Study On Anther Culture And Isolated Microspore Culture In Cucumber (Cucumis Sativus L.)

Cucumber is an important avegetable crop, commonly grown worldwide. Howere, progress in breeding new watieties only by conventional breeding has benn slow because the genetic base of cucumber is narrow. Production of haploids in cucumber anther or microspore culture woule allow cucumber breeders to release new lines more quickly and screen for particular traits more efficiently, shorten the breeding period. In this paper, anther culture and isolated microspore culture in cucumber were studied. And embryoids and regenerated plants were obtained from'7333','7551','Ningja3','Ningjia6','caoshi2','7447'and'7443' through anther;Cytoledanry embryoids and regenerated plants were produced from '7447' and 'Poinsett97' through isolated microspore for the first time.1.Studies on embryogenesis and plant regeneration of cucumber (Cucumis sativus L) through anther cultureAnther culture in cucumber was studied by using 15 different cucumber varieties. Embryogenesis and plantlets were obtained from 7 out of the 15 genotypes used. The results showed that growth regulators had markedly promote the induction of embryonic callus and embryoids. The best medium for embryonic callus induction was MS+1.0 mg·L^-1 2,4-D+0.5 mg·L^-1 6-BA+3% sucrose+0.8% agar. The embryoid induction medium was MS +0.5 mg·L^-1 6-BA+3% sucrose+0.8% agar. MS and B₅ medium could both be used as the base medium. The highest embryoid yield was obtained when the anthers were cultured on the embryonic callus induction medium for 14 days. Pretreatment at 4℃for 2 dincreased the embryoid induction ratio significantly higher than other treatments. Dark inocubation for a period of time (14 d) was proved necessary for the re-differentiation and plant regeneration. The buds connected from flourishing florescence had highest embryoid induction ratio. The embryonic callus ratio was recorded between 54.5%-83.8% on medium containing MS supplemented with 2,4-D (1.0 mg·L^-1) and 6-BA (0.5 mg·L^-1), whesea embryoid induction ratio was between 3.6%-52.4% on medium containing MS supplemented with 6-BA (0.5 mg·L^-1).2.Study on embryogenesis and plant regeneration of cucumber {Cucumis sativus L) through isolated microspore culrurer2.1 Microspore isolation and purification and microspore suspension preparationFor studing on embryogenesis and plant regeneration in isolated microspore culture of cucumber (Cucumis sativus L),'Changchunmici','Jinyou 31' and 'Ningjia 3'were used to study the microspore isolation and purification , and microspore suspension preparation method. The results showed that: After sterilization, the buds were placed in 10 mL centrifuge tubes, adding appropriate extract B5-13 (B5 +13% sucrose) medium, and pinched by glass to release microspores. Then the homogenate was filtered through a 76μm nylon filter into a centrifuge tube. The tube with microspore suspension was centrifuged at 600 r min^-1 for 5 min.Then the upper liquid was removed. Repeated centrifugal for 3 times. Later, the microspore pellet was resuspended in NLN-13 medium (13% sucrose, pH 8.0). Finally, microspore density was adjusted by haemacytometer, and the pure microsporesuspension to be incubated was obtained.2.2 Studies on the factors effect embryo induction and plant regeneration of cucumber(Cucumis sativus L) through isolated microspore cultureIsolated microspore culture in cucumber was studied by using 10 different varieties of cucumber. Cotyledon embryoids and plantlets were obtained from '7447' and 'Poinsett97' genotypes for the first time. The results showed that genotype and the development stage of microspore were the key factors in embryoid induction. The embryoid yields were significantly different among different genotypes. Embryoid yield per plate was between 1.5 and 33.4. The late-uninucleate stage of microspore was found optimum for isolated microspore culture in cucumber. Cold-pretreatment (4℃) for 2-4 days also promoted embryoid induction, while the highest embryoid yield was obtained form microspore pre-treated at 4℃for 2 days.The results also indicated that low concentration of exogenous hormones (0.5 mg·L^-1 2,4-D & 0.2 mg·L^-1 6-BA) were conducive to embryogenesis, whereas NLN and B₅ medium had no significant difference on embryoid induction. The regenerated plantlets were obtained only from cotyledon embryoids and noplantlet was produced by the embryoids at other developmental stages. 3.Ploid identification of regenerated plantsRoot tips of 21 regenerated plantlets were abtained from anther culture was used to check ploidy level. After chromosome counting, it was noted that number of haploid plants produced by '7333' and '7551' was 9 and 3 respectively. Wheseas '7551', 'Ningjia3', 'Ningjia6','Chaoshi2',and '7443' produced only diploid plants. In microspore culture, all regenerated plants obtained from genotypes '7447' (11 plants ) and 'Poinsett97' (3 plants) were found diploid after poloid identification.