Agrobacterium Mediated Genetic Transformation of Potato

Abstract

Potato (Solanum tuberosum L.) is one of the most economically important food crops worldwide for both consumers and farmers. The key objective of this work was to transform potato plants with rolA and rolC genes and to study the defense response of these rol transgenic plants by determining their antifungal, antibacterial and antioxidant activities. Stable transformation of potato genotype Desirée with rol genes was achieved by Agrobacterium mediated transformation. In order to accomplish this, an efficient, rapid and reproducible in vitro regeneration system was developed as a pre-requisite for genetic transformation. In vitro regeneration of internodal segments, leaf strips and microtuber discs from three important potato genotypes viz. Diamant, Desirée and Altamash have been compared to select the best explant type from one of the three genotypes for further gene manipulation experiments. In an attempt to select the best combination of callus, shoot and root induction media, six callus induction and shoot induction media (CIM and SIM respectively) while three root induction media (RIM) reported earlier, having different types and combinations of plant growth hormones for tissue culture of potato were evaluated. Internodes of Desirée proved to have a higher potential for callus formation (96.11%) on MS medium supplemented with 0.2 mg/l NAA + 0.02 mg/l GA3 + 2.5 mg/l zeatin riboside (CIM3) in 12.67 days. Similarly, the highest percentage of shooting (95.55%) was observed from the internodal calli of Desirée on MS medium containing 0.02 mg/l NAA + 0.02 mg/l GA3 + 2 mg/l zeatin riboside (SIM3) in 20.33 days. Finally, best rooting was achieved on 1⁄2 MS + 1.0 mg/l IBA (RIM2). Based on the protocol developed for in vitro regeneration of potato cultivar Desirée, biolistic gene transfer and Agrobacterium mediated transformation were optimized and compared using plasmid vector p35SGUSint containing gus reporter gene. Optimization of transient gus expression for biolistic transformation showed that helium pressure of 1100 psi, 6 cm target distance and 1.0 μm gold particle size was the best combination for transforming internodal explants while, the use of different osmoticum treatments had a little effect on transient gus expression and callus formation. In case of Agrobacterium-mediated transformation bacterial density of OD600 1.0, inoculation time of 15 minutes and co- cultivation duration of 48 hours for internodal explants proved to be the best combination of variables which produced highest transformation efficiency on the basis of transient gus expression. The final concentration of 500 mg/l cefotaxime for elimination of Agrobacterium and 50 mg/l kanamycin to select transgenic explants were used in CIM3. The comparison of biolistic gene transfer and Agrobacterium mediated transformation demonstrated that the later method remained more suitable for potato transformation and was further employed to produce rolA and rolC transgenic plants of potato cultivar Desirée using vectors pLBR29 and pLBR31 respectively under the transcriptional control of 70S promoter. Transformation efficiencies of 34.17%, 28.00% and 36.00% were recorded for gus, rolA and rolC genes on the basis of polymerase chain reaction (PCR). Southern blotting revealed the insertion of one or two copies of transgenes into the genome of transgenic plants. All the rolA and rolC transgenic plants exhibited distinct morphological characteristics as compared to the control plants. The shape, number and weight of tubers harvested from rolA and rolC transgenic plants also differed from the control plants. Both the rolA and rolC transgenic plants were evaluated for their antifungal, antibacterial and antioxidant activities in addition to the determination of total phenolic and flavonoid contents. Antifungal assay of crude methanolic extracts of rol transgenic plants showed that all the rolA and rolC transgenic lines gave antifungal activities better than gus gene transformed plants and untransformed wild type Desirée plants used as control. The rolC transgenic lines gave higher activities against Fusarium solani as compared to rolA transgenic lines whereas both rolA and rolC lines were equally active against Alternaria solani. Antibacterial assay revealed that most of the rolA and rolC transgenic lines proved better than both gus gene transformed plants and untransformed wild type Desirée plants. Among all the transgenic lines, rolC lines largely produced promising inhibitory result against bacterial strain Pseudomonas syringae when compared with rolA. However, the overall effectiveness of rolC transgenic lines was almost similar with that of rolA against Agrobacterium tumefaciens strain AT 10. Moreover, the activities of rolA lines remained lowest against Xanthomonas compestris as compared to the rolC lines. Antioxidant assay exhibited better free radical scavenging activity of all the rol transgenic lines as compared to the control plants. Antioxidant activity of transgenic lines revealed a maximum relative increase of 75.35% and 61.58% in the free radical scavenging for rolA and rolC transgenic lines respectively. An overall increase in total phenolics of rolA transgenic lines was almost three folds higher than rolC transgenic lines while, a comparable overall increase in total flavonoid contents was observed for both rolA and rolC transgenic lines. These studies suggest that the enhanced production of phenolics and flavonoids in rol gene transformants increased the antimicrobial and antioxidant activities which synergistically could improve the plant defense response.