WO2017120986A1 - 一种摩帝类兜兰优质种苗组织培养快速繁殖方法 - Google Patents

一种摩帝类兜兰优质种苗组织培养快速繁殖方法 Download PDF

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WO2017120986A1
WO2017120986A1 PCT/CN2016/072150 CN2016072150W WO2017120986A1 WO 2017120986 A1 WO2017120986 A1 WO 2017120986A1 CN 2016072150 W CN2016072150 W CN 2016072150W WO 2017120986 A1 WO2017120986 A1 WO 2017120986A1
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medium
buds
days
culture
paphiopedilum
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PCT/CN2016/072150
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English (en)
French (fr)
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曾宋君
罗白雪
江南
傅艳燕
周慧君
吴坤林
张建霞
段俊
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中国科学院华南植物园
东莞市农业科学研究中心
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Priority to JP2016574460A priority Critical patent/JP6483163B2/ja
Publication of WO2017120986A1 publication Critical patent/WO2017120986A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture

Definitions

  • the invention belongs to the field of plant biotechnology, and particularly relates to a rapid propagation method for tissue culture of high quality seedlings of the Mori.
  • Paphiopedilum Maudiae is a cross between P. lawrenceanum and P. callosum. With this as a parent, many hybrid offspring have been bred.
  • P.Maudiae type a type of Paphiopedilum with a similar pattern to the flowers of Paphiopedilum, which is called “P.Maudiae type”, is very rich in color. From purple black, reddish brown to greenish white, the spots and lines are also very rich. It is the main variety of Paphiopedilum in the international and domestic markets.
  • the conventional breeding of the Modi Paphiopedilum can be used as a ramets, but the breeding speed is slow and the reproduction rate is low, which is far from meeting the needs of the commercial market.
  • the object of the present invention is to provide a rapid propagation method for the tissue culture of the high-quality seedlings of the Paphiopedilum, and to carry out the seedlings of the clonal traits of the excellent strains of the Paphiopedilum.
  • Induction of adventitious buds and proliferation of cluster buds The lateral buds of Paphiopedilum moth orchids were excised as explants. After disinfection, they were inoculated into induction medium to induce adventitious buds, and then the induced adventitious buds were transferred to Proliferation culture is carried out on the proliferation medium to obtain cluster buds, and the induction medium and the proliferation medium are 3.0 to 10.0 mg per liter, 0.5 to 1.0 mg of naphthaleneacetic acid, and 100 to 200 ml of coconut milk.
  • Rooting and strong seedling culture inoculation of the obtained shoot buds into the strong seedling medium for strong seedlings, obtaining thick buds, then cutting them into single buds, transferring them to rooting medium for rooting and obtaining
  • the strong seedling medium contains 1.0-2.0 mg of 6-benzyl hydrazine, 0.5-1.0 mg of naphthaleneacetic acid, 100-200 ml of coconut milk, 150-200 mg of sodium dihydrogen phosphate, and 20-30 of sucrose.
  • the balance is 1/2MS medium, pH 5.8-6.0;
  • the rooting medium contains 0.3-benzyl hydrazine 0.3-0.5 mg per liter, naphthaleneacetic acid 0.5-1.0 mg, coconut juice 100-200 ml, sodium dihydrogen phosphate 150-200 mg, banana homogenate 50-150 g, activated carbon 0.1-1.0 g, sucrose 20-30 g and 6-7 g agar, the balance is 1/2 MS medium, pH 5.8 to 6.0;
  • the step a is inoculated into the induction medium, and transferred to the proliferation medium for proliferation culture, and the step b is used to inoculate the shoot buds into the strong seedling medium to grow the seedlings and cut the cluster buds into single buds.
  • the rooting culture is carried out in a rooting medium, and the culture temperature is 24 to 28 ° C, the illuminance is 1500 to 2000 lx, and the illumination is 12 to 16 hours/day.
  • the side shoots of the Morin type Paphiopedilum are cut out as explants, and after being disinfected, the explants are obtained by the following methods:
  • the disinfection is specifically as follows: the excised explants are firstly soaked in a 75% alcohol aqueous solution for 10 to 30 seconds on a super clean bench, and then soaked for 5 to 10 minutes with a 1.0% effective chlorine hypochlorous acid solution. Rinse with sterile water for 4 to 5 times, then disinfect with a mass fraction of 0.1% mercury solution for 5 to 10 minutes, rinse with sterile water for 4 to 5 times, then cut off the leaves and place them in a 1500-2000 dilution of streptomycin. Oscillating in an aqueous solution for 24 hours, Disinfected explants. This treatment can make the sterilization success rate 50-60%, thus effectively solving the problem of exhumation of explants.
  • the MS medium is an internationally-used medium, and its composition and configuration method are described in Murashige T, Skoog F (1962) (A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15: 473-497).
