CN102884980A

CN102884980A - Quick tissue propagation method of paphiopedilum bellatulum

Info

Publication number: CN102884980A
Application number: CN2012103436259A
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: 2013-01-23
Anticipated expiration: 2032-09-17
Also published as: CN102884980B

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 the 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 have 700 belong to, 20000 kinds-30000 kinds, be distributed widely in from the temperate zone, the subtropics is to the vast zone in the torrid zone.China has found and the orchid named approximately more than 170 belongs to more than 1300 and plants, and wherein 2/3 be epiphytic orchid, 1/3 life orchid be.At present, whole orchid is put into " CITS " appendix I or II.

Guizhou is the province, plateau of a karst landform extensive development, belong to subtropical climate at geographical environment, 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 approximately 76 genus more than 320 kinds of orchid within the border, comprises a large amount of mutation and kind.

Paphiopedilum (Paphiopedilum Pfitz) plant is one of species of the tool enjoying value of orchid, pocket is blue owing to unique flower moulding, gorgeous flower color, the lasting florescence of viewing and admiring have high ornamental value, be to cultivate in the world the most widely one of orchid, the blue market of China recent years pocket is also in rapid expansion.China is the important distribution ground of world's Paphiopedilum and 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 the stone, leaf 4-5 piece, the above has the green alternate grid spot of the depth; Spend common 1, white or cream are yellow, and the thick spot of tool puce is kind rare in the 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 the 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 the cypripedium, is germ plasm resource rare in the horticultural gardening, has been in the edge of extinction in imminent danger.

Because the blue embryonic development of huge lobe pocket is incomplete, the breeding difficulty exists seed germination rate extremely low, the difficulty that planting percent is low under nature; Traditional ramificatiom breeding method, the cycle is long, and reproduction coefficient is low, can only breed 1～3 times every year, and the 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 profound realistic meaning far away in its research and development at short notice.

Summary of the invention

The objective 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, method by 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 the aseptic seeding seedling to breed, obtain at short notice a large amount of uniform test-tube plantlets, cultivate by strengthening seedling and rooting and can form whole plant.By inventor's effort, finished 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 may further comprise the steps:

(1) the blue maternal plant of huge lobe pocket of robust growth is chosen in the foundation of Aseptic culture material, carries out artificial pollination when blooming, and pollinates rear 180 days, will grow to being mature on the whole uncracked fruit as explant.

(2) protocorm is sprouted the stage: select full fruit with 75% alcohol-pickled 30 seconds, place 20 minutes kinds of mercuric chloride solution sterilization of 0.1%, 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 on the medium, cultivated about 60 days, and formed protocorm and bud.

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

(4) the strengthening seedling and rooting stage: what will grow up to grows thickly plant separately, is inoculated on the medium of 1/4MS interpolation heteroauxin (IBA) 0.4 mg/litre, cultivates 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 grew to 3-4 centimetre, then the natural lighting lower refining seedling before the bottle outlet in the greenhouse 7 days took out it from vial, cleaned the medium of root, moved in the mixed-matrix of broken bark and sphagna.The greenhouse keeps 15-28 ℃ the sheltering from heat or light and ventilate of temperature, the humidity more than 70%, 78-80%, and huge lobe pocket orchid gets final product normal growth, and the transplanting survival rate of seedling can reach more than 90%.Watered a fertilizer (the oil cake water of fermentation) or foliage fertilizer in common about 15 days, 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 production of orchid provides an effective approach, 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, place 20 minutes kinds of mercuric chloride solution sterilization of 0.1%, aseptic water washing 4-5 time, cut fruit, brown powder shape embryo is inoculated on the medium of 1/2MS interpolation α-naphthaleneacetic acid (NAA) 2 mg/litre with transfer needle, cultivated about 60 days, and formed protocorm and bud.

(3) propagation cultivation stage: for obtaining more seedling, bud and protocorm line and staff control are seeded on additional 6-benzylaminopurine (6-BA) the 20 mg/litre medium of 1/2MS, cultivated through 120 days, 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 grew to 3-4 centimetre, then the natural lighting lower refining seedling before the bottle outlet in the greenhouse 7 days took out it from vial, cleaned the medium of root, moved in the mixed-matrix of broken bark and sphagna.The greenhouse keeps 15-28 ℃ the sheltering from heat or light and ventilate of temperature, the humidity more than 70%, 78-80%, and huge lobe pocket orchid gets final product normal growth, and the transplanting survival rate of seedling can reach more than 90%.Watered a fertilizer (the oil cake water of fermentation) or foliage fertilizer (diluting 1000 times) in common about 15 days, 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 may further comprise the steps:

1.The blue maternal plant of huge lobe pocket of robust growth is chosen in the foundation of Aseptic culture material, carries out artificial pollination when blooming, and pollinates rear 180 days, will grow to being mature on the whole uncracked fruit as explant;

2.Protocorm is sprouted the stage: select full fruit with 75% alcohol-pickled 30 seconds, place 20 minutes kinds of mercuric chloride solution sterilization of 0.1%, 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 on the medium, cultivated about 60 days, and formed protocorm and bud;

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

4.The strengthening seedling and rooting stage: what will grow up to grows thickly plant separately, is inoculated on the medium of 1/4MS interpolation heteroauxin (IBA) 0.4 mg/litre, cultivates 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 grew to 3-4 centimetre, then the natural lighting lower refining seedling before the bottle outlet in the greenhouse 7 days took out it from vial, cleaned the medium of root, moved in the mixed-matrix of broken bark and sphagna; The greenhouse keeps 15-28 ℃ the sheltering from heat or light and ventilate of temperature, the humidity more than 70%, 78-80%, and huge lobe pocket orchid gets final product normal growth, and the transplanting survival rate of seedling can reach more than 90%; Watered a fertilizer (the oil cake water of fermentation) or its concentration of foliage fertilizer in common about 15 days and be 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.