The problem of regeneration through callus culture in graminaccous crop plants has been a serious challenge to the workers in the beginning of plant tissue culture techniques. However, later several cercals and forage grasses have been successfully regenerated from tissue culture yet lack of multiplication procedures have long been a serious problem in sugarcane breeding programme. Moreover, in Pakistan, environmental conditions do not favour flower production in sugarcane, hence variability resulting from sexual crossing mayor may not provide sufficient room to permit greater chanees of improvement in important characters of the crop. However. for such crop. for the last 30 years, occurrence of uncontrolled variation during in vitro callus regeneration i.e., somaclonal variations proved to be quite promising.
Therefore. in the present study, attempts were made to develop protocols for the induction of somaclonal variation through callogenesis, organogenesis and somatic embryogenesis in sugarcane. Later on, such somaclonal variants were characterized on the basis of morphological. biochemical and molecular markers. Present study highlighted the response of different explants i.e., apical meristem, pith parenchyma and young innermost Leaves for callus formation. Among them, young leaf explants showed more capability to initiate and proliferate callus. Role of various concentrations of 2,4-D supplemented in MS medium was also optimized. For all ex plants most of the callus formation took place at 3.0 mg/I. Effect of kinetin and 2,4-D for call1ls initiation was also cheeked. No role of kinetin for callus induction alone or in combination with 2,4-0 was found. Attempts were also made to optimize the temperature and light/dark conditions for callus formation. The optimum temperature for callus growth was observed to be the 28°C Dark conditions were round best for callus initiation and proliferation. However. maximum regeneration was achieved in light condition.
In the present study, attempts were also made to regenerate plants through direct and indirect somatic embryogenesis. MS medium supplemented with 1.52.0 mg/I 2,4-D and 200 mg/I casein hydrolysate was found the best medium for direct somatic embryogenesis. However, MS medium with 3.0 mg/I 2,4-0 was found hest medium for indirect somatic embryogenesis. Shoot induction was achieved on MS basal medium free from all growth hormones. However, rooting was done on MS basal medium free of hormone +6% sugar.
Since the main object or the study was to get somaclonal variants. Therefore. old calli were preferred for plant regeneration and about 1500 plantlets were shi lied to ex vitro conditions. During hardening process, almost 25% mortality rate was observed. So, in this way almost 1175 plants were successfully shifted to soil after the process of hardening and acclimatization. Among these plants. early screening based on morphological markers i.e., tillering capacity, cane diameter and skin colour of cane was done and only 26 variant lines were selected for further study. Such variant lines on maturity were further characterized and compared with parent plant based on morphological characters i.e., tillering capacity. tiller length. tiller diameter, internodal length, susceptibility to borer and sugar recovery. In this way only three of the variant lines i.e., V7, V9 and V19 were proved to be better almost in all characters in comparison to parent plant and were finally selected and designated as V 1, V 2 and V 3 respectively for further biochemical and molecular study.
In tiller numbers, variant 1 with 37 tillers was found to be better as compared to parent plant with 33 tillers so variant I showed 12.1'% increase over the parent plant. Although variant" and III with 36 and 35 tillers showed 9.1 and 6.1% increase respectively over the parent plant yet the maximum tillering capacity was found in variant
In tiller length, variant III with 11.4 feet tiller length was excellent among the variants and also showed 11.8% increase over the parent plant. Although variant I and II with tiller length 10.9 and 10.7 showed 6.9 and 4.9% increase respectively over the parent plant but less than the variant III.
In tiller diameter, variant I with diameter of 3.4 cm was found better among the variants, showing 13.3% increase over the parent plant. However, variant II and III with tiller diameter of 3.1 and 3.2 revealed 3.3 and 6.6% increase respectively over the parent plant. In internodal length, variant I with 18.2 cm internodal length was maximum among the variants and showed 13.0% increase over the parent plant. Variant II and III with 17.5 cm and 16.9 cm also showed 8,7 and 4.9% increase in internodal length. However, variant I showed maximum increase in internodal length. However, variant I showed maximum increase in internodal length.
In the present study extent of susceptibility to borer was also studied. It was found the ll variants were moderately resistant to borer whereas parent plant was moderately susceptible. However, variant I showed maximum resistance as compared to variant II and III, which showed resistance to lesser extent.
In the present study, sugar recovery of the variants as well as parent plant was also checked. It was found that variant I showed maximum sugar recovery i.e 11% and hence revealed 7.8% increase over the parent plant. Variant II and III with 10.8% and 10.5% also showed 5.9 and 2.9% increase in sugar recovery over the parent plant. However, variant I showed maximum sugar recovery.
In the present study, all variants selected based on morphological characters, were also investigated on the basis of biochemical characterization. Various biochemical markers used in the present study are, total soluble protein content and acid phosphatases (quantitatively), peroxidases and esterases (quantitatively and qualitatively). of the soluble protein content, variant I showed almost 66% deerease as compared to parent plant so, showing maximum dissimilarity in comparison to other variants. Of the peroxidases conent, variant II showed almost 8% increase, while variant I and III showed almost 21 and 8% decrease. respectively. Same results were obtained when qualitative study was conducted on PAGE (Polyacrylamide gel electrophoresis).
Of the esterase content, variant I showed almost 37% deerease in comparison to parent plant, while variant II and III showed 3% and 46%J increase. Such quantitative results were reflected when intensity of bands on the gel was studied.
The major aim or the present study was to find out the changes at DNA level (using DNA as molecular marker) when cells pass through the callus cycle. For this, in the present study, PCR based RAPD technique was used. In the study, 8 primers, out of 50 detected polymorphism. Using Nie and Lis coefficient, Dendrogram was constructed to assess the extent of similarity of the variants with the parent plant. Of the variants, variant I showed 84% similarity, which was least among all the variants. Hence to most extent RAPD study supported the morphological and biochemical results although not 100%.