  • 1/2 MS medium means that the amount of a large amount of elements in the MS medium is 1/2 of the original amount, and the remaining components are unchanged.
  • the invention adopts a series of chemical treatments on the mother plant of the Modi type, adopting the new lateral bud as the explant, adopting the unique medium for the induction of adventitious buds, the proliferation of the buds and the rooting culture to obtain the high-quality seedlings quickly.
  • the method of breeding provides an effective way to meet the needs of the market for high-quality seedlings of the Modi type Paphiopedilum.
  • the technology is practical and has high application value.
  • the Paphiopedilum SCBG Miracle is a mother plant with strong plant growth and rounded shape. 35 days before the explants (side buds) were cut, the plants were irrigated with a 500-fold diluted aqueous solution of carbendazim and sprayed with the leaves. After 5 days, they were watered with a 500-fold diluted aqueous solution of 35% ketobacter wettable powder. The leaves were sprayed, and after another 5 days, the leaves were sprayed once with a 3000-fold diluted aqueous solution of 72% agricultural streptomycin soluble powder and sprayed with leaves.
  • the above steps were repeated once every 5 days, and 5 days after the last irrigation of the agricultural streptomycin, the lateral buds having a length of 2.5 cm were excised as explants with a scalpel that had been sterilized by a volume fraction of 75% alcohol solution.
  • the excised explants were firstly soaked in a 75% alcohol solution for 30 seconds on a clean bench, soaked in 1.0% effective chlorine in hypochlorous acid solution for 10 minutes, rinsed with sterile water for 5 times, and reused. The mass fraction of 0.1% liters of mercury was sterilized for 10 minutes and rinsed 5 times with sterile water. The sterilized explants were obtained, and then the leaves were cut off, and the remaining lateral buds were placed in a 2000-fold diluted aqueous solution of streptomycin in a shaker for 24 hours, and then inoculated into an induction medium at a culture temperature of 28 ° C.
  • the illuminance is 2000 lx
  • the light is 16 hours/day
  • the induction medium is 10.0 mg of 6-benzyl hydrazine (6-BA) per liter, naphthaleneacetic acid (NAA).
  • 6-BA 6-benzyl hydrazine
  • NAA naphthaleneacetic acid
  • the balance is 1/2 MS medium, pH 5.8-6.0.
  • the preparation method is to mix the ingredients in the above medium uniformly. Sterilization to obtain induction medium (the same below).
  • the disinfection success rate is 60%.
  • the explants After inoculation, the explants showed obvious brown secretion formation in about 10 days. At 30 days, they were transferred to a new induction medium, and then cultured for about 25 days to form adventitious buds on the explants. The adventitious buds were then inoculated into a proliferation medium containing 4.0 mg of 6-benzyl hydrazine (6-BA), 1.0 mg of naphthaleneacetic acid (NAA), and 200 g of coconut juice per liter.
  • 6-BA 6-benzyl hydrazine
  • NAA naphthaleneacetic acid
  • the multiplication coefficient can reach 3.0 times, and the shoots are obtained.
  • the shoot buds were inoculated into the strong seedling culture medium, the culture temperature was 28 ° C, the illumination was 2000 lx, the light was 16 hours/day, and after 60 days, the cluster buds with a height of 3.0 cm were cut into single buds and then transferred to the rooting medium for rooting culture.
  • the strong seedling medium contains 6-benzyl hydrazine (6-BA) 1.5 mg per naliter, naphthalene Acetic acid (NAA) 0.75 mg, coconut milk 150 ml, sodium dihydrogen phosphate 175 mg, sucrose 20 g and agar 6 g, the balance is 1/2 MS medium, pH 5.8-6.0, the preparation method is the ingredients in the above medium After mixing uniformly, sterilization and sterilization are used to obtain a strong seedling medium (the same applies hereinafter); the rooting medium contains 0.5 mg of 6-benzyl hydrazine (6-BA) per liter, 1.0 mg of naphthalene acetic acid (NAA), and 200 g of coconut water.
  • 6-benzyl hydrazine (6-BA) 1.5 mg per naliter
  • NAA naphthalene Acetic acid
  • coconut milk 150 ml sodium dihydrogen phosphate 175 mg
  • sucrose 20 g and agar 6 g the balance is 1/2 MS medium, pH 5.
  • the rooting medium is obtained by sterilization and sterilization (the same below).
  • the whole bottle with a height of about 6 cm will be transferred to a greenhouse with natural light for 20 days, then taken out from the glass bottle, the root medium is washed, and the phytolith is imported (imported from Japan): bark press In a mixed matrix with a mass ratio of 3:1, proper ventilation and sufficient humidity can be achieved, and the survival rate of transplanting can reach 95%.
