WO2020063955A1 - 一种提高组培植株诱导率的培养基及制备方法与应用 - Google Patents

一种提高组培植株诱导率的培养基及制备方法与应用 Download PDF

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WO2020063955A1
WO2020063955A1 PCT/CN2019/108963 CN2019108963W WO2020063955A1 WO 2020063955 A1 WO2020063955 A1 WO 2020063955A1 CN 2019108963 W CN2019108963 W CN 2019108963W WO 2020063955 A1 WO2020063955 A1 WO 2020063955A1
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parts
medium
induction rate
improving
tissue culture
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PCT/CN2019/108963
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English (en)
French (fr)
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宗树斌
王永平
顾立新
陈少卿
任焕焕
王静
仕明慧
沈凤
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江苏农林职业技术学院
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Publication of WO2020063955A1 publication Critical patent/WO2020063955A1/zh
Priority to ZA2021/02842A priority Critical patent/ZA202102842B/en

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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
    • A01H4/001—Culture apparatus for tissue culture
    • 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

Definitions

  • the invention belongs to the field of culture medium, and particularly relates to a culture medium for improving the induction rate of tissue culture plants, a preparation method and application thereof.
  • Blue Snowflake (Plumbago auriculata) is native to South Africa, also known as safflower, blue snow dan, safflower pine, blue jasmine and so on. It is a perennial evergreen shrub of the family safflower (blue snow family). Blue snowflakes are strong and strong, heat-resistant, more resistant to high temperature and humidity, less pests, simple management, and longer viewing period. The leaves are green, and the flowers are light and elegant. The hot summer gives a cool feeling. Potted plants can be used to decorate the living room and balcony. The mature plants are overhanging branches, suitable for large-scale container combination potting. Planting or decorating lawns. The blue snowflake flower period is the summer with few flowers, and it is a rare blue flower in nature.
  • Blue Snowflake is introduced and cultivated as a new type of excellent flower. It has been promoted and applied in some important green areas, and has played a very good landscape effect. The demand for its seedlings has increased year by year, and the market potential is huge. In addition, Blue Snowflake contains a large amount of Baihuatansu, which has a good therapeutic effect on rheumatoid joint pain, blood stasis amenorrhea, bruises, swollen poisonous malignant sore, and mange. It also has high ornamental and medicinal value. .
  • Blue Snowflake is a typical style heterotypic plant and has self-incompatibility, so it lacks pollinators.
  • the seed setting rate is very low. The seeds are relatively expensive, and the propagation coefficient is low, which makes the traditional breeding methods unable to meet the application requirements of Blue Snowflake.
  • the first object of the present invention is to provide a tissue culture medium capable of achieving a plant induction rate of more than 95%;
  • a second object of the present invention is to provide a method for preparing the medium
  • a third object of the present invention is to provide an application of the medium, which is applied to the primary cultivation of blue snowflakes, so that the blue snowflakes have a good growth state, and the number of effectively induced seedlings is greatly increased.
  • the medium for improving the induction rate of tissue culture plants of the present invention includes the following raw materials in parts by weight: 68-94 parts of WPM medium, 0.05-0.5 parts of zein, 0.1-1.5 parts of indole acetic acid, and brassinolide 0.3-1.8 parts, sucrose 23-56 parts, flavonoids 2-6 parts, calcium phosphate 6-18 parts and sodium dodecylbenzenesulfonate 32-68 parts.
  • the present invention By combining WPM medium, zeatin and sucrose, the present invention provides sufficient nutrients and improves the induction rate; at the same time, flavonoids are added, which are combined with indole acetic acid and brassinolide to increase the protein content of plants, Promote protein synthesis and increase the effective induction of seedling growth.
  • the present invention also adds calcium phosphate and sodium dodecylbenzenesulfonate. The addition of both can effectively slow down the antagonism between effective elements in WPM medium and avoid elements The occurrence of inter-inhibition effects promotes the mutual absorption of elements, so that the plant fully absorbs nutrients and improves the survival rate.
