影响甜樱桃离体小叶片再生的关键因子研究

The Keys of Generation Adventitious Buds from Leaves of Sweet Cherry in Vitro

适当提高蔗糖浓度能明显改善甜樱桃试管苗的状态,在F14培养基蔗糖浓度由原来2%增加到6%,增殖倍数由1～2增加到25～30,叶片由黄绿无光变为浓绿油亮,保持天数由原来的15d增加到70d。经过4步骤程序培养可以获得小叶片再生,即:①将普通继代试管苗接入F14(附加6-BA0.5mg/L+IBA0.05mg/L+GA30.2mg/L+蔗糖6%+琼脂7.0g/L,pH5.8)继代培养30d,获得小叶型高分化试管苗。②剪取并且横切2～3刀后将小叶片,先在0.1%VC无菌水中浸泡1到30min,然后接入MS或F14或WPM(附加6-BA2mg/L+2,4-D2mg/L+蔗糖4%+琼脂7.0g/L,pH5.8)进行光照培养3～4d,获得剪切口组织脱分化的小叶片。③转接1/2大量元素的WPM培养基(附加6-BA2mg/L+I-AA2mg/L+蔗糖4%+琼脂7.0g/L,pH5.8)及在环境控制(15h/24h日光周期变化,光照2000lux,温度20℃;黑暗,温度15℃)下培养20～30d,得到诱导出红色愈伤组织(含有花色苷)的小叶片。④转接F14培养基(附加6-BA0.5mg/L+IBA0.05mg/L+GA32mg/L+蔗糖6%+琼脂7.0g/L,pH5.8)培养20d后得到由红色愈伤组织中萌发出不定芽,同时颜色变淡。小叶片再生不定芽率可达91%。

The state of tube seedlings of cherry in vitro was improved remarkably by increasing concentration of sugar in medium properly. Proliferation rate was 1-2 times to 25-30 times that concentration of sugar from 2% to 6% in F14 medium and color of leaf from dim yellow-green into luster deep green, keep life from 15 days to 70 days. Adventitious shoots were induced from leaves after processed 4 stages culture. The regeneration rate is 91%. It was made a comprehensive survey of that ① The distinguishing feature tube seedlings which leaves were little and shoots were exuberant were gained by inducing common sweet cherry subcuhuring tube seedlings by planted them to F14 medium （addition 6-BA0.5mg/L＋IBA0.05mg/L＋GA30.2mg/L＋sucrose6%＋agar7.0g/L, pH5.8） for 30 /lays. ②The expanded and furled leaves were collected from the shoots. Each immature leaf was cut several times just across the midrib then dropped it in 0.1%VC sterile water immediately and kept 1-30minutes so as to absorb water and prevent to be oxidized, place them on MS,F14 or WPM medium （addition 6-BA2mg/L＋2,4-D2mg/L＋sucrose 4%＋ agar7.0g/L, pH5.8）for 3-4days. Then tissues of edge of leaves were dedifferentiated. ③placed leaves on WPM medium modified only with 1/2 macro （addition 6-BA2mg/L＋IAA2mg/L＋sucrose 4%＋agar7.0g/L, pH5.8）and environment control （light intensity at 2000lux and illuminated for 15h/d.temperature was controlled at duced in 20℃ in light period and 15℃ in dark）for 20-30 days. Colorless and translucent callus were inbeginning then it＇s color turn into red with progressively rich for endogenous anthocyanins were induced. ④placed leaves on F14 medium （addition 6-BA0.5mg/L＋IBA0.05mg/L＋GAC2mg/L＋ sucrose 6%＋ agar 7.0g/L, pH5.8）for 20 days. Then adventitious shoots were regenerated from red callus and callus＇s red color was fading.