Genetic Transformation Of Strawberry With LEA3 Gene

Strawberry (Fragaria ananassa Duch.) is one of the most valuable and most widely cultivated small fruits in the world. While the global soil salt stress and drought caused more and more serious affection on strawberry production. It is necessary to culture salt and drought resistant strawberry cultivars via foreign gene transformation.Call from strawberry anthers of Darslect and Toyonaka were induced on MS media with 6-BA and 2,4-D, or 6-BA and NAA, and the inducing frequency was very high. When anthers were cultured on MS medium with 2.0 mg/L 6-BA and 0.5 mg/L NAA, the inducing rate of Darslect and Toyonaka was as high as 98% and 85% respectively.Strawberry calli were transformed by particle bombardment (PDS-1000/He gene gun) with plasmid pBY520 containing late embryogenesis abundant protein gene, LEA3, from barley. LEA3 gene was regulated by Act I and PIN II, bar gene as a selected gene. The bombarded calli were transferred to LS medium with 2.0 mg/L 6-BA, 0.2 mg/L NAA, and 10.0 mg/L PPT for selection. After 4 times selected culture by PPT, 347 resistant calli were obtained. The PPT resistant calli were transferred to MS medium with 2.0 mg/L 6-BA, 0.1 mg/L NAA for regeneration. After 3 weeks, green buds were observed which developed into 2? cm tall plants 5 weeks later. Forty-eight resistant calli regenerated, and the regeneration rate was 13.8%.Plasmid DNA was digested by EcoR I and Xba I and 1.0 kb LEA3 segment was obtained. LEA3 gene probe and marker probe were labeled by DIG-Chem-Link. Twentymo transgenic plants were selected by DNA dot blot analysis, 15 of them detected hybridization signals and the positive rate was 68.2%. Hybridization bands were detected by Southern blot analysis using LEA3 gene probe labeled by digoxigenin The result showed that foreign LEA3 gene integrated into strawberry genome. Except for 1.0 kb hybridization band, other different hybridization bands were also detected which demonstrated that LEA3 gene integrated into strawberry genome in different loci or copies. Different hybridization bands were detected from different transgenic plants, which indicated that those transgenic plants came from different transformed events.The result of RNA dot blot analysis confirmed the transcription of LEA3 gene integrated into strawberry genome. 27KD protein bands were detected in transgenic plants by Western blot analysis, and the results showed that the foreign LEA3 gene could express in transgenic strawberry plants.Under normal condition, there was no visible difference in the growth between transgenic plants and untransgenic plants (CK). The difference of salt tolerance between transgenic strawberry and CK were proved by salt stress test. Under 50 mmol/L and 100 mmol/L NaCl salt stress conditions, the wilting rate of control plants was 60% and 100% respectively, while 16% and 40% for transgenic plants. After salt stress was removed, the survival rate of controls was 48% and 0 respectively, and for transgenic plants the survival rate was 80% and 56% respectively. The results showed that the salt tolerance of transgenic strawberry was increased, the development of damage symptoms (wilting) caused by salt stress was delayed, and the recovery improved when salt stress conditions were removed.