CN103070072A - Method for fast propagation of test tube water chestnut by tissue culture - Google Patents
Method for fast propagation of test tube water chestnut by tissue culture Download PDFInfo
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Abstract
The invention discloses a method for fast propagation of test tube water chestnut by tissue culture. A terminal bud or lateral bud of disease-free water chestnut is used as an explant and is subjected to stem tip starting, subculture multiplication, rootage and induction to form a test tube water chestnut. According to the method disclosed by the invention, the propagation of the test tube water chestnut is carried out by tissue culture; the test tube water chestnut has no disease, and thus high quality is obtained; the test tube water chestnut is high in growing speed, short in period and high in propagation coefficient; and culture conditions of the test tube water chestnut are controllable, so that anniversary industrial production can be realized and favorable social and economic benefits are created. According to the method disclosed by the invention, an induction technology for fast propagation of the test tube water chestnut by tissue culture provides a quick, convenient and effective water chestnut seedling propagation technology for the development of the water chestnut industry and greatly meets the market demand of the water chestnut.
Description
Technical field
The invention belongs to the agricultural breeding field, refer to particularly a kind of method of test tube water chestnut tissue-culturing quick-propagation.
Background technology
Water chestnut another name water chestnut, Di Li, black taro etc., be the perennial aquatic herbaceous plant of Eleocharis of sand-grass section, originate in southern china and India, cultivation history is long in China, and is edible with its underground bulb, contains rich in protein, carbohydrate and mineral element, both can do vegetables, also can make fruit, eat raw, prepared food all can, also can process canning and extract starch.
For a long time, vegetative propagation is adopted in the water chestnut cultivation, cause various infection processes, plant the kind sexual involution of shepherd's purse, plant the sick phenomenon of spherical zone serious, cause to yield poorly and unstable, its Major Diseases has bar rot, fusarium wilt, stem rot, gray mold etc., causing harm the heaviest on producing at present is bar rot and fusarium wilt, and two kinds of diseases can be propagated by bulb.When the bar rot was popular, common sick bar rate reached 30%～40%, weighs 100% most, causes cladodium withered, and underground part is not tied shepherd's purse, the general underproduction 40%～70%, even total crop failure.
Summary of the invention
The objective of the invention is sick for the kind spherical zone of vegetative water chestnut and kind sexual involution problem, a kind of method of test tube water chestnut tissue-culturing quick-propagation is provided, improve the quality of water chestnut.
The method of test tube water chestnut tissue-culturing quick-propagation of the present invention may further comprise the steps:
1) stem apex starts: terminal bud or the lateral bud of choosing anosis water chestnut are explant, behind cleaning and sterilizing, being inoculated in stem apex starts on the medium, it is the LS medium that described stem apex starts medium, add 6-benzyl aminopurine (6-BA) 0.5～2.6mg/L, methyl α-naphthyl acetate (NAA) 0.1～0.5mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.5～0.6% agar, the pH value is 5.8～6.0, cultivation temperature is 24～26 ℃, intensity of illumination is 2000～2500 lx, illumination every day 10h is until grow into simple bud or clump bud;
2) shoot proliferation: simple bud or clump bud are transferred to shoot proliferation cultivation on the shoot proliferation medium, described shoot proliferation medium is the LS medium, add 6-benzyl aminopurine (6-BA) 0.5～2.0mg/L, methyl α-naphthyl acetate (NAA) 0.1～1.0mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.5～0.6% agar, the pH value is 5.8～6.0, cultivation temperature is 24～26 ℃, intensity of illumination 2000～2500 lx, illumination every day 10h is to producing new clump bud;
3) culture of rootage: the clump bud is divided into simple bud, be transferred to root induction on the root media, described root media is the LS medium, add 6-benzyl aminopurine (6-BA) 0.5～1.0mg/L, indolebutyric acid (IBA) 0.1～0.5mg/L, active carbon (AC) 0.5～1.0g/L, mass fraction are 3.0% sucrose and mass fraction is 0.5～0.6% agar, the pH value is 5.8～6.0, cultivation temperature is 24～26 ℃, intensity of illumination is 2000～2500 lx, illumination every day 10h, the formation test-tube plantlet of taking root;
4) induce the test tube Water Chestnuts: test-tube plantlet is transferred to induce on the Water Chestnuts medium cultivates, described to induce the Water Chestnuts medium be the LS medium, add 6-benzyl aminopurine (6-BA) 0.1～1.0mg/L, methyl α-naphthyl acetate (NAA) 0.1～0.5mg/L, active carbon (AC) 0.5～1.0g/L, mass fraction are that 3.0～12.0% sucrose and mass fraction are 0.5～0.6% agar, the pH value is 5.8～6.0, cultivation temperature is 24～26 ℃, intensity of illumination 2000～2500 lx, illumination every day 8h induces to form the test tube water chestnut.
