Coastal Beet High Frequency Induction Of Regeneration Buds System To Establish And To Turn Beta Transgenic Plants

Salinity stress is one of the most serious worldwide factors that retarding the growth of plants and crops productivity. In China, there is nearly 1.5 billion mu saline soil. In order to make full use of saline soil, we should creat new varities that are more salt-tolerant in plants. Beta maritima L. is a wild beet which distrabute near the shore of Mediterranean Sea. Beta maritima L. shows moderate salt tolerance. In order to creat the varities with improved salt tolerance for Beta maritima L., we have done the experiments as follows.An efficient method has been developed in in vitro multiplication of Beta maritima from excised immature inflorescence tips of 5 ecotypes on MS medium supplemented with 1 mg/L 6-BA. Multiple bud clumps were induced from segments of inflorescence tips after inoculation for 2 weeks, and the proliferation frequency was 1: 20³0 after inoculation for 6 weeks. The speed and induction efficiency of bud varied from ecotypes > the development phase ^ the part of inflorescence and the season. In the middle ten days of May, we took inflorescence tips of phase I as explants, the induction efficiency of clumps was the highest, phase II took the second place, and the efficiency of phase III was the lowest. In contrast with the explants comes from low parts on inflorescence, the efficiency of explants comes from tips of inflorescence (only 0-0.5mm) is higher, and clumps of buds are more. The development phase of inflorescence also affects induction of the clusters of buds. The induction efficiency was the highest when took explants in the middle of ten days of May, while the induction efficiency was reduced evidently in the last ten days of August.Once single shoot has grown out 3⁴ leaves, they were shifted to rooting medium supplemented with 1 mg/L NAA, and white and strong roots appeared in 2 weeks. The rooting rate could reach 90%. The maximum survival rate of regeneration plants transplanted to pots was 70%⁸0%.In 1987, Paul H discovered sugar beet was sensitivity in Agrobacteriumimmersing experiments. Because of effective receptor regeneration system was lacked, the transformation of sugar beet has always been restricted. We chose one ecotype as the receptor of the transformation that Agrobacterium tumefaciens infected. The highest transformation efficiency of 4.83% was obtained when 2-3 mm shoot tips were infected in Agrobacterium suspension (supplemented 100 MM AS) of OD₆00 0.3, vacuum infiltrated at 40 KPa for 6 min, and then co-cultured for 34 d. The average livability was 80%, after co-cultured for 3⁴ days on induction medium, the buds were transferred to medium supplemented with 100 mg/L cefotaxime to inhibit the growth of agrobacterium for ten days. After two further 15 days subcultures on medium containing lvhuanglong (sulfonylurea herbicide) at the concentration of 0.01 mg/L, the survival frequency of infected buds was about 30%. Then, survival buds were transferred to medium without lvhuanglong and proliferated to form multiple bud clumps. Larger buds from multiple clumps were rooted after one week on the rooting medium, and were transplanted into pots after 10-15 days. And the plantlets were about 20% of the transformed shoot tips.During the infection, the durantion and temperature of co-culture vacuum infiltration concentration of acetosyingone (AS) were evaluated respectively. Vacuum infiltration is helpful for immersing of Agrobacterium, during the immersion. We adopted a moderate pressure (40 KPa) and infected 6 min for transformation. The efficiency of transformation was increased along with the time of co-culture within 2-4 days. The optimize co-culture temperature was 22 ± 1℃. Transformation efficiency increased at the 100 μM of AS in the liquid infection medium.After selected by lvhuanglong, the resisted bud clumps cultured for 15-30 days on the medium without lvhuanglong for recovery. While this period some cells proliferated swiftly, a lot of clumps of bud were produced. On the media supplemented with different concentrations of NaCl (the concentration were 1.5%, 2%...