| Salix suchowensis,a shrub willow native to China,is early flowering,fast growth and widespread in diverse habitats.In addition,the whole genome sequence of S.suchowensis has been published recently.All of these characteristics make S.suchowensis highly ideal for genetic studies of woody species.Meanwhile,it is highly desirable to develop a stable and effective transformation system to carry out functional genomic studies.Agrobacterium-mediated transformation has been successfully applied for many plant species.It developes plant materials and provides an efficient way for researches on plant development,resistance related physiology and organogenesis.However,a practical method for regeneration of transgenic Salix species has not been established to date.In this study,we aimed to develop the tissue culture system of S.suchowensis.By using young leaves,stems with axillary buds and mature seeds as explants for shoot induction with different ratios of plant growth regulators,we established the regeneration protocol for.We also made a preliminary exploration on the genetic transformation of S.suchowensis by examing effects of different explants infected with Agrobacterium harbouring different constructs.The main results were listed as follows:1.If young leaves were used as explants,the optimum sterilizing conditions were 70% ethanol for 30 sec and 1.5% sodium hypochlorite(30% commercial bleach 84)for 10 min,and the survival rate was 98%;as for the young stems,the optimal sterilizing conditions were 70% ethanol for 30 sec and then treating with 1.5% sodium hypochlorite for 8min,and the survival rate was 92%,and for the seeds,the optimal sterilizing conditions were 70% ethanol for 10 sec and then treating with 1.5% sodium hypochlorite for 2min,and the survival rate was 77.5%.2.Using the basic media of B5?MS?WPM?1/2MS to induce axillary buds,the best treatment is from MS.The optimal shoot induction medium of young stems was: MS+6-BA(1 mg/L)+NAA(0.01 mg/L)+Sucrose(30 g/L)+Phytagel(4.4 g/L).The best medium for rooting was 1/4MS(macroelement)+1/2MS(ferric salt)+ IBA(0.2 mg/L)+ NAA(0.03 mg/L)+ Sucrose(30 g/L)+ Phytagel(4.4 g/L),and the rooting rate reached 100%.The most optimal proliferation medium was 6-BA(0.5 mg/L)+NAA(0.05 mg/L)+Sucrose(30 g/L)+Phytagel(4.4 g/L),and the proliferation coefficient was 8.2.The adventitious buds were directly produced from the base of the single shoots.3.Compared to darkness,light cultivation is more suitable for the callus induction from mature seeds,and the optimum medium was 6-BA(0.5 mg/L)+NAA(0.3 mg/L)+Sucrose(30 g/L)+Phytagel(4.4 g/L).The rate of callus inductio n was 92.5%.Calluses were firstly induced from the hypocotyl of seeds,and then multiple buds could be regenrated from the calli on the medium of 6-BA(0.5 mg/L)+TDZ(0.2 mg/L)+Sucrose(30 g/L)+Phytagel(4.4 g/L).The regeneration efficiency was 100%,and the most number of buds on calli derived from one seed is 4.23.The rooting medium was MS+NAA(0.01 mg/L)+Sucrose(30 g/L)+Phytagel(4.4 g/L),and the average rooting rate peaks at 97.5%.Considering proliferation rate and browning,the callus proliferation medium was 6-BA(0.5 mg/L)+ NAA(0.5 mg/L)+Sucrose(30 g/L)+Phytagel(4.4 g/L)or 6-BA(0.5 mg/L)+NAA(0.8 mg/L)+Sucrose(30 g/L)+Phytagel(4.4 g/L).4.By examing the effects of different plas mid transformation vectors and explants on the genetic transformation,we identified the callus derived from hypocotyl was suitable for Agrobacterium infection,and the optimal plastid transformation vector is pCAMBIA1305.1?... |
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