虎掌和猕猴桃组织培养的研究
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摘要
以虎掌(Pinellia pedatisecta)下胚轴、子叶和秦美猕猴桃(Actinidia deliciosa (Qinmei))叶片、叶柄等外植体为材料，开展其组织培养和植株再生的研究。
(一)虎掌组织培养的研究
以虎掌下胚轴、子叶等外植体进行组织培养，结果显示：(1)下胚轴是进行虎掌快速繁殖的较好材料。下胚轴外植体在MS附加BA0.5mg/L、NAA0.5mg/L的培养基上，经7d培养可产生愈伤组织，进一步培养分化出不定芽，25d时观察显示虎掌下胚轴不定芽分化率达100％，平均每个下胚轴外植体产生不定芽4.2个。当不定芽长2-3cm高时，将其切下，转入1/2MS附加NAA0.2mg/L的生根培养基上，两周后试管苗基部生根成苗。生根试管苗移栽到蛭石：腐殖质土=1：1的基质中，试管苗成活率达72％。研究同时表明在虎掌下胚轴不定芽诱导过程中，与KT、ZT相比，BA对下胚轴不定芽分化的促进作用强。在虎掌生根过程中，1/2MS培养基、暗培养利于虎掌试管苗生根。(2)实验研究了不同激素种类及组合对子叶外植体愈伤组织形成的影响，结果显示：子叶外植体在MS附加2,4-D1.0mg/L、IBA0.5mg/L的培养基上，100％的外植体可产生愈伤组织，进一步的分化试验尚在进行之中。
(二)秦美猕猴桃组织培养的研究
以秦美猕猴桃叶片、叶柄为外植体进行组织培养，结果显示：(1)接种猕猴桃叶片切块在MS附加ZT1.0mg/L、NAA0.5mg/L培养基上，先形成愈伤组织，进而分化出不定芽，不定芽分化率达100％，平均每个叶片外植体可产生不定芽3.54个。当不定芽长至2-3cm高时，将其切下，转入培养基MS+BA0.3mg/L+IBA0.2mg/L上，即可获得完整的再生植株。对于猕猴桃叶片不定芽的分化，ZT的诱导作用最强，BA次之，KT的诱导效果最不明显。另外，叶片在MS附加BA(0.5-4.0mg/L)、2,4-D(0.5-4.0mg/L)培养基上均可产生淡黄色或绿色的愈伤组织。这种愈伤组织在MS附加ZT1.0mg/L+NAA0.5mg/L培养基上经2-3次继代培养后，愈伤组织也可分化出不定芽。在叶片愈伤组织的诱导过程中，BA/2,4-D比值高有利于芽的分化，BA/2,4-D比值低有利于愈伤组织的形成。(2)接种猕猴桃叶柄在MS附加BA1.0mg/L、IBA0.5mg/L的培养基上，经10d培养，100％的外植体形成愈伤组织，再经20d培养，愈伤组织可产生不定芽，不定芽分化率为37.5％，平均每个叶柄外植体可产生不定芽1.76个。当不定芽长至2-3cm高时，将其转入生根培养基中，经两周培养可生根成苗。
虎掌、秦美猕猴桃组织培养和植株再生系统建立不仅可为进一步利用现代
生物技术改良其品种、加速育种进程、促进优良种苗的快速繁殖与利用等奠定
基础，更能为进一步研究虎掌、猕猴桃的发育生理、细胞分化以及形态建成等
许多基础理论问题提供新的实验系统。
The tissue culture and plant generation were studied in the present paper by using hypocotyls and cotyledons of Pinellia pedatisecta and the leaves and petioles of Actinidia deliciosa(Qinmei). 1. Studies on the tissue culture of Pinellia pedatisecta
The hypocotyls, cotyledons of Pinellia pedatisecta were used as explants for tissue culture and the results showed:
(1) The hypocotyls were the better explants for propagation of Pinellia pedatisecta. The explants of hypocotyls could produce calli when cultured on MS medium supplemented with BA 0.5 mg/L, NAA 0.5 mg/L for 7 days and then the calli could differentiate into adventitious buds later. The differentiation rate of adventitious buds could be up to 100 % after 25 days, and there were about 4.2 adventitious buds produced from each hypocoryl. When the adventitious buds grew to 2-3 cm in height, they were excised and inoculated on 1/2 MS medium supplemented with NAA 0.2 mg/L. After cultured for two weeks, the plantlets rooted. They were transferred into the substrate (vermiculiterhumus = 1:1),and the survival rate of the regenerated plantlets was 72 %. The studies also showed that, BA had the better effects on the promoting of the adventitious buds differentiation of the hypocotyls than KT and ZT. 1/2 MS medium and darkness benefited the rooting of Pinellia pedatisecta in the process of tissue culture.
