Study On Anther Culture And Mechanism Of Embryogenesis In Pepper (Capsicum Annuum L.)

Pepper (Capsicum annuum L.) is one of the largest vegetable crops in our country. Anther culture can shorten breeding period and improve breeding efficiency. The protocols included influencing factors to anther culture, transplanting of anther plantlets and ploidy identification. In addition, cDNA-AFLP technique was used to isolate genes associated with embryogenesis in pepper. The results were as follows:1.17genotypes of pepper were used in anther culture to study the factors affecting embryo induction and formation. These influencing factors included genotypes, hormone combination, heat shock treatment and inoculation period. The process of anther embryogenesis was observed. The results showed that culture characteristics of pepper anthers among different genotypes showed large variation, and genotype "Haihua3" was an ideal material for embryo induction, whose induction ratio was28.70%; other8genotypes showed no embryo. Adding suitable concentration of hormones in MS medium could increase induction ratio of embryo, and the combination of6-BA at0.1mg·L-1and NAA at0.5mg·L-1and KT at1.0mg·L-1was suitable for indution.6～10d heat shock treatment after inoculation could increase the ratio of induction.In this experiment (Winter pepper cultivation), Feburay and March were the optimal time for embryo induction.2. The plantlets derived from anther-embryo were used as experimental material was used to study the influence of different concentration combinations of6-BA and NAA on generational proliferation, transplantation of anther culture plantlets and estimation of the ploidy level. The results showed that MS medium added the combination of6-BA at1.0mg·L-1and NAA at0.1mg·L-1was suitable for generational proliferation. The transplanting surviral ratio of plantlets could increase to90%through domesticating plantlets for2-3d. The discrepancy of the average chloroplast number between the haploid (8.78) and the diploid (17.26) was extremely significant. Comparing with chromosome tableletting of root tip, the reliability of ploidy identification by counting chloroplast number of stoma guard cell was high, with the average accurate rate of90%. It showed that measurement of the number of stoma chloroplast in guard cell could serve as a fast and accurate method to determine the haploids and the diploid in pepper. 3. To explore the molecular mechanism of anther embryogenesis on pepper, cDNA-AFLP technique was employed to analyze the gene fragments associated with anther embryogenesis for pepper cultivar "Haihua3".113ESTs were isolated by64pairs of AFLP primer combinations,21of which were cloned and sequenced. Sequence and BLAST analysis indicated that5ESTs had higher similarity to enzymes and membrane-anchored ubiquitin-fold protein of Solanum tuberosum,2ESTs had similarity to eIF3and uncharacterized mRNA of Solanum lycopersicum,2ESTs had similarity to potassium channel both in Nicotiana tabacum and Solanum tuberosum,3ESTs had similarity to CaCM328A06, CaCM642F15and RH190I20, and3ESTs had similarity to sequence with irrelevant embryogenesis. Other6ESTs showed no homology to the known sequences in GenBank.