CN102884980B

CN102884980B - Quick tissue propagation method of paphiopedilum bellatulum

Info

Publication number: CN102884980B
Application number: CN201210343625.9A
Authority: CN
Inventors: 王莲辉; 姜运力; 潘德权; 冯育才; 李丛瑞
Original assignee: GUIZHOU PROVINCIAL ACADEMY OF FORESTRY SCIENCES
Current assignee: Qianxinan Lvyuan Animal Science and Technology Development Co.,Ltd.
Priority date: 2012-09-17
Filing date: 2012-09-17
Publication date: 2014-04-02
Anticipated expiration: 2032-09-17
Also published as: CN102884980A

Abstract

The invention discloses a quick tissue propagation method of paphiopedilum bellatulum. The quick tissue propagation method comprises the following steps of (1), building explants; (2), protocorm germinating stage; (3), propagation culturing stage; (4), seedling strengthening and rooting stage; (5), acclimating and transplanting; when paphiopedilum bellatulum test-tube plantlets grow to 3-4cm, the test-tube plantlets are acclimated for about 10 days under natural light in the greenhouse before being transplanted; in the greenhouse, the temperature is 15-25 DEG C, the humidity is more than 70% and the shade area is 78-80% on the ventilation condition; the Paphiopedilum bellatulum can grow normally; the transplant survival rate of the test-tube plantlets reaches more than 93%; with the adoption of the quick tissue propagation method of the paphiopedilum bellatulum, the propagation coefficient of seedlings can be drastically improved, seedlings with uniform quality are obtained; and the quick tissue propagation method of the paphiopedilum bellatulum is convenient for factory plantation on large scale, and has important significance in searching and developing arethusa, searching specific property of the variety, selecting a specific method, protecting, developing and sustainably utilizing rare plants.

Description

The blue tissue culture and rapid propagation method of huge lobe pocket

Technical field

The present invention relates to bionic Plant Tissue Breeding mating system, particularly relate to the blue tissue culture and rapid propagation method of a kind of huge lobe pocket.

background method

Orchid is one of family of natural world maximum, is one of high monoid of the hugest and Evolutionary Levels in flowering plant, is only second to composite family and grass family and is positioned at the third-largest section in the middle of angiosperm.Global orchid approximately has 700 genus, 20000 kinds-30000 kinds, is distributed widely in from temperate zone, subtropics is to the vast region in the torrid zone.China has found and the orchid approximately more than 170 named belongs to more than 1300 and plants, and wherein 2/3 be epiphytic orchid, and 1/3 be gives birth to orchid.At present, whole orchid has been put into < < CITS > > appendix I or II.

Guizhou is the province, plateau of a karst landform extensive development, on geographical environment, belong to subtropical climate, have unique complicated various and good hydrothermal condition of topography and geomorphology, vegetation pattern, suitable various plants is multiplied and lived, and has also bred more rich orchid resource simultaneously.Guizhou Province is distributed with orchid approximately 76 within the border and belongs to more than 320 kinds, comprises a large amount of mutation and kind.

Paphiopedilum (Paphiopedilum Pfitz) plant is one of species of the tool enjoying value of orchid, pocket orchid is because unique flower moulding, gorgeous flower color, the lasting florescence of viewing and admiring have high ornamental value, be to cultivate in the world one of orchid the most widely, the blue market of China recent years pocket is also in rapid expansion.China be world's Paphiopedilum important distribution with the type locality of many novel species.

Huge lobe pocket blue (Paphiopedilum bellatulum (Rchb.f.) Stein) belongs to the herbaceos perennial of the orchid family (Orchidaceae) Paphiopedilum (Paphiopedilum), the ground such as distribution Guangxi, Yunnan and Guizhou, be born in In Limestone Area, epiphyte on ground life or stone, leaf 4-5 piece, has the green alternate grid spot of the depth above; Spend common 1, white or cream are yellow, and the thick spot of tool puce, is kind rare in Paphiopedilum, and its pocketed is spent large and look gorgeous, and attitude is graceful, fragrant odour, and the florescence is longer, has higher ornamental value.Huge lobe pocket orchid is classified as first class of protection species by international convention, is the distinctive first-grade state protection species that enjoy international concern of China, is top grade in cypripedium, is germ plasm resource rare in horticultural gardening, the edge in extinction in imminent danger.

