Studies On Anther&Microspore Culture In Pepper (Capsicum Annuum L.)

Pepper is an important vegetable crop, grown world wide. However, progress in breeding new varieties only by conventional breeding is limited because pepper is often cross-pollinated crops, which has high natural hybridization rate. Production of haploids in pepper anther and microspore culture allow pepper breeders to release new lines more quickly and screen for particular traits more efficiently, shorten the breeding period. However up to now, we had not established a general system to obtain haploid plants and DH plants. In this paper, anther culture and isolated microspore culture in pepper were studied. Embryoids and regenerated plants were obtained from sample ’003’,’065’,’009’,’081’,’O11’ and ’046’ through anther culture, and the anther culture system was further optimized; A better microspore isolation and purification method has also been set up.1. Embryoid induction and plant regeneration of pepper through anther culture(1) Studies on microspore developmental stages and the correlation with morphological characteristics of buds and the color of anthersTen varieties of pepper were used to study microspore developmental stages and the correlation with morphological characteristics of buds and the color of anthers. The results showed that microspore developmental stages demonstrated a correlativity with the morphological characteristics of buds and the color of anthers. The length of corolla was about the same as or slightly greater than that of the calyx, and the purple top accounted for1/4～1/3of the whole anthers were determined to contain microspores at the uninucleate stage mostly.(2) Studies on embryoid induction and plant regeneration of pepper(Capsicum annuum L.)through anther cultureTen varieties of pepper were used to study the effects of genotype, flower buds harvest time, cold-pretreatment, hot-shock treatment, carbon source and combination of hormones on the anther culture in pepper (Capsicum annuum L.). Through these tests, an effective system of embryoid production was established. Cotyledonary embryoids and plantlets were obtained from six genotypes. The results showed that genotype was the key factor in embryoid induction. The embryoid formation frequency was different significantly among different genotypes. Sample’003’had the highest embryoid formation frequency which was28.9%, Whereas ’046’ had the lowest which was0.42%. The flower buds at full-bloom stage were found to be optimum for anther culture in pepper. Cold-pretreatment (4℃) for1-3days also promoted embryoid induction, while the highest embryoid formation frequency was obtained from anther pretreated at4℃for2days. Embryoid and cotyledonary embryoid formation frequency could be remarkably improved by2%maltose than3%sucrose. The optimal culture medium which could effectively improve embryoid and regenerated plantlets formation was MS+0.5mg/L NAA+1.0mg/L KT+2%maltose.(3) Ploidy identification of regenerated plantsRoot tips of9regenerated plants abtained from anther culture were used to check ploidy level. After chromosome counting, it was noted that3regenerated plants produced by ’003’ were all haploid plants.2regenerated plants produced by ’009’ had1haploid plants. Whereas2regenerated plants produced by ’065’ and2by ’081’ proved only to be diploid. The frequency of haploid was44.4%. With the anther culture system used in this paper, haploid plants could be produced effectively.2Microspore suspension preparation in pepperSample ’003’,’065’,’009’,’081’,’011’,’046’ and ’336’ were used to study the microspore isolation and purification method, and microspore suspension preparation method. The results showed that:After sterilization, the buds whose length of corolla was about the same as or slightly greater than that of the calyx, and the purple top accounted for1/4～1/3of the whole anthers were placed in50mL glass beaker, containing B5-13medium (B5+13%sucrose), and pinched by glass rod to release microspores. The homogenate was filtered through a37μm nylon filter into a centrifuge tube. The microspore suspension was centrifuged at1000r/min for5min and the supestaut was removed. Repeated centrifugal for3times. Later the microspore pellet was resuspended in NLN-9medium (NLN+9%sucrose, PH5.8), then the pure microspore suspension was obtained. Finally, microspore density was adjusted by haemacytometer, and the pure microspore suspension to be incubated was obtained.