The liquid cultures were kept in lightlstatic, darklstatic and light/agitated conditions. Parameters recorded were the number of new roots induced, mean root length and the nature, colour and growth pattern of the main axis and laterals after 30 days in culture as per the culture protocol suggested by Reinert and Yeoman (1982). Data were recorded from six replicates.

2.3.2.4 Regeneration studies b

A two-step approach was adopted for exploiting the totipotency of the plants studied. A preliminary screening with different permutations of PGRs was set up followed by optimization of the selected combination.

2.3.2.4.1 Selection of PGR

To regenerate whole plants, leaf, stem and one-month old calli were used. Four cytokinins - Kn, BAP, aiP and TDZ - at 1 mg/l concentration were examined for their regeneration potential either alone or in combination with different auxins such as 2,4-D, NAA and IAA at 0.5 mg/l concentration. The concentrations selected were well within their reported biologically active range of concentrations

2.3.2.4.2 Optimization of PGR concentration

Experiments for obtaining optimum PGR concentrations of the responding PGR combination for indirect caulogenesis and direct caulogenesis in S. hilobaturn and somatic embryogenesis in S, wendlandii were carried out. Varying


concentrations of the respective cytokinins at 0.05,0.1, 0.5, I, 2, 3, 4 and 5 mg/l were tried while keeping the auxin concentration constant. Appropriate growth parameters were recorded and assessed for selection of the most productive media.

2.3.2.4.3 Rooting and hardening

To initiate roots from the adventitious shoots in S. trilobatum, salt concentration at full (MS) and quarter strength (0.25~ MS) were used. PGRs used were NAA and IBA at 0.5 mg/l and 2 mg/l concentrations. Effect of absence of PGRs was also tested. The competence of these media was evaluated after 15 days.

Hardening of the rooted plantlet was done in Soilrite (Keltec Energies Ltd. Bangalore) initially in micro chambers for two weeks and then in plastic cups in the green house. The plants were provided with liquid MS medium once in a week.

n .3 .2.4.4 Maturation and germination of somatic embryos

The competence of&, BAP, nip (all at I mg/l), adenine sulphate (lo mg/l), GA3 and ABA (both at 1 mg/l)and MS basal media without any growth regulator on maturation of embryoids was tested. For germination of the mature embryos, G&, ABA and MS basal media without any growth regulator were used. Both liquid and solid media were employed. Liquid cultures were agitated at go r.p.m.

2.3.2.5 Subculture

To get sufficient material for experiments subcutlure was done at regular intervals. Active and proliferating calli, root and shoot cultures were subcultured after 20-30 days onto fresh medium with the same composition under aseptic conditions. For suspension cultures the interval was 10-15 days.

2.3.2.6 Internal structure .

Free-hand sections of fresh samples of approximately 20 pm thickness were taken and observed under a light microscope for the developmental stages of different structures in vitro, after staining with Toluidiene Blue 0 (O'Brien

86 Part I1 - Establishment of in vitro svstems

et al., 1964). These sections were later preserved in FAA (formalin : acetic acid : alcohol) for later examination.

2.3.2.7 Statistical analysis

The standard norms for replication in each experiment were followed. All experiments were repeated and the data presented are of one representative experiment. The statistical methods adopted to analyse and present the data collected were as suggested by Mize and Chun (1988) and Compton (1994). The experiments were of completely randomized designs. The figures are presented as the mean and its standard deviation (SD). Analysis of Variance (ANOVA)

was used to determine the significance of variations in results. Log transformation of the data was done before analysis whenever necessary. Processing of data for presentation of the results were carried out on Microsoft Excel software. Mean separation using multiple range test was done according to Duncan's Muliple Range Test (DMRT) (Montgomery, 1997).

2.3.2.8 Photography p

Digital images of cultures were recorded using a Sony MVC FD 83 digital still camera (Japan) onto a floppy disc and transferred to a computer. Extreme close- up photographs of small structures were taken through a Leica-SqE Stereozoom microscope using a Cannon Powershot S4oE camera (USA). Photos of cells in

suspension and anatomical sections were taken as magnified by an Olympus CK2 binocular microscope (Japan), fitted with a C-35 AD camera (Olympus, Japan) on 200 and 400 ASA colour films.

2.4 Results

The diverse in vitro responses in the experiments conducted to develop protocols for the establishment and maintenance of culture systems are described below.

2.4.1 Callus cultures

All the three auxins - 2,4-D, NAA and IAA - could successfully induce callus from tender leaf explants of both S. trilobaturn and S. wendlandii, though by varying degrees.

2.4.1.1 Effect of 2,4-D on callogenesis t

From S. hilobaturn leaf explants, callus started forming from the seventh day onwards at the lowest concentration of 0.5 mg/l (Table 5). As the concentration increased to 5 mg/l, more time for callus formation was needed. All the cultures fully responded to 2,q-D in producing callus (100% response). Initially the callus manifested as irregular, soft cellular masses arising from the cut edges and veins of the laminar region. This mass was the main source of further callus proliferation with relatively less transformation of the explant during the course of time. The progress of callus formation is presented in Figure 18. The highest quantity of callus mass (p<o.oi) after 30 days reckoned as FGT was obtained in the medium supplemented with img/l 2,4-D. As the concentration increased, growth lessened considerably. Older calli were watery with ballooned cells. The callus produced under the influence of n,4-D was generally watery, soft masses of loosely connected cells, changing colour from dull white to pale brown on maturation.

Part 11 - E

stablishment of in vitro system

s

aM

*m

9

2 w

X

NN

NN

Results 89

2.4.1.1.2 S. wendlandii

In S. wendlandii, s,q-D was much more productive especially at 0.5 mg/l, where the increase in callus mass after 30 days was the highest (Table 6). When the concentration was raised to 5 mg/l the growth declined sharply. As in S. trilobatum, 2,4-D successfully induced callus in all cultures, but the time taken to produce it was less and more uniform at all concentrations. Callus originated as vigorously growing white mass of cells both from the cut ends, and by rupture, from the midrib and laminar regions (Figure 19). In o.gmg/l, after 40-45 days, the explant was completely covered with callus. Even a major portion of the explant dedifferentiated into callus. This was the most productive concentration (p<o.og) for callogenesis in S. wendlandii.

2.4.1.2 Effect of NAA on callogenesis

Leaf of S. h'lobatum under the influence of NAA, produced callus after one week in culture (Table 7). Curling and expansion of explant took place prior to callogenesis. This callus growth was accompanied by rhizogenesis from the midrib and lateral veins of the explants at lower concentrations of the auxin. Only low concentrations of NAA (0.5 and img/l) could induce callus from all cultures. As the concentrations increased, the number of cultures yielding to callogenesis fell. The maximum amount of callus was produced in 4 mg/l NAA

but was not significantly (p=o.og) higher than the callus yield in other concentrations. The callus was mostly loose aggregations of dull gray or brown coloured watery mass of cells (Figure 20). Proliferation was maintained by the initially formed calli only and a major portion of the explant, which did not dedifferentiate into callus, remained. The roots induced were feeble but with numerous long root hairs (Figure a m . During subsequent transfers, these roots were disturbed and did not grow further in solid media.

Table 6. Effect of 2, 4-D on callogenesis from leaf of S. wendlandii

compact, soft, whitish cream

1 38.57 420.33 797.05 1094.90 969.33 21.23 4 1.00 I00 4 compact, soft, creamish ab

2 36.96 436.20 807.05 1165.37 1171.61 20.71+0.64 100 3 compact, hard, dark brown a bc

3 37.53 521.72 658.14 905.37 863.35 1 7.39 f 0.54"~ 100 3 friable, dark brown, soft

4 36.48 490.36 596.09 735.60 71 9.50 15.87 f 0.46a-e 300 4 friable, dark brown, soft

5 30.20 281.67 474.77 628.54 559.59 15.70 f 0 . 4 8 ~ ' ~ 100 4 friable, dark brown, soft

* - Fresh Growth Index calculated after 30 days Means followed by the same letters are not significant1 y different (P4.05) according to Duncan's Multiple Range Test (DMRT)

Table 7 . Effect of NAA on callogenesis from leaf of S-trilobatum

Growth Rate PGR Callus mass (mg) FGI* Percentage

No. of days taken for Nature of callus

of response (mgA) 0 15days 30days 45days 60days induction

0.5 23.10 256.83 640.20 1033.37 2004.91 I00 7 dark brown, soft, 90% cultures 27.49 f 3.28 with roots

1 22.52 379.77 602.78 1 204.24 I 085.50 100 7 brown, soft, all cultures produced

25.80 .t. 0.91 roots

2 21.57 229.31 612.18 895.10 1271.78 28.3920.70 90 7 greyish brown, all cutlure produced roots

3 21.41 201.55 476.56 650.68 764.17 80 7 blackish brown, soft, 50

21.50 f 0.44 cultures with roots

4 21 -97 252.89 696.62 1047.65 11 99.34 31.82 k 1.21 60 7 brown, soft, 50 % cultures produced roots

5 23.76 231.68 544.20 779.07 821.45 21 -86 f 0.42 85 8 dark brown, soff, no roots

* - Fresh Growth Index calculated after 30 days

92 Part I! - Establishment of in vifro systems -


For S. wendlandii, callus started proliferating from the margins of the explant from the fifth day onwards in all concentrations of NAA, which were tested (Table 8). Even though 100% response was displayed by explants in yielding callus, ,

the amount of callus was much less (Figure 21). Among the six concentrations used, the highest quantity of callus was found in 0.5 mg/l, which was not a significant (p=o.og) increase from the other concentrations. The callus formed was with more compactly adhering cells than those induced by 2,4-D.

2.4.1.3 Effect of IAA on callogenesis

2.4.1.3.1 S. trilobatum

It took 5-10 days for callus to be induced from leaf explants of S. trilobatum in medium supplemented with IAA (Table 9) . The longest induction time was in the lowest concentration of 0.5 mg/l. Induction time diminished as the concentration increased to 5 mg/l. All the concentrations were equally productive in inducing callus. The highest callus yield (p<o.o~) was in the medium supplemented with 4 mg/l 'MA. Dense, profusely multiplying white cellular aggregates originated from the cut margins of the explant and completely enveloped it within one month (Figure 22). The explant also dedifferentiated into callus to a large extent. By the second month, yield of massive, friable, pale yellow to brown, slightly granular callus was obtained. At lower concentrations of IAA, 50-90 percent of the cultures put forth white roots with numerous root hairs. Rhizogenesis also occurred at higher concentrations albeit with lesser frequency (Figure 228. These were discernable only in the initial stages of callus induction up to one month. Rapid callus formation overtook the explant and caused cessation of growth in roots. Moreover, repeated subcultures also disturbed the roots.


Cell proliferation started much earlier in S. wendlandii on the third day itself under the influence of IAA (Table lo). At 4 mg/l IAA, significantly (p<o.oi) higher mass of green nodular callus was obtained (Figure 23). However the capacity of IAA to induce callus from leaf decreased as the concentration

Table 8. Effect of NAA on callogenesis from leaf of S. wendlandii

Growth Rate PGR FGI*

No. of days Callus mass (mg)

I rna I I \ nf Percentage racnnnca taken for Nature of callus Ul I FI3pUI IaCZ indu

0.5 69.79 588.28 1060.14 j076.69 1104.14 16.47 f 0.71 100 5 compact , brown

1 56.34 592.93 899.08 959.32 1145.19 14.86 f 0.36 100 5 compact, hard, light brown

2 59.71 547.30 840.56 91 6.23 892.25 13.85 f 0.67 100 5 friable, dark grey

3 63.10 577.62 771.99 788.02 843.73 11.88 f 0.48 100 5 friable, dark grey

4 54 -24 467.24 674.10 746.83 749.61 13.30 f 0.62 100 5 friable, dark grey

5 53.54 404.54 588.32 620.13 699.33 32.14 -t- 0.31 100 5 friable, dark grey

* - Fresh Growth Index calculated after 30 days Variations in FGI was not significant at P=0.05 level

Table 9. Effect of IAA on callogenesis from leaf of S. trilobatum

Growth Rate Percentage No. of days

PGR Callus mass (mg) FGI* taken for Nature of callus of response

(mgll) 0 15days 30days 45days 60days induction

0.5 29.37 310.35 1967.98 2042.91 1973.80 6 6 . 3 7 & ~ , 0 2 ~ - ~ 100 10 dark brown, friabel, granular 90% culture with roots

1 32.97 372.72 2221.68 2605.97 2763.58 6 8 . 4 6 f 0 . 8 ~ ~ ~ ~ 100 8 dark brown with profuse roots, 70% of culture with roots

2 27.94 51 7.23 2565.34 2849.42 2921 -53 9, -74 f (-y34a-d 100 8 brownish cream callus, friable, WOh cultures with roots

3 28.59 652.92 2647.54 3162.48 3052.46 ~ ~ 4 . 0 8 f 1 , 9 1 ~ ~ ~ 100 6 light brown, friable, 5O0k cultures with roots

4 29.45 877.97 2917.68' 3913.66 4581.18 101.06 f a 100 5 yelllowish cream, compact, 70% cultures with roots

5 31.50 893.70 2980.89 4003.84 5635.30 95.58+2.26 ab 100 5 brownish cream callus, more compact, 30% cultures with roots

* - Fresh Growth Index calculated after 30 days Means followed by the same letters are not significantly different (P<0.01) according to Duncan's Multiple Range Test (DM RT)

..

