Optimization On Isolated Microspore Culture For Rice And Barley

The technology of microspore culture is the ideal experiment system in researchingembryonic development and it can overcome the shortcomings of anther culture inimproving the efficiency of breeding. However, some barriers such as genotypes, lowefficiency of callus induction and plantlet regeneration and high albinos, obstructed itswidely application in cereal breeding and genetic research.In order to improve the callus quantity and green plantlet number, cropped riceand barley were used as materials, and the pretreatment method, carbon and nitrogensource and exogenous hormone combinations in induction medium and differentiationmedium, and transition time to differentiation were studied. The major results weresummarized as below:1. The anther and microspore isolated from4cropped rice varieties werepretreated to investigate the effects on callus yield and green plantlet regeneration. Theresults showed that3days separate pretreatment on isolated anthers with6%mannitolor10mg/L colchicines could obviously improve the callus yield and green planlet yield.Meanwhile,32℃heat shock on isolated microspores for2days also increased thecallus yield and green plantlet yield.2. Five cropped rice varieties were used as materials, the maltose,2,4-D and PEGused in induced medium were studied to improve the callus incudtion and plantlet regeneration. Of the results showed that75g/L maltose achived largest green plantlet yield,2.0mg/L2,4-D was most effective for callus induction and resluted in largest greenplantlet yield, and2%PEG had promoted callus induction and green plantletregernation while higher PEG lead to death of microspores (no callus apperaed at morethan8%PEG). The concentrations of hormones in differentiation medium were testedand the optimum concentraions for6-BA, NAA and KT were1.0mg/L,0.5mg/L and2.0mg/L, respectively.3. Three cropped barley varieties were used to study the effect of low temperaturepretreatment time, the organic and inorganic nitrogen type and concentration in inducedmedium on callus yield and green plantlet regeneration. The results showed that the best time of low-temperature pretreatment was21days. In terms of inorganic nitrogen levelin N6medium witout organic nitrogen addition, the1/2-level was better than1-levelfor callus differentiation. As to organic nitrogen, the effect on callus differentiation wasrelated to the genotype. For the material of BI45, the highest green plantlet regenerationwas obtained without Glu and CH addition, but approriate Glu and CH could increasecallus yield. For H30, the green plantlet regeneration was higher in medium without CHaddition, but the average green plantlet number per dish was highest in the medium with800mg/L CH. For H11, the highest yields of callus and green plantlets were obtained inthe medium with800mg/L Glu.4. Three cropped barley varieties were used to study the effect of four hormonecombinations of6-BA, KT, NAA and IBA in differentiation medium, transition time todifferentiation meidum on green plantlet regeneration. The resluts showed that differentgenotypes had different responses to the medium. For H30, the optimum differntiationmedium was1.5mg/L6-BA+1.0mg/L KT+0.05mg/L NAA; For BI45, the optimumdifferntiation medium was0.5mg/L6-BA+1.5mg/L KT+0.05mg/L NAA+0.5mg/L IBA,which is the differentiation medium coded No.2; For H11, the optimum differntiationmedium was1.5mg/L6-BA+2.0mg/L KT+0.05mg/L NAA in three tested media.Regarding the transition time to differentiation medium, the best time was18-d and15-d for1-dose level induction meidum and M90induction medium, respectively.