Used minimal medium is the LS medium among the present invention, because through experimental study, the water chestnut test-tube plantlet is more vigorous with respect to the growth of MS medium in the LS medium.
The present invention starts in medium and the shoot proliferation medium at stem apex and has all added auxin substance NAA, is more conducive to startup and the subculture growth of water chestnut stem apex.
The present invention is in culture of rootage and induce in the test tube Water Chestnuts medium in two stages and all added AC, and AC can hestening rooting, alleviate the browning in the tissue culture procedures.
Select to be 2000～2500Lx in each cultivation stage intensity of illumination among the present invention, stem apex startup, shoot proliferation and take root the stage, every day, light application time was 10h, induced the test tube water chestnut stage, every day, light application time was 8h.Plant is different to the demand of light at different growing stages, and the present invention is in the selection of each cultivation stage to intensity of illumination and light application time, more meets in the plant growth and development process demand to light.
The present invention cultivates by tissue and carries out the breeding of test tube water chestnut, has the following advantages:
1, the test tube water chestnut not in spite of illness, quality is high.By the water chestnut Shoot Tip Culture, carry out Fast-propagation, obtain regeneration plant, and inducing formation test tube water chestnut, at first can obtain anosis bacterial classification shepherd's purse, solve the kind spherical zone disease of water chestnut and plant the sexual involution problem, improve large fruit rate and regularity, improve quality, thereby improve commodity, improve output.The test tube water chestnut that obtains by tissue-culturing rapid propagation not in spite of illness, the genetic background of water chestnut seedling is consistent simultaneously, thereby has guaranteed the purity of water chestnut kind.
2, the growth of test tube water chestnut is fast, and the cycle is short, and reproduction coefficient is high.Conventional water chestnut breeding in year generation of planting; And the test tube water chestnut can breed for 8～10 generations in 1 year, had improved greatly the reproduction speed of water chestnut, was conducive to applying of excellent new water chestnut kind, providing with way far away accent of new varieties is provided plants.
3, test tube water chestnut condition of culture is controlled, but preferably Social benefit and economic benefit is created in anniversary batch production production.
4, the stripped indoor preservation of resource be can carry out, other physiology, the research work of biochemical aspect carried out, for these researchs provide an exercisable single system.Thereby, in theory with in the production practices extremely important meaning and application prospect are widely arranged.
In a word, by inductive technology tissue-culturing quick-propagation test tube water chestnut for the development of water chestnut industry provide a kind of fast, convenient, effective water chestnut sapling multiplication technology, greatly satisfied the market demand of water chestnut.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment.