Table 10. Effect of IAA on callogenesis from leaf of S. wendlandii

Growth Rate No. of days PGR Callus mass (mg) FGI* taken for Nature of callus Percentage

0 15days 30days 45days 60days of response induction

0.5 20.14 21 5.9~4 305.29 453.26 566.03 16-04 f 1-02 90 4 d hard, nodular, 90% cultures with thick white roots

d 90 1 32.26 21 6.83 493.44 730.59 906.18 16.1 7 0.89 4 hard, nodular, 75% cultures with roots

2 34.30 449.29 1235.98 1439.68 2023.94 35.74 f 60 4 watery, soft, brown, 20% cultures with rmts

3 26.80 574.87 1562.14 1774.46 1855.35 60.89f1.91ab 80 3 very hard, nodular, greenish

4 23.29 90 3 a hard , brown with green 669.52 1485.67 1503.11 1762.81 64.49f1.53

nodules

5 26.76 748.38 1363.19 1406.91 1591.73 57.13f2.26 70 3 hard , granular abc

* - Fresh Growth Index calculatd after 30 days V

Means followed by the sam e letters are not significantly different (P<0.01) according to Duncan's Multiple Range Test (DMRT)

96 Part I1 - Establishment of jn vitro systems

increased. Thick white roots also were found in medium fortified with 0.5 mg/l and I mg/l of IAA. The significance of rhizogenesis for obtaining starting material for root culture at this concentration is presented at a later stage. The yield of callus at o.grng/l and 1 mg/l was meagre compared to that obtained in other concentrations of IAA. The explant underwent extensive curling during callus induction. From the inception itself the callus formed hard, white nodules. The nodules were larger and green on the lower side of the callus mass (Figure 2$) where it was in direct contact with the nutrient medium.

2.4.1.4 Growth patterns of calli

Monitoring the growth every fifteen days from the day of inoculation till the end of two months helped to establish the pattern of growth and proliferation in both the plants.


A comparative display of growth patterns of calli in media supplemented yith the three auxins is given in Figure lo. In a,4-D, most of the calli in concentrations ranging from o.gmg/l to gmg/l attained steady phase or showed a tendency to reach it soon, in 40 days. In NAA, the callus continued slow but steady growth even after 60 days. Only the callus at 0.5 mg/l showed a sharp spurt in growth after 50 days. In IAA, the majority of calli in most of the concentrations reached steady state by the 30" day. Calli growing in 4 mg/l and 5 mg/l IAA, however, showed accelerated growth, almost doubling the mass in the 30-60 day period. Figure 12 shows a comparison of relative 'inductiveness' and growth of callus as a function of auxins in S. trilobaturn. IAA produced consistently massive quantities of calli (2 - 3 g) in one month from an explant of ca. 25 mg fresh weight. z,4-D comes next with only half the potential (400 mg - I g). NAA's acumen was much less at all concentrations in terms of biomass (500 - 700 mg).

Based on the information gathered from the data on callogenesis, the ideal medium that induced the maximum amount of continuously proliferating callus in the shortest possible time was 4 mg/l IAA for S. trilobatum. It was selected as the callus induction medium for S. trilobatum (CIMT).

Resu tts 97


The growth rate of calli of S. wendlandii under the influence of the three auxins showed similar initial response as in S. tn'lobatum with a short lag phase (Figure 11). All the three auxins were able to jumpstart the process of callogenesis followed by actively proliferating calli showing the exponential growth early. In IAA and NAA, the log phase lasted 30 days and callus showed growth at a steady pace till the end of the study period on the 6 0 ~ day. 2,4-D influenced callus growth in such a way that it reached steady state after 45 days.

In Figure 13, a comparison of the callus mass produced by the three auxins in S. wendlandii is presented. It was seen that IAA imparted an effect similar to that seen in S. trilobatum, being the auxin responsible for producing the largest quantity of callus (300 mg - 1.5 g). NAA was not far behind, with comparable range of production (500 mg - I g) of callus in one month. 2,4-D also was on par with NAA producing callus in the same range (400 mg - 800 mg).

Considering the most crucial aspect of continuous proliferatipn, which is necessary for the maintenance of callus, 0.5 mg/l2,4-D was selected as the ideal concentration of the callus induction medium for S. wendlandii (CIMW). Even though IAA showed better performance in terms of callus yield, a tendency for chlorophyll synthesis and nodulation proved disadvantageous.

2.4.2 Cell suspension cultures

Cell suspension culture of both plants was successfully established in MS basal medium containing 0.5 mg/l2,4-D and 0.5 mg/l Kn.

2.4.2.1 Establishment of primary suspension cultures

Calli of S. trilobatum fell into two distinct categories. Type I callus was the brown coloured callus produced as a result of serial subculture, (Figure 240). Vigorously proliferating freshly induced callus with pale yellow to greenish colour was designated as type I1 callus (Figure 24b). Primary suspensions were

established using both type I and type I1 calli.


In S. wendlandii, callus cultures, which were 2-3 weeks old with actively dividing cells, were used for initiating suspension cultures. They disintegrated easily into cells and small clumps since the cells were loosely bound to each other.

Changes in the mother cultures were monitored visually every day. Measurements indicating the rate of growth were not recorded during the period. Periodic evaluation of viability showed high proportion of live cells up to go percent in the first 3 days but decreasing to 60 percent after that and it remained more or less steady at 60 percent for the rest of the period.

When the cultures became suficiently populated with actively dividing cells, it was bulked and lo ml of the culture consisting of free floating cells and cell clumps were introduced into the media with varying sugar and salt concentrations to establish suspension cultures.

2.4.2.2 Selection of medium b

2.4.2.2.1 S. trilobatum '

The two types of calli in S. trilobatum gave rise to two distinct cell suspension cultures (Table 11) . The cultures established from type I1 callus appeared healthier with pale to dark brown colour and a high incidence of callus cell aggregates (CCA) (Figure 24). Suspension cultures of type I1 callus were with

a lesser number of CCA and appeared dark brown to black in colour. Free floating cells and cell clumps continued to grow in both types of cultures, though at a different pace. After two weeks in culture, type I suspension cultures showed better growth in medium supplied with 1.5% sucrose and half-strength MS salt concentration. Type I1 cultures showed remarkable growth of both free cells and CCA at 3 % sucrose + MS. The CCA were large, numerous and actively enlarging. The actively growing cultures became thick and viscous with accumulation of cells. Based on these results, the type I1 callus in media supplied with 3% sucrose and full addendum of MS salt concentration was selected for further studies.

Resu Its 99

Table 11. Response of callus types I and II of S. trilobatum to different media supplemented with 0.5 mgll 2,4-D and 0.5mgll Kn

Initiating Viscous nature Colour of Diameter of Type/cotour of call us Medium of culture culture nodules (mm) nodules

3% sucrose + 0.5 x Type 1 MS ++ blackish 0.75 - 2.50 medium / bream

brown

(habituated 1.5% sucrose + 0.5 x blackish medium to large callus)

MS ++

brown 1.00 - 5.00 , cream

3% sucrose + MS

1 -5% sucrose + MS +

small 1 dark dull brown 0.50 - 1.75

brown

blackish medium to large brown 0m77 - / dark brown

3% sucrose + 0.5 x medium to large Type11 MS ++ lightbrown 0.65-4.00

(freshly 1.5% sucrose + 0.5 x

induced MS ++ small to medium light brown 0.50 - 3.50 / cream

callus)

++++ dark 3% sucrose + MS 1.00-6.00 large/cream

brown

1.5% sucrose + MS ++ light brown 0.25 - 1.00 tiny)/ brown

2.4.2.2.1.1 Formation of CCA in S. trilobatum cell suspension cultures

The small cell clumps formed during initiation of suspension cultures continued to divide in multiple planes to enlarge and give rise to uniformly rounded firm structures designated as callus cell aggregates or CCA (Figure 25a). The diameter of these aggregates ranged between 0.25 mm and 5 mm. The weight of these bodies ranged from 0.4 mg to 61 mg. Drying caused a loss of 20-30 percent reduction in mass due to water loss. The aggregates were fully developed by the second week in culture and did not grow more than 6 mm. Newly formed aggregates were pale yellow, which later turned dark brown on maturation (Figure 25b).

Internally CCA was revealed as two distinct layers in large aggregates

(Figure 2 5 c ) A dense white and compact inner cortex enveloped by a thinner soft pale brown outer cortex consisting mostly of elongated cells even extending

. . , ' - ,

I I '

I ' 4 ' . I

. . '! . 100 '.

1 . . . , Part I1 - Establishment of in vitro svstems

< % 9

outwards'as short arms, was observed. The central core was occupied by a cavity ., ' . .

filled with loo&' &11S. Small-sized CCA did not possess this central vacuole (Figure 254. The inner compactly arranged isodiametric cells cut off cells to outside in a radial pattern. These outer elongated cells were seldom released into the liquid medium (Figure 25e).

The growth of CCA was maintained by groups of actively dividing meristematic cells (Figure a f l . These CCA were isolated and maintained separately for further experimentation.

2.4.2.2.2. S. wendlandii

S. wendlandii cultures showed very few visually distinguishable peculiarities for assessment. However, cultures having 3% sucrose and full MS salt concentration were more viscous with higher cell density than other cultures (data not shown). Other cultures were lighter and without much active division. In S. wendlandii cell suspension cultures, the cell aggregates comprised groups of 4-12 cells. They did not undergo compact aggregation as in S. bilobatum.

2.4.2.3 Growth curve

Identical procedures were followed for establishing homogenous cell suspensions (Figure 246) consisting of only free floating cells or microscopic clumps for both plants. Using the cell cultures in MS medium supplemented with 0.5 mg/l a,4-D and 0.5 mg/l Kn, the growth curve of cells of both plants was plotted.


The cells were characteristically elongated (Figure 26a) and twisted types

(Figures 26c and 266) were common after a few days growth. Cells had large vacuoles and were thin-walled. Aggregations were many-celled and compact (Figure 26b).

The cells in S. trilobaturn suspension cultures showed a sigmoid pattern of growth (Figure 14). The lag phase was very short with duration of possibly 2-3

days soon after initiation and went undetected in the present data. The cell

Results

suspension entered peak growth or log phas phase by the lqth day following a decline in g the quantity of cells doubled. A transient increase in the mass was noticed after 28 days in cultures, which could be due to the beginning of another cycle of growth. These changes were reflected both in fresh weight and dry weight measurements. Statistical analysis of data revealed strong relation (r = o .g) between packed cell volume and fresh weight of cell suspensions. Relation between fresh weight and dry weight however was less strong in the cell population (r=o.8). Viability of the cells was highest at 80-90 percent at the peak period after which it declined.

Table 12. Growth pattern of S. trilobatum cells in suspension cultures

Days PCV (ml/l) Fresh wt. (g) Dry wt.(g)


The cells of S. wendlandii in liquid agitated medium'(~i~ure z7a) were rounded, sometimes oval or elongated (Figures 276 and 2 7 c ) The cell aggregates in S. wendlandii suspension cultures were smaller (4-15 cells) and loosely bound (Figures 27d and 27e). Dead cells appeared highly vacuolated with shrunken cytoplasm or were ruptured owing to the shearing forces of other cells in the agitated medium. Different stages of cell division and septum formation were

also noticed (Figure 2 f l . The cells were fragile and rapidly lost viability. The

viability fluctuated between 50 percent at times to 80 percent during peak growth

phase. The cells took more than 7 days to start active division (lag phase) and

1 02 Part 11 - Establishment of in vitro svstems

reached peak growth on the fourtheenth day (Table 13). By the 21" day the growth declined drastically due to massive death of cells. The momentum was

picked up again by the 28'h day. These changes were reflected in dry weight measurements also (Figure 15). High correlation (r=o.g) between fresh weight and dry weight of the cells in suspension was found. While correlation (r=o.7) ,

between packed cell volume and fresh weight was not significant at p<0.05.

