Optimization Of Flax Efficient Regeneration System And Bar Genetic Transformation

Flax(Linum usitatissimum L.) is an important economic crop for fiber and oil. The textiles which are made of flax were deeply subjected to a large consumer approbation fancy. The development of flax production is mainly attribute the success to breeding of new cultivars with high yield, superior quality and poly-resitance. With the development of science and bio-techonology, Flax genetic transformation is becoming an effective way in germplasm innovation and breeding of flax. In this study, Paikesi, Zhongya2 and Huaxing009 were used as the recipient plants to develop an efficient Agrobacterium-mediated flax transformation system, and transgenic flax with bar gene were also obtained. The main results as follows:(1)The optimization of efficient protocols for plant regeneration from hypocotylsAn efficient flax regeneration system was established through optimization of genotype, explants, basic medium and hormone combination. Results show that: hypocotyls from plant surface-sterilized first with 75% ethanol for 3 min, followed by 0.1% HgCl2 for 3 min is the optimal sterilize condition. The best medium for callus induction and differentiation was Y4 (MS1+0.02mg/l NAA+1mg/l 6-BA), the efficiency of induction and differentiation were 98.89% and 95.56%, respectively. The optimum medium for root formation of regeneration shoot is 1/2MS+0.001mg/l NAA. We also find that Zhongya2 have the highest regeneration efficiency comparing with Paikesi and Huaxing009.(2)The optimization of efficient Agrobacterium-mediated flax transformation systemBased on above efficient protocol of plant regeneration, Agrobacterium strain LBA4404 containing the pCAMBIA3301 vector were used to develop the Agrobacterium-mediated flax transformation system. In this experiment we mainly focused on preparing of explant, effect of infection time, the OD value of Agrobacterium, pre-cultivation time, co-cultivation time, selection pressure, concentration of Ampicillin and Ceftriaxone. Results show that the best concentration of PPT for selection is 1.5mg/l, for Kanamycin the concentration is 50mg/l. The optimum concentration of Ampicillin Sodium and Ceftriaxone Sodium which were used for inhibit the growth of Agrobacterium are 300mg/l and 400mg/l, respectively. GUS transient assay results show that the GUS expression level reached 96% after 3 days pre-culture and peeling the epidermis layer of hypocotyls. Agrobacterium tumefaciens was resuspended in MS1 liquid medium and with the culture was at density of 0.8 OD600 is the most appropriate condition for infection, the optimum infection time is 30 min; the best co-cultivation time is 3 days.(3)PCR and GUS expression analysis of transgenic plantsThe transgenic flax plants were identified by PCR and stable GUS expression analysis, results have showed that bar gene was stably introduced into the genome of flax.