CN103782811B - A kind of watermelon plantlet in vitro test tube micro-grafting method - Google Patents
A kind of watermelon plantlet in vitro test tube micro-grafting method Download PDFInfo
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- CN103782811B CN103782811B CN201410065302.7A CN201410065302A CN103782811B CN 103782811 B CN103782811 B CN 103782811B CN 201410065302 A CN201410065302 A CN 201410065302A CN 103782811 B CN103782811 B CN 103782811B
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Abstract
The invention discloses a kind of watermelon plantlet in vitro test tube micro-grafting method.Step comprises: stock preparation, scion preparation, grafting, graft cultivation, grafting are transplanted.With the watermelon of aseptic germination, cucurbit or pumpkin seedling for stock, with aspermous watermelon plantlet in vitro for scion, cleft graft carries out grafting, graft is placed in solid culture medium cultivate, transplant in seedling medium after the grafting domestication survived, then be transplanted to land for growing field crops in good time, planting type management routinely.Invention removes stock cotyledon and growing point, without the need to removing the lateral bud on stock, saving of work and time after between transplant field; Namely plantlet in vitro about 1cm can be used for grafting, without the need to taking root, saves time; Easy to implement the method, easy and simple to handle, survival rate is high, have wide range of applications; Can carry out in the anniversary, manual control condition, solve the problem that watermelon tissue culture regeneration plantlet of transplant survival rate is low; For the application of graft technology provides new way, for the research of grafting correlation theory provides the Study system of science.
Description
Technical field
The present invention relates to graft technology field, more specifically relate to a kind of watermelon plantlet in vitro test tube micro-grafting method.Easy to implement the method, easy and simple to handle, survival rate is high, have wide range of applications.For the application of graft technology provides new way, for the research of grafting correlation theory provides the Study system of science.
Background technology
Watermelon [
citrullus Lanatus(Thunb.) Matsum. et Nakai] Curcurbitaceae, Citrullus, the annual herbaceous plant that overgrows.Originate in Africa, be one of large fruit in the world five, in Chinese national economy, occupy critical role.Grafting is the unique effective ways solving the soil-borne diseases such as watermelon blight at present, but graft growth course is complicated, is subject to the impact of environmental condition, lacks desirable Study system, causes the research of grafting correlation theory delayed.Meanwhile, watermelon cultured in vitro regeneration plant is taken root undesirable, and transplanting survival rate after taking root is not high yet.
Micrografting is a kind of technology of aseptically stock and scion being carried out grafting, cultivation, the combination of Plant Tissue Breeding and graft technology, have the cycle short, occupy little space, instant effect, result of study reliability are strong, graft growth conditions can the advantage such as manual control.(1) it can be used as the method for preserving special breeding intermediate materials, the direct planting survival rates of aseptic seedling such as Regenerated Plants From Somatic Cells, transfer-gen plant, Shoot Tip Culture plant is low, constrain the application in genetic modification of plants etc. of distant hybridization, biotechnology and cell engineering to a certain extent, micrografting technology can improve this situation.As Agricultural University Of Hebei Wang Yan rosy clouds etc. work out extensive efficient graft technology-" bonding method " preserved cotton heredity segregation population, rare germ plasm resource and distant hybridization sterile material and acquire a special sense, utilize this technology extensive grafting transgenic cotton plant, hereditary segregation population, conventional cotton plant etc.(2) integrenric grafting hybrid (chimera) can also be cultivated, plant between genus as far away in many affiliations, though can not sexual hybridization be carried out, but can obtain by mutual grafting the distant hybrid adopting sexual hybridization to obtain, the distant grafting hybrid that cultivation stock scion compatibility is good, can improve the quality and yield etc. of fruit.Various on flowers market " skill chrysanthemum ", generally all adopts yellow wormwood artemisia, sweet wormwood, Sievers wormwood, Chinese mugwort etc. to do the cultivation of stock grafting cultivation chrysanthemum and forms.(3) what grafting also can be utilized to produce can genetic variation breeding, although grafting is vegetative propagation theoretically, can not cause the change of scion characters of progenies, it is found that some grafting can cause hereditability to make a variation.Such as in the integrenric grafting of the different plant of Solanaceae of chromosome number, observe chromosomal proterties and have very large variation: the destruction of reproductive process, the change of chromosome quantity in sexual cell, the change of chromosome form, the appearance etc. of chromosome coloured differently degree and chromosome translocation.(4) can be the Study system that the theoretical research of grafting relevant rudimentary provides science.
