Introduction. Plant growth regulators are the critical media components in determining the developmental pathway of the plant cells.

Transcription

1 Introduction Callus is defined as an unorganized tissue mass growing on solid substrate. Callus forms naturally on plants in response to wounding, infestations, or at graft unions (Bottino, 1981). Callus formation is central to many investigative and applied tissue culture procedures. Callus can be multiplied and later used to clone numerous whole plants. Since extensive callus formation can be induced by elevated hormone levels, tissue culture media designed to produce callus contain pharmacological additions of cytokinins and auxins. During callus formation, there is some degree of dedifferentiation (i.e. the changes that occur during development and specialization are, to some extent, reversed), both in morphology (a callus is usually composed of unspecialized parenchyma cells) and metabolism. One major consequence of this dedifferentiation is that most plant cultures lose the ability to photosynthesize. This has important consequences for the culture of callus tissue, as the metabolic profile will probably not match that of the donor plant. This necessitates the addition of other components such as vitamins and, most importantly, a carbon source to the culture medium, in addition to the usual mineral nutrients. Callus culture is often performed in the dark (the lack of photosynthetic capability being no drawback) as light can encourage differentiation of the callus. During long term culture, the culture may lose the requirement for auxin and/or cytokinin. This process, known as habituation, is common in callus cultures from some plant species (such as sugar beet).callus cultures are extremely important in plant biotechnology. Plant growth regulators are the critical media components in determining the developmental pathway of the plant cells. The plant growth 53

2 regulators used most commonly are plant hormones or their synthetic analogues. There are five main classes of plant growth regulator used in plant cell culture, Namely:(1) auxins; (2) cytokinins; (3) gibberellins; (4) abscisic acid; (5) ethylene. Auxins promote both cell division and cell growth. The most important naturally occurring auxin is indole-3-acetic acid (IAA), but its use in plant cell culture media is limited because it is unstable to both heat and light. Occasionally, amino acid conjugates of IAA (such as indole-acetyl-lalanine and indole-acetyl-l-glycine), which are more stable, are used to partially alleviate the problems associated with the use of IAA. It is more common, though, to use stable chemical analogues of IAA as a source of auxin in plant cell culture media. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and is extremely effective in most circumstances. Other auxins are available (see Table 2.3), and some may be more effective or potent than 2, 4-D in some instances. Cytokinins promote cell division. Naturally occurring cytokinins are a large group of structurally related purine derivatives. Of the naturally occurring cytokinins, two have some use in plant tissue culture media zeatin and42 Plant tissue culture. Auxins and cytokinins are the most important hormones for transgenic work. Commonly used auxins, their abbreviations and chemical names Abbreviation/name Chemical name 2, 4-D 2, 4-Dichloro phenoxy acetic acid 2, 4, 5-T 2, 4, 5-Trichloro phenoxy acetic acid Dicamba 2-Methoxy-3, 6-dichlorobenzoic acid IAA Indole-3-acetic acid IBA Indole-3-butyric acid MCPA 2-Methyl-4-chloro phenoxy acetic acid NAA 1-Naphthyl acetic acid NOA 2-Naphthyloxy acetic acid 54

3 There are two kinds of cytokinenins one is the naturally occurring cytokinenis which includes zeatin, 6-ϒ, ϒ-dimethyl allyl amino purine (2iP) and adenine. There is another type which is synthetic cytokinines. This group consists of substituted purines i.e. 6-benzyl amino purine (BAP) and 6- furfural amino purine (kinetin) and phenyl ureas such as thiodiazuron. The synthetic analogues kinetin and 6-benzylaminopurine (BAP) are therefore used more frequently. Non-purine-based chemicals, such as substituted phenylureas, are also used as cytokinins in plant cell culture media. These substituted phenylureas can also substitute for auxin in some culture systems Picloram 4- Amino-3, 5, 6-trichloro- 2 pyridine carboxylic acid Their use is not widespread as they are expensive (particularly zeatin) and relatively unstable. There are numerous, naturally occurring, structurally related compounds termed gibberellins. They are involved in regulating cell elongation, and are agronomically important in determining plant height and fruit-set. Only a few of the gibberellins are used in plant tissue culture media, GA3 being the most common. Abscisic acid (ABA) inhibits cell division. It is most commonly used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis. Ethylene is a gaseous, naturally occurring, plant growth regulator most commonly associated with controlling fruit ripening in climacteric fruits, and its use in plant tissue culture is not widespread. It does, though, present a particular problem for plant tissue culture. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture. The type of culture vessel used and its means of closure affect the gaseous exchange between the culture vessel and the outside atmosphere and thus the levels of ethylene present in the culture. Explants, when cultured on the appropriate medium, usually with both an auxin and a cytokinines, can give rise to an unorganized, growing, and dividing mass of cells. It is thought that any plant tissue can be used as an explant, if the correct conditions are found. In culture, this proliferation can be 55

