Reproducible in vitro regeneration system for purifying sugarcane clones
Genome purification of a selected clone of sugarcane is the key to developing homogenous lines. Generally, regenerated plants after transformation are heterogeneous at genome level, and several successive rounds of selection on antibiotic-containing medium and regeneration cycles are required to purify the genome to develop a homogenous population. Sugarcane is a vegetatively propagated plant and hence it requires to be grown in the field to harvest mature cane tops carrying meristematic tissues. In the present studies, stems of in vitro grown plants of four indigenous genotypes namely; HSF-242, US-778, HSF-243 and HSF-240, were subjected to regeneration. Five days post incubation at various levels of 2,4-D, the segments were placed on regeneration medium containing a combination of casein hydrolysate (500 mg/L), kinetin (0.5 mg/L) and benzylaminopurine (BAP, 0.5 mg/L). Response to regeneration was varied from basal to top sections. Nevertheless, more than 137 shoots were regenerated from basal segment, suggesting that the segment consisting of meristematic tissues responded well to in vitro conditions. This procedure may be considered as one of the best ever published report on regeneration from in vitro grown plants to purify clones without subjecting the plants to field conditions and harvesting the mature cane. This technique was used to purify transgenic sugarcane plants carrying Bacillus thuringiensis gene.
Key words: In vitro explant, basal stem segment, callus induction, proficient regeneration, Saccharum officinarum.