  • the Paphiopedilum SCBG Yunzhijun is a mother plant with strong plant growth and rounded shape. 35 days before the explant (side bud) was cut, the carbendazim 600-fold diluted aqueous solution was used to irrigate and spray the leaves, and after 5 days, it was watered with a 600-fold diluted aqueous solution of 35% ketobacter wettable powder. The leaves were sprayed, and after another 5 days, the leaves were sprayed once with a 3500-fold diluted aqueous solution of 72% agricultural streptomycin soluble powder and sprayed with leaves.
  • the above steps were repeated every 5 days, and 5 days after the last application of the streptomycin for agricultural use, the lateral buds having a length of 2.5 cm were excised as explants with a scalpel having a volume fraction of 75% alcohol aqueous solution.
  • the excised explants were firstly soaked in a 75% alcohol solution for 20 seconds on a clean bench, then immersed in 1.0% effective chlorine in hypochlorous acid solution for 7.5 minutes, rinsed with sterile water for 5 times, and reused. The mass fraction of 0.1% liters of mercury was sterilized for 7.5 minutes and rinsed 5 times with sterile water. The sterilized explants were obtained, and then the leaves were cut off. The remaining lateral buds were placed in a 1750-fold diluted aqueous solution of streptomycin in a shaker for 24 hours, and then inoculated into an induction medium at a culture temperature of 24 ° C. The illuminance is 1500 lx, and the illumination is 12 hours/day.
  • the induction medium contains 7.5 mg of 6-benzyl hydrazine (6-BA), 0.75 mg of naphthaleneacetic acid (NAA), 150 ml of coconut juice, and sodium dihydrogen phosphate 175 per liter.
  • 6-BA 6-benzyl hydrazine
  • NAA naphthaleneacetic acid
  • Mg 30 grams of sucrose and 7 grams of agar, the balance is 1/2 MS medium, pH 5.8 ⁇ 6.0
  • the preparation method is to mix the ingredients in the above medium, disinfection and sterilization to obtain induction medium (the same below) .
  • the disinfection success rate is 55%.
  • the explants After inoculation, the explants showed obvious brown secretion formation in about 10 days. At 30 days, they were transferred to a new induction medium, and then cultured for about 28 days to form adventitious buds on the explants. The adventitious buds were then inoculated into a proliferation medium containing 10 mg of 6-benzyl hydrazine (6-BA), 0.5 mg of naphthaleneacetic acid (NAA), and 100 g of coconut water per liter.
  • 6-BA 6-benzyl hydrazine
  • NAA naphthaleneacetic acid
  • the shoot buds were inoculated into the strong seedling culture medium, and the culture temperature was 24 ° C, and the illuminance was 1500 lx. After 12 hours of light, the clustered shoots with a height of 2.5 cm were cut into single shoots after 60 days, and then transferred to rooting medium for rooting culture at a temperature of 24 ° C, an illuminance of 1500 lx, a light of 12 hours/day, and a height of 5.5 cm after 60 days.
  • the strong seedling medium contains 6 mg of 6-benzyl hydrazine (6-BA), 1 mg of naphthaleneacetic acid (NAA), 200 ml of coconut juice, 200 mg of sodium dihydrogen phosphate, 30 g of sucrose per liter.
  • the balance is 1/2 MS medium, pH 5.8-6.0;
  • the rooting medium contains 0.6 mg of 6-benzyl hydrazine (6-BA) per liter, 0.75 mg of naphthaleneacetic acid (NAA), coconut 150 ml of juice, 175 mg of sodium dihydrogen phosphate, 100 g of banana homogenate, 0.5 g of activated carbon, 30 g of sucrose and 7 g of agar, the balance being 1/2 MS medium, pH 5.8-6.0;
  • the Paphiopedilum SCBG Red Jewel is a mother plant with strong plant growth and rounded shape. 35 days before the explants (side buds) were cut, firstly sprayed with 800 times diluted aqueous solution of carbendazim and sprayed the leaves, and after 5 days, watered with 800 times diluted aqueous solution of 35% ketobacter wettable powder. The leaves were sprayed, and after another 5 days, the leaves were sprayed once with a 4000-fold diluted aqueous solution of 72% agricultural streptomycin soluble powder and sprayed with leaves.
  • the excised explants were firstly soaked in a 75% alcohol solution for 10 seconds on a clean bench, then soaked in 1.0% effective chlorine in hypochlorous acid for 5 minutes, rinsed 4 times with sterile water, and reused. The mass fraction was diluted with 0.1% liters of mercury for 5 minutes and rinsed 4 times with sterile water. The sterilized explants were obtained, and then the leaves were cut off. The remaining lateral buds were placed in a 2000-fold diluted aqueous solution of streptomycin in a shaker for 24 hours, and then inoculated into an induction medium at a culture temperature of 26 ° C, illuminance.
