Studies On The Anther Culture Technology In Processing Tomato

In this study, six different combinations of processing tomato were used as materials, suitable nuclear stain was screened by using acetic acid and phenol fuchsin magenta dye, the appropriate anther inoculation time through observing the developmental stages of microspores pollen was determined, studied the low temperature pretreatment, high temperature pretreatment, centrifugation pretreatment, and mannitol pretreatment, and screened the optimal media of different additives and plant growth regulators proportions.Developmental pathways, microspore callus morphology and cytology, and histology of anther were also studied by observing the anther culture in vitro of processing tomato in order to establish the anther culture technology system of processing tomato and optimize the anther haploid breeding techniques. The results are as follows:1. Correlation studies on the microspore developmental stage and bud, anther of processing tomato.Six crosses were used as tested materials in this study. We found that the bud, anther sizes were different in the microspore of processing tomato in various developmental stages of different combinations. The anther was in the tetrad period when its length was 2-3mm; the pollen was in the middle single-core when anther length was 4-5mm; the pollen was nuninucleate stage when the anther length was 6-7mm.Anther used as testing standards seemed more accurate in the developmental stages of microspore due to the length difference of sepals buds in different combinations.2、Effects of pretreatment on the induction of processing tomato anther callus. The results of anther pretreatment in six crosses of processing tomato showed that 2 days of low temperature had a better effect;2 days or 6 days of heat pretreatment seemed better due to different genotypes; there were significant differences in the centrifugal pretreatment due to different combinations. The overall effect of mannitol pretreatment was better than low temperature, high temperature and centrifugal pretreatment. Pretreatment effect of different genotypes inconsistent took different pretreatment, so different varieties could choose different pretreatments.3、Effects of different additives on the processing tomato anther callus induction. We selected the optimal induction medium ratio by studying the effect of different additives on the processing tomato anther callus induction. Callus induction rates of all the six crosses were better when anther was cultured in treatment 29(MS + 0.1g/L activated carbon + 1.0mg/L NAA + 2.0mg/L ZT + 1.0mg/L 6-BA + 0.25 mg/L AgNO3 + 30g/L Sucrose + 10g/L Glucose + 80ml/L coconut milk), which was the optimal medium ratio.Callus induction rates of five crosses were also high when the anther was cultured in treatment 8(MS +0.1mg/L NAA + 1.5mg/L KT + 3.0mg/L ZT + 0.5mg/L 6-BA + 0.5mg/L AgNO3 + 10g/L sucrose + 10g/L glucose) and treatment 32(MS + 0.1g/L activated carbon + 1.0mg/L NAA + 1.5 mg/L KT + 1.0mg/L ZT +0.5mg/L AgNO3 + 20g/L glucose + 30g/L maltose + 40ml/L coconut milk), which were also good medium ratios. In the research on mixing different exogenous substances and growth regulators proportions of processing tomato anther, we not only found that the anther callus induction rates of same combinationwere different in different treatment, but also the anther callus induction rates of different combinations were different even in the same treatment.4、Cytology and histology observation of anther development in vitro culture of processing tomato.Through the study on the whole developmental process of anther microspores of processing tomato, results showed that there were three ways in microspore split in the processing tomato anther culture. They were multicore pollen way, equal split way, unequal split way, respectively. In the morphology and cytology observation of callus in the processing tomato anther culture, results indicated that there were four types in the formation of anther callus. First, the callus growth was slow, the color was green, the shape was circular block or granular; the second callus type had strong growth, the color was white and the texture was soft;the third callus type had relatively slow growth, dark green color, flat character, and hard texture; Fourth,the embryonic callus grew more vigorously, the color was bright yellow, the shape was crisp. The four types of callus can form four kinds of callus cells in the microscopic examination: A: cells closely packed together with deep staining; B: cells arranged loosely with dark staining; C: cell arranged more closely with shallow staining; D: cells arranged relatively loose with lighter staining.