Study On Salt-resistance Variety Selection,Tissue Culture And Genetic Transformation Of Sweet Sorghum,An Energy Plant

Sweet Sorghum,a kind of C4 plant with advantages in fast growth,high yield,vast sugar content,stress resistance,is suitable for large-scale cultivation in marginal,saline-alkali soil in coastal wetland and drought area.This study focuses on the salt-resistance variety selection,tissue culture and genetic transformation of Sweet Sorghum,an energy plant,which is meaningful for relieving food pressure and developing marginal lands in our country.Four varieties of Sweet Sorghum,Supper so-20(DLW)/ Big dragoon(NMW)/ Monster(HN)/ Paker(PK),salts resistance abilities were preliminarily appraised and evaluated during their germination period.The results of germination and seedlings growth changes under salt stress show that NMW is the most salt-resistance type,the next is DLW,while PK and HN are easily affected by salt stress.Our research shows that under low concentration of salt stress(80 mM),protective enzyme activities and osmotic regulation substances of plants increased to improve the salt resistance ability,while the osmotic regulation substances played an important role to protect plants under high concentration of salt stress(160 mM),as protective enzyme system in plants suffered irreversible damage.Mature seeds of four varieties Sweet Sorghum were employed as explants to establish tissue culture system: the best concentration of 2,4-D for callus induction is 4 mg/L,IAA and 6-BA have negative effects on callus induction and callus quality.Considering the quality of four varieties Sweet Sorghum callus,DLW is the best type to establish tissue culture system,the next is NMW,HN and PK are the latest types for callus induction.The optimum callus differentiation medium is MS + 4 mg/L 6-BA + 0.5 mg/L IBA and the best rooting culture medium is MS + 3 mg/L IAA when DLW callus used as expalnts in redifferentiation and rooting experiments.The successful establishment of DLW tissue culture system in this experiment will facilitate the further research of genetic transformation.The vacuolar Na+/H+ antiporter gene(HcNHX1)from Halostachys capsica was successfully transformed into DLW with agrobacterium-mediated methods.To optimum the transgene protocol,factors such as concentrations of the acetyl clove ketone(AS),hygromycin,agrobacterium bacteria(OD600)and time for vacuum infection,co-cultivation,were taken into consideration.Our results show that the best combination condition for agrobactium-mediated transgene is 100 ?M AS,OD600=0.6,vacuum infection 5 min,co-cultivation 3 d,20 mg/L hygromycin for selection.Under this condition,five transgene plants contain HcNHX1 gene were obtained and identified by molecule detection.Cell vigor and ROS of root tips as well as the contents of Na+/K+ in transgenic plants and wild type plant were compared to further study plant salt-resistance ability.In comparison with wild type plant,transgenic plants displayed high root tip cell vitality,less reactive oxygen content in root tip and lower Na+ content as well as Na+/K+,thus transgene plants had strong salt resistance ability compared to wild type plant.