Table 73. Growth pattern of S. wendlandii cells in suspension cultures

Days PCV (%) Fresh wt. (g) Dry uvtrn (g)

2.4.3 Root cultures

Initial experiments were conducted to obtain sufficient adventitious roots as starting material for raising root cultures.

2.4.3.1 Rhizogenesis

Roots were induced from S. tn'lobatum leaves both in dark and light conditions when medium was supplemented with low concentrations (0.5 mg/l) of NAA

and IBA (Table 14). In the absence of PGRs, half-strength MS salts and half- strength sugar, the explants turned yellow a few days after inoculation and underwent necrosis. In dark conditions, only rhizogenesis occurred (Figure 28a). Under light conditions, the rhizogenesis was followed by callogenesis from the explant (Figure 28b). NAA was better than IBA in inducing roots in terms of the percentage of response and also the number of roots. The roots generally started

Results 1 03

originating by the fourth or fifth day. Under dark conditions, the number of roots induced in medium supplemented with IBA and NAA were more. To

initiate root cultures of S. h'lobatum, the medium supplemented with 0.5

mg/l NAA under dark conditions (Figure 28c) was selected(RIMT).

Table 74. Rhizogenesis from S. trilobatum leaves under light and dark conditions

Condition Medium Percentage of No. of roots Nature of response

response

Dark 0.5~ MS turned yellow , necrosis

1.5% sucrose turned yellow , necrosis

0.5 mgll NAA 90 2-5 thick, white roots

0.5 mgll IBA 20 0-3 short roots

Light

1.5% sucrose

turned yellow , necrosis +

turned yellow , necrosis

0.5 mgll NAA 60 0-5 callogenesis

0.5 mgll IBA 20 0-2 callogenesis

Numerous thick white roots were produced in 7-9 days from leaf explants under the influence of 0.5 mg/l IAA (Figure 286), a result already established during callus studies. So 0.5 mg/l IAA was selected as the medium for inducing roots in S. wendlandii (RIMW).

2.4.3.2 Selection of medium for root culture


In the beginning of culture period, differentiation of inoculated roots in the form of lateral roots from the inoculated main axis took place in all the tested media in three different physical conditions (Table 15). A comparative visual


assessment of roots in S. trilobatum of the differentiation and growth as a function of physical and chemical conditions of the media is shown in Figure 29. The largest number of new roots induced was in cultures, which were agitated under light conditions (=14 roots) followed by those in static cultures (=lo roots) also kept in light. The number of new roots differentiating in dark was the lowest ,

( ~ 1 8 roots). Elongation of roots indicating growth of the newly formed roots were high ("3.5 cm) in darkjstatic conditions while it was uniformly low in light/ static (a cm) and lightlagitated (=I cm) conditions. Among the physical conditions, since elongation of roots was very important for sustaining cultures, dark condition was found to be the most suitable one for root cultures.

The nutritional, chemical and hormonal influences affecting the twin aspects of differentiation and growth of roots were also considered for the selection of optimal medium composition. The number of new laterals was the highest in media having the full addendum of ammonium nitrate (14-15 roots) but elongation of those same roots was poor in it (0.57-0.92 cm). The negative effect of ammonium nitrate was somewhat lessened in dark condition by IAA. Even though IAA induced highest number of roots in dark, elongation ,was inhibited by it simultaneously (Figure 28e). IAA and light were the most unproductive combination resulting in the lowest number of roots with minimal elongation.

Media containing half and quarter of the usual amounts of ammonium nitrate, promoted formation of new roots especially in dark (6 and 8 roots respectively). Complete absence of ammonium nitrate was conducive for both differentiation and growth more in light than in dark. Low salt concentration (0.25~ MS) favoured differentiation and growth irrespective of the physical condition. Agitation was more desirable than static conditions especially for root differentiation.

There was also morphological variation in roots formed in different conditions. External features were mostly abnormal in cultures grown in the presence of ammonium nitrate. Roots were distended, swollen, tufted and stunted. Those induced in its absence were uniformly thin and appeared normal.

Thus, taking into account the root growth (elongation), external morphology and root differentiation (induction of new laterals), MS+o.25x

Results

Part I1 - E

stablishment of in vitro system

s

Results 107

NH4N0, was selected as the best root culture medium for S. trilobatum (RCMT).


Adventitious roots did not grow equally in all the media tested (Table 16). In light conditions, new roots could be induced in MS liquid media only in the absence of ammonium nitrate in static cultures and half salt concentration in agitated cultures (Figure 30). In other media, continuation of the elongation of the tip of the main axis (inoculated root) took place. 1 mg/l IAA (Figure 28f)

and MS+NH4N0, failed to evoke any response. Under dark conditions, roots differentiated and elongated well in media with half and quarter of the normal amount of ammonium nitrate.

The roots formed in such cultures formed an intertwining mat of numerous laterals. So in S. wendlandii, MS media with 0.25~ NH4N0, was selected as the root culture medium (RCMW).

In both plants no correlation was found between root differentiation and >

root growth.

2.4.4 Regeneration studies

Since the trials for regeneration and the results were distinct, they are presented separately for both plants.

2.4.4.1 Regeneration by organogenesis in S. trilobatum

2.4.4.1.1 Selection of PGR combination

Out of the sixteen combinations tried, shoots were induced directly from leaf in the media supplemented with Kn, BAP, 2iP and TDZ used singly (Table 17).

When these cytokinins were used in combination with an auxin, 3 combinations - IAA+Kn, IAA+2iP and W + T D Z - produced adventitious shoots. Adventitious shoots* arose from stem in media having BAP, 2iP, TDZ and IAA+TDZ. Shoots originated indirectly from one-month old callus in the presence of 2iP, TDZ,

* The term adventitious buds/shoots refers to buds of shoots arising from any place other

than leaf axil or the shoot apex.


IAA+2iP, NAA+TDZ and IAA+TDZ after 30 days exposure. In the rest of the

media, only callus was observed. The typical responses to each type and combination of calli were easily distinguishable and are presented in detail, for easy visual assessment, in the series of figures starting from Figure 31, till Figure

35.

Table 7 7. Regeneration response of leaf, stem, and callus of S. trilobatum to different PG R's *

Treatment PGR (rng/l) No.

Leaf Stem Callus

1 1 Kn shoots (4) call us call us

2 1 BAP shoots (2) shoots (6) call us

3 1 2iP shoots (8) shoots (1 0) shoots (9)

4 1 TDZ shoots ( 2) shoots (6) shoots (20)

5 0.5 NAA + 1 Kn call us call us

6 0.5 2,443 + 1 Kn callus call us

7 0.5 IAA + 1 Kn shoots (8) call us

8 0.5NAA+IBAP - . callus

9 0.5 2,4-D + 1 BAP callus

10 0.5 IAA + 1 BAP callus

callus

callus

call us

call us

call us

callus >

call us

call us

callus

11 0.5 NAA + 1 2iP shoots (2) call us callus

12 0.5 2,443 + 1 2iP call us callus callus

q3 0.5 IAA + I 2iP shoots (12) callus shoots (7)

14 0.5 NAA + 1 TDZ call us call us ' shoots (3)

15 0.5 2,4-D + ITDZ call us callus callus

16 0.5 IAA + ITDZ shoots(4) shoots (5) shoots (3)

*the observations were taken after 30 days The values in parenthesis represent the average number of shoots

BAP and Kn were not able to consistently induce regeneration in S. trilobatum. Only TDZ and 2iP were successful in strongly influencing all the three types of explants -.leaf, stem and callus - to put forth shoots. Shoots developed and elongated normally in the combination M+2 iP . Those produced

Results 109

under the influence of TDZ were small and stunted. So the combination of IAA and 2iP was selected for further optimization.

2.4.4.1.2 Optimization of PGR concentration

Direct caulogenesis from leaf and indirect caulogenesis from callus of S. trilobatum was optimized with varying concentrations of the more critical cytokinin (2iP) while the concentration of the auxin (IAA) was kept constant at 0.5 mg/l.

2.4.4.1.2.1 Direct caulog enesis

Adventitious shoots arose from leaves only in the media supplemented with 2iP

in the ranges 0.5-4.0 mg/l (Table 18). In low concentrations (0.01 and 0.1 mg/l) and high concentration (5 mg/l) shoots failed to form. Maximum number of shoots (19) was found in media fortified with 0.5 mg/l IAA+3 mg/l2iP which was significantly (pco.01) higher than those produced in other combinations. This result was shown by all the cultures (loo% response) in the shortest time (12 days). The length of the shoots also was greatest (1.06 cm) in this combination after 30 days. Wheir 2iP alone was used at I mg/l the shoots took longer time to form and were considerably short.

Table 18. Effect of 2iP on direct caulogenesis from leaves of S.trilobatum

Trial PGR (mgll) Percentage Days taken for No. of response induction No.of shoots Shoot length

IAA 2 i P

9 0 1 .O 40 28 13 * 4-53 ab 0.44 * 0.24

Means followed by the same letters are not significantly different (P<0.01) according to Duncan's Multiple Range Test (DMRT)

110 Parf I1 - Establishment of in vifro systems

The shoots appeared as small green bulbs (Figure 36a) from the cut margins of the explant (Figure 366) 12 days after inoculation. The stages of development of these shoots were monitored at regular intervals. Soon, the apex of the bulbs differentiated into a clearly distinguishable shoot apex (Figure 27b). One week after induction, each shoot primordia started to elongate and put forth leaves (Figure 36c). The green nodules were restricted to the margins of the explants and were rarely produced from its surface (Figure 36e). After 20-25 days, thick cluster of developing shoots could be seen (Figure 36f). Thereafter, these shoots elongated to a length of 2-5 cm carrying fully expanded normal leaves (Figure 369).

The clusters were parted into small bunches comprising 3-4 shoots and subcultured in the same media. These triggered new shoots from the base of the other shoots and allowed the already formed small shoots to grow and elongate. However, the multiplication rate declined after two subsequent subcultures, where the number of fully formed shoots was reduced to four or five. Mass shoot cultures were obtained by conducting cycles of inoculation of explant, induction of shoots and subculture (Figure 37). b

Certain abnormalities were noticed in shoot clusters during the second and third subcultures. Basal callus was a major hindrance to shoot formation in some cultures (Figure 3 8 ~ ) . In cultures with basal callus, only one or two shoots could develop fully (Figure 38b). Some cultures with small shoots and malformed leaves turned to callusing (Figure 38c). The leaves of such cultures, which did not yield to callusing, abruptly turned white, loosing all its chlorophyll content (Figure 38d). A very small percentage of shoot cultures were vitrified, displaying dark green, crumpled and water-soaked leaves. Also, the stems appeared fasciated in these cultures (Figure 38e). Occasionally, adventitious roots originated from the basal shoot/callus portion that had no direct connection with the shoots (Figure 38B.

These abnormalities were restricted to 17% of the total shoot cultures raised and were detected early and discarded.

2.4.4.1.2.2 Indirect caulog enesis

Adventitious shoots were produced from the callus of S. trilobatum after 10-20

Results 11 1

days when exposed to different concentrations of 2iP (Table 19). Higher concentrations of 2iP at 4 and 5 mg/l could not produce shoots. Maximum shooting at a significant level (p=o.oi) was obsewed in medium fortified with 0.5 mg/l IAA and 0.5 mg/l IAA+i mg/l2iP. When 2iP was tried alone at 1 mg/l concentration, even though shoots were induced early, their number and length were far below the desirable level.

Table 19. Effect of 2ip on indirect caulogenesis from leaves of S.triobatum

Trial PGR (mgil) Percentage Days taken for No. of response induction No.of shoots Shoot length

I AA 2iP

1 0.5 0.05 60 20 3.2 + 0.8 de 0.81 + 0.19

Mens foYwd by ihe smw le(tsn am nd signitkantiy differan (Pc0.01) acoxlhg to Duncnh MulWple Rage Test (DMRT)

By the tenth day after introduction into regeneration medium, small green protuberances (Figure 3ga) could be seen on the surface of compact callus. These structures developed shoot apex andleaf primordia by the day (Figure39b). These young shoots were seen singly or in clusters (Figure 3gc) and later produced expanded leaves (Figure 394 . Within lo days after the formation of shoot primordia, well-developed shoots with sufficient length and 4-5 leaves could be witnessed (Figures 3ge and 398. A comparison of the external and internal morphological stages of development of shoots indirectly from callus is shown in Figure 40.