Embodiment 1
Take the terminal bud of water chestnut kind Shayang County water chestnut as explant, through stem apex startup, shoot proliferation, take root, induce and form the test tube water chestnut.Comprise the steps:
1) stem apex starts: take Shayang County water chestnut as test material, get the anosis Shayang County water chestnut terminal bud that cleans up, through 75% after alcohol-pickled 30 seconds, use 0.1%HgCl
2Solution soaked 0.5 minute, use again aseptic water washing 3 times, each 3 minutes, be inoculated in stem apex and start on the medium, it is the LS medium that used stem apex starts medium, add 6-benzyl aminopurine (6-BA) 1.5mg/L, methyl α-naphthyl acetate (NAA) 0.2mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.6% agar, and the pH value is 5.8, it is 26 ℃ in temperature, under intensity of illumination 2500 lx, illumination every day 10h produces the clump bud behind the 30d;
2) shoot proliferation: the clump bud is transferred to shoot proliferation cultivation on the shoot proliferation medium, used shoot proliferation medium is the LS medium, add 6-benzyl aminopurine (6-BA) 1.0mg/L, methyl α-naphthyl acetate (NAA) 0.2mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.6% agar, the pH value is 5.8, cultivation temperature is 26 ℃, intensity of illumination 2000 lx, illumination every day 10h, 35d produce new clump bud;
3) take root: the clump bud is divided into simple bud, be transferred to root induction on the root media, used root media is the LS medium, add 6-benzyl aminopurine (6-BA) 0.5mg/L, indolebutyric acid (IBA) 0.5mg/L, active carbon (AC) 0.5g/L, mass fraction are 3.0% sucrose and mass fraction is 0.6% agar, the pH value is 5.8, cultivation temperature is 26 ℃, intensity of illumination is 2500 lx, illumination every day 10h, the formation test-tube plantlet of taking root about 20d;
4) induce the test tube water chestnut: test-tube plantlet is transferred to induce on the Water Chestnuts medium cultivates, used to induce the Water Chestnuts medium be the LS medium, add 6-benzyl aminopurine (6-BA) 0.6mg/L, methyl α-naphthyl acetate (NAA) 0.3mg/L, active carbon (AC) 0.5g/L, mass fraction are that 5.0% sucrose and mass fraction are 0.6% agar, the pH value is 5.8, cultivation temperature is 25 ℃, intensity of illumination 2500 lx, illumination every day 8h, 30d induce later on and form the test tube water chestnut.
Embodiment 2
Take the lateral bud of water chestnut kind Shayang County water chestnut as explant, through stem apex startup, shoot proliferation, take root, induce and form the test tube water chestnut.Comprise the steps:
1) stem apex starts: take Shayang County water chestnut as test material, get the anosis Shayang County water chestnut lateral bud that cleans up, through 75% after alcohol-pickled 30 seconds, use 0.1%HgCl
2Solution soaked 1 minute, use again aseptic water washing 5 times, each 5 minutes, be inoculated in stem apex and start on the medium, it is the LS medium that described stem apex starts medium, add 6-benzyl aminopurine (6-BA) 1.0mg/L, methyl α-naphthyl acetate (NAA) 0.3mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.6% agar, and the pH value is 5.8, it is 24 ℃ in temperature, under intensity of illumination 2200 lx, illumination every day 10h produces simple bud behind the 30d;
2) shoot proliferation: simple bud is transferred to shoot proliferation cultivation on the shoot proliferation medium, described shoot proliferation medium is the LS medium, add 6-benzyl aminopurine (6-BA) 0.8mg/L, methyl α-naphthyl acetate (NAA) 0.1mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.6% agar, the pH value is 5.8, cultivation temperature is 24 ℃, intensity of illumination 2300 lx, illumination every day 10h, 35d produce new clump bud;
3) take root: the clump bud is divided into simple bud, be transferred to root induction on the root media, described root media is the LS medium, add 6-benzyl aminopurine (6-BA) 1.0mg/L, indolebutyric acid (IBA) 1.0mg/L, active carbon (AC) 0.7g/L, mass fraction are 3.0% sucrose and mass fraction is 0.6% agar, the pH value is 5.8, cultivation temperature is 26 ℃, intensity of illumination is 2000 lx, illumination every day 10h, the formation test-tube plantlet of taking root about 20d;
4) induce the test tube water chestnut: test-tube plantlet is transferred to induce on the Water Chestnuts medium cultivates, described to induce the Water Chestnuts medium be the LS medium, add 6-benzyl aminopurine (6-BA) 0.3mg/L, methyl α-naphthyl acetate (NAA) 0.1mg/L, active carbon (AC) 0.5g/L, mass fraction are that 8% sucrose and mass fraction are 0.5% agar, pH value 5.8, cultivation temperature is 26 ℃, intensity of illumination 2500lx, illumination every day 8h, 30d induce later on and form the test tube water chestnut.