Watermelon crop is carried out the technical research of test tube micrografting and has not yet to see report.The present invention can solve the problem that in the training of watermelon group, the current Transplantation of Regenerated Plantlets survival rate existed is low to a certain extent, application for graft technology provides new approach, simultaneously for the researchs such as the research of Grafting watermelon correlation theory, breed improvement, cell engineering provide technology to ensure, also significant on Grafted Watermelon Seedlings is produced.
Summary of the invention
The object of the invention is to propose a kind of watermelon plantlet in vitro test tube micro-grafting method, with aseptic seed germination seedling for stock, the grafting method being scion with watermelon plantlet in vitro, easy to implement the method, easy and simple to handle, survival rate is high, have wide range of applications.
In order to realize above-mentioned object, the present invention by the following technical solutions:
A kind of watermelon plantlet in vitro test tube micro-grafting method, the steps include:
A, stock prepare, and watermelon or cucurbit or Pumpkin Seed, through surface sterilization, are then inoculated in the MS solid culture medium of autoclave sterilization and cultivate, and can be used for grafting during the open and flat and tool a slice true leaf of stock seedling age 15 days-20 days, two panels cotyledon;
B, scion prepare, and elongation medium grows to 25 days about-30 days, the plantlet in vitro that grows to the growing way of 1.5 cm-2 cm consistent does scion;
C, grafting, by stock cotyledon and cut with upper part, and become otch by rip cutting in the middle of its hypocotyl, for subsequent use; To be cut sth. askew out in scion lower end " V " shape wound, for subsequent use; Scion is inserted in stock otch, make the formation layer close contact of stock and scion;
D, graft are cultivated, and graft is placed in autoclaved solid culture medium, seals moisturizing with sealed membrane, cultivate under 28 DEG C of conditions, humidity requirement more than 95%;
E, grafting are transplanted, after grafting about 25 days, and the grafting survived, after domestication room tames 3 days-4 days, is transplanted and in seedling medium, is cultured to tool 4 blades can be transplanted to land for growing field crops, planting type management routinely.
Described stock prepares, watermelon or cucurbit or Pumpkin Seed surface sterilization process: after watermelon or cucurbit or Pumpkin Seed shell, superclean bench carries out sterilization treatment: in the liquor natrii hypochloritis of 5%, soak 5 min, thoroughly clean up with sterile water, then blot the surface of the seed moisture with aseptic filter paper;
Culture environment is: temperature 26 DEG C, illumination 2500 lux-3000 lux, every day 16 h light, cultivate under 8 h dark conditions.
During described grafting is transplanted, about 25 d after grafting, the grafting survived is in the domestication of domestication chamber opening sealed membrane, day temperature 22-25 DEG C, night temperatures 20 DEG C, after 3 days-4 days, takes out regrowth with tweezers from triangular flask, clean root medium, do not injure root hair, be then transplanted in sterilized vermiculite nutritive cube, temperature remains on 25 DEG C as far as possible, land for growing field crops can be transplanted to during tool 4 blades, planting type management routinely after plant to be planted delays seedling;
Described domestication room is greenhouse by solar heat.
Innovation of the present invention there are provided a kind of method of preserving the special breeding intermediate materials of watermelon.The direct planting survival rates of aseptic seedling such as Regenerated Plants From Somatic Cells, transfer-gen plant, Shoot Tip Culture plant is low, and constrain the application in genetic modification of plants etc. of distant hybridization, biotechnology and cell engineering, micrografting technology can improve this situation.
The invention provides the method for a kind of Watermelon Germplasm innovation simultaneously.Cultivate integrenric grafting hybrid (chimera), plant between the genus that many affiliations are far away, can not carry out sexual hybridization, can be obtained the distant hybrid usually adopting sexual hybridization to obtain by grafting.The distant grafting hybrid that cultivation stock scion compatibility is good, can improve the quality and yield etc. of Watermelon Fruit, what grafting can be utilized to produce can genetic variation breeding.
The present invention compared with prior art, has the following advantages and effect:
1, eliminate stock cotyledon and growing point, grafting operation is simple, without the need to removing the lateral bud on stock, saving of work and time after between transplant field, and makes nutrition fully supply scion;
2, namely plantlet in vitro 1 about cm can be used for grafting, without the need to taking root, saves time;
3, easy to implement the method, easy and simple to handle, survival rate is high, has wide range of applications, and can preserve the breeding intermediate materials that watermelon is special, can carry out Watermelon Germplasm innovation again, can also carry out seeling industry;
4, can carry out in the anniversary, manual control condition, the application for graft technology provides new approach, also for the theoretical research of grafting relevant rudimentary provides the Study system of science.