(2) All the explants could produce calli in 25 days after they were cultured on differentiation medium supplemented with MS+ 2,4-D 1.0 mg/L+ IBA 0.5 mg/L. The effects of hormones on the callus formation were also studied in this paper. The studies of the differentiation of adventitious buds are undergoing.
2.Studies on the tissue culture of Actinidia deliciosa(Qinmei)
The leaves and the petioles of Actinidia deliciosa(Qinmei) were used as explants and the result showed:
(1) Calli were produced and further differentiated when the leaves had been cultured on MS medium supplemented with ZT 1.0 mg/L, NAA 0.5 mg/L for 20 days. The differentiation rate was 100 % and there was about 3.54 adventitious buds produced
per explant. For the induction rate of adventitious buds of the leaves, the effect of ZT was the best, BA followed, but KT was not obvious. The leaves could produce light-yellow or green calli when cultured on MS medium supplemented with BA ( 0.5 -4.0mg/L) and 2,4-D(0.5-4.0 mg/L). The calli produced adventitious buds after they had been subcultured on MS medium supplemented with ZT 0.5 mg/L and NAA 0.5mg/L for 2-3 times culture. The higher ratio of BA and 2,4-D benefited the formation of the buds and the lower ratio of BA and 2,4-D benefited the formation of the calli in the process of leaf callus induction.
(2) All the'petioles produced the calli when cultured on MS medium supplemented with BA 1.0 mg/L and IBA 0.5 mg/L after 10 days. When the calli were subcultured on this medium for 20 days, the adventitious buds were differentiated and the rate was 37.5 %. About 1.76 adventitious buds were produced per petiole. The effect of BA on the regeneration of adventitious buds was better than KT and ZT. The combination of BA and IAA, IBA or NAA could significantly improve the regeneration rate of the petioles.
The studies on the tissue culture and the plant regeneration of Pinellia pedatisecta and Actinidia deliciosa(Qinmei) supply not only the bases for the improvement of breed, acceleration of breeding and rapid propagation of Pinellia pedatisecta and Actinidia deliciosa(Qinmei).established, but also a new experimental system for many foundational theory study, such as physiologic development, cell differentiation and morphogenesis etc.
(一)虎掌组织培养的研究
以虎掌下胚轴、子叶等外植体进行组织培养，结果显示：(1)下胚轴是进行虎掌快速繁殖的较好材料。下胚轴外植体在MS附加BA0.5mg/L、NAA0.5mg/L的培养基上，经7d培养可产生愈伤组织，进一步培养分化出不定芽，25d时观察显示虎掌下胚轴不定芽分化率达100％，平均每个下胚轴外植体产生不定芽4.2个。当不定芽长2-3cm高时，将其切下，转入1/2MS附加NAA0.2mg/L的生根培养基上，两周后试管苗基部生根成苗。生根试管苗移栽到蛭石：腐殖质土=1：1的基质中，试管苗成活率达72％。研究同时表明在虎掌下胚轴不定芽诱导过程中，与KT、ZT相比，BA对下胚轴不定芽分化的促进作用强。在虎掌生根过程中，1/2MS培养基、暗培养利于虎掌试管苗生根。(2)实验研究了不同激素种类及组合对子叶外植体愈伤组织形成的影响，结果显示：子叶外植体在MS附加2,4-D1.0mg/L、IBA0.5mg/L的培养基上，100％的外植体可产生愈伤组织，进一步的分化试验尚在进行之中。
(二)秦美猕猴桃组织培养的研究
以秦美猕猴桃叶片、叶柄为外植体进行组织培养，结果显示：(1)接种猕猴桃叶片切块在MS附加ZT1.0mg/L、NAA0.5mg/L培养基上，先形成愈伤组织，进而分化出不定芽，不定芽分化率达100％，平均每个叶片外植体可产生不定芽3.54个。当不定芽长至2-3cm高时，将其切下，转入培养基MS+BA0.3mg/L+IBA0.2mg/L上，即可获得完整的再生植株。对于猕猴桃叶片不定芽的分化，ZT的诱导作用最强，BA次之，KT的诱导效果最不明显。另外，叶片在MS附加BA(0.5-4.0mg/L)、2,4-D(0.5-4.0mg/L)培养基上均可产生淡黄色或绿色的愈伤组织。这种愈伤组织在MS附加ZT1.0mg/L+NAA0.5mg/L培养基上经2-3次继代培养后，愈伤组织也可分化出不定芽。在叶片愈伤组织的诱导过程中，BA/2,4-D比值高有利于芽的分化，BA/2,4-D比值低有利于愈伤组织的形成。(2)接种猕猴桃叶柄在MS附加BA1.0mg/L、IBA0.5mg/L的培养基上，经10d培养，100％的外植体形成愈伤组织，再经20d培养，愈伤组织可产生不定芽，不定芽分化率为37.5％，平均每个叶柄外植体可产生不定芽1.76个。当不定芽长至2-3cm高时，将其转入生根培养基中，经两周培养可生根成苗。
虎掌、秦美猕猴桃组织培养和植株再生系统建立不仅可为进一步利用现代
生物技术改良其品种、加速育种进程、促进优良种苗的快速繁殖与利用等奠定
基础，更能为进一步研究虎掌、猕猴桃的发育生理、细胞分化以及形态建成等
许多基础理论问题提供新的实验系统。
The tissue culture and plant generation were studied in the present paper by using hypocotyls and cotyledons of Pinellia pedatisecta and the leaves and petioles of Actinidia deliciosa(Qinmei). 1. Studies on the tissue culture of Pinellia pedatisecta