Embodiment 3
Take the terminal bud of water chestnut kind Shayang County water chestnut as explant, through stem apex startup, shoot proliferation, take root, induce and form the test tube water chestnut.Comprise the steps:
1) stem apex starts: take Shayang County water chestnut as test material, get the anosis Shayang County water chestnut terminal bud that cleans up, through 75% after alcohol-pickled 30 seconds, use 0.1%HgCl
2Solution soaked 0.8 minute, use again aseptic water washing 3 times, each 3 minutes, be inoculated in stem apex and start on the medium, it is the LS medium that described stem apex starts medium, add 6-benzyl aminopurine (6-BA) 2.2mg/L, methyl α-naphthyl acetate (NAA) 0.5mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.6% agar, and the pH value is 5.8, it is 24 ℃ in temperature, under intensity of illumination 2500 lx, illumination every day 10h produces the clump bud behind the 30d;
2) shoot proliferation: the clump bud is transferred to shoot proliferation cultivation on the shoot proliferation medium, described shoot proliferation medium is the LS medium, add 6-benzyl aminopurine (6-BA) 2.0mg/L, methyl α-naphthyl acetate (NAA) 0.3 mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.5% agar, the pH value is 6.0, cultivation temperature is 24 ℃, intensity of illumination 2500 lx, illumination every day 10h, 25d produce new clump bud;
3) take root: the clump bud is divided into simple bud, be transferred to root induction on the root media, described root media is the LS medium, add 6-benzyl aminopurine (6-BA) 1.0mg/L, indolebutyric acid (IBA) 1.0mg/L, active carbon (AC) 1.0g/L, mass fraction are 3.0% sucrose and mass fraction is 0.5% agar, the pH value is 6.0, cultivation temperature is 26 ℃, intensity of illumination is 2000 lx, illumination every day 10h, the formation test-tube plantlet of taking root about 20d;
4) induce the test tube water chestnut: test-tube plantlet is transferred to induce on the Water Chestnuts medium cultivates, described to induce the Water Chestnuts medium be the LS medium, add 6-benzyl aminopurine (6-BA) 1.0mg/L, methyl α-naphthyl acetate (NAA) 0.5mg/L, active carbon (AC) 1.0g/L, mass fraction are that 10.0% sucrose and mass fraction are 0.5% agar, the pH value is 6.0, cultivation temperature is 24 ℃, intensity of illumination 2000 lx, illumination every day 8h, 30d induce later on and form the test tube water chestnut.
Embodiment 4
Take the lateral bud of water chestnut kind Shayang County water chestnut as explant, through stem apex startup, shoot proliferation, take root, induce and form the test tube water chestnut.Comprise the steps:
1) stem apex starts: take Shayang County water chestnut as test material, get the anosis Shayang County water chestnut lateral bud that cleans up, through 75% after alcohol-pickled 30 seconds, use 0.1%HgCl
2Solution soaked 1 minute, use again aseptic water washing 4 times, each 4 minutes, be inoculated in stem apex and start on the medium, it is the LS medium that described stem apex starts medium, add 6-benzyl aminopurine (6-BA) 1.3mg/L, methyl α-naphthyl acetate (NAA) 0.2mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.5% agar, and the pH value is 5.8, it is 26 ℃ in temperature, under intensity of illumination 2400 lx, illumination every day 10h produces simple bud behind the 30d;
2) shoot proliferation: simple bud is transferred to shoot proliferation cultivation on the shoot proliferation medium, described shoot proliferation medium is the LS medium, add 6-benzyl aminopurine (6-BA) 1.0mg/L, methyl α-naphthyl acetate (NAA) 0.2mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.5% agar, the pH value is 5.8, cultivation temperature is 25 ℃, intensity of illumination 2000 lx, illumination every day 10h, 30d produce new clump bud.
3) take root: the clump bud is divided into simple bud, be transferred to root induction on the root media, used root media is the LS medium, add 6-benzyl aminopurine (6-BA) 0.8mg/L, indolebutyric acid (IBA) 0.6mg/L, active carbon (AC) 0.7g/L, mass fraction are 3.0% sucrose and mass fraction is 0.5% agar, the pH value is 6.0, cultivation temperature is 26 ℃, intensity of illumination is 2500lx, illumination every day 10h, the formation test-tube plantlet of taking root about 20d;
4) induce the test tube water chestnut: test-tube plantlet is transferred to induce on the Water Chestnuts medium cultivates, used to induce the Water Chestnuts medium be the LS medium, add 6-benzyl aminopurine (6-BA) 0.3mg/L, methyl α-naphthyl acetate (NAA) 0.1mg/L, active carbon (AC) 0.7g/L, mass fraction are that 4% sucrose and mass fraction are 0.5% agar, the pH value is 5.8, cultivation temperature is 25 ℃, intensity of illumination 2500lx, illumination every day 8h, 30d induce later on and form the test tube water chestnut.