  • the induction medium is 3 mg of 6-benzyl hydrazine (6-BA), 0.5 mg of naphthaleneacetic acid (NAA), 100 ml of coconut milk, 200 mg of sodium dihydrogen phosphate, 30 g of sucrose and 6.5 g of agar per liter.
  • the balance is 1/2 MS medium, pH 5.8-6.0, and the preparation method is that the components in the above medium are uniformly mixed, and then sterilized to obtain an induction medium (the same below).
  • the disinfection success rate is 50%.
  • the explants After inoculation, the explants showed obvious brown secretions in about 10 days. At 30 days, they were transferred to a new induction medium, and after 30 days of culture, adventitious buds were formed on the explants. The adventitious buds were then inoculated into a proliferation medium containing 3.0 mg of 6-benzyl hydrazine (6-BA), 1.0 mg of naphthaleneacetic acid (NAA), and 200 g of coconut juice per liter.
  • 6-BA 6-benzyl hydrazine
  • NAA naphthaleneacetic acid
  • the multiplication coefficient can reach 2.5 times, and the cluster buds are obtained.
  • the shoots were inoculated into the strong seedling culture medium, the culture temperature was 26 ° C, the illumination was 1800 lx, the light was 14 hours/day, and after 60 days, the 2 cm-high cluster buds were cut into single buds and transferred to rooting medium for rooting culture.
  • the strong seedling medium contains 6-benzyl hydrazine (6-BA) 1 mg per naliter, naphthalene Acetic acid (NAA) 0.5 mg, coconut milk 100 ml, sodium dihydrogen phosphate 150 mg, sucrose 30 g and agar 6 g, the balance is 1/2 MS medium, pH 5.8-6.0; the rooting medium contains liters per liter 6-benzyl hydrazine (6-BA) 0.3 mg, naphthaleneacetic acid (NAA) 0.5 mg, coconut milk 100 ml, sodium dihydrogen phosphate 150 mg, banana homogenate 50 g, 0.1 g activated carbon, sucrose 30 g and agar 6.5 g The balance is 1/2 MS medium, pH 5.8-6.0;

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Abstract

