The Further Optimization Research Of Indica Rice (Oryza Sative L.) Anther Culture System

Anther culture was seen as a useful technique in plant breeding to enhance selection efficiency, stabilize hybrid separation, enrich genetic diversity and rapidly develop homozygous lines. However the anther culture efficiency was too low to meet the current breeding needs, especially for the indica rice. The main reasons why anther culture cannot be utilized for indica rice breeding was attribute to the strong genotype-specific feature, low callus induction rate, and high albino rate. The low efficiency can be improved by employing sensitive rice lines or modifying the culture medium. Nowadays, modifications of the genetic background and optimizations of culture medium have brought about substantial improvements in indica rice anther culture. In our study,10 kingds of rice varieties were used,39 different kinds of induction mediums were conducted, which containing different kinds of basic mediums, different carbon source, different hormones, and so on. The research studied the different anther culture effect between different materials, the effect of different sowing time in anther culture, the correlation of anther culture induction force and the composition of indica-japonica, and the most suitable basic medium, carbon source, hormones, additives for indica rice anther culture. The main results of the study were as follows:1. Indica-japonica composition of 10 tested materials were analyzed through TnDel markers, the results were as follows:indica gene frequency of A, C, shuhui’527, 9311, minghui63, Y58S were greater than or equal to 0.9, these materials could be considered as typical indica rice varieties; the indica gene frequency of D and xianhui207 were 0.774 and 0.839, these two varieties could be considered as indica rice; the indica gene frequency of linglun was 0.419, which could be considered as inter indica-japonica type; the indica gene frequency of nipponbare was 0, it was condidered as typical japonica rice. Besides the two japonica varieties （linglun and nipponbare）, the other eight varieties were indica rice. The results were consistent with Rice data （the national rice data center）, that indicate that there was a high accuracy to identify indica-japonica composition with InDel markers.2. Optimization of indica anther culture system by combining different basic culture medium, carbon source, and hormones. We can conclude that:（1） Modified Mg medium had the best effect on indica anther culture and wide adaption for different varieties; SK3 and Hes mediums were genotype-sensitive mediums which had better effects on anther callus induction on certain varieties; NB medium was not suitable for indica rice anther culture. （2） Sucrose as carbon source were greater than maltose as carbon source in callus induction rate, which indicating that sucrose had better effects on indica rice callus induction. （3） There was no significant difference between single hormone and composite hormones, which indicating that single hormone was also feasible for indica rice anther culture. （4） By analyzing the correlationthe composition of indica-japonica with anther culture induction rate and the green seedling rate, found that indica gene frequency had a significant negative correlation （-0.89*） with green seedling rate, so the more the indica component a variety had, the lower green seedling rate it would have. There was no significant correlation between the healing rate with the indica-japonica composition. （5） The average induction rate of the induction medium （modified Mg+ sugarcane+2,4-D: NAA:KT（2:1:1）） was 5.7%. In this medium, the callus induction rate was high, the green seedling rate was relatively high and the lower rate of white seedling.3. Further optimization of the indica rice anther culture system through different rice varieties, dosages of carbon source, basic media additions and light temperature conditions. The results showed that:（1） The callus induction rates of variety E （Shuhui 527）, H （9311） and B （Linglun） were significantly higher than other varities among 10 varities. （2） 30 g·L^-1 sucrose was significantly better than the mixture of 30·gL^-1 sugar and 30g·L^-1 maltose on callus induction rate; 30 g·L^-1 sucrose was better than 50g·L^-1 sucrose, but there was no significantly difference. （3） High concentration of coconut juice or potato extract was unfavourable for rice anther culture, low concentration of coconut juice promoted the anther culture. （4） Adding AgNO3 was conducive to callus induction, but light should be avoided strictly in the process of anther induction. （5） The growth and development of rice anther was easily influenced by the light and temperature during the different sowing times, thus influenced the anther culture effect. The sunny weather and the average daily temperature 25℃ or so before sampling, were beneficial to the growth and development of rice anther, which be better for anther culturet. Late sampling time, continued rainy weather and the higher temperature before sampling would affect the normal growth of anther. （6） There was no significant correlation between the healing rate and the indica-japonica composition. （7） The basic culture medium Ⅱ was better than I and III, but there was no significant difference.These results show that the medium of GMg/Ⅱ+30g·L-sucrose+2,4-D:NAA: KT （2:1:1）+low concentration coconut juice/AgNO3 has the best effect for anther culture induction rate in our experiment; choosing the higher japonica genotype varieties would improve the green seedling rate; and controlling the light and temperature conditions to ensure the good growth of anther before sampling would also be beneficial for the anther cluture ability.