樱桃远缘杂交种离体叶片再生植株的研究

Study on Regeneration Adventitious Buds from Leaves of F8 Cherry in Vitro

在F14培养基蔗糖浓度由原来2%增加到8%培养下,F8继代试管苗增殖倍数由2-3增加到15-20,叶片由黄绿色变为浓绿油亮,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剪取苗顶端未展开小叶片并且横切至叶中脉2-3刀后将小叶片,先在0.1%VC无菌水中浸泡5到30m in,然后接入MS或F14或WPM(附加6-BA2.0mg/L+2,4-D2.0mg/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-BA2.0mg/L+IAA2.0mg/L+蔗糖4%+琼脂7.0g/L,pH5.8)及在环境控制(15h/24h光周期变化,光照2000 lx,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+GA32.0mg/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87.1%。

The state of tube seedlings of cherry in vitro was improved remarkably by increasing concentration of sugar in medium properly. Proliferation rate was 2 -3 times to 15 -20 times that concentration of sugar from 2% to 8% 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 87. 1%. 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. 05rag/ L ＋ GA30. 2mg/L ＋ sucrose6% ＋ agar7.0g/L, pH5.8） for 30 days. （2）The expanded and furled leaves were collected from the shoots. Each immature leaf was cut several incisions just across the midrih then droped it in 0. 1% VC sterile water immediately and kept 5 -30minutes so as to absorb water and prevent to be oxidized,place them on MS,F14 or WPM medium （ addition 6 - BA2.0mg/L ＋ 2,4 - D2. 0mg/L ＋ sucrose 4% ＋ agar7.0g/L, pH5.8 ） for 3 - 4days. then tissues of edge of leaves were dedifferentiated. （3）placed leaves on WPM medium modified only with 1/2 marco（ addition 6 - BA2.0mg/L ＋ IAA2.0mg/L ＋ sucrose 4% ＋ agar7.0g/L, pH5.8 ） and enviroment control （ light intensity at 20001ux and illuminated for 15h/d. temperature was eontroled at 20℃ in light period and 15℃ in dark）for 20 -30 days. Colorless and translucent callus were induced in beginning then it＇s color turn into red with progressively rich for endogenous anthocyanins were induced. （A） placed leaves on F14 medium（addition 6 - BA0. 5rag/1, ＋ IBA0. 05mg/L ＋ GA32.0mg/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.