一种摩帝类兜兰优质种苗组织培养快速繁殖方法，在对摩帝类兜兰母株进行一系列药剂处理的基础上，以新生侧芽为外植体，采用独特的培养基进行不定芽的诱导、丛生芽增殖和生根培养获得优质种苗。该方法切实可行，应用价值高。

Description

一种摩帝类兜兰优质种苗组织培养快速繁殖方法 技术领域：
本发明属于植物生物技术领域，具体涉及一种摩帝类兜兰优质种苗组织培养快速繁殖方法。
背景技术：
摩帝兜兰(Paphiopedilum Maudiae)是由劳氏兜兰(P.lawrenceanum)和胼胝兜兰(P.callosum)杂交育成。以其为亲本又育成了许多杂交后代，市场上把具有与摩帝兜兰花朵形态相似的一类兜兰，统称为“摩帝类兜兰(P.Maudiae type)”，他们的花色十分丰富，从紫黑色、红褐色至绿白色均有，斑点和线条形态也十分丰富，是国际、国内市场上商品兜兰的主要品种。摩帝类兜兰的常规繁殖可采用分株，但繁殖速度慢，繁殖率低，远不能满足商业市场的需要。目前，世界市场上销售的摩帝类兜兰多是采用无菌播种而来。但由于摩帝类兜兰是杂交种，无菌播种后代分离大，不能保持亲本的优良性状获得性状统一的种苗，严重地制约了它的规模化生产。同时，由于兜兰在组织培养时，外植体去污难及增殖速度慢等原因，其组织培养难度极大，世界上还没有从温室中选取优良株系作外植体进行摩帝类兜兰组织培养快速繁殖的报道。
发明内容：
本发明的目的是提供一种摩帝类兜兰优质种苗组织培养快速繁殖方法，从而进行摩帝类兜兰优良株系的无性克隆获得性状统一的种苗。
本发明的摩帝类兜兰优质种苗组织培养快速繁殖方法，其特征在于，包括以下步骤：
a、不定芽的诱导和丛生芽的增殖：切取摩帝类兜兰的侧芽作为外植体，经消毒后，接种到诱导培养基中，诱导出不定芽，然后将诱导出的不定芽转移到增殖培养基上进行增殖培养，获得丛生芽，所述的诱导培养基和增殖培养基都为每升含有6-苄基嘌呤3.0～10.0毫克、萘乙酸0.5～1.0毫克、椰子汁100～200毫升、磷酸二氢钠150～200毫克、蔗糖20～30克和6～7克琼脂，余量为1/2MS培养基， pH 5.8～6.0；每隔60天继代一次，至第6代时，增殖系数可达到2.5～3.0倍。
b、生根壮苗培养：将增殖获得的丛生芽接种到壮苗培养基中进行壮苗，获得粗壮的丛生芽，然后将其切成单芽后转接到生根培养基中进行生根培养，获得完整植株，所述的壮苗培养基每升含有6-苄基嘌呤1.0～2.0毫克、萘乙酸0.5～1.0毫克、椰子汁100～200毫升、磷酸二氢钠150～200毫克、蔗糖20～30克和6～7克琼脂，余量为1/2MS培养基，pH 5.8～6.0；所述的生根培养基每升含有6-苄基嘌呤0.3～0.5毫克、萘乙酸0.5～1.0毫克、椰子汁100～200毫升、磷酸二氢钠150～200毫克、香蕉匀浆50～150克、活性炭0.1～1.0克、蔗糖20～30克和6～7克琼脂，余量为1/2MS培养基，pH 5.8～6.0；
c、将完整植株转移到自然光下炼苗，炼苗后，洗净其根部培养基移入植金石：树皮按质量比1-3：1的混合基质中培养获得摩帝类兜兰种苗。一般保持适当通风和足够的湿度，移栽的成活率均可达90％以上。
所述的步骤a的接种到诱导培养基中，以及转移到增殖培养基上进行增殖培养、步骤b的将丛生芽接种到壮苗培养基中进行壮苗和将丛生芽切成单芽后转接到生根培养基中进行生根培养，其培养温度24～28℃，光照度1500～2000lx，光照12～16小时/天。
所述的切取摩帝类兜兰的侧芽作为外植体，经消毒后，其外植体是通过以下方法获得的：
选择植株生长势强、花型圆整的摩帝类兜兰为母株，在切取外植体消毒的35天前，先用多菌灵的500～800倍稀释水溶液浇灌一次并喷施叶片，5天后用35％尅土菌可湿性粉剂的500～800倍稀释水溶液浇灌一次并喷施叶片，再过5天后，用72％农用链霉素可溶性粉剂的3000～4000倍的稀释水溶液浇灌一次并喷施叶片，然后每过5天，重复上述步骤一次，最后1次浇灌农用链霉素后5天，用体积分数75％酒***溶液消过毒的解剖刀切取长度1.5～2.5厘米的侧芽为外植体。
其消毒具体为：在超净工作台上将切下的外植体先用体积分数75％酒***溶液中浸泡10～30秒后，用1.0％有效氯的次氯酸溶液浸泡5～10分钟，无菌水冲洗4～5次，再用质量分数0.1％升汞溶液消毒5～10分钟，无菌水冲洗4～5次，然后切掉叶片，放置于医用链霉素的1500～2000倍稀释水溶液中振荡24小时，得到 消毒后的外植体。如此处理可以使消毒成功率为50～60％，从而有效的解决了外植体去污难的难题。
