柔枝松和红锥的组织培养研究
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摘要
木本植物具有重要的经济、生态和社会价值。但是由于木本植物生长缓慢,生长周期长,种子生产受自然环境影响大等特性,极大地限制了木本植物繁殖和优树选育。植物组织培养不仅能够实现苗木的快速高效生产,还作为林木优良品种选育的新技术,大大缩短了林木育种的周期,实现了林木遗传增益的最大化。然而木本植物,特别是优树的组织培养过程中,仍然存在许多难以解决的问题。本研究以柔枝松(Pinus flexilis James)和红锥(Castanopsis hystrix A. DC)为研究对象,以柔枝松的合子胚和幼嫩顶芽及红锥优树根蘖枝条的幼嫩茎段为外植体,对不同外植体在器官直接发生途径的组织培养过程中存在的问题、解决的方法及其作用机理进行了系统研究,最终获得不同外植体类型在不同培养阶段的最佳培养方法为：
(1)表面灭菌：柔枝松合子胚的最佳灭菌方法是以20%的NaCIO溶液浸泡20min；柔枝松幼嫩顶芽是在15%的NaCIO溶液中浸泡20 min；红锥幼嫩茎段是在冰水混合物中冷藏1 d后,在0.1%HgCl2溶液中浸泡13 min。
(2)芽分生组织诱导：将表面灭菌后的柔枝松合子胚接种于含有BA2.0 mg·L-1的GD培养基中,暗培养2周后,于光下培养2周；柔枝松幼嫩顶芽宜采用含有BA0.5mg·L-1和NAA0.1 mg·L-1的SH培养基,于光下培养4周；为了抑制红锥幼嫩茎段的褐化,可将其接种于附加PVP1.0g·L-1和BA0.5 mg·L-1的SH培养基中,暗培养1周后,转至光下培养3周,或者接种于含有AC0.5 g·L-1和BA0.5 mg·L-1的SH培养基中,并于接种后的第二天将幼嫩茎段转至培养基的另一端,第2周和第4周分别继代至含有同样成分的培养基中,共培养6周。
(3)芽分生组织的继代和增殖：对柔枝松合子胚的不定芽进行继代分化时,最佳培养基配方为附加AC0.5g·L-1、蔗糖25 g·L-1和琼脂6 g·L-1的SH培养基,pH值调整至5.2；柔枝松幼嫩顶芽的不定芽在SH基本培养基中即可伸长生长；对柔枝松合子胚和幼嫩顶芽产生的不定芽进行增殖时,将不定芽接种于含有BA2.0 mg·L-1的SH培养基中诱导4周,然后转至SH基本培养基或含有AC0.5 g·L-1的SH培养基中,进行伸长生长；红锥幼嫩茎段的不定芽继代宜采用含有BA 0.5 mg-L-1、NAA 0.1 mg·L-1和0.1 g·L-1 AC的SH培养基。
(4)不定根发生和组培苗移栽：对柔枝松的合子胚和幼嫩顶芽起源的不定芽进行不定根诱导时,将1.0-1.5 cm长的不定芽接种于1／2GD培养基中,暗培养2周后,于光下培养4周,即可。组培苗在室内炼苗1周后,洗去基部培养基,移栽于灭菌后的基质(V草炭土：V蛭石=1：1)中,成活率为64.86%。
(5)木本植物组织培养的成功是一系列因素共同作用的结果。外植体自身的个体发育阶段和生理状态是决定组织培养成功的关键,培养基种类和植物生长调节剂决定了外植体的生长和发育方向,培养基中的其他物质对外植体的生长起优化调节作用。
Woody plants have important economic, biologicl and social value, however, their multiplication and superior breeding were restricted by the characteristics of poor growth, long growth cycle, and unprecdictable seed production which was mainly depending on environmental change. Plant tissue culture could achieve the rapid, high-efficiency production of nursery stock. On the other hand, as a new technology for the breeding of superior tree species, plant tissue culture has shortened breeding period, and maximized its genetic gain. But there were still a lot of problems which were hard to solve in woody plant tissue culture, especially for superior tree species. In this paper, limber pine (Pinus flexilis James) and Castanopsis hystrix A. DC were taken as objects, the zygotic embryo and tender apical bud of limber pine and the tender stem segment derived from the stump sprouting of C. hystrix A. DC were used as explants, in order to investigate systematically the exsiting problems, their solutions, and mechinisms between them. Finally, the optimal culture methods of different explants during different culture phases were obtained:
(1) Surface sterilization:the optimal method for zygotic embryos of limber pine were soaked 20 min in 20%NaClO solution; the tender buds of limber pine were soaked 20 min in 15%NaClO solution; the tender stem segements of C. hystrix A. DC were soaked 13 min in 0.1%HgCl2 solution after storing in ice-water mixture for 1 d.