Because the blue embryonic development of huge lobe pocket is incomplete, under nature, breeding difficulty, exists seed germination rate extremely low, the difficulty that planting percent is low; Traditional ramificatiom breeding method, the cycle is long, and reproduction coefficient is low, can only breed every year 1～3 times, and Sterile culture difficulty is large.Therefore how to use plant biological to facilitate the huge lobe pocket of method Fast-propagation orchid to become the emphasis of orchid scientific research project.The tissue culture and rapid propagation method of huge lobe pocket orchid is exactly to adopt the aseptic seeding of orchid and the method for the quick seedling of test tube, to carry out implant mass to meet the need of market, has far deep realistic meaning in its research and development at short notice.

Summary of the invention

The object of the invention is to develop the tissue culture and rapid propagation method of a kind of huge lobe pocket orchid, on the medium of special preparation, by the method for artificial aseptic seeding, through propagation steps such as aseptic seeding, seed germination, strong seedling culture and test-tube seedling transplantings, obtain protocorm and bud mixture, utilize aseptic seeding seedling to breed, obtain at short notice a large amount of uniform test-tube plantlets, by strengthening seedling and rooting, cultivate and can form whole plant.By inventor's effort, completed the research work of the tissue culture and rapid propagation method of huge lobe pocket orchid.

The tissue culture and rapid propagation method of the huge lobe pocket of the present invention orchid, its process comprises the following steps:

(1) foundation of Aseptic culture material, chooses the blue maternal plant of huge lobe pocket of robust growth, carries out artificial pollination when blooming, and pollinates latter 180 days, will be developed to be mature on the whole and uncracked fruit as explant.

(2) protocorm is sprouted the stage: select full fruit with 75% alcohol-pickled 30 seconds, be placed in 0.1% mercuric chloride solution, the 20 minutes kinds of sterilizing, aseptic water washing 4-5 time, cut fruit, with transfer needle, brown powder shape embryo being inoculated into 1/2MS adds in α-naphthaleneacetic acid (NAA) 2 mg/litre on medium, cultivate about 60 days, form protocorm and bud.

(3) propagation cultivation stage: for obtaining more seedling, Jiang Yahe protocorm line and staff control is seeded on additional 6-benzylaminopurine (6-BA) the 20 mg/litre medium of 1/2MS, cultivates through 120 days, grows up to the seedling of growing thickly with short.

(4) the strengthening seedling and rooting stage: grow thickly plant separately by what grow up to, be inoculated on the medium of 1/4MS interpolation heteroauxin (IBA) 0.4 mg/litre, cultivate the whole plant that grows up to tool 2-3 bar root, 3-4 sheet leaf after 90 days.

(5) practice seedling and transplanting: when test-tube plantlet grows to 3-4 centimetre, the natural lighting lower refining seedling before bottle outlet in greenhouse 7 days then takes out it from vial, cleans the medium of root, moves in the mixed-matrix of broken bark and sphagna.Greenhouse keeps the sheltering from heat or light and ventilate of temperature, more than 70% humidity, 78-80% of 15-28 ℃, and huge lobe pocket orchid gets final product normal growth, and the transplanting survival rate of seedling can reach more than 90%.Within common about 15 days, water a fertilizer (the oil cake water of fermentation) or foliage fertilizer, its concentration is 1000 times of mass ratioes of dilution, and thin fertile duty is executed, and strengthens damage by disease and insect and manages and protects the industrialization breeding method system that measure can be set up the blue seedling of huge lobe pocket.

The invention has the beneficial effects as follows: the tissue culture propagation method of the huge lobe pocket orchid of this invention; can improve reproduction coefficient; it is the high efficiency method for test tube propagation that obtains the blue seedling of a large amount of huge lobe pockets; have germination rate high, seedling is fast, the advantage such as seedling quality better, growth potential are strong; can access the seedling of neatization; and a large amount of seedlings is provided at short notice, for the seedling of orchid, produce an effective approach is provided, thereby better protect wild resource.

