大丽花叶片离体再生体系的建立

Establishment of Leaf in vitro Regeneration System in Dahlia pinnata

为了建立大丽花高效遗传转化体系及解决今后通过植物基因工程选育新种质的问题,以大丽花为试材,研究光照条件、叶片生理状态、激素浓度等因素对叶片再生的影响,建立以大丽花离体叶片为外植体的高频再生体系。研究结果表明：以顶部充分展开的25天叶龄的无菌苗叶片为外植体,在含KT7 mg/L＋NAA 0.05 mg/L的MS分化培养基上,暗培养15天后转到光下培养,20天后开始有不定芽直接从叶片上分化产生,出现的高峰期在接种后30~35天,芽分化率最高可达86%,平均叶片再生芽位点数为5.0。待不定芽长至2 cm以上时,将其剪下转到生根培养基1/2MS＋NAA0.1 mg/L上培养后得到生根的完整植株。

In order to establish Dahlia pinnata efficient genetic transformation system and to solve the problem of breeding of new varieties in the future through genetic engineering.In this paper,the D.pinnata leaf regeneration system was discussed.The leaves of D.pinnata were used as the experimental material.Several factors such as illumination condition,explant condition,hormone concentration and so on were investigated to optimize the regeneration system in vitro.The experimental results showed that leaf situation could greatly affect D.pinnata regeneration frequently.The best explants were those 25 days apical leaves from the cultured plantlets.The high frequency of shoot regeneration was observed when explants were cultured on MS medium supplemented with 7 mg/L KT and 0.05 mg/L NAA.The leaves were cultured in dark for 15 days firstly,and then they were transferred under light and began to directly regenerate adventitious buds in about 20 days.The maximum number of the adventitious buds was observed within 30-35 days.Maximum shoot differentiation frequency was 86%.Shoot site number per explant was 5.0.When the shoots grew to more than 2 cm high,they were cut from and transferred to the root differentiation medium 1/2MS＋0.1 mg/L NAA and developed into the whole plants.