Embodiment
Scion preparation, stock prepare and grafting procedures is all carried out at aseptic operating platform, and graft is cultivated and carried out at climatic cabinate, and grafting is transplanted and carries out at green house.Concrete enforcement:
A kind of watermelon plantlet in vitro test tube micro-grafting method, the steps include:
A, stock prepare, and watermelon or cucurbit or Pumpkin Seed, through surface sterilization, are then inoculated in the MS solid culture medium of autoclave sterilization and cultivate, and can be used for grafting during the open and flat and tool a slice true leaf of stock seedling age 15 days-20 days, two panels cotyledon;
B, scion prepare, and elongation medium grows to 25 days about-30 days, the plantlet in vitro that grows to the growing way of 1.5 cm-2 cm consistent does scion;
C, grafting, by stock cotyledon and cut with upper part, and become otch by rip cutting in the middle of its hypocotyl, for subsequent use; To be cut sth. askew out in scion lower end " V " shape wound, for subsequent use; Scion is inserted in stock otch, make the formation layer close contact of stock and scion;
D, graft are cultivated, and graft is placed in autoclaved solid culture medium, seals moisturizing with sealed membrane, cultivate under 28 DEG C of conditions, humidity requirement more than 95%;
E, grafting are transplanted, after grafting about 25 days, and the grafting survived, after domestication room tames 3 days-4 days, is transplanted and in seedling medium, is cultured to tool 4 blades can be transplanted to land for growing field crops, planting type management routinely.
Described stock prepares, watermelon or cucurbit or Pumpkin Seed surface sterilization process: after watermelon or cucurbit or Pumpkin Seed shell, superclean bench carries out sterilization treatment: in the liquor natrii hypochloritis of 5%, soak 5 min, thoroughly clean up with sterile water, then blot the surface of the seed moisture with aseptic filter paper;
Culture environment is: temperature 26 DEG C, illumination 2500 lux-3000 lux, every day 16 h light, cultivate under 8 h dark conditions.
During described grafting is transplanted, about 25 d after grafting, the grafting survived is in the domestication of domestication chamber opening sealed membrane, day temperature 22-25 DEG C, night temperatures 20 DEG C, after 3 days-4 days, takes out regrowth with tweezers from triangular flask, clean root medium, do not injure root hair, be then transplanted in sterilized vermiculite nutritive cube, temperature remains on 25 DEG C as far as possible, land for growing field crops can be transplanted to during tool 4 blades, planting type management routinely after plant to be planted delays seedling;
Described domestication room is greenhouse by solar heat.
Innovation of the present invention there are provided a kind of method of preserving the special breeding intermediate materials of watermelon.The direct planting survival rates of aseptic seedling such as Regenerated Plants From Somatic Cells, transfer-gen plant, Shoot Tip Culture plant is low, and constrain the application in genetic modification of plants etc. of distant hybridization, biotechnology and cell engineering, micrografting technology can improve this situation.
The invention provides the method for a kind of Watermelon Germplasm innovation simultaneously.Cultivate integrenric grafting hybrid (chimera), plant between the genus that many affiliations are far away, can not carry out sexual hybridization, can be obtained the distant hybrid usually adopting sexual hybridization to obtain by grafting.The distant grafting hybrid that cultivation stock scion compatibility is good, can improve the quality and yield etc. of Watermelon Fruit, what grafting can be utilized to produce can genetic variation breeding.
Claims (3)
1. a watermelon plantlet in vitro test tube micro-grafting method, the steps include:
A, stock prepare, and watermelon or cucurbit or Pumpkin Seed, through surface sterilization, are then inoculated in the MS solid culture medium of autoclave sterilization and cultivate, and can be used for grafting during the open and flat and tool a slice true leaf of stock seedling age 15 days-20 days, two panels cotyledon;
B, scion prepare, and elongation medium grows to 25 days-30 days, the plantlet in vitro that grows to the growing way of 1.5 cm-2 cm consistent does scion;
C, grafting, by stock cotyledon and cut with upper part, and become otch by rip cutting in the middle of its hypocotyl, for subsequent use; To be cut sth. askew out in scion lower end " V " shape wound, for subsequent use; Scion is inserted in stock otch, make the formation layer close contact of stock and scion;
D, graft are cultivated, and graft is placed in autoclaved solid culture medium, seals moisturizing with sealed membrane, cultivate under 28 DEG C of conditions, humidity requirement more than 95%;
E, grafting are transplanted, after grafting about 25 days, and the grafting survived, after domestication room tames 3 days-4 days, is transplanted and in seedling medium, is cultured to tool 4 blades is transplanted to land for growing field crops, planting type management routinely.