4 maintained more or less indefinitely, provided that the callus is subculture on to fresh medium periodically. Manipulation of the auxin to cytokinin ratio in the medium can lead to the development of shoots, roots, or somatic embryos from which whole plants can subsequently be produced. Callus cultures can also be used to initiate cell suspensions, which are used in a variety of ways in plant transformation studies. Additionally, various genetic engineering protocols employ callus initiation procedures after DNA has been inserted into cells; transgenic plants are then regenerated from transformed callus. In other protocols callus is generated for use in biotechnological procedures such as the formation of suspension cultures from which valuable plant products can be harvested. Explant tissues generally show distinct planes of cell division, various specializations of cells, and organization in to specialized structures such as the vascular system. Callus formation from explant tissue involved the development of progressively more random planes of cell division, less frequent specialization of cells, and loss of organized structures. (Wagley et al.1987). When sub-cultured regularly on agar media, callus culture will exhibit an S-shaped or sigmoid pattern of growth. Hence it is importance to study the callogenesis with different growth regulators like auxins and cytikinins in different mulberry varieties. RESULTS Time Taken for callus initiation (days) from the auxiliary buds at different concentrations of 2,4 D were significantly variable. In M 5 variety (Table 4.1 and Fig 4.1) time taken for callus initiation significantly decreased with the concentrations from 0.5 to 2.0. The order of decrease at different concentrations was 0.5 mg -l 2, 4 D > 1.0 mg - 2, 4 D, > 1.5 mg -l 2, 4 D > 2.0 mg -l. A similar trend was also observed in V 1 (Table 4.2 and Figure 4.2), S 36 (Table 4.3 and Figure 4.3) and Anantha (Table 4.4 and Figure 4.4). 56

5 From the data presented table 4.5 and figure 4.5, it is observed that the percent frequency of callus initiation after 20 days at different concentrations of 2, 4-D (MS medium) in M 5. Percent frequency of callus initiation (days) from the auxiliary buds at different concentrations of 2,4 D were significantly increased with increased concentrations (0.5mg -l to 2.0 mg -l ). The order of increase Percent frequency of callus initiation (days) at different concentrations was 0.5 mg -l 2, 4 D < 1.0 mg - 2, 4 D, < 1.5 mg -l 2, 4 D < 2.0 mg -l. A similar trend was also observed in V 1 (Table 4.6 and Figure 4.6), S 36 (Table 4.7 and Figure 4.7) and Anantha (Table 4.8 and Figure 4.8). DISCUSSION In the present investigation auxiliary buds were used as the source material. Shoot culture (shoot tips or buds) is often sufficiently reliable with herbaceous plants to be used commercially (Holdgate, 1977). Oka and Ohyama (1975) performed material for invitro propagation of mulberry. They used three kinds of explants and various aged auxiliary buds for their study. Young greenish buds with long or short stem grew into leafy shoots on MS + NAA or MS + NAA + BA medium respectively. Excised greenish buds did not grow in var. Ichinose, but they grow in var. Kenmochi when supplemented with BAP. Excised brownish buds that were more aged than greenish buds did not show any growth in both varieties. The physiological status of the explant has been of prime importance with adult trees. Extensive studies at the Association Forest - Cellulose (AFOCEL) research institute in France indicated that explants from adult forest trees were frequently slow to commence growth invitro, or failed completely unless selected from rejuvenated tissues. When natural rejuvenation is absent, it has been induced by various treatments such as grafting, shoot pruning, maintenance of high fertilizer levels, vegetative propagation or spraying with cytokinines (Franclet, 1979). Hence Two months after pruning, young 57

6 rejuvenated twigs up to 5th node were collected in the present study. The response of explants varied depending upon the variety. The choice of a particular medium depends mainly on the species of the plant, the tissue or organ to be cultured. In the present investigation, MS media was tested for callus initiation in M 5, V 1, S 36 and Anantha, days required for callus initiation and percent frequency of callus in M 5, V 1, S 36 and Anantha differed considerably in different varieties of Morus Spp. M 5, V 1, S 36 and Anantha (Plate 1to 4). All mulberry variety responded well on MS medium fortified with 2mg/l 2, 4-D. This may be due to high nitrate requirement of M 5, V1, S 36 and Anantha and nitrate is relatively high in. The present study clearly demonstrates that there was 100% response in M5 and V1.Callus was yellow and smooth in M 5, V 1, S 36 and Anantha. During subsequent cultures callus ceased to grow and appeared brown in colour due to accumulation of phenolic compounds. This was reported by Tewary et al., (1989) in mulberry and also in other angiosperm taxa by Shah and Mehta (1976) and Singh et al., (1982). By using 0.1% activated charcoal and this was prevented in the present investigation. Activated charcoal adsorbs many organic and inorganic molecules from the culture medium (James, 1971) By using 10M 2, 4-D Oka and Ohyama (1973) obtained two types of calli from stem segments of Mulberry varieties. As for the effect of auxins better callus formation was observed on medium with 2, 4-D. Oka and Ohyama (1976) reported that the addition of 2,4-D remarkably enhanced the callus formation at the lower part of the explants, but the shoots developed rarely. The effect of different concentrations of 2,4-D (0.5, 1, 1.5, 2mg/l) on sprouting, rooting and callus proliferation was studied in the present investigation. High concentration of 2, 4-D (2mg/l) showed remarkable suppression of both sprouting and rooting. Gamborg et al. (1976) stated that 2, 4-D is a powerful suppressant of organogenesis and it would not be used in experiments involving root and shoot initiation. 58

