Induction Of Embryogenic Callus Of Bingtang Orange (Citrus Sinensis Osbeck) And The Regeneration Of Citrus Tatter Leaf Virus-free Plantlets

Bingtang orange (Citrus sinensis Osbeck) ( BT ), selected out from Hongjiang of Hunan in 1970s, was a very seedless sweet orange cultivar, and had been widely spreaded in the south of China. The yield and quality were getting down since 1990 because of infecting of citrus tatter leaf virus and the other reasons. It was very important to find a way by using biotechnology to overcome these problems. Present, as the lack of the fundamental research about the tissue culture, the application of biotechnology on the improvement of BT had been limited. Therefore, establishing the system of induction of embryogenic calli of BT and regeneration of plantlets, then obtaining the citrus tatter leaf virus-free plantlets or improving this cultivar are significant the sustainable development of the citrus industry.In this study, the undeveloped ovules of mature fruit of BT were used as explants to induce embryogenic calli and plantlets regeneration. What's more, we identified the regenerated plantlets whether there were citrus tatter leaf virus in them by indicator (Rusk Citrange) and PAS-ELISA. The system of production of citrus tatter leaf virus-free plantlets from undeveloped ovules of BT was established. The main results are as follows:1. The regenerateratio of embryogenic calli and embryoids of BT varied on different medium and culture conditions (light or darkness). Malt extract, GAs and darkness were beneficial for induction of embryogenic callus.2. Silver nitrate was not beneficial to the induction of embryogenic callus, and it controlled the formation of embryoids. During the subculture, silver nitrate (5mg/L) was suitable for embryogenic callus from the embryoids, and 28 percent could be obtained. The embryogenic cell line of BT was established by several subcultures.3. The regeneration rate of plantlets from embryoids varied on different medium. The rate was higher (88.2%) on the MKBN (MT+ KT 0.5mg/L + BA0.5mg/L + NAA0.1mg/L) medium than on the EME1000 (MT+ ME1000mg/L)(80.9%) and EME500 (MT+ ME500mg/L)(78.7%) media.4. The rooting rate was 76.7 percent on the medium with NAA (0.5mg/L). Grafted on sour orange in vitro, the survival rate was 88.9 percent.5. The regenerated plantlets were identified whether there were citrus tatter leaf virus in them by using Rusk citrange as indicator and by PAS-ELISA. The results showed that the citrus tatter leaf virus-free plantlets of BT were obtained. Of course, it needs further study to examine citrustatter leaf virus by RT-PCR.The advantage and disadvantage of this system of virus-free plantlets from undeveloped ovules of citrus, the examining technique of citrus tatter leaf virus and the application of the virus-free plantlets in citrus industry were also discussed at the end of this paper.