  • the flavonoid compound may be 3 to 4.5 parts, and may be isoflavone, isoflavone, chalcone, or flavone glycol (3, 4).
  • Sodium dodecylbenzenesulfonate may be 45-60 parts.
  • the WPM medium of the present invention includes, by mass percentage, 83.1-91.3% of a large number of elements, 3.4-5.7% of a trace element, 0.3-0.5% of ethylenediamine iron diglycolate, 2.7% -4.6% of calcium chloride dihydrate, Organic matter 2.3-6.1%.
  • a large number of elements include KNO 3 900-1100 mg / L, (NH 4 ) 2 SO 4 270-330 mg / L, MgSO 4 ⁇ 7H 2 O 360-440 mg / L, and KH 2 PO 4 180-220 mg / L.
  • Trace elements include KI 1.0-1.1 mg / L, MnSO 4 ⁇ 4H 2 O 18-22 mg / L, ZnSO 4 ⁇ 7H 2 O 6-10 mg / L, and H 3 BO 3 4-6 mg / L.
  • Organic substances include inositol 80-120mg / L, niacin 0.8-1.2mg / L, vitamin B1 0.4-0.6mg / L, vitamin B6 0.8-1.2mg / L, and vitamin C 4-6mg / L.
  • the method for preparing a medium for improving the induction rate of tissue culture plants of the present invention comprises the following steps: after mixing and dissolving raw materials according to the amount, adjusting the pH value to 5.4-5.6, and sterilizing for 15-30 minutes.
  • the medium for improving the induction rate of tissue culture plants is applied to the primary culture of blue snowflake.
  • the medium can effectively increase the induction rate of plants to about 95%, and can increase the robustness of subsequent cultured seedlings, improve the survival rate, and adapt It has strong sex; at the same time, its preparation method is simple, environmental protection and safety; In addition, it is applied to the primary culture of blue snowflake, which effectively improves the induction rate, and the blue snowflake grows robustly.
  • Raw material components of the culture medium 80 parts of WPM medium, 0.25 parts of zein, 0.8 parts of indole acetic acid, 1.2 parts of brassinolide, 35 parts of sucrose, 4.5 parts of isoflavones, 12 parts of calcium phosphate, and dodecylbenzene 45 parts of sodium sulfonate.
  • the WPM medium includes 90% of a large amount of elements, 3.6% of trace elements, 0.4% of ethylenediamine iron diglycolate, 3.0% of calcium chloride dihydrate, and 3.0% of organic matter.
  • a large number of elements include KNO 3 1000 mg / L, (NH 4 ) 2 SO 4 300 mg / L, MgSO 4 ⁇ 7H 2 O 400 mg / L, and KH 2 PO 4 200 mg / L.
  • Trace elements include KI 1.0 mg / L, MnSO 4 ⁇ 4H 2 O 20 mg / L, ZnSO 4 ⁇ 7H 2 O 8 mg / L, and H 3 BO 3 5 mg / L.
  • Organic substances include inositol 100mg / L, niacin 1.0mg / L, vitamin B1 0.5mg / L, vitamin B6 1mg / L, and vitamin C 5mg / L.
  • the preparation method includes the following steps: mixing and boiling the above-mentioned raw materials, so that all materials are thoroughly mixed and dissolved, adjusting the pH of the system to 5.4-5.6, and packaging them in a container for high-temperature and high-pressure sterilization for 20min.
  • the medium can be autoclaved.
  • the above-mentioned medium is used for the initial cultivation method of blue snowflakes, which includes the following steps:
  • the culture medium prepared above was placed in a culture flask, and 10 explants were inoculated in each culture flask, and then the culture flask was placed at a temperature of (25 ⁇ 1) ° C and a light intensity of 36-54 ⁇ mol ⁇ m -2 ⁇ s -1 , cultured under light conditions of 12h light / 12h for 60 days, and the number of effectively induced seedlings was recorded statistically.
  • an effective induced seedling refers to an induced bud seedling having a height of more than 2 cm and containing at least one stem node.