MS培养基为国际通用的培养基，其成份和配置方法见Murashige T,Skoog F(1962)(A revised medium for rapid growth and bioassay with tobacco tissue cultures.Physiol Plant 15:473–497)。1/2MS培养基是指将MS培养基中大量元素用量为原来的1/2，其余成份不变。
本发明在对摩帝类兜兰母株进行一系列药剂处理的基础上，以新生侧芽为外植体，采用独特的培养基进行不定芽的诱导、丛生芽增殖和生根培养获得优质种苗快速繁殖的方法，为满足摩帝类兜兰优质种苗市场的需要提供一条有效的途径。技术切实可行，应用价值高。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：红摩帝奇迹(Paphiopedilum SCBG Miracle)的组织培养
(1)材料选择和处理
选择植株生长势强、花型圆整的摩帝类兜兰[红摩帝奇迹(Paphiopedilum SCBG Miracle)]为母株。在切取外植体(侧芽)消毒的35天前，先用多菌灵的500倍稀释水溶液浇灌一次并喷施叶片，5天后用35％尅土菌可湿性粉剂的500倍稀释水溶液浇灌一次并喷施叶片，再过5天后，用72％农用链霉素可溶性粉剂的3000倍稀释水溶液浇灌一次并喷施叶片。然后每过5天，重复上述步骤一次，最后1次浇灌农用链霉素后5天，用体积分数75％酒***溶液消过毒的解剖刀切取长度2.5厘米的侧芽为外植体。
(2)外植体消毒
在超净工作台上将切下的外植体先用体积分数75％酒***溶液中浸泡30秒后，用1.0％有效氯的次氯酸溶液浸泡10分钟，无菌水冲洗5次，再用质量分数0.1％升汞水溶液消毒10分钟，无菌水冲洗5次。得到消毒后的外植体，然后切掉叶片，剩余的侧芽放置于医用链霉素的2000倍稀释水溶液中在摇床中振荡24小时后，再接种到诱导培养基中，培养温度28℃，光照度2000lx，光照16小时/天，所述的诱导培养基为每升含有6-苄基嘌呤(6-BA)10.0毫克、萘乙酸(NAA) 1.0毫克、椰子汁200毫升、磷酸二氢钠150毫克、蔗糖20g和琼脂6g，余量为1/2MS培养基，pH 5.8～6.0，其配制方法是将上述培养基中的成份混合均匀后，消毒灭菌得到诱导培养基(下同)。消毒成功率60％。
(3)不定芽诱导和丛生芽增殖
接种后的外植体在10天左右可见明显的褐色分泌物形成，在30天时要将转入新的诱导培养基，再培养25天左右能在外植体上形成不定芽。然后将不定芽接种到增殖培养基中进行继代增殖培养，所述的增殖培养基为每升含有6-苄基嘌呤(6-BA)4.0毫克、萘乙酸(NAA)1.0毫克、椰子汁200毫升、磷酸二氢钠150毫克、蔗糖20克和琼脂6克，余量为1/2MS培养基，pH 5.8～6.0，培养温度28℃，光照度2000lx，光照16小时/天，每隔60天继代一次，至第6代时，增殖系数可达到3.0倍，获得丛生芽。
(4)生根壮苗培养
将丛生芽接种到壮苗培养基中壮苗培养，培养温度28℃，光照度2000lx，光照16小时/天，60天后将高度3.0厘米的丛生芽切成单芽后转到生根培养基中生根培养，培养温度28℃，光照度2000lx，光照16小时/天，60天后以形成6厘米高的完整植株，所述的壮苗培养基每升含有6-苄基嘌呤(6-BA)1.5毫克、萘乙酸(NAA)0.75毫克、椰子汁150毫升、磷酸二氢钠175毫克、蔗糖20g和琼脂6g，余量为1/2MS培养基，pH 5.8～6.0，其配制方法是将上述培养基中的成份混合均匀后，消毒灭菌得到壮苗培养基(下同)；所述的生根培养基每升含有6-苄基嘌呤(6-BA)0.5毫克、萘乙酸(NAA)1.0毫克、椰子汁200毫升、磷酸二氢钠200毫克、香蕉匀浆150克、1.0克活性炭、蔗糖20克和琼脂6克，余量为1/2MS培养基，pH 5.8～6.0，其配制方法是将上述培养基中的成份混合均匀后，消毒灭菌得到生根培养基(下同)。
(5)试管苗移栽
将具6厘米左右高完整植株将培养瓶转移到具自然光的温室中炼苗20天，然后将其从玻璃瓶中取出，洗净根部的培养基，移入植金石(日本进口)：树皮按质量比为3：1的混合基质中，保持适当通风和足够的湿度，移栽的成活率均可达95％。
实施例2：绿摩帝云之君(Paphiopedilum SCBG Yunzhijun)的组织培养
(1)材料选择和处理
选择植株生长势强、花型圆整的摩帝类兜兰[绿摩帝云之君(Paphiopedilum SCBG Yunzhijun)]为母株。在切取外植体(侧芽)消毒的35天前，先用多菌灵的600倍稀释水溶液浇灌一次并喷施叶片，5天后用35％尅土菌可湿性粉剂的600倍稀释水溶液浇灌一次并喷施叶片，再过5天后，用72％农用链霉素可溶性粉剂的3500倍稀释水溶液浇灌一次并喷施叶片。然后每过5天，重复上述步骤，最后1次浇灌农用链霉素后5天，用体积分数75％酒***溶液消过毒的解剖刀切取长度2.5厘米的侧芽为外植体。