112 Pad I1 - ESt8bli~hmnf of in I&O systems

2.4.4.1.3 Rooting

Well-developed shoots derived through direct and indirect caulogenesis having 4-7 leaves were transferred to the liquid media. The competence of IBA, NAA and the absence of PGRs as well as reduced concentration of MS nutrients to induce rooting is presented in Table 20. The absence of PGR favoured root induction from the base of the shoots in S. trilobatum (Figure 414. Combined with low salt concentration - only a quarter of the normal level - ideal medium for rooting was established (Figure d c and 416). Roots originated as thick white prolongations covered with root hairs (Figure 4 b ) . The shortest time required for rooting was between 4 and 7 days. However, IBA succeeded in inducing rooting from the maximum number of cultures especially in low salt concentration. NAA could induce only the smallest number of short stumpy roots. In NAA, the shoot base also showed a tendency for callusing, which was highly undesirable (Figure @). Roots produced in the absence of PGRs were better in terms of number, length and appearance. In IBA, the number of lateral roots was large. Shoot elongation, leaf expansion and formation of new leaves were better in the absence of PGR In NAA, the shoots turned yellow and growth virtually ceased.

Table 20. Rooting from adventitious shoots of S. trilobatum

Basal PGR Concentration Days taken Percentage of No. of roots per Root length

Medium (mgll) for induction rooting shoots shoot (em)

0.5 4 90 5.13 t 2.64 1.75 i 0.58 IBA

2.0 8 50 4.38 i 3.29 2.13 + 1.43

0.5 5 80 3.63 21.06 0.82 t 0.32 NAA

2.0 0 0 0 0

0.0 5 20 5.50 t 2.07 3.91 i 0.78

The observations were recorded after 15 days

Results 113

2.4.4.1.4 Hardening

Plantlets with sufficient number of 5-8 roots (Figure 4 3 ) were planted in Soilrite in glass micro chambers (Figure #b) for acclimatization for 2 weeks. During this period, a dense mat of new root system was formed and the aerial parts were satisfactorily hardened. Then they were transferred to plastic cups and exposed to external environment (Figure 43c and 436). This step-by-step transfer ensured loo percent survival of the plantlets. In greenhouse conditions, the plants grew well for two months (Figure 43e and 43f) and were later planted in soil in the field (Figure 439).

The complete cycle of direct regeneration in S. trilobatum is represented in Figure

44

2.4.4.2 Regeneration by somatic embryogenesis in S. wendlandii

2.4.4.2.1 Selection of PGR combination

The preliminary experiment conducted to assess the morphogenetic response of S. wendlandii to various auxins and cytokinins yielded somatic embryogenesis (from only leaf explants) in media fortified with TDZ alone (1 mg/l) and TDZ (I mg/l) in combination with 2,4-D, NAA and IAA (all at 0.5 mg/l) (Table 21). The results are presented in the series of Figures 45 - 49 to enable easy visual comparison. When leaf explants were exposed to the action of cytokinin alone, the explants remained green without any morphogenetic changes. In all the other treatments using leaf and stem, only callus was formed and in the case of one-month old calli, further proliferation of callus occurred.

The globular structures on the leaf lamina, based on the external and internal scrutiny of structures developed from it later, were identified as embryoids or proembryos. Since the number of these embryoids was maximum in ~A-D+TDZ combination, it was selected for further optimization.

2.4.4.2.2 Opthiastion of PGR concentration

The results on the effect of TDZ on embryoids from leaf explants of S. wendlandii are presented in Table 22. The embryoids were initiated over a period of time

114 Part I/ - Establishment of h vitm systems

Table 21. Regeneration response of leaf, stem and callus of S.wendlandii to different PGR's *

treatment &I-. PGR (fv') Leaf Stem Callus

1 1 Kn callus

2 1 BAP callus

3 1 2iP callus

4 1 TDZ somatic embryos (15) callus

5 0.5 NAA + lKn callus

6 0.5 2.4-D + 1Kn callus

7 0.5 IAA + 1 Kn callus

8 0.5 NAA + 1BAP callus

9 0.5 2,4-D + 1 BAP callus

10 0.5 IAA + 1 BAP callus

11 0.5 NAA + 1 2iP callus

12 0.5 2,4-D + 1 2iP callus

13 0.5 IAA + 1 2iP callus

callus

callus

callus

callus

callus

cauus

callus

callus

callus

14 0.5 NAA + ITDZ somatic embryos (25) callus

15 0.5 2,CD + 1TRL rromatk ombfyos (38) callus

16 0.5 IAA + ITDZ m a t i c embrvos (18) callus

callus

callus

callus

callus

callus

callus

callus

callus

callus

callus

callus

callus

callus

callus

callus

callus . . . The observations were taken alter 30 days The values in parenthesis represent the average number of somatic emblyos

ranging from 15 days to almost one month. The shortest time necessary for full development of embryoids was in medium supplemented with 0.5 mg/l2,4-D and irngll TDZ. Significantly large (p<o.oi) number of these globular structures was also obtained in this combination averaging ca. 38 embryoids per explant after a period of one month since induction. So this was selected as the somatic embryo induction medium for S. wendlandii (SEIMW). The second highest number of embryoids, 27, was found in medium supplemented with 1 mg/l TDZ alone. But both treatments produced the result in the same period of time (15- 20 days). But the potentid of TDZ to induce embryogenesis in all the cultures

Results 115

uniformly was better when it was used alone. In concentrations higher and lower than 1 mg/l of TDZ, the d t was significantly lower in terms of the number of embryoids, the percentage of response and the time taken.

TaMe 22. Effect of TDZ on somatic embyrogenesis from leaf explants of S. wendlandii

Trial No. PGR (m@) P-tage of Days taken for No.of embryoids

2 .kD TDZ responSe induction

2 0.5 0.10 60 15-28 9.7 * 6.25 b-f

6 0.5 3.00 80 15 - 30 16.2 * 8.89 abc

7 1.00 100 15 - 20 27.0 * 11.63 ab

~ t d D n d b y l k m a ~ ~ n d s i g n i l l c 8 n l f y d m a r r n t ( P 4 . o l ) ~ t o - ' s M u l t i p l e Range Test (DMRT)

The explant lost its dark green colour and appeared 'burnt' prior to embryogenesis (Figure 50a) Embryoids were produced en masse initially as protuberances from near the margins (Figure 50b), which later spread to the whole lamina (Figure 50c). Somatic embryos always formed from the adaxial surface, arising mostly from near the cut surface, in groups or as isolated rounded structures. There were also non-embryogenic regions on the explant, which were dead by the time the embryo induction process was over. One month after the induction, these embryoids enlarged and resembled shiny, green blobs (Figure sod and 50e) Most of them were in groups covering an entire region, but isolated embryoids with basal attachment to the explant were not uncommon (Figure 505). No vascular connection between the embryo and the subjacent tissues was noticed.

116 Part I1 - Esteblshment of in Mm systems

In the second month, the embryoids matured into embryos. The basal connecting tissue disintegrated and in&vidual embryoids could be separated (Figure 51a) and analysed. Embryos enlarged into distinct bipolar structures and passed through globular (Figure sib), elongated (Figwe SIC), heart-shaped (Figure 516), torpedo-shaped (Figure sic) and cotyledonary stages, which a somatic embryo passes through during the normal course of development. Globular and heart-shaped stages were commonly met with and growth was synchronous during these stages. The occurrence of advanced stages such as torpedo-shaped, walking-stick shaped arid cotyledonary stages were limited in number. Before the majority of embryoids reached these stages, abnormalities began to appear in cultures.

An internal structure of the somatic embryos as seen in the longitudinal sections of the different stages of embryo development is presented in Figure 52. The origin of somatic embryos appeared to be kom epidermal cells, since there was no rupture of the tissues (Figure %a). Globular and elongated globular stages did not possess any tracheary elements (Figure ~b and pc) . The body of the embryo consisted of uniformly isotliametric cells enveloped by a distinct epidermis. Conducting elements were visible in torpedo-shaped and heart- shaped stages (Figurepc and 52d). These structures also had clearly demarcated shoot and root poles (Figure 52f). However, the xylem elements were not met with in most of the embryos in advanced stages. There was no vascular connection with the mother tissue. Even in the advanced stages root meristem of any kind failed to form in the root pole. The shoot pole followed more or less normal development until primary leaves were produced (Figure 52g), after which its growth also ceased. The frequency of somatic embryos producing normal shoot meristem and subsequent formation of shoot apex was 70 percent.

Aberrant behaviour of somatic embryos was seen in cultures even from their inception (Figure 53 a ton. Embryoids in the pre-globular stage coalesced to form larger structures owing to the close proximity of the embryo initiating sites/ cells. These structures did not convert to embryos. Budding of the globular stage embryo and shoot pale region of the elongated embryo was also common. These deviants comprised 20% of the somatic embryo cultures duringthe initial phase of synchronous development of enibryoids.

Results 117

2.4.4.2.3 Maturation of somatic embryos

To avoid formation of tuber-like growth of root pole, abnormal structural peculiarities and precocious germination, the embryoids were transferred to maturation media within 5 days after the appearance of embryoids (Table 23).

TaMe 23. Maturatiin of somatic embryos of S. wendandii

PGR M-ic Percentageof

responSe mature embyros

Kn (Im@) callus

BAP (1 mdl) callus

2iP (I mgll) callus Kn (I mul) + BAP (I mu) + 2iP (lmgll)

callus Kn (lmgh) + BAP (lmgll) + Ads (I Omg/l)

callus

GA3 tuber-like formation

ABA (Imdl) tuber-like formation

No PGR maturation of somatic emblvos 85

The embryoids in the absence of any regulatory chemical matured into easily separable round, oblong, or heart-shaped somatic embryos. These cultures had to be kept undisturbed for 3-4 months for full maturation. Simultaneously, the remnants of the explants were dead and turned black (Figure ~ e ) . An examination of internal structure revealed absence of organized active root meristem while the shoot pole was well developed. The structure resembled those already described. Cytokinins, singly or in combination of two or three, and adenine sulphate triggered only callusing (Figure 54a and 54b) while in presence of both GAS and ABA somatic embryos developed into peculiar hard callus. The callus was a green or yellowish hard mass carrying leafy shoots of the already formed embryos. (Figure 54 c and 54d). Only growth regulator-free medium was suitable for embryo maturation. Callusing was also seen to some extent wen in this medium (Figure 54B.

118 Pert /I - Eshbtishment of h vitro systems

If the embryoids were allowed .to remain in the induction medium comprising 2,4-D and TDZ, hypomtyl and the root pole region enlarged into a hard, brown tuber-like growth (Figure55a). The shoot pole remained unaffected, continued its growth and put forth leaves (Figure 55c), suggesting precocious germination. It was also observedthat the basal region of the embryoid clusters coalesced to form one single hard callus/tuber-like structure with the small leaves of the shoot poles dispersed over the surface (Figure 55d and 552). Hard callus also formed de novo from the sides of the embryonic mass and did not consist of portions of embyros. In rare cases, the shoots over these hard masses developed 2-3 leaves but they were too short to be excised for rooting or separation from the basal mass (Figure m.

Summarizing, the whole process of embryo development and that of the associated structures could be categorized into two phases. During the primary phase synchronous induction of embryoids and their development into globular and heart-shaped embryos occurred. Embryogenic cultures remained in this phase for two months. The second phase was marked by asynchronous growth and abnormalities. Deviation h m the normal proam of embryogenesis occurred during this period.

2.4.4.2.4 Germination of somatic embryos

The mature somatic embryos in both solid and liquid media failed to germinate in MS basal's medium (Table 24). In liquid agitated conditions, the somatic embryos formed large yellow coloured amorphous nodules (Figure 56b). In solid MS medium, the embryos remained dormant even after two months. Under the influence of GA, (1 mgll) in solid media also the embryos were dormant (Figure 5612). But in agitated liquid media, the shoot pole developed abnormally without any activity in the root pole region (Figure 56e). When 1 mg/l ABA was used to fortify solid MS medium, the embryo callused vigorously (Figure 56d). In liquid media with the same PGR ( 1 mg/l ABA) they formed green to brown coloured nodules/shoots (Figure 56f).

