The present research work includes studies for callus induction and plant regeneration from nine sugarcane elite varieties named, S96SP-302, S96SP-571, S96SP-574, HSF-240, CP722086, CP77-400, SPF-213, S97US-183 and S97US-102.Protocols for embryogenic and nonembryogenic (organogenic) calluses as well as direct somatic embryos were optimized (or these varieties. Embryo development was achieved directly on young leaf roll discs in vitro (on callus induction medium i.e. MS3) and on calluses when calluses were transferred to MS2 medium at second subculture. Seven 2,4-0 levels (1-5mgr1) singly and five 2,4-0 levels (1-5mgr1) in combination with Kn (I mgr1) were used for callus induction. All the genotypcs responded good to 2,4-0 alone as well as in combination with Kn and maximum callus formation occurred on different 2,4-D levels ranging from 1.5 to 3mgrl whereas 3mgr1 2,4-D with I mgr1 Kn combination proved best for all genotypes for callus induction except S97US-183 in which optimum callogenesis occurred at 2mgrl 2,4-D with 1 mgr1 Kn. Maximum shoot induction was observed in all the genotypes at different 2,4-D levels in combination with 500mgrl casein hydrolysate. Among different root induction media tried, R5 (NAA, 5mgrl) effective to develop profuse roots. A two step system, for efficient regeneration of sugarcane plants was devised for all the above said varieties, comprising a faster route of regeneration from young leaf rolls to minimize the time spent generating embryogenic callus. This system consists of two phases (preparatory phase on MSCD medium and regeneration phase on Msvit medium) to regenerate the whole plants in vitro. Additional work was done to study the callogenic and morphogenic response ofHSF-240.
Synthetic B.t gene, crylab developed using gradient thermocycler was characterized through tobacco chloroplast transformation, using established protocols. The synthetic PCR product was sequence confirmed and cloned under the light regulated psbA promoter and UTR (or hyper expression. The final transformation vector, containing synthetic cry 1 Ab gene and antibiotic resistcmce conferring gene, (we/A under Prrn promoter and psbA terminator sequences, was used to transform tobacco chloroplasts. The successful expression of the toxin was confirmed in tobacco chloroplasts using OAS-ELISA.
Two final transformation vectors were developed to tailor sugarcane with insect- resistance and target GFP to chloroplasts as a reporter. The transformation vector TG 1 carries a selectable marker, neo that confers resistance to kanamycin and genetic in, and a reporter gene, gfp from jellyfish Aeqllorea victoria. The neo gene was expressed from 35S promoter and Nos terminator for mRNA stability, however, the expression cassette carrying rbcS promoter along with transit peptide and 3-sequence was used to express and target gfp to chloroplasts. The synthetic crylAb was cloned under promoter PEP-C for tissue-specific expression to control the sugarcane top borers. Thus TG-2 carries neo gene for selection of transformation events, gfp for l1uorescence and synthetic cry1Ab for insect resistance. Restriction analysis confirmed the presence of neo, clylAb and gfp genes.
After successful expression of synthetic crylab gene in tobacco chloroplasts, four-month-old sugarcane calli (cv. HSF-240) maintained at MS2 medium were used in nuclear transformation experiments using transformation vectors TG-1 and TG-2 in separate experiments. After bombardments, the proliferating fluorescent chunks of cells were proliferated on MS2 medium containing geneticin were unable to regenerate due to prolonged incubation on proliferation medium. Therefore, an alternate regeneration protocol was followed to recover drug-resistant and fluorescent fully developed plant.
Based on the previous findings. a highly regenerable system was developed in which two weeks old calli were used to bombard with the gold microprojectiles, carrying plasmid constructs TG-1 and YG-2. The bombarded calli were maintained on geneticin (45mg 1-1) containing MS2 medium for proliferation for longer than was practically required, to ensure a true frequency of geneticin resistant plants. Most of the callus pieces (non transformed) and the control ones turned out to be brown and their growth ceased within 2-3weeks on the medium containing antibiotic whereas the transformed cells multiplied into visible white callus outgrowths. These outgrowths eventually developed into green spots as shoot embryoids, giving rise to shoots on Sl medium containing geneticin (45mgrl-1). These putative sugarcane transformant shoots on transfer to root induction medium (R5) developed roots within 2-3weeks. The presence of insecticidal gene in the putative transgenic plants was detected by PCR using primers specific to cry1AB amplifying 400 bp fragments. The endotoxin was detected using lmmuno-Strips for rapid screening of the transgenic plants. Genetically analyzed clones were subjected to second round of selection and regeneration for transgene purity following 2-step regeneration protocol. Shoots developed were rooted on antibiotic-containing R5 medium. The regenerated plants were analyzed for transgene through PCR using the same cry1AB-specific primers. Tissue-specifically expressed cry1AB coded toxins were quanti1ied through DAS-ELISA. Four first generation, PCR positive plants, 11, 12, 18, 21 and three plants from second round of regeneration of clone 21, namely 21 a, 21 band 21c were subjected to DAS-ELISA tests. The amount of toxin protein in the first generation PCR positive plants, 11, 12, 18, 21, as calculated through DAS-ELISA test was 2.165, 2.008, 2.336,1.709 ng ml-1 whereas in the second generation plants i.e. 21a, 21b, 21c, the toxin was 32.72, 30.16,42.8 ng ml-1 respectively. The manifolds increase in the amount of Cry1Ab toxin protein after second round of selection and regeneration leads towards homozygosity and stability of the transgene.