2. a kind of watermelon plantlet in vitro test tube micro-grafting method as claimed in claim 1, is characterized in that:
Described stock prepares, watermelon or cucurbit or Pumpkin Seed surface sterilization process: after watermelon or cucurbit or Pumpkin Seed shell, superclean bench carries out sterilization treatment: in the liquor natrii hypochloritis of 5%, soak 5 min, thoroughly clean up with sterile water, then blot the surface of the seed moisture with aseptic filter paper;
Culture environment is: temperature 26 DEG C, illumination 2500 lux-3000 lux, every day 16 h light, cultivate under 8 h dark conditions.
3. a kind of watermelon plantlet in vitro test tube micro-grafting method as claimed in claim 1, is characterized in that:
During described grafting is transplanted, about 25 d after grafting, the grafting survived is tamed in domestication chamber opening sealed membrane, day temperature 22-25 DEG C, night temperatures 20 DEG C, after 3 days-4 days, with tweezers, regrowth is taken out from triangular flask, clean root medium, then be transplanted in sterilized vermiculite nutritive cube, temperature remains on 25 DEG C, is transplanted to land for growing field crops after plant to be planted delays seedling during tool 4 blades, planting type management routinely;
Described domestication room is greenhouse by solar heat.
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| CN106688589A (en) * | 2017-01-16 | 2017-05-24 | 山西省农业科学院棉花研究所 | Cotton test tube seedling trench transplanting method |
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| CN106106149A (en) * | 2016-06-23 | 2016-11-16 | 四川农业大学 | Efficient hot pepper genetic transformation and the method for plant regeneration |
| CN106417025A (en) * | 2016-10-14 | 2017-02-22 | 山西农业大学 | Test tube micro-grafting method for seedless grape embryo-rescued malformed plantlets |
| CN107493926A (en) * | 2017-09-27 | 2017-12-22 | 界首市菜盟家庭农场 | A kind of high yield high-quality implantation methods of watermelon |
| CN108848994A (en) * | 2018-04-18 | 2018-11-23 | 宁夏林业研究院股份有限公司 | A kind of method of suitable grape micrografting seedling Liquid Culture |
| CN113796219A (en) * | 2021-09-06 | 2021-12-17 | 河北农业大学 | Watermelon tissue culture seedling out-of-tube grafting and transplanting method |
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| CN101731089B (en) * | 2009-12-04 | 2011-11-30 | 武汉市农业科学研究所 | Method for producing seedless watermelon seedlings |
| US20130298273A1 (en) * | 2012-05-07 | 2013-11-07 | Nunhems B.V. | Watermelon Double Grafting Methods |
| CN102972190A (en) * | 2012-12-07 | 2013-03-20 | 北海市种子管理站 | Method for grafting watermelons and cultivating grafted seedlings |
| CN103229672A (en) * | 2013-04-23 | 2013-08-07 | 安徽省农业科学院园艺研究所 | Mini watermelon grafting and seedlings raising method |
| CN103314790B (en) * | 2013-06-05 | 2015-12-23 | 南京市蔬菜科学研究所 | A kind of seedling-cultivating method of autumn in summer watermelon |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106688589A (en) * | 2017-01-16 | 2017-05-24 | 山西省农业科学院棉花研究所 | Cotton test tube seedling trench transplanting method |
| CN106688589B (en) * | 2017-01-16 | 2019-11-15 | 山西省农业科学院棉花研究所 | Cotton test tube seedling trench method for transplanting |
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Address after: 430345 Hubei Province, Wuhan city Huangpi District Wu lake agricultural agricultural ecological park in Wuhan city of Wuhan City Patentee after: INSTITUTE OF CROP SCIENCES OF WAAST Address before: 430345 Hubei Province, Wuhan city Huangpi District Wu lake agricultural agricultural ecological park in Wuhan city of Wuhan City Patentee before: Wuhan Institute of Agricultural Science |
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Effective date of registration: 20161206 Address after: 430345 Hubei Province, Wuhan city Huangpi District Wu lake agricultural agricultural ecological park in Wuhan city of Wuhan City Patentee after: INSTITUTE OF CROP SCIENCES OF WAAST Patentee after: WUHAN GUYIFENG TECHNOLOGY SERVICE CO., LTD. Address before: 430345 Hubei Province, Wuhan city Huangpi District Wu lake agricultural agricultural ecological park in Wuhan city of Wuhan City Patentee before: INSTITUTE OF CROP SCIENCES OF WAAST |