Agrobacterium-mediated Transformation Of Potato (Solanum Tuberosum) With MSI-99 Gene And Plant Regeneration

Potato is a world-cultured crop and ranked 4th in tube production following wheat, maize and rice. It can be used as food and vegetable. However, in potato culture, diseases often cause huge loses. Creating a new broad-spectrum resistant variety has become the goal of potato breeders. MSI-99, a analog of antimicrobial peptide magainin II, is a broad-spectrum antibacterial peptide. It can effectively inhibit the growth of many species of bacteria, fungi and viruses which infect plants. The introduction of MSI-99 gene into potato cultivars could improve the resistance of the cultivars to diseases. This study includes the screening of high effective regeneration systems and the transformation of potato with MSI-99 gene by Agrobacterium.1. Screening of high effective regeneration systemsThe leaf discs, stem segments and tuber pieces of 3 potato cultivars, Superior, Shepody and Favorita, were used as the experimental materials. The high effective regeneration systems were obtained by the comparison of 7 different regeneration systems:(1)The system used for leaf discs and stem segments culture: MS base medium complemented with 2.25mg /L 6-BA + 0.18mg /L NAA (for callus induction); MS base medium complements with 2.25mg /L 6-BA+5.0mg /L GA3 (for shoots generation);(2)The system used for tuber pieces culture: MS base medium complements with 0.9mg /LIAA + 1.8mg /L ZT (for callus and shoots induction)2. Transfer of gene MSI-99 to potatoes mediated by AgrobacteriumThe binary vector pBIN-MSI99 carrying MSI-99 gene was transferred to Agrobacterium LBA4404 by electroporation apparatus. Then the Agrobacterium LRA4404/pBIN-MSI99 was cocultured with the leaf discs, stem segments and tuber pieces cut from Superior, Shepody and Favorita respectively. The callus and shoots were induced on the regeneration system and 36 adventitious shoots were gained. In these adventitious shoots, 2 shoots couldn't root. PCR analysis gave 13 positive regeneration plants and Southern blot analysisconfirmed the MSI-99 gene has been integrated into the genome of 7 regenerated plants. After being transplanted to flowerpot, the transgenic plants have smaller and more leaves than the control and the color of the leaves is darker also. The shape of oval transgenic microtubers revealed thinner and longer than the control.