  • the results obtained are shown in Table 1 below.
  • Example 1 From Table 1, it can be seen that the medium of Example 1 was subjected to three repeated experiments, and the average induction rate obtained was 95%. It was found through observation that the induced seedlings grew robustly.
  • Raw material components of the culture medium 85 parts of WPM medium, 0.3 parts of zeatin, 1.2 parts of indole acetic acid, 0.8 parts of brassinolide, 45 parts of sucrose, 3 parts of isoflavone, 10 parts of calcium phosphate, and dodecane 60 parts of sodium benzene sulfonate.
  • the WPM medium includes 85% of a large amount of elements, 4.5% of trace elements, 0.35% of ethylenediamine o-diglycolate, 4.35% of calcium chloride dihydrate, and 5.8% of organic matter.
  • a large number of elements include KNO 3 950 mg / L, (NH 4 ) 2 SO 4 280 mg / L, MgSO 4 ⁇ 7H 2 O 420 mg / L, and KH 2 PO 4 190 mg / L.
  • Trace elements include KI 1.1 mg / L, MnSO 4 ⁇ 4H 2 O 19 mg / L, ZnSO 4 ⁇ 7H 2 O 9 mg / L, and H 3 BO 3 4.5 mg / L.
  • Organic matter includes 115 mg / L of inositol, 1.1 mg / L of niacin, 0.45 mg / L of vitamin B1, 0.9 mg / L of vitamin B6, and 4.5 mg / L of vitamin C.
  • the preparation method includes the following steps: mixing and boiling the above raw materials, so that all materials are thoroughly mixed and dissolved, adjusting the pH value of the system to 5.4-5.6, and packaging in a container for high temperature and high pressure sterilization for 25min.
  • the medium can be autoclaved.
  • Raw material components of the culture medium 68 parts of WPM medium, 0.5 parts of zeatin, 0.1 parts of indole acetic acid, 1.8 parts of brassinolide, 23 parts of sucrose, 6 parts of chalcone, 6 parts of calcium phosphate, and dodecyl 32 parts of sodium besylate.
  • the WPM medium includes a large amount of elements 83.1%, trace elements 5.7%, ethylene diamine o-dihydroxyacetate 0.5%, calcium chloride dihydrate 4.6%, and organic matter 6.1%.
  • a large number of elements include KNO 3 900 mg / L, (NH 4 ) 2 SO 4 270 mg / L, MgSO 4 ⁇ 7H 2 O 440 mg / L, and KH 2 PO 4 180 mg / L.
  • Trace elements include KI 1.1 mg / L, MnSO 4 ⁇ 4H 2 O 18 mg / L, ZnSO 4 ⁇ 7H 2 O 6 mg / L, and H 3 BO 3 6 mg / L.
  • Organic substances include inositol 80mg / L, niacin 1.2mg / L, vitamin B1 0.4mg / L, vitamin B6 1.2mg / L, and vitamin C 4mg / L.
  • the preparation method includes the following steps: mixing and boiling the above-mentioned raw materials, so that all materials are thoroughly mixed and dissolved, adjusting the pH of the system to 5.4-5.6, and packaging in a container for high-temperature and high-pressure sterilization for 30 minutes.
  • the medium can be autoclaved.
  • Example 3 As can be seen from Table 3, the medium of Example 3 was subjected to three repeated experiments, and the average induction rate obtained was 94%. It was found through observation that the induced seedlings grew robustly.
  • Raw material components of the culture medium 94 parts of WPM medium, 0.05 parts of zeatin, 1.5 parts of indole acetic acid, 0.3 parts of brassinolide, 56 parts of sucrose, 2 parts of flavanediol (3, 4), and 18 parts of calcium phosphate And 68 parts of sodium dodecylbenzenesulfonate.
  • the WPM medium includes 91.3% of a large amount of elements, 3.4% of trace elements, 0.3% of ethylenediamine iron diglycolate, 2.7% of calcium chloride dihydrate, and 2.3% of organic matter.