The hypocotyls, cotyledons of Pinellia pedatisecta were used as explants for tissue culture and the results showed:
(1) The hypocotyls were the better explants for propagation of Pinellia pedatisecta. The explants of hypocotyls could produce calli when cultured on MS medium supplemented with BA 0.5 mg/L, NAA 0.5 mg/L for 7 days and then the calli could differentiate into adventitious buds later. The differentiation rate of adventitious buds could be up to 100 % after 25 days, and there were about 4.2 adventitious buds produced from each hypocoryl. When the adventitious buds grew to 2-3 cm in height, they were excised and inoculated on 1/2 MS medium supplemented with NAA 0.2 mg/L. After cultured for two weeks, the plantlets rooted. They were transferred into the substrate (vermiculiterhumus = 1:1),and the survival rate of the regenerated plantlets was 72 %. The studies also showed that, BA had the better effects on the promoting of the adventitious buds differentiation of the hypocotyls than KT and ZT. 1/2 MS medium and darkness benefited the rooting of Pinellia pedatisecta in the process of tissue culture.
(2) All the explants could produce calli in 25 days after they were cultured on differentiation medium supplemented with MS+ 2,4-D 1.0 mg/L+ IBA 0.5 mg/L. The effects of hormones on the callus formation were also studied in this paper. The studies of the differentiation of adventitious buds are undergoing.
2.Studies on the tissue culture of Actinidia deliciosa(Qinmei)
The leaves and the petioles of Actinidia deliciosa(Qinmei) were used as explants and the result showed:
(1) Calli were produced and further differentiated when the leaves had been cultured on MS medium supplemented with ZT 1.0 mg/L, NAA 0.5 mg/L for 20 days. The differentiation rate was 100 % and there was about 3.54 adventitious buds produced
per explant. For the induction rate of adventitious buds of the leaves, the effect of ZT was the best, BA followed, but KT was not obvious. The leaves could produce light-yellow or green calli when cultured on MS medium supplemented with BA ( 0.5 -4.0mg/L) and 2,4-D(0.5-4.0 mg/L). The calli produced adventitious buds after they had been subcultured on MS medium supplemented with ZT 0.5 mg/L and NAA 0.5mg/L for 2-3 times culture. The higher ratio of BA and 2,4-D benefited the formation of the buds and the lower ratio of BA and 2,4-D benefited the formation of the calli in the process of leaf callus induction.
(2) All the'petioles produced the calli when cultured on MS medium supplemented with BA 1.0 mg/L and IBA 0.5 mg/L after 10 days. When the calli were subcultured on this medium for 20 days, the adventitious buds were differentiated and the rate was 37.5 %. About 1.76 adventitious buds were produced per petiole. The effect of BA on the regeneration of adventitious buds was better than KT and ZT. The combination of BA and IAA, IBA or NAA could significantly improve the regeneration rate of the petioles.
The studies on the tissue culture and the plant regeneration of Pinellia pedatisecta and Actinidia deliciosa(Qinmei) supply not only the bases for the improvement of breed, acceleration of breeding and rapid propagation of Pinellia pedatisecta and Actinidia deliciosa(Qinmei).established, but also a new experimental system for many foundational theory study, such as physiologic development, cell differentiation and morphogenesis etc.
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