Studies On Agrobacterium Tumefacinens-mediated Transformation Of Alfalfa (Medicago Sativa L.) With H~+-ATPases Genes

Salinity is a very important environmental factor constraining world plants growth and agricultural production. Alfalfa is the biggest for the growth area and the highest quality forage grass, and it is difficult to grow under salinity and alkaline conditions for its lower salt and saline-alkali tolerance. Using the gene-transfer technology is one of the most effective methods to breed anti-salt and anti-saline-alkali variety alfalfa in the present of forage grass breeding. The purpose of this research was to improve the salt-tolerance of alfalfa by means of transfering the anti-salt and anti-saline-alkali V-H+-ATPase subunits B, C, H (ScVHA-B, ScVHA-C and ScVHA-H) from Suaeda corniculata into it.1. Alfalfa (Medicago sativa L.) species named "Gong-nong No.1" was used in this study. The ScVHA-B, ScVHA-C and ScVHA-H genes were transferred into alfalfa by Agrobacterium-mediated method. Finally, we got 36,32,33 transgenic plantlets separately. The result of PCR analysis showed that 12,8 and 10 transgenic plantlets were positive lines, and transformation efficiency rates reached up to 33%,25%,30% respectively.2. Southren blot analysis demonstrated that the V-H+-ATPase subunits B, C, H had been integrated into the chromosome. Furthermore, plants from line B, C, H carried a single active T-DNA locus and were applied for further analysis.3. The transcription of foreign genes in transformants was also confirmed by semi-quantitive RT-PCR. Expression analysis by RT-PCR clearly indicated the V-H+-ATPase genes are transcribed into mRNA in positive transgenic plants.4. Northern blot analysis indicated that V-H+-ATPase subunit B, C and H transcripts were stably detected in salt-treated and saline-alkali-treated transgenic plants. However, as expected, the V-H+-ATPase transcripts could not be detected in the WT plant.5. Testing of the salt and saline-alkali tolerance indicated:the transgenic plants enhanced seed germination, root length and seedling growth than the WT plant under the salt and saline-alkali stresses. These results showed that overexpression of the subunits B, C and H in transgenic alfalfa were able to significantly improve the salt and saline-alkali tolerance of the transgenic alfalfa plants.6. The physiological indices of transgenic plants showed:under salt and saline-alkali stress, the transgenic plants showed an increase of free proline content and SOD activities, while MDA content was not significantly affected. So that the transgenic plants can better cope with salt stress and alkaline stress.