  • a large number of elements include KNO 3 1100 mg / L, (NH 4 ) 2 SO 4 330 mg / L, MgSO 4 ⁇ 7H 2 O 360 mg / L, and KH 2 PO 4 220 mg / L.
  • Trace elements include KI 1.0 mg / L, MnSO 4 ⁇ 4H 2 O 22 mg / L, ZnSO 4 ⁇ 7H 2 O 10 mg / L, and H 3 BO 3 4 mg / L.
  • Organic substances include inositol 120mg / L, niacin 0.8mg / L, vitamin B1 0.6mg / L, vitamin B6 0.8mg / L, and vitamin C 6mg / L.
  • the preparation method includes the following steps: mixing and boiling the above raw materials, so that all materials are thoroughly mixed and dissolved, adjusting the pH value of the system to 5.4-5.6, and packaging in a container for high temperature and high pressure sterilization for 15min.
  • the medium can be autoclaved.
  • Example 2 The basic steps are the same as in Example 1, except that isoflavones are not added to the raw materials.
  • the specific components and contents are shown below.
  • Raw material components of the culture medium 80 parts of WPM medium, 0.25 parts of zeatin, 1.5 parts of indole acetic acid, 1.8 parts of brassinolide, 35 parts of sucrose, 12 parts of calcium phosphate and 45 parts of sodium dodecylbenzenesulfonate .
  • Raw material components of the culture medium 80 parts of WPM medium, 0.25 parts of zein, 0.8 parts of indole acetic acid, 1.2 parts of brassinolide, 35 parts of sucrose, 4.5 parts of isoflavones, and 18 parts of calcium phosphate.
  • the addition of sodium dodecylbenzenesulfonate can effectively slow down the antagonism between the effective elements in the WPM medium through the interaction with calcium phosphate, avoid the generation of inhibition between the elements, and promote the mutual absorption between the elements.