Results 119

TaM 24. Germination of somatic embryos of S. wendandii

Percentage of PGR Concentration Medium germinating

(m@) embwos

solid G& 1

liquid

ABA solid

liquid

solid

liquid

2.5 Discussion

2.5.1. Basal medium

The selection of only MS medium as the source of nutrients was dictated by the fact that it is the most popular medium for culture for not only Solanum species (refer Table 25 ) but also for most other dicot species (Bhojwani and Razdan, 1983) and many crop plants (Werbrouck. and Debergh, 1994). To cite specific examples, among B5, White's and modified MS media, MS was found to be most suitable for producing maximum biomass from fruit explants of S. platanifolium (Jaggi et al., 1987). The stimulating effect of MS medium in comparison to B5 was also quite evident in callus cultures of S. malacoxylon (Suardi et al., 1994). Thus the selection of MS as the basal meclium was adequately justified.

2.5.2 Role of auxins on dogenesis

The order of effectiveness of auxins in inducing callus from the data obtained in the present study was IAA>2,4-D>NAPL. Thus surprisingly in both plants, 2,4-D was found to be inferior to IAA in producing maximum biomass in 30 days. 2,4-D had to be selected for callus induction medium for S. wendlandii only because of the fact that IAA produced green nodular calli, which was unfit for callus studies. 2,4-D at 0.5 mg/l produced continuously proliferating calli. IAA was reported to produce green compact calli in S. xanthocarpum also by Heble et al. (1971).

These results do not agree with most of the reports on Solanurn where the superiority of 2,4-D in inducing callus has been loudly accalimed (Jaggi et al., 1987; Nigra et al., 1989; El Badaoui et al., 1996; Villarreal et al., 1997; Shahzad et al., 1999; Bhalsing et al., 2000).

Discussion 121

It is to be noted that in reports where only the best growth regulator was mentioned (2,4-D), there is no way to ascertain whether the selection was made from among different auxins and 54-D was in fact truly superior to other auxins for callogenesis as it appears to be. This is the case with most of the reports.

In S. aculeatissimum, equal effectiveness of IAA and 2,4-D was reported (Manjula and Nair, 2002). IAA was the preferred auxin for callogenesis in S. nigrum (Bhatt et al., 1983) and in S. surattense (Malpathak and David, 1992,1994) in combination with BA.

An earlier report on S. trilobatum recommended lo pM NAA + 1 pM BA which was produced in the third week after inoculation (Krishnarnurthy and Parabia, 1995). The present study has wme up with a better protocol where earlier induction (in 3 days) and rapid growth was achieved by using only a single awin (IAA 4 mg/l). The biomass after 30 days was also more in the present study.

The inferior nature of NAAin producing callus is in agreement with report of callus formation in other Solanum species, where when 2,4-D and NAA were tested together, 2,4-D was found to be superior (Jaggi et al., 1988; Nigra et al., 1989; Shahzad et al., 1999; Ikenaga et al., 2000). Contrastingly there were also instances where NAAwas most suited for callogenesis in some species (Zacharius and Osman, 1977; Barnabas and David 1986; Banerjee and Ahuja, 1993; Krishnamurthy et al., 1996; Syahrani et al., 2000; Jaggi and Singh, 2000). Roots produced under the influence of NAA was seen in S. eleagnifolium (Nigra et al., 1989). NAA produced roots from explants along with callus in Scutellaria discolor (Sinha et al., 1999), Withania somnifera (Govindaraju et al., 2003) and Coffea bongalensis (Mishra and Sreenath, 2003).

2,4-D was active at low concentrations (0.5 - 1 mg/l) and IAA at high levels (4 mg/l). The active range of 2,4-D is in agreement with the active range reported earlier in S. platanifolium (Jaggi et al., 1987) and S. aculeatissimum (Ikenaga et al., 2000).

Another interesting feature was that S. wendlandii produced callus faster (3-5 days) than S. trilobatum (5-11 days). But the latter produced more biomass

1 22 P d l I - EstaWishment of of in systems

in two months in the optimized media than the former. The performance of the single auxin was satisfactory as callus was produced in the shortest time with almost complete dedifferentiation of the explant in the optimized media. The supplementary cytokinin, as used in most studies was not required since sufficient callus for subculture and experiments were generated in the simplest media with single auxin.

The establishment of suspension cultures proved difficult with S. wendlandii. The cultures showed low viability and were subject to frequent contamination. On the other hand, suspended cells of S. hilobaturn thrived in the media. They also showed high viability. Results suggest that cells of S. trilobatum - characteristically elongated in callus and more so in liquid medium- was more tolerant to variabilities in culture eorlditions and abrupt state changes. Suspension cultures of another, woody perennial species of Solanum, S. mauritianum, were also difficult according to Drewes andvan Staden ( 1 ~ 5 a ) . It is possible that the low success rate in woody species such as S. wendlandii was because of its inherent reluctance and low potential of cells to multiply in liquid media.

Tbis is the &st report of suspension eulhwa~ of S. trilobatum and S. medtmwW. The values obtained for maximum biomass, doubling time and the time to reach the stationary phase were consistent with the values reported for other species of the genus Solanum (Nigra et al., 1990; Alvarez et al., 1993; Villarreal et al., 1997). The suspension cultures c o d be maintained for about six months. The arbitrary selection of 2,4-D and Kn for suspension cultures was justified by earlier reports where the general trend was to use an auxin and cytokinin at low concentrations to maintain the suspension. 2,4-D was the auxin of choice for suspension cultures, irrespective of the growth regulator used for callus initiation for species of Sdanum (Khanna et al., 1976; Khanna and Manot, 1976; Jain and Sahoo, 1981; Chandler and lhdds, 1983a; Ehrnke and Eilert 1986; Lemmonier et al., 1989; Subramani et al.. 1989, Nigra et al., lggo; Quadri and Giulietti, 1993, Makand Doran, 1993; Drewes andvan Staden, 1gg5a). According to Szyeykowska (1974), most cell suspensions have an absolute requirement for auxin especially 2,4-D, to maintain the dispersed nature of cells.

Discussion 123

Kn is the most preferred cytokinin for addition in cell suspension cultures of S. aviculare (Jirku et al., 1981). S. surattense (Barnabas and David, 1988), S. xanthocarpum (Subnunani et al., 1989), S. eleagnifolium (Quadri and Giulietti, 1993), S. carolinense (Reynolds, 1987), S. aculeatissimum (Manjula and Nair, 2002), S. aviculare (Tsoulpha and Doran, 1993) andS. dulcamara (Ehmke and Eilert, 1986). It has been found after extensive studies in suspension cultures of Acer pseudoplatanus, that although an external supply of cytokinins was not essential for continuous culture of sycamore cells, the presence of kinetin and zeatin in the medium caused a distinct shortening of thelag phase (Szyeykowska,

1974).

In cell suspension cultures of cytokinin dependent tobacco strains, cell division was strictly dependent on the presence of both Kn and auxin (Szyeykowska, 1974). The absolute necessity of an auxin and kinetin for growth or maintenance of suspension cultures in the present study, though not conclusively proven owingtolack of data, couldbe accepted as having a stabiing if not promotory effect on the cell cultures, in the light of information gathered from existing literature.

The unique structures formed in suspension cultures of S. trilobatum with an average size of 0.25-6 mm was similar to those obtained in Rhodiola sachalinensis (2-8 mm) (Xu et al., 1999) and 1-5 mm (Wu et al., 2003) smaller than those obtained from S. aviculare (4-20 mm) (Tsoulpha and Doran, 1991).

These aggregates of S. trilobatum shared similar characteristics with those already reported, in having pale yellow colour, absence of internal xylem or cambium, central cavities in large aggregates, peripheral mass of meristematic cells and inner parenchymatous cells with secondary wall formation. In compact cell aggregates of R. sachalinensis, an outer epidermis was visible, which was not seen in the aggregates of S. trilobatum. Under conditions of nutrient depletion, autolysis began to take place and the aggregates became soft and friable in R. sachalinensis (Wu et al., 2003) and S. aviculare (Tsoulpha and Doran,

1 24 Pati N - Esf&Mm& of in vitro systems

1991). Similar decline of the quality of CCA was observed in the present study also. G m n pigmentation observed in aggregates of S. aviculare (Tsoulpha and Doran, 1991) and S. dulcamara (Ehmke and Eilert, 1986) was not seen in S. trilobatum.

As the CCA increased in diameter, the cells at the interior must have been deprived of nutrients and oxygen. In Tagetespatula, oxygen concentration was zero at the center of the aggregates which were greater than 3 mm in diameter (Hulst et al., 1989). The nutrient depletion at the center may cause cell death and lysis leading to the formation of cavities. Similar observations were also made by Tsoulpha and Doran (1991) in S. aviculare. Nutrient depletion clearly explains the presence of cavities in only larger CCA of S. trilobatum.

Usually the presence of an auxin and cytokinin (in equal amounts or high cytokinin/auxin) was required for the formation of aggregates as pointed out on the aforementioned studies. Presence of equal amounts of auxin and cytokinin in the medium may have played a role in S. trilobatum cultures. Whether it is a major factor in the hrmation of aggregates is doubtful since an identical condition had no aggregate-forming effect on S. wendlandii cultures. Lack of cohesiveness between the cells, and absence of the redisp is position factor' for spontaneous aggregation might have prevented the cells of S. wendlandii from forming aggregates.

Callus cell aggregates capable of maintaining morphological integrity and continuous proliferation is a good candidate for bioreactor studies. But CCA of S. trilobatum did not show any significant activity of growth or proliferation on prolonged culture. It could be because of the presence of 2,4-D. The auxin, which has always been believed to effectvely support proliferation of dispersed cells was found to adversely affect CCA biomass and dry weight accumulation in R. sachalinensis (Xu et al., 1998,1999).

The role of Kn in the growth of CCA was however promotory. In S. aviculare, Kn was reported to promote aggregation and maintenance of the integrity of CCA (Tsoulpha and Doran, 1991). In R. sachalinensis also high concentration of Kn enhanced CCA grow.th (Wu et al., 2003).

Discussion 125

2.5.4 Root CUb lVS

S u c e e d inihtion and continuous culturing of untransformed roots S. trilobatum md S. wendlamdii in a medium devoid of growth regulators up to seven months was possible in the present study and probably the first report of its kind in both S. trilobaturm and S. wendlandii. In S. eleagnifolium, root cultures were established, but wuld not be maintained beyond a few weeks (Nigra et al., 190). Hairy root cultures were preferred to normal root cultures by most workers as it was stable and grew faster than untransformed ones without much decrease in metabolite production (Flores et al., 1987).

2.5.4.1 Medium

White's medium was by far the earliest wmmonly used medium for root cultures (Street, 1973a; Bhojwani and Razdan, 1983; George and Sherrington, 1984). White's medium is low in nutrients compared to MS medium (George et al., 1988). Later many workers adopted other media such as MS, Bg (Gamborg's) and LS (Linsamier and Skoog's) media for root cultures and they were found to sustain satisfactory root growth. In the present study also besides the concentration of ammonium nitrate, MS medium was found suitable for the growth of roots. MS medium was used for root cultures of Duboisia (Yoshimatsu et al., iggo), Cephaelis ipecacuanha (Jha et al., 1991), Nicotiana alata (Friesen et aL, 1992), Panax ginseng (Yu et al., 2002).

Werbrouck and Debergh (1994) had suggested that two separate media or conditions may be required for root initiation and root elongation as the differentiation (characterized by induction of new roots) and growth (characterized by elongation of roots) were two distinct processes. This was clearly evidenced in the present study also. To improve the performance of root cultures a two-stage culture is recommended. The first would be a differentiation phase in the presence of light and ammonia ions and the second, a growth phase in darkness in low concentrations of ammonium ions. In S. wendlandii, enough data wuld not be gathered in all the media tried so as to enable a comparison.

1 26 Pert I1 - Estsblishment of in kib-0 systems

But it could be said that the morphology of the roots was apparently determined by the concentration of ammonium ions rather than by the presence of light.

2.5.4.3 Light and dark

The d t s obtained prove convincingly that darkness had a profound promotive effect on both differentiation and elongation of roots while light was inhibitory for elongation.

In intact plants, roots grow in the soil in complete darkness. It is only natural that the roots grow well in dark in in vitro conditions also. Darkness, either at a particular stage of growth or throughout the period of culture, promoted root growth inAsparagus, Prunus, parsley and several Rosaceous plants (George and Sherrington, 1984). Most recent experiments on root culture were perfomed in dark conditions such as in rcmt cultures of Duboisia (Yoshimatsu et al., lago), Cephaelis ipecacuanha (Jha et al., 1991), Nicotiana alata (Friesen et al., 1992) and Panax ginseng (Yu et al., 2002). There were also cases when darkness inhibited root growth as in the cultures of Cephalotaxus harringtonia (Wickremesinhe and Arteca, 1993).

