Establishment Of The Regeneration System And Study On Genetic Transformation Of Thebroma Cacao L.

In this experiment, we established regeneration system and genetic transformation of Theohroma Cacao L. .The main results were as follows.Flower buds,seeds(with and without seed capsule), shoot tip and stem fragment derived from field grown plant were sterilized by different concentration(4-6%,v/v) of javal water(NaC1O) for different time.This paper draw conclusions according to the contamination rate,browning rate and germination capacity: (1) The proper concentration of sterilizing solution for flower buds was 5% NaC1O with sterilizing time 20 minutes (2)The proper concentration and time of sterlization for seeds with capsule were 5% NaCIO and 30 mintus;(3)The seeds without capsule were easily contaminated and brown after sterilization;(4)shoot tip and stem fragment brown severely for sterilization,This could be eased when they were treated with Vatamin C.The proper concentration and time was 4% NaCIO and 20 min.Plants were regenerated through somatic embryogenesis using staminodes of floral parts and cotyledon as explants.The optimized culture medium and condition for each stage were as follows, (1)Primary calli medium (PCG) was DKW + 2,4-D 3.0 mg/L+KT 1.0 mg/L+TDZ 0.0lmg/L +Glucose 20g/L+glutamine 250mg/L +l%agar. Explants cultured on PCG medium in the dark at 28 ?1) for 20 day .(2) Callus propagation medium (SCO) was DKW + 2,4-D 3.0 mg/L+ KT 1.0 mg/L-+Glucose 20g/L+10%CW +l%agar, callus cultured on SCG medium for 30 days in the dark ;(3) Subculture embryogenic callus onto ED medium which was DKW +Sucrose 30g/L+ Glucose 20g/L+l%agar in the dark for 150 day, and embryos at different developmental stages (globular, beart torpedo and mature) were present on individual explants at the same time.(DTransfer mature embryos to fresh PEC medium every 30 days until shoots with one or two leaves were developed.The contents of PEC medium was 1/2DKW+ KN03 0.3 g/L+AA 1000X stock solution Iml/L+Glucose320g/L+Sucrose 10g/L+l%agar. Transfer shoot- producing embryos with 2 leaves onto RD medium.Maintain cultures under light with a 16/8 photoperiod for 30 to 90 days,and regcnered plants were obtained. While RD medium were 1/2DKW+ KNOa 0.3 g/L +AA 1000X stock solution Iml/L +Glucose lOg/L+Sucrose 5g/L+IBA Img/L+IAA lmg/L+0.8%agar.Regenerated plants were obtained through fast propagation pathway using shoot tip and stem fragment axemic plant as explants.The medium for each stage were optimized. (?Multiplication medium was DKW+Glucose 10g/L+ Sucrose 20g/L+ NAA O.lmg/L+vitaminC 0.1mg/L+l%agar (2) Rooting medium was 1/2 DKW + Sucrose 20g/L +IBA 1.5mg/L+IAA lmg/L+l%agar.In the research of genetic transformation of cacao, GUS gene was used as reporter gene. Immersion time and co-culture days were optimized through Agrobacterium-mediated transfer system (LBA4404/PBI121) for genetic transformation. The proper immersion time was 20 min while proper co-culture time was 3 days.Optional pressure research was conducted on Kan calli.The optinal pressure of calli (75mg/L ) inducing was determined according to calli inducing rate and browning of explants. GUS gene of 20 resistance calli was examined by PCR,and five of the calli had positive reaction.The result showed that GUS genes had already introduced into cacao calli.