金钗石斛转基因体系的建立

The Establishment of Genetically Modified Dendrobium nobile Tissues

以金钗石斛种子为外植体建立组织培养体系,在含1.5mg/L6-BAMS培养基中添加0.5mg/LNAA和2%蔗糖能有效地诱导原球茎,诱导率达91.67%;添加0.25mg/LNAA和3%蔗糖则更适于原球茎分化形成不定芽;添加0.10mg/LNAA和2%蔗糖适宜于不定根的诱导.同时建立了根癌农杆菌(Agrobacterium tumefaciens)转化金钗石斛的方法和体系:使用根癌农杆菌EHA105侵染30min,共培养3d时,金钗石斛的根段、根尖和组培幼苗的转化率最高,而含腋芽的茎节则需要共培养5d.文章所建立的组培体系与转基因体系将为探讨金钗石斛的基因功能和调控机制等生物学问题及建立稳定转化的再生植株提供技术手段.

This study attempts the establishment of the tissue culturing system using Dendrobium nobile seeds as the starting materials. It is found that the MS medium supplemented with 1.5 mg/L 6-BA+0.5 mg/L NAA+2% sucrose is the most suitable for the induction of protocorm,showing an induction rate of 91.67% after being cultured for 15 days . Adding 0.25 mg/L NAA and 3% sucrose to the MS medium with 1.5 mg/L 6-BA is more suitable to form adventitious seedlings with dark green sturdy leaves,and the addition of 0.10 mg/L NAA and 2% sucrose to the MS medium with 1.5 mg/L 6-BA is suitable for root emergency and growth. The transgenic system for Dendrobium nobile tissues is also studied. It is found that compared with GV1301 strain,the Agrobacterium tumefaciens EHA105 strain can be used to obtain higher transformation rates. The rate of transformation using root segments,root tips and tissue-cultured seedlings as the starting materials reaches the highest when they are soaked with EHA105 for 30 minutes and then co-cultured for 3 days,but the rate of transformation using nodes with axillary buds reaches the highest if co-cultured for 5 days. The results of the present study can provide meaningful techniques for further research on Dendrobium nobile and the breeding of Dendrobium varieties.