2.54.4 Role of ammodurn ions

Inorganic nitrogen is supplied in the medium in two forms - as nitrates and ammonium compounds. Ammonium ions are utilized directly and nitrates are reduced to ammonium by plant cells itself. Keeping this fact in mind, if ammonium ions are supplied in high amounts, the acidity of the medium increases (Martini et al., 197) and proves detrimental to roots, which are generally very sensitive to ammonium ion concentration (Street, i973b). Moreover, ammonium ions in free form are toxic to roots (George et al., 1988).

MS medium has both nitrate and ammonium as sources of nitrogen but the ammonium ion concentration in MS medium is above the average concentration used in other formulations (George et al., 1988). Earlier, MS medium as such, was feared to be unsuitable for root growth in its full strength of ammonium ions. In the present study, the strategy to selectively reduce the

Discussion 1 27

level of ammonium ions by using full, half, quarter amounts along with complete absence of ammonium ions proved successful. This effectively reduced the ammonium ion concentration while the nitrate ions supplied by potassium nitrate remained comparably high.

In the present study, the effect of reduced levels of ammonium ions was clearly discernable in dark conditions only. Another interesting feature noticed was that high amount of ammonium ions promoted differentiation of roots but inhibited growth. Low levels of ammonium promoted growth but adversely affected differentiation.

Similar effect of ammonium nitrate ratio on roots was reported in Cephalotaxus harringtonia (Wickremesinhe and Arteca, 1993) where doubling the amount of ammonium inhibited the formation of lateral roots whereas doubling the amount of nitrate increased fresh and dry weights. It is also interesting to note that complete withdrawal of ammonium ions from the medium had adverse effect on root growth of both the plants. It showed that concentrations of ammonium ions were a crucial limitiw factor for root growth.

2.5.4.5 Auxins

Exogenous auxins are normally a prerequisite for root induction but not for root elongation as they may even inhibit the process, as suggested by Werbrouck and Debergh (1994). IAA showed a similar effect on roots of S. trilobatum in dark conditions while no such effect could be discernable in the presence of light. The effect of IAA was most pronounced in dark, where it promoted root growth even in high ammonium environment. In light conditions IAA was more or less ineffective.

In the present study it was found that roots did not require exogenous supply of auxin owing to sufficient levels of endogenous auxin.

To conclude, the results from root culture prove beyond doubt that root cultures have special requirements of physical and chemical nature for optimum growth (Yu et al., 1996) and it is much more sensitive than other types of cultures.

1 28 Part /I - Establishment of in vitro systems

2.5.5 Morphogenetk response oiS. tribhztum and S. wmdlandii

On assessing the morphogenetic response regeneration of S. trilobatum took place through adventitious shoots and S. ~oendlandiiby somatic embryogenesis. When the morphogenic potential of leaf, stem and calli was evaluated in both the plants, leafwas found to be a better explant than stem or caUus, The capacity for totipotency of leaf was more apparent in trials where the other two tissues tried - stem and callus - produced only callus while the leaf alone produced shoots. In trials with most ideal combinations, all the three tissues managed to produce shoots. Somatic embryogenesis in S. wendlandii also was formed only on leaf explants while stem and callus tissues invariably produced callus. The difference in the caulogenic response from the explants of some species or genera may be due to the presence of varying concentration of endogenous hormones and their interaction with exogenously supplemented hormones (Cacho et al., 1991; Ray et a[., 1992; Patra et al., 1998; Nagaraja et a[., 2003).

The major aspect of cytokinin efficiency in adventitious shoot formation was also assessed. In S. trilobatum, the order of efficiency was 2iP>TDZ>Kn. BAP was totally ineffective in S. wendlandii; regeneration took place via somatic embryogenesis. Since it is a different pathway, it is discussed under another heading later on.

An interesting feature that came up after an overall scan of the different

trials was that the cytokinins, in general, were more successful when used alone, both in terms of the number of effective cytokinin types (2iP, TDZ and Kn) and the number of explants producing shoots (leaf, stem and callus). Out of the twelve trials where cytokinins were used alone on different tissues, shoots originated from nine. But when an auxin was added to the cytokinin in the other thirty-six trials, only eight could successfully produce shoots. When auxin is added to a cytokinin, it either complements the action of cytokinin by increasing the number of shoots or reduces it by producing a typical 'auxin- effect'. After addition of auxin, caulogenesis was observed only in leaf explants cultured on media fortified with IAA along with 2iP, TDZ or Kn. This result suggests that auxins other than IAAproved antagonistic to caulogenesis by tilting the delicate balance of exogenous and endogenous hormones in favour of callussing. The cytokinins were able to exert the morphogenetic action only when

Discussion 129

weak auxin such as IAA was present. The equilibrium was determined by the endogenous levels of hormones especially auxin in the plant system, which was variable with tissue type, plant type and the phase of plant growth (Bhojwani and Razdan, 1983; George, 1993).

Low concentration of auxin when supplied with cytokinin does help in increasing the number of shoots only in the case of the ideal combination of IGA+2iP selected for S. trilobatum. With all other cytokinins combinations, addition of auxin resulted in callus only. The typical effect of each auxin was also discernable in the qualitative difference of calli produced (see Figures 31 to

35).

The two types of morphogenetic response (organogenesis and embryogenesis) produced by the two plants under the influence of the same set of PGRs was strong evidence of the genetic control of regeneration1 embryogenesis pathways. These can be expressed during hormonal manipulation only if the heritable control was actively present in the tissue undergoing trial for regeneration. A similar view was expressed by Birbman et al. (1994) that media modification with PGRs has only an enhancing role. Thus genetic p ~ p o s i t i o n of cultured tissue becomes an unpredictable limiting factor in regenerative pathways.

The importance of genotype of the donor plant/tisue on regenerability has been widely recognized (Henry et al., 1994). Birhman et al. (1994) put forward the hypothesis of in vitro response being under the control of a heritable genetic system involving three genes while explaining the variability of in vitro response from leaf explants of S. chacoense. Later confirmatory evidence obtained during the studies on tissue culture responsiveness of S. tuberosum, also suggested a two or three gene system to be responsible for regenerability (Van Stint Jan et al., 1996). In vitro responsiveness is a heritable character and the existence of wide genetic variation for this trait is now well documented in many plant species (Cappadocia, 1990; Armstrong et al., 1992; Cowen et al.,

1992).

The genetic mechanism was evidently expressive in S. trilobatum. Contrastingly, in S. wendlandii, this genetic trait was not evidenced in the cultured tissue. In S. wendlandii a separate genetic mechanism favouring

130 Part /I - Estsblishment of in vitm systems

embryogenesis should have been operative. It is under the influence of this genetic control that most plant species regenerate either by organogenesis or embryogenesis while a few species can regenerate by both the process (Bhojwani and Razdan, 1983).

2.5.5.1 Direct and indirect caulogenesis in S. tTilobahrm

Of the three approaches for mimpropagation, two (indirect and direct shoot formation) were achieved successfully in the present study. The third one - axillary bud multiplication - was not attempted. This is probably the first

report of simultaneous direct and indirect caulogenesis in S. hh&batum in medium supplemented with IAA and r ip .

The response of S. trilobatum was a classic example of typical response of in vitro regeneration from different tissues to different PGRs. During initial investigation, a combination of auxin and cytokinin was selected on the basis of its in vitro performance. The findings are in a c c o h c e with many other reports on other species of Solunum where a concentration of low auxin with cytokinin at relatively high potency yielded shoots (Lernrnonier et al., 1989; Shahzad et al., 1999; Manjula and Nair, 2002). The combination of IAA and 2iP, though not reported in Solanum species so far, was optimized for shoot formation at 0.1

mg/l and lo mg/l respectively in Spathiphyllum cv. Svend Neilsen (Dabski and

KO*, 1997).

Repeated subculture evidently caused a decline in the number and quality of the shoots and showed increased tendency for callwing. Morphogenetic potential declines with time as the tissues are subcultured, according to George and Sherrington (1984).

In the present study shoot bud formation from callus was faster from callus requiringlower amounts of cytokinin than fromleaf tissue, but the number of shoots produced were fewer. So in S, trilobatum formation of shoot buds directly from leaf is a more efficient method.

The requirement of lower potency of cytokinin for de novo formation of buds from callus (1 mg/l) than from leaf (3 mg/l) could be due to the endogenous

Discussion 131

production of zip. It was reported that callus cells of tobacco, another Solanaceous plant, were able to synthesize 2iP (Szweykowska, 1974).

In the only earlier report of regeneration of S. trilobatum (Emmanuel et al., 2000) among the cytokinins, TDZ at 1.25 mgll was found to be the best to initiate shoot buds continuously. The study did not include zip. A comparison of the in vitro response of the plant to TDZ showed similarity in both studies. In the earlier report, the short shoots produced in TDZ had to be elongated separately in GA3 2 mg/l. In the present study shoots formed in IAA+ziP elongated normally in the induction medium itself without any need for an elongation stimulus.

The present protocol is a single step procedure and undoubtedly better than the previously reported two-step procedure. The number of shoots obtained was also the same (= 19) in both studies. Also the present protocol is superior, when the quality of shoots, known from their performance of plantlets during rooting and hardening stages was considered.

Based on the information gathered from a cross-section of representative protocols, the average concentration of IAA and 2iP for direct shoot formation were 0.8 mg/l and 4 mg/l respectively and for indirect organogenesis, 0.7 mg/l and 1 mg/l respectively (George, 1993). In the present study, the optimized concentrations of IAA and d P are quite similar to the aforesaid values. For direct regeneration, it was 0.5 mg/l and 3 mg/l respectively and for indirect organogenesis it was 0.5 mg/l and 1 mg/l respectively. The requirement of lower concentrations of cytokinin (2iP) for indirect regeneration than the concentration required for direct regeneration is in agreement with its general trend in most of the earlier studies.

2.5.5.1.1 Role of 2iP

A comparison of shoots obtained in 2iP and TDZ showed difference in the quality of shoots. 2iP was definitely superior in producing better quality shoots with normal leaves and sufficiently elongated stem, while shoots formed under the influence of TDZ, even though larger in number, were stunted with thickleaves. This particular 'stunting' effect of TDZ on adventitious shoots is well known (Huetteman and Preece, 1993). In Gardenia jasminoides also, longer shoots

1 32 Part 11 - Estebk'shment d i n vitro systems

were produced in presence of 2iP was compared to those in presence of TDZ (Al- Juboory et al., 1998).

2iP is considered a weaker cytokinin than BAP (Werbrouck and Debergh, 1994). The reason for a result contrary to the established fact is not known. It can only be attributed either to the chemical nature of 2iP in comparison with BAP, which makes it preferable to S. tilobatum or genetic predisposition of the plant.

Regeneration took place successfully in S. commersonii (Iapichino et al., 1991) and S. melongena (Billings et al., 1997) in presence of 2iP. It was more effective than BAP or kinetin for Rhododendron, blueberry and garlic (Bhojwani and Razdan, 1983).

2.5.5.1.2 Role of IAA

IAA was very crucial to direct regeneration in S. trilobatum. Exactly similar tindings of IAAplaying a key role in regeneration were reported in S. commersonii (Iapichino et al., 1991). More shoots werc: produced from leaf and stem explants by the synergistic effect of IAA with either 2iP and zeatin.

In S. sarrachoides, however, evidence to the contrary was found as IAA when combined with BAP reduced the number of shoots by 50 percent. However, for the second stage of elongation and growth of these shoots IAA was found necessary (Banerjee et al., 1985).

IAA was more suitable for direct shoot initiation than 2,4-D. 2,4-D is not recommended for regeneration because of its strong callusing tendency (Bhojwani and Razdan, 1983). Addition of IAA promoted adventitious shoots than when cytokinin was supplied alone in Coffea bengalensis (Mishra and Sreenath, 2003). In Withania somnipera, IAA in combination with BAP produced multiple shoots directly from leaf explants (Govindaraju et al., 2003).

2.5.5.1.3 Vitrescence and albinism

Vitrescence is commonly encountered in micropropagation work (George and

Discussion 1 33

Sherrington, 1984). Two reasons have been pointed out as the possibilities for the appearance of 'glass-like' or 'watery' shoots in culture. Earlier, vitrescence was believed to be due to high osmotic (water) potential of MS medium (George and Sherrington, 1984). Later evidences pointed to the occurrence of high ammonium ion concentrations as the major cause for vitrescence. The phenomenon occurs due to high amino acid production and subsequent failure of lignin biosynthesis (George et al., 1988).