(2)外植体消毒
在超净工作台上将切下的外植体先用体积分数75％酒***溶液中浸泡20秒后，用1.0％有效氯的次氯酸溶液浸泡7.5分钟，无菌水冲洗5次，再用质量分数0.1％升汞水溶液消毒7.5分钟，无菌水冲洗5次。得到消毒后的外植体，然后切掉叶片，剩余的侧芽放置于医用链霉素的1750倍稀释水溶液中在摇床中振荡24小时后，再接种到诱导培养基中，培养温度24℃，光照度1500lx，光照12小时/天，所述的诱导培养基为每升含有6-苄基嘌呤(6-BA)7.5毫克、萘乙酸(NAA)0.75毫克、椰子汁150毫升、磷酸二氢钠175毫克、蔗糖30克和琼脂7克，余量为1/2MS培养基，pH 5.8～6.0，其配制方法是将上述培养基中的成份混合均匀后，消毒灭菌得到诱导培养基(下同)。消毒成功率55％。
(3)不定芽诱导和丛生芽增殖
接种后的外植体在10天左右可见明显的褐色分泌物形成，在30天时要将转入新的诱导培养基，再培养28天左右能在外植体上形成不定芽。然后将不定芽接种到增殖培养基中进行继代增殖培养，所述的增殖培养基为每升含有6-苄基嘌呤(6-BA)10毫克、萘乙酸(NAA)0.5毫克、椰子汁100毫升、磷酸二氢钠200毫克、蔗糖30克和琼脂7克，余量为1/2MS培养基，pH 5.8～6.0，培养温度24℃，光照度1500lx，光照12小时/天，每隔60天继代一次，至第6代时，增殖系数可达到2.8倍，获得丛生芽。
(4)生根壮苗培养
将丛生芽接种到壮苗培养基中壮苗培养，培养温度24℃，光照度1500lx， 光照12小时/天，60天后将高度2.5厘米的丛生芽切成单芽后转到生根培养基中生根培养，培养温度24℃，光照度1500lx，光照12小时/天，60天后以形成5.5厘米高的完整植株，所述的壮苗培养基每升含有6-苄基嘌呤(6-BA)2毫克、萘乙酸(NAA)1毫克、椰子汁200毫升、磷酸二氢钠200毫克、蔗糖30克和琼脂7克，余量为1/2MS培养基，pH 5.8～6.0；所述的生根培养基每升含有6-苄基嘌呤(6-BA)0.4毫克、萘乙酸(NAA)0.75毫克、椰子汁150毫升、磷酸二氢钠175毫克、香蕉匀浆100克、0.5克活性炭、蔗糖30克和琼脂7克，余量为1/2MS培养基，pH 5.8～6.0；。
(5)试管苗移栽
将具5.5厘米左右高完整植株从培养瓶转移到具自然光的温室中炼苗15天，然后将其从玻璃瓶中取出，洗净根部的培养基，移入植金石(日本进口)：树皮按质量比为2：1的混合基质中，保持适当通风和足够的湿度，移栽的成活率均可达93％。
实施例3：摩帝红宝石(Paphiopedilum SCBG Red Jewel)的组织培养
(1)材料选择和处理
选择植株生长势强、花型圆整的摩帝类兜兰[摩帝红宝石(Paphiopedilum SCBG Red Jewel)]为母株。在切取外植体(侧芽)消毒的35天前，先用多菌灵的800倍稀释水溶液浇灌一次并喷施叶片，5天后用35％尅土菌可湿性粉剂的800倍稀释水溶液浇灌一次并喷施叶片，再过5天后，用72％农用链霉素可溶性粉剂的4000倍稀释水溶液浇灌一次并喷施叶片。然后每过5天，重复上述步骤，最后1次浇灌农用链霉素后5天，用体积分数75％酒***溶液消过毒的解剖刀切取长度1.5厘米的侧芽为外植体。
(2)外植体消毒
在超净工作台上将切下的外植体先用体积分数75％酒***溶液中浸泡10秒后，用1.0％有效氯的次氯酸溶液浸泡5分钟，无菌水冲洗4次，再用质量分数0.1％升汞水溶液消毒5分钟，无菌水冲洗4次。得到消毒后的外植体，然后切掉叶片，剩余的侧芽放置于医用链霉素的2000倍稀释水溶液中在摇床中振荡24小时后，接种到诱导培养基中，培养温度26℃，光照度1800lx，光照14小时/天， 所述的诱导培养基为每升含有6-苄基嘌呤(6-BA)3毫克、萘乙酸(NAA)0.5毫克、椰子汁100毫升、磷酸二氢钠200毫克、蔗糖30克和琼脂6.5克，余量为1/2MS培养基，pH 5.8～6.0，其配制方法是将上述培养基中的成份混合均匀后，消毒灭菌得到诱导培养基(下同)。消毒成功率50％。
(3)不定芽诱导和丛生芽增殖
