Establishment And Optimization Of Regeneration System Of Tissue Culture Of Rong'an Kumquat

Kumquat(Fortunella crassifolia Swingle)is a kind of citrus plants belonged to the genus Fortunella in subfamily of Rutaceae.Because the juvenile period is short,which its seedling plant can flower one year after planted,it is a good material for studying on genes related to functional verification of woody plants,especially for the excellent material transgenic research on flower development and fruit traits related to gene function research.Although the research about Kumquat tissue culture and regeneration system have been published,there is no reports about establishment and optimization of cultivation organization efficient regeneration system about Rong'an kumquat.Rong'an kumquat used as the materials in vitro,the author studied the effects of different explants on callus induction and different concentration combinations of 6-BA and NAA on callus induction,the induction of adventitious buds,bud proliferation and rooting.The study established the efficient and fast method of the regeneration system of Rong'an Kumquat,and the results laid the foundation for the subsequent validation of related gene and molecular biology about kumquat.The main conclusions were as follow:1.The seeds sterilized in 0.1%HgCl2 for 8 min could be best sterilization effect in the sterile seeding test.The germination rate cultured in MT Medium was higher than that in MS medium.When the seeds without seed coat were cultured in MT medium adding 2.0 mg/L GA3 under dark condition,24±2? and 60%the relative air humidity,the germination rate reached 93.67%,the average length of bud was 6.45cm,and the average length of roots was 4.23 cm.2.The induction rate was the highest on callus induction with the epicotyl used for explants,which reached 100%after 30 days cultured.The best medium was the MT medium containing 6-BA 2.00 mg/L + NAA 1.00 mg/L+ Sugar 30 g/L+ Agar 8 g/L.The results showed that the induction rate reached 97.67%after 20 days.The optimal hormone combination for adventitious bud induction was 6-BA 3.00 mg/L +NAA 0.50 mg/L,and the average number of adventitious buds reached 11.90 after 30 days.The optimal hormone combination for adventitious bud induction by callus was 6-BA 2.00 mg/L+NAA 1.00 mg/L,the average number of adventitious buds was up to 8.80.The optimal hormone combination for bud induction of tender stems was 6-BA 3.00 mg/L+NAA 0.50 mg/L.The germination rate was up to 48.33%after 15 days,and the number of adventitious buds was about 2.26.3.In sterile bud proliferation,the best culture medium was MT basic medium supplemented with 6-BA 1.00 mg/L and NAA 0.40 mg/L,add 5%Coconut Juice,sugar 30 g/L,agar 8 g/L.The bud proliferation could reach about 15.11 times after 20d culture;sterile buds grew strong with dark green leaves.4.MT basic medium added NAA 0.50 mg/L+IBA 0.10 mg/L+IAA 0.40 mg/L hormone combination effect is best for rooting,the rooting rate was up to 81.11%,the average number of root strips was 3.38.In vitro grafting cultivation,Rong'an kumquat used as rootstocks in vitro grafting of buds,graft survival rate was 50%after 15 d culture.