Albino plants were frequently observed in cereal and grass tissue cultures but were sometimes produced also in broad-leafed species. The reason for albinism appearing occasionally in S. trilobatum cultures is unknown. It could be considered a random phenomenon as the appearance of albino plants was sporadic and only a few shoots in a culture vessel turned white while others continued normal growth.

As this abnormal phenomenon did not affect the overall productive nature of cultures and the end result, it could be safely ignored as random occurrences.

2.5.5.1.4 Rooting

A low salt medium is adequate for root initiation from shoots in a large number of plant species (Bhojwani and Razdan, 1983). In agreement with these findings, shoots of S. trilobatum also required only quarter-strength MS medium for producing maximum shoots. In an earlier study on the same species, contrary to the present findings, reduced salt concentration of half MS was found better than quarter-strength MS medium or full-strength MS medium (Emmanuel et al., 2000). Reduced salt concentration has favoured other species of Solanum also, such as S. commersonii (Iapichino et al., 1985). The presence of low salt concentration was found to promote rooting in Withania somnifera (Govindaraju et al., 2003) and Coffea bengalensis (Mishra and Sreenath, 2003).

Generally, for most dicotyledonous species, either NAA or IBA were required for rooting (Bhojwani and Razdan, 1983). For S. trilobatum medium without any growth regulators was found most stimulatory. IBA was inferior and NAA was totally unsuitable. Similarly in many Solanum species, hormone-

1 34 Part 11 - Establishment of in vitm systems

free medium was reported to be ideal in producing maximum roots (George et

al., 1987).

2.5.5.1.5 Hardening

Efficient rooting of in vitro regenerated plants and subsequent field establishment constituted the last and cn~cial stage of rapid clonal propagation. For S. trilobatum, this phase was a resounding success. No plantlets were lost either during hardening or transplantation to soil. In an earlier study in S. trilobatum by Ernmanuel et al., (2000) only 50-60 percent plants survived during hardening and 70-%O percent on transplantation to the field. This proved the better quality of the shoots obtained in the protocol developed for S. hilobaturn in the present study.

2.5.5.2 Somatic embryoge~~~is in S. w e n d l d i

A three-stage development of somatic embryogenesis was observed in S. wendlandii consisting of initiation, maturation and germination. Of the three stages, the last one was not successful as recovery of plantlets from somatic embryos could not be achieved. However, curredy there are no published reports of somatic embry~neeis in S. wendI&, that too under the in&lemn of a -potent eytokidn web as TDZ. In many plants, initiation and maturation occurs in two or more steps and involves specific media designs (Eapen and George, 1993; Binzel et al., 1996).

2.5.5.2.1 Role of TDZ and 2,4-D in emblyogenesis

The use of TDZ and 2,4-D for embryo initiation brought to light certain unique aspects of interest. The data obtained in the initial screening for morphogenesis clearly suggest that in S. wendlandii the stimulus for somatic embryogenesis was provided exclusively by TDZ and 2,4-D had only a secondary role. The fact that somatic embryogenesis occurred only in presence of TDZ irrespective of the absence or presence of auxin or the type of auxin if present, is yet another proof of the dramatic embryo-inducer potential of TDZ. The role of TDZ in the

Discussion 1 35

present study appears to be that of a trigger for embryogenically competent cells to follow the developmental path of somatic embryogenesis.

TDZ is the most potent of all adenine type of cytokinins (Mok et al., 1982; Huetteman and Preece, 1993). It is an effective synthetic growth regulator for the induction of somatic embryogenesis in a number of crops ranging from herbaceous species to woody perennials, used either alone (Gill and Saxena, 1992; Visser et al., 1992; Murthy and Saxena, 1998 and Chen et al., 1999) or in combination with an auxin, usually 2,4-D (Huetteman and Preece, 1993; Zhou et al., 1994; Binzel et al., 1996). The present findings are in full accordance with the dramatic embryogenic potential of TDZ, where most conventional cytokinins failed.

TDZ is employed for multiple shoot proliferation at very low concentrations (0.08 - 0.4 mg/l) (Huetteman and Preece, 1993; Wilhelm, 1999). For adventitious shoot formation and embryogenesis, 0.05 - 2 mg/l is usually used (Huetteman and Preece, 1993; Werbrouck and Debergh, 1994). In the present study also, the concentrations used (0.05 - 3 mg/l) and the optimum concentration of 1 mg/l were in agreement with the existing knowledge about the active range of concentration for TDZ.

According to earlier reports, TDZ was most effective for trees and woody climbers (Huetteman and Preece, 1993). In the present study also, S. wendlandii, which is also a woody climber, responded to TDZ treatment, substantiating the earlier observations.

The combination of 2,4-D and TDZ has been the most effective one in many plants. In fad, TDZ has been most successful with 2,4-D than any other auxin. In Cayratia japonica, the best results for somatic embryogenesis from callus were obtained on medium containing 0.5 - 1 mg/l2,4-D and TDZ (Zhou et al., 1994). The concentrations required for somatic embryogenesis in Capsicum annum was 1 - 4 mg/l2,4-D and 2 mg/l of TDZ (Binzel et al., 1996). The same combination of 2,4-D and TDZ produced somatic embryos in Cymbidium ensifolium (Chang and Chpng, 1998). However, in contrast, addition of 2,4-D or NAA was found to decrease the frequency of somatic embryogenesis in Azadirachta (Murthy and Saxena, 1998). In general, the optimum

1 36 Part / I - Establishment of m vitro systems

concentrations of 2,4-D and TDZ used in the present study are in agreement with the already published reports.

The presence of 2,4-D in the medium definitely enhanced the number of TDZ-induced embryos. The data suggest the role of 2,4-D as being complementary to TDZ. TDZ seems to he most successful with 2,4-D when an auxin is required to supplement its ernbryogenic action. The two growth regulators seem to interact synergistically at a deeper level than most auxin:cytdbin interactions to initiate the cascade of events that is called somatic emb-. In the present study, however, 2,4-D was found to be not a prerequisite for embryogenesis as somatic embryos formed even whenTDZ alone was supplied.

Thew b a separate category of embryogenic response where 2,4-D plays the active role as an inducer. For such a response 2,4-D is an absolute necessity, that too at high concentrations (Rao et czl., 1985; Yeh and Chang, 1987; Binzel et al., 1996).

The present results in terms of concentration agree with the finding of Tripathi and Tiwari (20031, who reported the requirement of only a low auxin concentration in Glycine max for somatic embryogenesis. But the results of a later study contradict it as a high auxin requirement for enhanced embryogenesis in the same phnt reported in it (Yeh, 1989).

In agraanent with the generally accepted view of auxin playing a very important role in somatic embryogenesis (Evans et al., 1981; Ravindra and Nataraja, 2003). 2,443 was found to play a very important role in somatic embryogenesis ot S. ursnhndii in the present study also.

The cytokinin TDZ on account of its strong biological activity exerted a strong morpho-regulatory role on the somatic embryos of S. wendlandii, if the post-induction dwdopment of embryoids was considered. It is to be believed that TDZ deeply idluences (much more than the conventional cytokinins) on the metabolism and cellular events and morphological characters of somatic embryos. Evidence of such a strong influence of TDZ has been observed and reported by many researchers.

Discussion 137

Very soon after initiation the somatic embryos of S. wendlandii began to exhibit abnormalities in the form of fused embryos, embryos with multiple shoot poles, enlarged and tuber-like root pole, etc. The longer the embryos were allowed to continue in the induction medium comprising 2,4-D and TDZ, the higher was the frequency of oanuTence of such abnormalities.

In the light of the information available on the action of TDZ on other plants, it can be safely implicated for inducing such abnormalities. The negative role of TDZ in proper elongation of adventitious shoots is already known (Huetteman and Preece, 1993).

According to Zhou et al. (1994), strong morpho-regulatory activity of TDZ was observed in somatic embryos of Cayratia japonica so much so that the embryos never converted to whole plants regardless of the subsequent media. Occurrence of morphologically abnormal (Ranch et al., 1986) distorted and fasciated embryo6 (Binzel et al., 1996) reported among TDZinduced embryos confirms the suspicion of a negative role of TDZ in the post-inductive phase.

Prdonged exposure to 24-D also was pointed out as a reason for improper development of somatic embryos in carrot and soybean (Merkle et al., 1995). 2,q-D had to be removed from the medium for proper development in carrot embryos (Zimmerman, 1993). Exogenous auxin was believed to disrupt normal auxin transport (Koh and Loh, 2000) and ionic currents, which control polarity (Brawley et al., 1984). Continued presence of 2,q-D was also reported to inhibit the synthesis of embryo-specific proteins (Sung and Okimoto, 1981).

The formation of abnormal embryos might be due to overexposure to both TDZ and 2,q-D. The best possible solution to overcome this problem would be to induce somatic embryogenesis in the lowest effective TDZ concentration. The embryogenic cells should be exposed to TDZ only for the least minimum time to prevent 'carry over effect' of the cytokinin on cultures (Huetteman and Preece, 1993). Avague but critical distinction between competence and induction of somatic embryos has been recognized (Christiansen and Warnick, 1985). The present data are inadequate to characterize the competence phase and distinguish it from the induction phase in terms of time required for the

1 38 Part 11 - EsteMishment of in wtm systems

completion of each. It seems likely that TDZ and possibly 2,4-D, may be required only during the competence phase and may turn antagonistic to embryo development if continued into induction and differentiation phases.

2.5.5.2.2 Maturation and germination of somatic embryos

The results of experiments for maturation and germination of TDZ+2,4-D- induced embryos should be viewed in the light of the dubious role played by both the auxin and the cytokinin in embryo development of S. wendlandii. Removal of both the growth regulators from the medium facilitated proper maturation of somatic embryos but the development was not satisfactory, as the root pole remained quiescent.

In S. wendlandii, wmplete plantlets were not formed as the result of somatic embryogenesis owing to the absence of a functional root pole or normal vasculature. It appears that TDZ hwersiblyblocks somatic embryo development soon after induction at a metabolic level. TDZ was reported to increase the levels of endogenous cytokinin by, at least partly, inhibiting the action of cytokinin oxidase (Hare and van Staden, 1994). It was therefore possible that inability to form a functional root pole or its inhibition was because of the elevated levels of endogenous cytokinins (Magioli et al., 1998). TDZ most probably disrupts the internal gradient of hormones, which is responsible for initiation and maintenance of polarized growth and subsequent embryo development. In Cassava, absence of active root pole or mot meristem in 2,4-D induced somatic embryos has been reported (Sofiari et a1 ,1997).

The normal, if not successful, development of somatic embryos in complete absence of PGR is also an indication of the heightened endogenous levels of cytokinin in them. Acwrding to Huetteman and Preece (1993) the problem caused by TDZ wuld be overcome by transfer to a second medium devoid of TDZ.

Both ABA and GA3 are known to promote normal embryo development, maturation and germination of somatic embryos of a widevariety of plant species (Chang and Hsing, 1980; Emons et al., i!)93; Dong et al., 1997; Bela et al., 1999; Ignacimuthu et al., 1999). The observations in the present study of the failure of

Discussion 139

ABA and G& on maturation can point to the 'residual effect' that TDZ might have produced on them. During germination trials, G& was able to exert a positive effect on the embryos. This is in agreement with the positive results obtained in Eryngium foetidum at 1 mg/l G& which was effective for conversion of 70 percent of embryos to plantlets (Ignacimuthu et al., 1999). Incidentally it was the same concentration that was used in the present study.

It is to be noted that, during maturation trials, ABA and G& were acting on somatic embryoids that were still under the effect of TDZ. After maturation in the absence of 2,4-D and TDZ the lingering effect of TDZ would have been much reduced and the embryos were found responding to GA3 treatment. The role of GA3 in promoting normal embryo development has been only rather rare (Chang and Hsing, 1980; Bhojwani and Razdan, 1983; Ignacimuthu et al., 1999). It is known more as an inhibitor of somatic embryogenesis (Fujimura and Komamine, 1975; Nomaet a[., 1982; George and Shenington, 1984; Hutchinson et al., 1997).

On the other hand, ABA - a well known promoter of maturation of somatic embryos - inhibited it in S. wendlandii. Most of the earlier reports recommend the use of ABA for differentiation and further germination of somatic embryos (Becwar et al., 1989; Roberts et al., 1990; Bama and Wakhlu 1993; Suhasini et al., 1994; Sumathi et al., 2003). However, ABA has been found to inhibit somatic embryogenesis in Medicago sativa (Kepczynski et al., 1997).