(2) Bud meristem induction:the disinfected zygotic embryos of limber pine were inoculated in GD medium supplemented with BA2.0 mg·L-1, after being cultured in darkness for 2 weeks they were exposed in light for other 2 weeks; the tender buds of limber pine were cultured in SH medium containing BA0.5 mg·L-1 and NAA0.1 mg·L-1 for 4 weeks in light; in order to reduce browning, the tender stem segments of C. hystrix A. DC were inoculated in SH medium containing PVP1.0 g·L-1and BA0.5 mg·L-1, cultured in darkness for 1 week, then exposed to lightness for another 3 weeks. Or inoculated in SH medium supplemented with AC0.5 g·L-1和BA0.5 mg·L-1, the explants were moved to the other side of medium at the 2nd d, and subcultured to the same medium at the 2nd and 4th weeks, respectively, and totally cultured for 6 weeks.
(3) Subculture and multiplication of bud meristem:SH medium supplemented with AC0.5 g·L-1, sugar25 g·L-1, agar6 g·L-1, pH was adjusted to 5.2, was suitable for the differentiation of zygotic embryos of limber pine; the adventitious shoots induced from the tender buds of limber pine were elongated in SH basal medium; and the adventitious shoots differentiated form zytotic embryos and the tender buds were multiplicated in SH medium containing BA2.0 mg·L-1, for 4 weeks. Then transferred to SH basal medium or containing AC0.5 g·L-1 for elongation; the tender stem segments of C. hystrix A. DC were subcultured in SH medium supplymented with BA 0.5 mg·L-1, NAA 0.1 mg·L-1 and AC0.1 g·L-1.
(4) Rhizogenesis and plantlet transplantation:the adventitious shoots derived from zygotic embryos and the tender buds of limber pine,1.0-1.5 cm in length, were induced for rooting in 1/2GD medium, following 2 weeks cultured in darkness and 4 weeks in light. After being acclimated 1 week in room, the rooted shoots were removed from medium, and transplanted in sterilised substrate (Vpeat: Vvermiculite=1:1), the survival rate was 64.86%。
(5) The success of woody plants tissue culture was the consequence of a series of factors co-acting. The ontogenic phase and physiological status of explant was the key of plant tissue culture, medium type and plant growth regulator controled the development direction of explant, and the other additions had an adjustment function on growth.
(1)表面灭菌：柔枝松合子胚的最佳灭菌方法是以20%的NaCIO溶液浸泡20min；柔枝松幼嫩顶芽是在15%的NaCIO溶液中浸泡20 min；红锥幼嫩茎段是在冰水混合物中冷藏1 d后,在0.1%HgCl2溶液中浸泡13 min。
(2)芽分生组织诱导：将表面灭菌后的柔枝松合子胚接种于含有BA2.0 mg·L-1的GD培养基中,暗培养2周后,于光下培养2周；柔枝松幼嫩顶芽宜采用含有BA0.5mg·L-1和NAA0.1 mg·L-1的SH培养基,于光下培养4周；为了抑制红锥幼嫩茎段的褐化,可将其接种于附加PVP1.0g·L-1和BA0.5 mg·L-1的SH培养基中,暗培养1周后,转至光下培养3周,或者接种于含有AC0.5 g·L-1和BA0.5 mg·L-1的SH培养基中,并于接种后的第二天将幼嫩茎段转至培养基的另一端,第2周和第4周分别继代至含有同样成分的培养基中,共培养6周。
(3)芽分生组织的继代和增殖：对柔枝松合子胚的不定芽进行继代分化时,最佳培养基配方为附加AC0.5g·L-1、蔗糖25 g·L-1和琼脂6 g·L-1的SH培养基,pH值调整至5.2；柔枝松幼嫩顶芽的不定芽在SH基本培养基中即可伸长生长；对柔枝松合子胚和幼嫩顶芽产生的不定芽进行增殖时,将不定芽接种于含有BA2.0 mg·L-1的SH培养基中诱导4周,然后转至SH基本培养基或含有AC0.5 g·L-1的SH培养基中,进行伸长生长；红锥幼嫩茎段的不定芽继代宜采用含有BA 0.5 mg-L-1、NAA 0.1 mg·L-1和0.1 g·L-1 AC的SH培养基。
(4)不定根发生和组培苗移栽：对柔枝松的合子胚和幼嫩顶芽起源的不定芽进行不定根诱导时,将1.0-1.5 cm长的不定芽接种于1／2GD培养基中,暗培养2周后,于光下培养4周,即可。组培苗在室内炼苗1周后,洗去基部培养基,移栽于灭菌后的基质(V草炭土：V蛭石=1：1)中,成活率为64.86%。
(5)木本植物组织培养的成功是一系列因素共同作用的结果。外植体自身的个体发育阶段和生理状态是决定组织培养成功的关键,培养基种类和植物生长调节剂决定了外植体的生长和发育方向,培养基中的其他物质对外植体的生长起优化调节作用。
