Micropropagation and isozyme studies on interclonal and somaclonal variations in in vitro regenerated sugar cane (saccharum officinarum)

Abstract

An in vitro protocol for sugar cane micropropagation was developed through callus induction and plant regeneration of ten commercial sugar cane clones in Kenya. The explants were prepared from young shoot tips and the surrounding 2-3 whorls obtained from 6-8 week-old young plants grown in wooden boxes outside the laboratory. The shoot tips were transversely cut into small sections 2- 2.5 mm wide and about 4 mm long and surface sterilized in 0.5 % sodium hypochloride solution for five minutes and then rinsed in sterile distilled water. These were then aseptically cultured into MS medium in combination with 0, 1.5,3.0 and 4.5 mg/I 2,4-0 and then solidified with 109/I agar in universal bottles. Callus induction was realised within one week of incubation in growth chambers illuminated with fluorescent lighting of 16 - hour photoperiod at 25 "C. Analysis of variance (ANOVA) for calli fresh weights obtained at the four concentrations of 2,4-D indicated that they were significantly different (p = 0.5) with a concentration of 3.0 mg/I giving the best results for callus induction. Caulogenesis was obtained by transfering the callus to the above MS medium without 2,4-0 but supplimented with 10 % coconut water. After two weeks, shoot regeneration from callus was evident arising from compact calli with green islands. Shoot multiplication of the regenerants was obtained by supplementing the culture medium with 2 mg/l BAP whereas rhizogenesis was achieved by supplementing the culture medium with 2 mg/l IL3J\. Enzymatic browning was controlled by introducing 5 g/l of activated charcoal to the medium. The presence of activated charcoal also led to a faster rhizogcnesis and rootlets could be noticed even after only three days of incubation. Plantlets were transfered into a soil/white pellets growing medium and successfully acclimated to the greenhouse conditions. isozyme variation was used to identify the biochemical markers of potential utility in sugar cane genetics and breeding. Peroxidase and esterase isozyme patterns from leaf extracts of the in vitro regenerated plants were analysed using porosity gradient gel electrophoresis (PoroP AGE). Electrophoretic polymorphism identified all the clones analyscd. Assays of isozymes revealed that whereas both enzyme systems could be reliably used to identify the clones based on basic bands, peroxidase isozymcs proved to be the most appropriate in detecting sornaclonal variation. Upto 20 % sornaclonal variation was detected with over 15 % being detected by peroxidase patterns alone. Apart from the increased band intensity and clarity, there was an increase in the number of bands in established plants in the greenhouse when compared with in vitro plantlets in the laboratory. No variation was observed in individual established plants in the greenhouse at various growth stages. Morphological variations in tillering abilities and errectness were noticed in threemonth old somaclones. No variations were observed in plants resulting from multiplication of individual plantlets.