140 Pati I1 - EsteMishment of in vitro systems -

Illustrations 141

3 0 40 Days

Figure 70. Growth pattern of callus from leaf explants under the influence of auxins in S , frilobafum

Illustrations 143

3 0 4 0 Days

Figure I-I. Growth pattern of callus from leaf explants under the influence of auxins in S. wendlandii

Illustrations 145

2 3 Concentration of auxin (mgll)

Figure 12. Effect of auxins on callogenesis from leaf explants of S. ttilobatum

2 3 Concentration of auxin (mgll)

Figure 13. Effect of auxins on callogenesis from leaf explants of S. wendlandii

Illustrations 147

l4 Time ID) 2 1

. . EZEl Fresh wt 6 Dry wl t --

Figure 74. Growth of cells in S.trilobatum suspension cultures

l4 Time (Dl 2 1

. . - ESl Fresh wt. +- Dry wt. 1

Figure 15. Growth of cells in S. wendlandii suspension cultures

Illustrations

Figure 16. Effect of darkflight and agitation on differentiation of new roots in different media in S. trilobatum ,

p+ ~ ~ 4 ~ 0 3 m0.5xMS + NH4W-3 OMS+ 0 . 5 ~ NH4N03 OMS + 0.25~ NH4lW HMS- MN03 MIFA 2m@l j

Figure 17. Effect of darkllight and agitation on growth of new roots in different media in S. wendlandii

Illustrations 151

Fbwe 78. Callus growth from leaf of S. fn'lobatum in medium supplemented with 1 mgll 2,4-D. The explant a) after inoculation; b) callus induction after 8 days; c) 5 days; d) 30 days; e) 45 days and 9 80 days.

illustrations 153

Figure 79. Callus growth from leaf of S. wndiandii in medium supplemented with 0.5 m/l 2,443. The a) explant after inoculation; b) callus indudion after 4 days; c)15 days; d) 30 days; e) 45 days and f) 60 days.

Illustrations 155

Figure 20. CaHus growth from leaf of S. tri lobafm in medium suppbmnted with 4 mgA NAA. The explant a) after inoculation; b)$5 days, c) 30 days (d) 45 days, (0)

after 60 days and (f) rhizogenesis.

Figwe 2l.Callus growth from leaf of S. wendlandii in medium supplemented with 0.5 m@ NAA. The a) explant after inoculation; b) callus induction afler 5 days;^) 15 days; d) 30 days; e) 45 days and f) 60 days.

Figure 22. Callus growth h m leaf of S, tn'lobatum in mdlum supplemented WM 4 IT@ IAA. The a) explant after inoculation; b) Mer 15 days; c) 30 days, d) 45 days; e) 60 days and f) rhizwenesls in 0.5 mg/l tAA.

illustrations 161

Figure 23. Callus growth from leaf of S. wnd&nbli in medium supplamntetd with 4 mgR IAA. The explant after a) 5 days; b) 15 days with white roots (at armw);~) 30 days; d) 45 days: e) 60 days and f ) green nodular calli at the region in contact wWI the medium.

Hlustrations 163

Figure 24 . Establishment of suspension cultures in S. mobaturn. a) Callus type I-habituated callus; b) callus type Il-newly induced callus; c) cell suspension cultures with callus cell aggregates (CCA) and (d) fine suspension for growth studies. ,

illustrations 165

Figure 25. Callus cell aggregates (CCA) of S. trilobatum. a) close-up view x 6.3; b) external morphology; c)T.S. of CCA x 30; d) T.S. of CCA-cellular view x SO(OC-outer cortex,lC-inner cortex,V-vacuole); e) inner isodiametric cells and marginal elongated cells x I00 and f) dense meristematic zones (at amw) x 50.

Illustrations 167

Figure 26 . Cells of S. Mobaturn in suspension culture. x 100 a) single cell b) cell aggregate c) elongated cells; d) twisted cells and cells in loops.

Illustrations 169

Figure 27. Cells of S. wendlandii in suspension cutlure x 200 a) suspension culture; b) single round cell; c) elongated cells; d and e) cell cluster and f) dividing cells.

illustrations 171

Figure 28. (a lo d) Rhizogenesis from leaf in S. trilobatum and S. wendlandii . In S. trilobatum induction of only roots in a) 0.5 mgll NAAIdark and b) along with callogenesis in 0.5 mgll NAA/light. Well developed roots of c) S. trilobatum in 0.5 mgll NAAIdark (RIMT) and d) S. wendlandii in 0 .5 mgll IAA (RIMW). Growth of excised roots of e) S. trilobafum and 9 S. wendlandii under the influence of 2 mgll IAA.

full NH4N03 reduced level of NHANO~

Figure 29. Morphology of S. trilobafum roots grown in lightldark and agitation affer 30 days. a and b) darwstatic cultures ; c and d) lighffstatic cultures; e and f) light/ agitated cultures.

Illustrations 175

- ---- full NH4N03

I reduced level of NH4NOs

=igure 30. Morphology of S. trilobafirm roots grown in lighffdark and agitation after 10 days. a and b) dark/static cultures ; c and d) IighUstatic wliures; e and 9 light1 igitated cultures.

Figure 31.Effect of individual cytokinins (1 mgll) on leaf, stem, and one-month old calli of S. trilobatum on medium supplemented with (a, b and c) Kn; (d, e and f) BAP; (g, h and i ) 2iP and (j, k and I) TDZ.

illustrations 179

Figure 32. Effect of three auxins (0.5 rngll) and Kn (1 mgll) on leaf, stem and one- month old callus of S. trilobatum. (a, b and c) on medium supplemented with NAA + Kn; (d,e and f) 2,4-D + Kn and(g, h and i) IAA + Kn. .

Figure 33. Effect of three auxins (0.5 rngtl) and BAP (Imgll) on leaf, stem and one- month old callus of S, trilobafum. (a, b and c) on medium supplemented with NAA + BAP ( d , e and f) 2,4-D+BAP and (g, h and i) IAA + BAP.

Illustrations 183

Figure 34. Effect of three auxins (0.5 mg/l) and 2iP (1 mgll) on leaf,stem and one- month old callus of S. trilobatum. (a, b and c) on medium supplemented with NAA + 2iP; (d, e and f) 2,4-D + 2iP and (g, h and i) IAA + 2iP:

ltlustrations 185

Figure 35. Effect of three auxins (0.5 mgll) and TDZ (1 mg/l) on leaf, stem and one- month old callus of S. trilobatum. (a, b and c) on medium supplemented with NAA +

TDZ; (d, e and f) 2,4-D + TDZ and (g , h and i) IAA + TDL.

Figure 36. Direct caulogenesis in S. Mobaturn, a)) shoot primordia x 45; b) development of shoot apex x 42; c) welldeveloped shoot; d) induction directly from explant after 12 days; e) shoats arising 7 days after induction; f) thick cluster of developing shoots and g)welldeveloped shoots after 45 days in medium supplemented with0.5 mg/l IAA + 3 mgIl2iP.

Illustrations 189

Figure 37. Multiplication and subculture of adventitious shoots in S. trilobatum. a) adventitious shoots in optimized media; b) sub cultured shoots; c) growth and elongation of shoots; d) production of new shoots; e) second subculture and 9 reduced number of shoots in second subculture.

lllustralions 191

Figure 38.Abnormal growth during proliferation of adventitious shoots of S.trilobatum. a) basal callus afler subcutlure; b) dark, senescent basal callus; c) green chlorophyllous callus from shoots; d) non-chlorophyllous shoots; e)vitrified shoots and f) roots from basal callus.

Figure 39. Indirect caulogenesis in S.trilobatum. a) induction of shoot bud (at arrow) b) formation of primary leaves; c) after 15 days in culture; d) expansion of leaf; e) shoot formation in optimized media (0.5 mg/l IAA + 0.5 mg/l 2iP) and f) growth and elongation of shoots.

Illustrations -

Figure 40.Development of adventitious shoots from callus in S.tri/obatum (a and b) 5 days afler induction; (c and d) development of well-developed shoot 15 days afler induction; (e and f) elongation of shoots from callus (S-shoot, C-callus).

illustrations 197

Figure 41. Rooting from adventitious shoots of S. Mobaturn. a) nwts growing ~TWI

base of the shoot eight days after inoculation; b) dose-up of young rods with root hairs; c) in 0.25 x MS basal medium after5 days and d) full MS basal medium after 15 days.

illustrations 199

Figure 42. Rooting from adventitious shoots of S. trilobatum in media supplemented with IBA and NAA.

(a) 0 25 MS + 0.5 mgll IBA (b) 0.25 MS + 2 0 mgll IBA (c) 0.25 MS + 0.5 mgll NAA (d) 0.25 MS + 2.0 mgA NAA

(e) MS + 0.5 mgA IBA (f) MS + 2.0 mgA IBA (g) MS + 0.5 mgA NAA (h) MS + 2.0 mgll NAA

Illustrations 203

Figure 44. Plant regeneration in S. trilobatum

illustrations 207

Figure 46. Effect of three auxins (0.5 m g A ) and Kn (1 mg4) on leaf, stern and one-manth old callus of S. wendlandii. (a, b and c) on medium supplemented with NAA + Kn (d, e and f) 2,4D +

Knand(g, handi) IAA+Kn.

illustrations 209

Figure 47.Effed of three auxins (0.5 mgll) and BAP (1 mgll) on leaf, stem and one-month old callus of S, Wendlandii. (a, b and c) on medium supplemented with N U + BAP (d, e and f) 2,4-D + BAP and (g, h and i) IAA + BAP.

Illustrations 21 1

Figure 48.Effect of three auxins (0.5 mgll) and 2iP (1 mgA) on leaf, stem and one-month old callus of S, wendlandii. (a, b and c) on medium supplemented with NAA + 2iP (d, e and f ) 2,4-D + 2iP and (g, h and i) IAA + 2iP.

Illustrations 21 3

Figure 49. Effect of three auxins (0.5 rngll) and TDZ (1 mg/l) on leaf, stem and one-month old callus of S. wendlandii. (a, b and c) on medium supplemented with NAA+ TDZ (d, e and f) 2,4-D + TDZ and (g, h and i) IAA + TDZ.

Illustrations 21 5

Figure 50. Direct somatic embryogenesis in S. wendlandii. (a) 'burnt' appearance of explant; b) induction after 15 days; c) whole explant covered with embryoids (d and e) close-up of embryoids and f) single embryoid with basal connection.

Figure 51. Stages of somatic embryogenesis in S. wendlandii. a) series of embryo development stages; b) globular embryo x 42; c) elongating globular embryo x 30;d) hearl-shaped embryo x 36 and e) torpedo-shaped embryo x 42.

Illustrations 21 9

Figure 52 . L.S. of different stages of embryogenesis in S. wendlandii. a) T.S. of explant with globular embryo x 25 b) globular embryo c) elongating globular embryo;d) torpedo-shaped embryo; e)heari-shaped embryo with apical notch; f) walking-stick shaped embryo; f) torpedo-shaped embryo and g) cotyledonary-stage embryo (all images x 35).

Illustrations 22 1

Figure 53. Aberrant embryo development in early stages of embryogenesis in S. wendlandii - comparison of external appearance and internal structure. ( a and b) initiation of cleavage in budding embryo (c and d) developing bud (e and f) budding during elongated stage of the embryo.

illustrations 223

Figure 54. Maturation trials of embryoids of S. wendlandii. a) callusing in 1 mgA Kn+ 1 mg/l BAP+1 mg/l 2iP; b) callusing in I mgll Kn+l mgll BAP+10 mg/l adenine sulphate; hard green callus in; c) 0.1 mg/l GA3; d) in 0.1 mgll ABA; e) maturation of ern- bryoids in MS medium and f) callusing in MS medium.

Illustrations 225

Figure 55. Tuber-like formation of root pole of somatic embryos in S. wendlandii. a) enlargement of root pole x 25; b) multiple shoot primordia sites (at arrow) x 25; c) development of leaves (d, e and 9 advanced stages of tuberous structures on subculture in SEIMW.

Illustrations 227

Figure 56. Germination trials of embryoids of S, wendlandii. a) mature somatic embryo in germination media; b) nodules in MS liquid medium; c) in 0.1 mgll GA3 1 solid; d) in 0.1 mg/l ABAIsolid; e) in 0.1 mgll GA3/liquid, f) 0.1 mg/l ABAJliquid.

ollshment of in vitro syshodhganga.inflibnet.ac.in/bitstream/10603/337/10/10_chapter 2.pdf ·...

Documents