Woody plants have important economic, biologicl and social value, however, their multiplication and superior breeding were restricted by the characteristics of poor growth, long growth cycle, and unprecdictable seed production which was mainly depending on environmental change. Plant tissue culture could achieve the rapid, high-efficiency production of nursery stock. On the other hand, as a new technology for the breeding of superior tree species, plant tissue culture has shortened breeding period, and maximized its genetic gain. But there were still a lot of problems which were hard to solve in woody plant tissue culture, especially for superior tree species. In this paper, limber pine (Pinus flexilis James) and Castanopsis hystrix A. DC were taken as objects, the zygotic embryo and tender apical bud of limber pine and the tender stem segment derived from the stump sprouting of C. hystrix A. DC were used as explants, in order to investigate systematically the exsiting problems, their solutions, and mechinisms between them. Finally, the optimal culture methods of different explants during different culture phases were obtained:
(1) Surface sterilization:the optimal method for zygotic embryos of limber pine were soaked 20 min in 20%NaClO solution; the tender buds of limber pine were soaked 20 min in 15%NaClO solution; the tender stem segements of C. hystrix A. DC were soaked 13 min in 0.1%HgCl2 solution after storing in ice-water mixture for 1 d.
(2) Bud meristem induction:the disinfected zygotic embryos of limber pine were inoculated in GD medium supplemented with BA2.0 mg·L-1, after being cultured in darkness for 2 weeks they were exposed in light for other 2 weeks; the tender buds of limber pine were cultured in SH medium containing BA0.5 mg·L-1 and NAA0.1 mg·L-1 for 4 weeks in light; in order to reduce browning, the tender stem segments of C. hystrix A. DC were inoculated in SH medium containing PVP1.0 g·L-1and BA0.5 mg·L-1, cultured in darkness for 1 week, then exposed to lightness for another 3 weeks. Or inoculated in SH medium supplemented with AC0.5 g·L-1和BA0.5 mg·L-1, the explants were moved to the other side of medium at the 2nd d, and subcultured to the same medium at the 2nd and 4th weeks, respectively, and totally cultured for 6 weeks.
(3) Subculture and multiplication of bud meristem:SH medium supplemented with AC0.5 g·L-1, sugar25 g·L-1, agar6 g·L-1, pH was adjusted to 5.2, was suitable for the differentiation of zygotic embryos of limber pine; the adventitious shoots induced from the tender buds of limber pine were elongated in SH basal medium; and the adventitious shoots differentiated form zytotic embryos and the tender buds were multiplicated in SH medium containing BA2.0 mg·L-1, for 4 weeks. Then transferred to SH basal medium or containing AC0.5 g·L-1 for elongation; the tender stem segments of C. hystrix A. DC were subcultured in SH medium supplymented with BA 0.5 mg·L-1, NAA 0.1 mg·L-1 and AC0.1 g·L-1.
(4) Rhizogenesis and plantlet transplantation:the adventitious shoots derived from zygotic embryos and the tender buds of limber pine,1.0-1.5 cm in length, were induced for rooting in 1/2GD medium, following 2 weeks cultured in darkness and 4 weeks in light. After being acclimated 1 week in room, the rooted shoots were removed from medium, and transplanted in sterilised substrate (Vpeat: Vvermiculite=1:1), the survival rate was 64.86%。
(5) The success of woody plants tissue culture was the consequence of a series of factors co-acting. The ontogenic phase and physiological status of explant was the key of plant tissue culture, medium type and plant growth regulator controled the development direction of explant, and the other additions had an adjustment function on growth.
引文
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