Optimization Sugarcane Explants Transformation of P5CS Gene using Agrobacterium tumefaciens

Abstract

Genetic transformation is one of the attempt in generating sugarcane (Saccharum officinarum L.) that are tolerant to drought stress. P5CS gene has a role in prolin biosynthesis, the amino acid that accumulating in water stress condition. Transformation of a P5CS gene construct into plant cells in conjunction with regeneration for transgenic plantlets should develop sugarcane tolerant to drought stress. The aim of this research is to generate drought-tolerant sugarcane through optimization transformation which includes the strain of Agrobacterium tumefaciens, a good source of sugarcane eksplant and varieties. This study was done through verification of IBRIEC (Indonesian Biotechnology Research Institute for Estate Crops) P5CS gene collection A. Tumefaciens transformation (Wang 2006), sugarcane explants transformation (Sain 1994) and sugarcane transformant regeneration. Verification of P5CS gene collection was PCR analysed by specific primers P5CS full lenght. PCR and gel electrophoresis showed a single band agarose and the same size of 2.4 kb with positive control. It is convinced that the P5CS gene has been isolated and transformed into A. tumefaciens. IBRIEC collection P5CS genes in plasmid pBI121 were arranged in a recombinant construct of PBI-P5CS transformed into A. tumefaciens strain GV3101, LBA4404 and AGL1. Recombinant constructs PBI- P5CS from Vigna aconitifolia (V. aconitifolia) successfully transformed into cell A. tumefaciens were grown with appropriate antibiotics. Strain GV3101 and LBA4404 used 50 ppm kanamycin and rifampicin respectively, while antibiotics were added in AGL1 strain is 50 ppm kanamycin, rifampicin and ampicillin respectively. The existence of P5CS gene in A. tumefaciens was tested by observing the growth of transformed colonies in media selection and colony PCR analysis using specific primers P5CS full lenght. The results showed that A. tumefaciens transformants were grown in selection media. PCR and gel electrophoresis analysis showed a single band agarose and same size of 2.4 kb with positive control. This showed the success of the transformation A. tumefaciens and used to explants sugar cane transformation. Sugarcane explants used in this research was included calli grown in solid medium, embryogenic calli and somatic embryos with MS medium derived from Temporary Immersion System (TIS) culture with three varieties Kidang Kencana, PS 881 and PS 891. Co-cultivation medium used solid MS medium with acetosyringon in room temperature for two days. Transgenes selection was done in solid MS with antibiotics 50 ppm kanamycin and 500 ppm cefotaxime. Explants were not transformed cultured in MS media as positive control negative control while planted in the media selection. Observation of sugarcane transforman in selection media was calculated the percentage growth in 16th weeks and 32th weeks. The existence of P5CS gene in transgenic shoots was tested with GUS histochemical assay, PCR using conserved regions of specific primers P5CS and NPTII. GUS histochemical test for the presence of P5CS gene constructs showed positive results in the presence of blue staining. DNA amplification showed that expected bent size at 1.2 kb and 700 bp for primers NPTII. The results of this examination proved that both transgenes were inserted in the sugarcane genomes. The test results are stable sugarcane transformants through histochemical GUS test and PCR analysis showed stable transformants sugarcane. Growth of transformants on selection media indicated P5CS gene via A. tumefaciens transformation has been successfully performed on sugarcane. Regeneration of transformed sugarcane in the MS medium with the addition of 1% glucose and putrescine with various concentration of 0 ppm, 10 ppm and 30 ppm. Green shoots transformant were increased after growth in regeneration medium for 2 months with the optimum concentration of putrescine was 30 ppm. Transformant plantlets ready tobe acclimatized and to be further tested stability of P5CS gene. It can be concluded that A. tumefaciens strain LBA4404 was the most effective transformation media of P5CS gene on sugarcane. The regeneration of Kidang Kencana transformants was better than the other two varieties, PS 881 and PS 891. Whilst, the best performance of transformants based on the source of explants was somatic embryo.