Embodiment

embodiment 1

(1) foundation of Aseptic culture material, select to grow be mature on the whole and uncracked fruit as explant, carry out aseptic culture.

(2) protocorm is sprouted the stage: select full fruit with 75% alcohol-pickled 30 seconds, be placed in 0.1% mercuric chloride solution, the 20 minutes kinds of sterilizing, aseptic water washing 4-5 time, cut fruit, with transfer needle, brown powder shape embryo is inoculated on the medium of 1/2MS interpolation α-naphthaleneacetic acid (NAA) 2 mg/litre, cultivate about 60 days, form protocorm and bud.

(3) propagation cultivation stage: for obtaining more seedling, Jiang Yahe protocorm line and staff control is seeded on additional 6-benzylaminopurine (6-BA) the 20 mg/litre medium of 1/2MS, through 120 days, cultivate, grow up to the seedling of growing thickly with short, reproduction coefficient can reach 3-4.

(4) the strengthening seedling and rooting stage: the plant that will grow thickly separately, is inoculated on the medium of 1/4MS interpolation heteroauxin (IBA) 0.4 mg/litre, cultivates the whole plant that can grow up to tool 2-3 bar root, 3-4 sheet leaf after 90 days.

(5) practice seedling and transplanting: when test-tube plantlet grows to 3-4 centimetre, the natural lighting lower refining seedling before bottle outlet in greenhouse 7 days then takes out it from vial, cleans the medium of root, moves in the mixed-matrix of broken bark and sphagna.Greenhouse keeps the sheltering from heat or light and ventilate of temperature, more than 70% humidity, 78-80% of 15-28 ℃, and huge lobe pocket orchid gets final product normal growth, and the transplanting survival rate of seedling can reach more than 90%.Within common about 15 days, water a fertilizer (the oil cake water of fermentation) or foliage fertilizer (diluting 1000 times), thin fertile duty is executed, strengthen damage by disease and insect and manage and protect the industrialization breeding method system that measure can be set up the blue seedling of huge lobe pocket, plant at present blue seedling more than 3000 strains of huge lobe pocket.

Claims

1. the tissue culture and rapid propagation method of huge lobe pocket orchid, is characterized in that method comprises the following steps:

1.the foundation of Aseptic culture material, chooses the blue maternal plant of huge lobe pocket of robust growth, carries out artificial pollination when blooming, and pollinates latter 180 days, will be developed to be mature on the whole and uncracked fruit as explant;

2.protocorm is sprouted the stage: select full fruit with 75% alcohol-pickled 30 seconds, being placed in 0.1% mercuric chloride solution sterilizes 20 minutes, aseptic water washing 4-5 time, cut fruit, with transfer needle, brown powder shape embryo being inoculated into 1/2MS adds on α-naphthaleneacetic acid (NAA) 2 mg/litre medium, cultivate about 60 days, form protocorm and bud;

3.propagation cultivation stage: for obtaining more seedling, Jiang Yahe protocorm line and staff control is seeded on additional 6-benzylaminopurine (6-BA) the 20 mg/litre medium of 1/2MS, cultivates through 120 days, grows up to the seedling of growing thickly with short;

4.the strengthening seedling and rooting stage: grow thickly plant separately by what grow up to, be inoculated on the medium of 1/4MS interpolation heteroauxin (IAA) 0.4 mg/litre, cultivate the whole plant that grows up to tool 2-3 bar root, 3-4 sheet leaf after 90 days;

5.hardening and transplanting: when test-tube plantlet grows to 3-4 centimetre, the natural lighting lower refining seedling before bottle outlet in greenhouse 7 days then takes out it from vial, cleans the medium of root, moves in the mixed-matrix of broken bark and sphagna; Greenhouse keeps the sheltering from heat or light and ventilate of temperature, more than 70% humidity, 78-80% of 15-28 ℃, and huge lobe pocket orchid gets final product normal growth, and the transplanting survival rate of seedling can reach more than 90%; Common oil cake water or the foliage fertilizer that waters one time fermentation for about 15 days, its concentration is 1000 times of mass ratioes of dilution, thin fertile duty is executed, and strengthens damage by disease and insect and manages and protects the industrialization breeding method that measure can be set up the blue seedling of huge lobe pocket.