  • the blue snowflake can fully absorb nutrients, improve survival rate, and grow robustly.

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Abstract

本发明公开了一种提高组培植株诱导率的培养基及制备方法与应用，包括WPM培养基68-94份、玉米素0.05-0.5份、吲哚乙酸0.1-1.5份、油菜素内酯0.3-1.8份、蔗糖23-56份、黄酮类化合物2-6份、磷酸钙6-18份及十二烷基苯磺酸钠32-68份；制法为按量将原料混合溶解后，调节pH值5.4-5.6，进行灭菌15-30min。其应用于蓝雪花的初代培养。本发明的培养基能够有效提高植株的诱导率，使其达到95％左右，且能够提高后续培养苗的健壮程度，成活率提高，适应性强；同时，其制备方法简单，环保安全；此外，应用于蓝雪花的初代培养，有效提高了诱导率，蓝雪花生长健壮。

Description

一种提高组培植株诱导率的培养基及制备方法与应用 技术领域
本发明属于培养基领域，尤其涉及一种提高组培植株诱导率的培养基及制备方法与应用。
背景技术
蓝雪花(Plumbago auriculata)原产南非，又名蓝花丹、蓝雪丹、蓝花矶松、蓝茉莉等，为白花丹科(蓝雪科)白花丹属多年生常绿灌木。蓝雪花长势强健，耐热，较耐高温、高湿，病虫害少，管理简单，观赏期长。叶色翠绿，花色淡雅，炎热的夏季给人以清凉感觉，可盆栽点缀居室、阳台；成熟植株枝条悬垂，适宜大型容器组合盆栽，可用于场馆周边道路、立交桥等主要路段的绿化美化，可地栽林缘种植或点缀草坪。蓝雪花花期正值少花的夏季，而且是自然界少有的蓝色花，受到园林工作者青睐。蓝雪花作为一种新优观花花卉被引种栽培，在一些重要绿化地段被推广应用，发挥了很好的景观效果，其种苗需求量逐年递增，市场潜力巨大。另外，蓝雪花含有大量白花丹素，对风湿关节疼痛、血瘀经闭、跌打损伤、肿毒恶疮以及疥癣均具有较好的治疗作用，兼具较高的观赏价值和药用价值。
目前国内对蓝雪花的研究刚刚起步，主要研究了蓝蓝雪花的观赏特性、引种栽培、生物学特性等。对蓝雪花的栽培管理、繁殖技术还主要是一些经验性的总结，在学术方面的系统研究则比较少。蓝雪花的繁育类型为异交型，开花强度不高，不利于吸引昆虫，不利于生殖成功，加之蓝雪花是典型的花柱异型植物，且具有自交不亲和性，故缺少传粉者，其结实率很低。而其种子价格较为昂贵，扩繁系数低，造成目前传统繁殖方式己无法满足蓝雪花的应用需求。蓝雪花的繁殖主要有两种方式：一是蓝雪花无性繁殖技术的研究，主要研究了扦插繁殖技术；二是组织培养快速繁殖技术，该技术能够克服传统繁殖方法的不足，具有繁殖系数大、速度快、保持优良性状，特别适合工厂化繁育等诸多优点。
因此，先亟需一种对蓝雪花进行组培的基质，以提高蓝雪花的组培诱导率。
发明内容
发明目的：本发明的第一目的是提供一种植株诱导率能够达到95％以上的组培培养基；
本发明的第二目的是提供该培养基的制备方法；
本发明的第三目的是提供该培养基的应用，其应用于蓝雪花的初代培育，使得蓝雪花具有良好的生长状态，有效诱导苗数量大大增加。
技术方案：本发明提高组培植株诱导率的培养基，按重量份数包括如下原料：WPM培养基68-94份、玉米素0.05-0.5份、吲哚乙酸0.1-1.5份、油菜素内酯0.3-1.8份、蔗糖23-56份、黄酮类化合物2-6份、磷酸钙6-18份及十二烷基苯磺酸钠32-68份。
本发明通过将WPM培养基、玉米素和蔗糖相结合，提供充足的营养物质，提高诱导率；同时添加黄酮类化合物，其与吲哚乙酸和油菜素内酯相结合，提高植物的蛋白质含量，促进蛋白质的合成，提高有效诱导苗的生长；此外，本发明还添加磷酸钙和十二烷基苯磺酸钠，两者的加入能够有效减缓WPM培养基中有效元素间的拮抗作用，避免元素间抑制作用的产生，促进元素间的相互吸收，使得植株充分地吸收营养物质，提高成活率。优选的，黄酮类化合物可为3-4.5份，可为异黄酮、异黄烷酮、查耳酮或黄烷二醇(3，4)。十二烷基苯磺酸钠可为45-60份。