接种后的外植体在10天左右可见明显的褐色分泌物形成，在30天时要将转入新的诱导培养基，再培养30天左右能在外植体上形成不定芽。然后将不定芽接种到增殖培养基中进行继代增殖培养，所述的增殖培养基为每升含有6-苄基嘌呤(6-BA)3.0毫克、萘乙酸(NAA)1.0毫克、椰子汁200毫升、磷酸二氢钠150毫克、蔗糖20克和琼脂6.5克，余量为1/2MS培养基，pH 5.8～6.0，培养温度26℃，光照度1800lx，光照14小时/天，每隔60天继代一次，至第6代时，增殖系数可达到2.5倍，获得丛生芽。
(4)生根壮苗培养
将丛生芽接种到壮苗培养基中壮苗培养，培养温度26℃，光照度1800lx，光照14小时/天，60天后将高度2厘米的丛生芽切成单芽后转到生根培养基中生根培养，培养温度26℃，光照度1800lx，光照14小时/天，60天后以形成5厘米高的完整植株，所述的壮苗培养基每升含有6-苄基嘌呤(6-BA)1毫克、萘乙酸(NAA)0.5毫克、椰子汁100毫升、磷酸二氢钠150毫克、蔗糖30克和琼脂6克，余量为1/2MS培养基，pH 5.8～6.0；所述的生根培养基每升含有6-苄基嘌呤(6-BA)0.3毫克、萘乙酸(NAA)0.5毫克、椰子汁100毫升、磷酸二氢钠150毫克、香蕉匀浆50克、0.1克活性炭、蔗糖30克和琼脂6.5克，余量为1/2MS培养基，pH 5.8～6.0；。
(5)试管苗移栽
将具5厘米左右高完整植株从培养瓶转移到具自然光的温室中炼苗10天，然后将其从玻璃瓶中取出，洗净根部的培养基，移入植金石(日本进口)：树皮按质量比为1：1的混合基质中，保持适当通风和足够的湿度，移栽的成活率均可达90％。

Claims (3)

  1. 一种摩帝类兜兰优质种苗组织培养快速繁殖方法，其特征在于，包括以下步骤：
    a、不定芽的诱导和丛生芽的增殖：切取摩帝类兜兰的侧芽作为外植体，经消毒后，接种到诱导培养基中，诱导出不定芽，然后将诱导出的不定芽转移到增殖培养基上进行增殖培养，获得丛生芽，所述的诱导培养基和增殖培养基都为每升含有6-苄基嘌呤3.0～10.0毫克、萘乙酸0.5～1.0毫克、椰子汁100～200毫升、磷酸二氢钠150～200毫克、蔗糖20～30克和6～7克琼脂，余量为1/2MS培养基，pH 5.8～6.0；
    b、生根壮苗培养：将增殖获得的丛生芽接种到壮苗培养基中进行壮苗，获得粗壮的丛生芽，然后将其切成单芽后转接到生根培养基中进行生根培养，获得完整植株，所述的壮苗培养基每升含有6-苄基嘌呤1.0～2.0毫克、萘乙酸0.5～1.0毫克、椰子汁100～200毫升、磷酸二氢钠150～200毫克、蔗糖20～30克和6～7克琼脂，余量为1/2MS培养基，pH 5.8～6.0；所述的生根培养基每升含有6-苄基嘌呤0.3～0.5毫克、萘乙酸0.5～1.0毫克、椰子汁100～200毫升、磷酸二氢钠150～200毫克、香蕉匀浆50～150克、活性炭0.1～1.0克、蔗糖20～30克和6～7克琼脂，余量为1/2MS培养基，pH 5.8～6.0；
    c、将完整植株转移到自然光下炼苗，炼苗后，洗净其根部培养基移入植金石：树皮按质量比1-3：1的混合基质中培养获得摩帝类兜兰种苗。
  2. 根据权利要求1所述的摩帝类兜兰优质种苗组织培养快速繁殖方法，其特征在于，所述的步骤a的接种到诱导培养基中，以及转移到增殖培养基上进行增殖培养、步骤b的将丛生芽接种到壮苗培养基中进行壮苗和将丛生芽切成单芽后转接到生根培养基中进行生根培养，其培养温度24～28℃，光照度1500～2000lx，光照12～16小时/天。
  3. 根据权利要求1所述的摩帝类兜兰优质种苗组织培养快速繁殖方法，其特征在于，所述的切取摩帝类兜兰的侧芽作为外植体，经消毒后，其外植体是通过以下方法获得的：
    选择植株生长势强、花型圆整的摩帝类兜兰为母株，在切取外植体消毒的 35天前，先用多菌灵的500～800倍稀释水溶液浇灌一次并喷施叶片，5天后用35％尅土菌可湿性粉剂的500～800倍稀释水溶液浇灌一次并喷施叶片，再过5天后，用72％农用链霉素可溶性粉剂的3000～4000倍的稀释水溶液浇灌一次并喷施叶片，然后每过5天，重复上述步骤一次，最后1次浇灌农用链霉素后5天，用体积分数75％酒***溶液消过毒的解剖刀切取长度1.5～2.5厘米的侧芽为外植体；
    其消毒具体为：在超净工作台上将切下的外植体先用体积分数75％酒***溶液中浸泡10～30秒后，用1.0％有效氯的次氯酸溶液浸泡5～10分钟，无菌水冲洗4～5次，再用质量分数0.1％升汞溶液消毒5～10分钟，无菌水冲洗4～5次，然后切掉叶片，放置于医用链霉素的1500～2000倍稀释水溶液中振荡24小时，得到消毒后的外植体。
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