Claims (1)
1. the method for a test tube water chestnut tissue-culturing quick-propagation is characterized in that, may further comprise the steps:
1) stem apex starts: terminal bud or the lateral bud of choosing anosis water chestnut are explant, behind cleaning and sterilizing, being inoculated in stem apex starts on the medium, it is the LS medium that described stem apex starts medium, add 6-benzyl aminopurine 0.5～2.6mg/L, methyl α-naphthyl acetate 0.1～0.5mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.5～0.6% agar, the pH value is 5.8～6.0, cultivation temperature is 24～26 ℃, intensity of illumination is 2000～2500 lx, illumination every day 10h is until grow into simple bud or clump bud;
2) shoot proliferation: simple bud or clump bud are transferred to shoot proliferation cultivation on the shoot proliferation medium, described shoot proliferation medium is the LS medium, add 6-benzyl aminopurine 0.5～2.0mg/L, methyl α-naphthyl acetate 0.1～0.5mg/L, mass fraction is 3.0% sucrose and mass fraction is 0.5～0.6% agar, the pH value is 5.8～6.0, cultivation temperature is 24～26 ℃, intensity of illumination 2000～2500lx, illumination every day 10h is to producing new clump bud;
3) culture of rootage: the clump bud is divided into simple bud, be transferred to root induction on the root media, described root media is the LS medium, add 6-benzyl aminopurine 0.5～1.0mg/L, indolebutyric acid 0.1～1. 0mg/L, active carbon 0.5～1.0g/L, mass fraction are 3.0% sucrose and mass fraction is 0.5～0.6% agar, the pH value is 5.8～6.0, cultivation temperature is 24～26 ℃, intensity of illumination is 2000～2500lx, illumination every day 10h, the formation test-tube plantlet of taking root;
4) induce the test tube Water Chestnuts: test-tube plantlet is transferred to induce on the Water Chestnuts medium cultivates, described to induce the Water Chestnuts medium be the LS medium, add 6-benzyl aminopurine 0.1～1.0mg/L, methyl α-naphthyl acetate 0.1～0.5mg/L, active carbon 0.5～1.0g/L, mass fraction are that 3.0～12.0% sucrose and mass fraction are 0.5～0.6% agar, the pH value is 5.8～6.0, cultivation temperature is 24～26 ℃, intensity of illumination 2000～2500lx, illumination every day 8h induces to form the test tube water chestnut.
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| CN103688862A (en) * | 2013-12-16 | 2014-04-02 | 广西壮族自治区农业科学院生物技术研究所 | Method for efficiently propagating water chestnut tissue culture seedlings |
| CN107637523A (en) * | 2017-10-31 | 2018-01-30 | 博白县富山水果种植专业合作社 | A kind of method for culturing seedlings of horseshoe |
| CN113207697A (en) * | 2021-06-18 | 2021-08-06 | 广西壮族自治区农业科学院 | Water chestnut stem tip tissue culture method |
| CN113455392A (en) * | 2021-07-13 | 2021-10-01 | 长江大学 | Culture method of water chestnut bud seedlings |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103688862A (en) * | 2013-12-16 | 2014-04-02 | 广西壮族自治区农业科学院生物技术研究所 | Method for efficiently propagating water chestnut tissue culture seedlings |
| CN103688862B (en) * | 2013-12-16 | 2016-01-27 | 广西壮族自治区农业科学院生物技术研究所 | A kind of method of water chestnut plantlet in vitro high efficiently multiplying |
| CN107637523A (en) * | 2017-10-31 | 2018-01-30 | 博白县富山水果种植专业合作社 | A kind of method for culturing seedlings of horseshoe |
| CN113207697A (en) * | 2021-06-18 | 2021-08-06 | 广西壮族自治区农业科学院 | Water chestnut stem tip tissue culture method |
| CN113455392A (en) * | 2021-07-13 | 2021-10-01 | 长江大学 | Culture method of water chestnut bud seedlings |
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Application publication date: 20130501 |