进一步说，本发明WPM培养基按质量百分比包括：大量元素83.1-91.3％、微量元素3.4-5.7％、乙二胺邻二羟基乙酸铁0.3-0.5％、二水合氯化钙2.7-4.6％、有机物2.3-6.1％。其中，大量元素包括KNO 3 900-1100mg/L、(NH 4) 2SO 4 270-330mg/L、MgSO 4·7H 2O 360-440mg/L及KH 2PO 4 180-220mg/L。微量元素包括KI1.0-1.1mg/L、MnSO 4·4H 2O 18-22mg/L、ZnSO 4·7H 2O 6-10mg/L、H 3BO 3 4-6mg/L。有机物包括肌醇80-120mg/L、烟酸0.8-1.2mg/L、维生素B1 0.4-0.6mg/L、维生素B6 0.8-1.2mg/L、维生素C 4-6mg/L。
本发明制备提高组培植株诱导率的培养基的方法，包括如下步骤：按量将原料混合溶解后，调节pH值5.4-5.6，进行灭菌15-30min。
本发明提高组培植株诱导率的培养基应用于蓝雪花的初代培养。
有益效果：与现有技术相比，本发明的显著优点为：该培养基能够有效提高植株的诱导率，使其达到95％左右，且能够提高后续培养苗的健壮程度，成活率 提高，适应性强；同时，其制备方法简单，环保安全；此外，应用于蓝雪花的初代培养，有效提高了诱导率，蓝雪花生长健壮。
具体实施方式
下面结合实施例对本发明的技术方案做进一步详细说明。
实施例1
培养基的原料组分：WPM培养基80份、玉米素0.25份、吲哚乙酸0.8份、油菜素内酯1.2份、蔗糖35份、异黄酮4.5份、磷酸钙12份及十二烷基苯磺酸钠45份。
其中，WPM培养基包括大量元素90％、微量元素3.6％、乙二胺邻二羟基乙酸铁0.4％、二水合氯化钙3.0％、有机物3.0％。具体的，大量元素包括KNO 31000mg/L、(NH 4) 2SO 4 300mg/L、MgSO 4·7H 2O 400mg/L及KH 2PO 4 200mg/L。微量元素包括KI 1.0mg/L、MnSO 4·4H 2O 20mg/L、ZnSO 4·7H 2O 8mg/L、H 3BO 35mg/L。有机物包括肌醇100mg/L、烟酸1.0mg/L、维生素B1 0.5mg/L、维生素B6 1mg/L、维生素C 5mg/L。
制备方法包括如下步骤：将上述原料混合煮沸，使得所有材料充分混匀溶解，调节体系的pH值为5.4-5.6，分装在容器中进行高温高压灭菌20min，此处的高温高压采用现有的培养基高温高压灭菌即可。
采用上述的培养基进行蓝雪花的初代培育方法，包括如下步骤：
(1)以带腋芽茎段为外植体，用75％酒精灭菌60s后，再用10％次氯酸钠灭菌8min，或者用0.1L汞灭菌6min进行外植体消毒；
(2)将上述制备的培养基置于培养瓶中，并且每个培养瓶接种10个外植体，随后培养瓶放置在温度(25±1)℃，光照强度36-54μmol·m -2·s -1，光周期12h光照/12h黑暗条件下进行培养60d，统计记录有效诱导苗数。
其中，有效诱导苗是指诱导芽苗高度2cm以上，至少含有1个茎节。计算各处理的诱导率，诱导率(％)＝(有效诱导苗数/接种苗数)×100％。获得的结果如下表1所示。
表1实施例1的培养基获得的诱导率
性能 重复I 重复II 重复III 平均诱导率
参数 94 96 95 95
通过表1可知，将实施例1的培养基进行3次重复试验，所获得的平均诱导率能够达到95％。且通过观察发现可知，诱导苗生长健壮。
实施例2
培养基的原料组分：WPM培养基85份、玉米素0.3份、吲哚乙酸1.2份、油菜素内酯0.8份、蔗糖45份、异黄烷酮3份、磷酸钙10份及十二烷基苯磺酸钠60份。
其中，WPM培养基包括大量元素85％、微量元素4.5％、乙二胺邻二羟基乙酸铁0.35％、二水合氯化钙4.35％、有机物5.8％。具体的，大量元素包括KNO 3950mg/L、(NH 4) 2SO 4 280mg/L、MgSO 4·7H 2O 420mg/L及KH 2PO 4 190mg/L。微量元素包括KI 1.1mg/L、MnSO 4·4H 2O 19mg/L、ZnSO 4·7H 2O 9mg/L、H 3BO 34.5mg/L。有机物包括肌醇115mg/L、烟酸1.1mg/L、维生素B1 0.45mg/L、维生素B6 0.9mg/L、维生素C 4.5mg/L。