7 At 2 mg/l there was no sprouting at all and it was represented in Tables 4.1 to 4.8 and figures 4.1 to 4.8. Sekih et al., (1974) reported highest growth rate of callus obtained with 2, 4-D followed by IAA and NAA in the light condition. In our present study highest callus initiation was observed at 2mg/l 2, 4-D concentration. The explants cultured on medium without any hormones, sprouted very little and did not show any further growth or callus proliferation. Patel et al., (1983) cultured stem, petiole segments, leaf discs on MS medium alone which has not showed any response, but when supplemented with IAA, IBA, NAA, 2, 4-D at 0.5, 1, 2 mg -l, they proliferated into callus tissue within 4 weeks. Callus tissue was green and friable on 2, 4-D medium, whereas on NAA and IAA medium brownish, compact callus was reported. Though the nature of callus in our present study was smooth and yellow in M5, V1, S36, Anantha, nodular and yellow colour was observed in M5 in the beginning, it has become compact and greenish patches appeared when sub-cultured on to the medium fortified with kinetin and IAA. Tewary et al., (1989) reported that cultured leaf explants of mulberry on BAP and Kinetin does not provided any result. However, they found that BAP (2 mg/l) + 2,4-D (0.5mg/1) was most suitable for callus proliferation and maintenance of callus for long term culture. Many investigators amply stressed the importance of 2, 4-D on both callus initiation and proliferation. There is every need to provide considerable interest on the type of media which also proved the significant effect on callus initiation and proliferation. Thus the present study provides a scope of rapid callusing of M 5, V1, S 36 and Anantha on MS + 2, 4-D (2mg/l). This in vitro technique for rapid callusing can be exploited for plant regeneration, in M 5, V1, S 36 and Anantha. 59

8 Table 4.1: Effect of different concentrations of 2, 4-D 1 (MS medium) time taken for callus initiation in M 5. Time Taken for callus initiation (days) Data represents an average of 10 replicates Fig 4.1: Effect of different concentrations of 2, 4-D 1 (MS medium) time taken for callus initiation in M 5. 60

9 Table 4.2: Effect of different concentrations of 2, 4-D 1 (MS medium) time taken for callus initiation in V1.. Time Taken for callus initiation (days) Data represents an average of 10 replicates Fig 4.2: Effect of different concentrations of 2, 4-D 1 (MS medium) time taken for callus initiation in V1. 61

10 Table 4.3: Effect of different concentrations of 2, 4-D 1 (MS medium) time taken for callus initiation in S36. Time Taken for callus initiation (days) Data represents an average of 10 replicates Fig 4.3: Effect of different concentrations of 2, 4-D 1 (MS medium) time taken for callus initiation in S36. 62

11 Table 4.4: Effect of different concentrations of 2, 4-D 1 (MS medium) time taken for callus initiation in Anantha. Time Taken for callus initiation (days) Data represents an average of 10 replicates Fig 4.4: Effect of different concentrations of 2, 4-D 1 (MS medium) time taken for callus initiation in Anantha. 63

12 Table 4.5: Effect of different concentrations of 2, 4-D 1 (MS medium) % frequency of callus initiation after 20 days in M 5. % frequency of callus initiation Data represents an average of 10 replicates and % frequency was recorded at the end of 20 days. Photo period of 16 hours light and 8 hours dark was maintained. Fig 4.5: Effect of different concentrations of 2, 4-D 1 (MS medium) % frequency of callus initiation after 20 days in M 5. 64

13 Table 4.6: Effect of different concentrations of 2, 4-D 1 (MS medium) % frequency of callus initiation after 20 days in V1. % frequency of callus initiation Data represents an average of 10 replicates and % frequency was recorded at the end of 20 days. Photo period of 16 hours light and 8 hours dark was maintained. Fig 4.6: Effect of different concentrations of 2, 4-D 1 (MS medium) % frequency of callus initiation, after 20 days in V1. 65

14 Table 4.7: Effect of different concentrations of 2, 4-D 1 (MS medium) % frequency of callus initiation after 20 days in S36. % frequency of callus initiation Data represents an average of 10 replicates and % frequency was recorded at the end of 20 days. Photo period of 16 hours light and 8 hours dark was maintained. Fig 4.7: Effect of different concentrations of 2, 4-D 1 (MS medium) % frequency of callus initiation, after 20 days in S36. 66

15 Table 4.8: Effect of different concentrations of 2, 4-D 1 (MS medium) % frequency of callus initiation after 20 days in Anantha. % frequency of callus initiation Data represents an average of 10 replicates and % frequency was recorded at the end of 20 days. Photo period of 16 hours light and 8 hours dark was maintained. Fig 4.8: Effect of different concentrations of 2, 4-D 1 (MS medium) % frequency of callus initiation after 20 days in Anantha. 67