A Preliminary Study On The Establishment Of Regeneration System Of Mature Embryo And Genetic Transformation System In Oryza Officinalis

The wild rice（Oryza officinalis）contains many excellent traits and valuable genes that cultivated rice does not have or has lost,including high yield,disease resistance,insect resistance,and high photosynthetic light potential.Transfer these beneficial genes from O.officinalis to cultivated rice varieties is an important goal for rice breeders.However,the genome type of O.officinalis is CC,and it is very difficult to cross with cultivated rice varieties.Constructing a mutant library is one of the most economic and effective means to mine and utilize the functional genes in O.officinalis,but it depends on an efficient genetic transformation system.For O.officinalis,the establishment of regeneration system is very difficult,and the genetic transformation system has not yet been reported.This study aims to establish a regeneration system for the callus from mature embryos of O.officinalis,and aims to make a primary exploration for its genetic transformation system.The main results are listed as follows:（1）Optimization of the callus induction conditions.The suitable conditions for inducing callus from mature embryos of O.officinalis were screened by comparing nine groups of 2,4-D concentrations,five different induction media,and two different surface sterilization methods for explants.The suitable 2,4-D concentration for callus induction is 5 mg/L,and the average callus induction frequency is 55%.The suitable induction media for O.officinalis are NB and NMB,and the additional additives in the media are 0.5 g/L Gln,0.5 g/L Pro,0.3 g/L CA,2.8 g/L phytagel and 30 g/L sucrose.Compared with 75%alcohol,the explants of O.officinalis are more suitable to be soaked in 0.5%TCCA solution for 3.5 h and then sterilized with 0.1%Hg Cl₂for8 minutes.This method of surface sterilization is more conducive to the growth of buds and the induction of callus.（2）Establishment of the regeneration system for O.officinalis.Embryogenic callus can be obtained from the induced callus after 2～3 subculture,and the frequency of embryogenic callus formation is high when subculture at low concentration of2,4-D（1～2 mg/L）.The suitable media for O.officinalis subculture are NB,NMB and N₆.The medium for the first two differentiations is NB supplemented with 3 mg/L6-BA,3 mg/L ZT,0.25 mg/L NAA,10μM Cu²⁺and 30 g/L sucrose.The subsequent differentiation medium is NMB supplemented with 2 mg/L 6-BA,2 mg/L ZT,0.25mg/L NAA,10μM Cu²⁺and 30 g/L sucrose.The rooting culture medium is 1/2NB supplemented with 10 g/L sucrose and 3.5 g/L phytagel.（3）Primary establishment of the genetic transformation system for O.officinalis.The sub-cultured embryogenic callus was used as the transgenic recipients.The callus was infected with Agrobacterium tumefaciens containing p FX-E24.2-15R vector with OD600 of 0.4 for 30 minutes,and then co-cultured in a dark incubator at 28℃for 36hours.Selective medium supplemented with 0.4 g/L cefotaxia and 15 mg/L hygromycin was used for screening.After isolating resistant callus,the transformed seedlings were successfully obtained by differentiation and rooting.The transgenic positive plants to be preliminarily verified using further GUS staining.