制备方法包括如下步骤：将上述原料混合煮沸，使得所有材料充分混匀溶解，调节体系的pH值为5.4-5.6，分装在容器中进行高温高压灭菌25min，此处的高温高压采用现有的培养基高温高压灭菌即可。
采用上述的培养基进行蓝雪花的初代培育方法，与实施例1相同，获得的结果如下表2所示。
表2实施例2的培养基获得的诱导率
性能 重复I 重复II 重复III 平均诱导率
参数 96 96 97 96.3
通过表2可知，将实施例2的培养基进行3次重复试验，所获得的平均诱导率能够达到96.3％。且通过观察发现可知，诱导苗生长健壮。
实施例3
培养基的原料组分：WPM培养基68份、玉米素0.5份、吲哚乙酸0.1份、油菜素内酯1.8份、蔗糖23份、查耳酮6份、磷酸钙6份及十二烷基苯磺酸钠32份。
其中，WPM培养基包括大量元素83.1％、微量元素5.7％、乙二胺邻二羟基乙酸铁0.5％、二水合氯化钙4.6％、有机物6.1％。具体的，大量元素包括KNO 3900mg/L、(NH 4) 2SO 4 270mg/L、MgSO 4·7H 2O 440mg/L及KH 2PO 4 180mg/L。 微量元素包括KI 1.1mg/L、MnSO 4·4H 2O 18mg/L、ZnSO 4·7H 2O 6mg/L、H 3BO 36mg/L。有机物包括肌醇80mg/L、烟酸1.2mg/L、维生素B1 0.4mg/L、维生素B6 1.2mg/L、维生素C 4mg/L。
制备方法包括如下步骤：将上述原料混合煮沸，使得所有材料充分混匀溶解，调节体系的pH值为5.4-5.6，分装在容器中进行高温高压灭菌30min，此处的高温高压采用现有的培养基高温高压灭菌即可。
采用上述的培养基进行蓝雪花的初代培育方法，与实施例1相同，获得的结果如下表3所示。
表3实施例3的培养基获得的诱导率
性能 重复I 重复II 重复III 平均诱导率
参数 95 94 93 94
通过表3可知，将实施例3的培养基进行3次重复试验，所获得的平均诱导率能够达到94％。且通过观察发现可知，诱导苗生长健壮。
实施例4
培养基的原料组分：WPM培养基94份、玉米素0.05份、吲哚乙酸1.5份、油菜素内酯0.3份、蔗糖56份、黄烷二醇(3，4)2份、磷酸钙18份及十二烷基苯磺酸钠68份。
其中，WPM培养基包括大量元素91.3％、微量元素3.4％、乙二胺邻二羟基乙酸铁0.3％、二水合氯化钙2.7％、有机物2.3％。具体的，大量元素包括KNO 31100mg/L、(NH 4) 2SO 4 330mg/L、MgSO 4·7H 2O 360mg/L及KH 2PO 4 220mg/L。微量元素包括KI 1.0mg/L、MnSO 4·4H 2O 22mg/L、ZnSO 4·7H 2O 10mg/L、H 3BO 34mg/L。有机物包括肌醇120mg/L、烟酸0.8mg/L、维生素B1 0.6mg/L、维生素B6 0.8mg/L、维生素C 6mg/L。
制备方法包括如下步骤：将上述原料混合煮沸，使得所有材料充分混匀溶解，调节体系的pH值为5.4-5.6，分装在容器中进行高温高压灭菌15min，此处的高温高压采用现有的培养基高温高压灭菌即可。
采用上述的培养基进行蓝雪花的初代培育方法，与实施例1相同，获得的结果如下表4所示。
表4实施例4的培养基获得的诱导率
性能 重复I 重复II 重复III 平均诱导率
参数 93 92 95 93.3
通过表4可知，将实施例4的培养基进行3次重复试验，所获得的平均诱导率能够达到93.3％。且通过观察发现可知，诱导苗生长健壮。
对比例1
基本步骤与实施例1相同，不同之处在于原料中不加入异黄酮，具体组分及含量如下所示。
培养基的原料组分：WPM培养基80份、玉米素0.25份、吲哚乙酸1.5份、油菜素内酯1.8份、蔗糖35份、磷酸钙12份及十二烷基苯磺酸钠45份。
