Genetic transformation of Amorphophallus paeoniifolius (Dennst) Nicolson

Abstract

A study on Agrobacterium-mediated genetic transformation of Amorphophallus paeoniifolius (Dennst.) Nicolson was conducted at the Central Tuber Crop Research Institute, Sreekariyam, Thiruvananthapuram during 2013- 2014. Calli were initiated using petiole and leaves of in vitro plantlets of elephant foot yam cv. Gajendra in callus induction media. After four weeks of incubation, actively dividing globular, hard and creamy white calli were developed. Subculture of developed calli was repeated periodically (20 days) in CIM with an approximate size of one cm2. 15 days old calli was found to be suitable for transformation study. Calli sufficient for the transformation study was obtained after 3 months of subculture. Experiments were conducted to evaluate the sensitivity of elephant foot yam calli to different doses of antibiotics viz. geneticin, hygromycin, ticarcillin. It was observed that complete death and discoloration of the calli obtained with 20 mgl-1 geneticin and 10 mgl-1 hygromycin from sixth week treatment. Statistical analysis of sensitivity response of calli indicated that LD100 was 20 mgl-1 and 5 mgl-1 with geneticin and hygromycin respectively. Sensitivity of the calli to ticarcillin was studied and the responses are analysed with ANOVA. The lowest lethal concentration of ticarcillin was found to be 650 mgl-1. So, concentration below 650 mgl-1 can be used for the successful elimination of Agrobacterium without affecting the regeneration potential of explant. 500 mgl-1 ticarcillin used in this study was observed sufficient for the successful elimination of Agrobacterium without affecting the regeneration potential of calli. For the optimization of parameters affecting transformation, experiments were conducted for the standardisation of optimum concentration of acetosyringone, time of co cultivation, temperature of co cultivation, and suitable Agrobacterium strain. In a study conducted for standardisation of optimum concentration of acetosyringone, increasing number of transformants was obtained with increase in acetosyringone. Significantly higher GUS staining of calli (21.5896) was achieved with the addition of 400μM acetosyringone in the co cultivation media. The effect of number of days of co cultivation on transformation was compared on the GUS expression of 14-day old selected calli. Two-three days of co-cultivation was determined to be the suitable for elephant foot yam because prolonged co-cultivation period (more than three days) was found to promote overgrowth of bacteria and subsequent death of the calli. Correspondingly the transformation percentage was found to decrease with the decrease (less than two days) of co-cultivation period. Investigation of the effect of temperature during co cultivation in elephant foot yam calli revealed that temperature plays an important role in transformation efficiency. Higher temperature, 28°C was found to be optimal to support the highest transient transformation frequency in elephant foot yam and dramatic transient expression reduction occurred when temperature decreased from 22 °C to 20°C. Transformation efficiency with respect to the different strain of Agrobacterium was investigated and the results showed that maximum percent of GUS stained tissue (24.5 percent) of transformants was obtained with the strain LBA4404 with pOYE153 vector followed by AGL0/pOYE153 (14 percent) and GV3103/pCAMBIA 1305.2 (6 percent). GUS assay of transformed callus showed blue colour and confirmation was done by PCR analysis with specific primers and southern blotting. PCR amplification of the DNA of the calli survived in selection medium yielded an expected band size of 280 bp for nptII primer, two bands of size 880bp and 700bp for GUS primer, 300 bp single band for hpt primer and GUSPlus primer. No amplification was obtained for untransformed calli DNA. Nucleic acid spot hybridisation of putative transformants of elephant foot yam further confirmation of the presence of transgene in the DNA. Hybridisation with nptII probe yield spots of varying intensity for all the transformants of AGL0/pOYE153 and LBA4404/pOYE153. Whereas only 5 out of the 8 transformants of GV3103/pCAMBIA1305.2 gave positive for hpt probe and the intensity of spot was low when compared to the spots obtained with nptII probe. Southern hybridisation with DIG labelled nptII probe yield a band for positive control (pOYE153 plasmid) whereas the bands in sample lane was not observed. It is possible that the concentration of DNA (10μl) used in the blot was too low for detection of T-DNA inserts. Hybridisation with hpt probe gave a single band corresponding to the putative transformants lane, which are visible after 30 min exposure indicated that successful hybridisation of the DIG-labelled hpt probe. But the absence of band for positive control was not expected.