采用上述的培养基进行蓝雪花的初代培育方法，与实施例1相同，获得的结果如下表5所示。
表5对比例1的培养基获得的诱导率
性能 重复I 重复II 重复III 平均诱导率
参数 85 89 86 86.6
通过表5可知，将对比例1的培养基进行3次重复试验，所获得的平均诱导率只达到86.6％，且通过观察发现可知，诱导苗长势较弱。由此可知，添加黄酮类化合物，其与吲哚乙酸和油菜素内酯能够相结合，提高植物的蛋白质含量，促进蛋白质的合成，提高有效诱导苗的生长。
对比例2
基本步骤与实施例1相同，不同之处在于原料中不加入十二烷基苯磺酸钠，具体组分及含量如下所示。
培养基的原料组分：WPM培养基80份、玉米素0.25份、吲哚乙酸0.8份、油菜素内酯1.2份、蔗糖35份、异黄酮4.5份、磷酸钙18份。
采用上述的培养基进行蓝雪花的初代培育方法，与实施例1相同，获得的结果如下表6所示。
表6对比例2的培养基获得的诱导率
性能 重复I 重复II 重复III 平均诱导率
参数 80 75 81 78.6
通过表6可知，将对比例2的培养基进行3次重复试验，所获得的平均诱导率只有78.6％，且通过观察发现可知，诱导苗长势非常较弱，这是由于本发明原料中加入了WPM培养基，而该WPM培养基中含有大量元素，大量元素间产生了拮抗，从而使得元素间不相互吸收，反而相互抑制，进而使得蓝雪花组培的营养物质缺失，诱导率大大降低。而加入十二烷基苯磺酸钠，其通过与磷酸钙的相互作用，进而能够有效减缓WPM培养基中有效元素间的拮抗作用，避免元素间抑制作用的产生，促进元素间的相互吸收，使得蓝雪花充分地吸收营养物质，提高成活率，生长健壮。

Claims (10)

  1. 一种提高组培植株诱导率的培养基，其特征在于按重量份数包括如下原料：WPM培养基68-94份、玉米素0.05-0.5份、吲哚乙酸0.1-1.5份、油菜素内酯0.3-1.8份、蔗糖23-56份、黄酮类化合物2-6份、磷酸钙6-18份及十二烷基苯磺酸钠32-68份。
  2. 根据权利要求1所述的提高组培植株诱导率的培养基，其特征在于：所述黄酮类化合物3-4.5份。
  3. 根据权利要求1所述的提高组培植株诱导率的培养基，其特征在于：所述黄酮类化合物为异黄酮、异黄烷酮、查耳酮或黄烷二醇(3，4)。
  4. 根据权利要求1所述的提高组培植株诱导率的培养基，其特征在于：所述十二烷基苯磺酸钠45-60份。
  5. 根据权利要求1所述的提高组培植株诱导率的培养基，其特征在于：所述WPM培养基按质量百分比包括：大量元素83.1-91.3％、微量元素3.4-5.7％、乙二胺邻二羟基乙酸铁0.3-0.5％、二水合氯化钙2.7-4.6％、有机物2.3-6.1％。
  6. 根据权利要求1所述的提高组培植株诱导率的培养基，其特征在于：所述大量元素包括KNO 3 900-1100mg/L、(NH 4) 2SO 4 270-330mg/L、MgSO 4·7H 2O 360-440mg/L及KH 2PO 4 180-220mg/L。
  7. 根据权利要求1所述的提高组培植株诱导率的培养基，其特征在于：所述微量元素包括KI 1.0-1.1mg/L、MnSO 4·4H 2O 18-22mg/L、ZnSO 4·7H 2O 6-10mg/L、H 3BO 3 4-6mg/L。
  8. 根据权利要求1所述的提高组培植株诱导率的培养基，其特征在于：所述有机物包括肌醇80-120mg/L、烟酸0.8-1.2mg/L、维生素B1 0.4-0.6mg/L、维生素B6 0.8-1.2mg/L、维生素C 4-6mg/L。
  9. 一种制备权利要求1所述的提高组培植株诱导率的培养基的方法，其特征在于包括如下步骤：按量将原料混合溶解后，调节pH值5.4-5.6，进行灭菌15-30min。
  10. 权利要求1所述的提高组培植株诱导率的培养基应用于蓝雪花的初代培养。
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