CN104026010B - A kind of abductive approach of mulberry tree cotyledon indefinite bud - Google Patents
A kind of abductive approach of mulberry tree cotyledon indefinite bud Download PDFInfo
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- CN104026010B CN104026010B CN201410243674.4A CN201410243674A CN104026010B CN 104026010 B CN104026010 B CN 104026010B CN 201410243674 A CN201410243674 A CN 201410243674A CN 104026010 B CN104026010 B CN 104026010B
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Abstract
The invention discloses a kind of abductive approach of mulberry tree cotyledon indefinite bud, belong to plant genetic engineering field.Specifically with cotyledon in embryo in Mulberry Seeds for explant, the seed that running water soaks is separated and obtains cotyledon after surface disinfection, and to be connected in inducing culture directly induction and to produce indefinite bud, indefinite bud grows to 1cm and moves in root media, formation whole plant.The regeneration plant time cycle adopting the inventive method to produce through indefinite bud is short, and result is stablized, and reproducible, being mainly used in Mulberry transgenic, is prerequisite and the basis of Mulberry transgenic breeding.
Description
Technical field
The present invention relates to the direct abductive approach of a kind of cotyledon indefinite bud, be the acceptor portion of Mulberry transgenic, belong to plant genetic engineering field.
Background technology
In numerous plant regeneration systems, belong to unicellular origin by the method for inducer blade indefinite bud, somatic embryo and protoplast regeneration, be suitable for the receptor system of plant transgene.In the transgenic technology of numerous maturation, the plant such as peanut, willow adopts mostly infects the mode that blade produces adventitious shoot regeneration plant, the crop such as cotton, paddy rice adopts mostly infects hypocotyl induction embryo callus subculture through body embryo mode regeneration plant or first induce embryo callus subculture, infect embryo callus subculture again, by body embryo germination mode regeneration plant.Protoplast system is used less as genetically modified receptor system, and main cause is that this method is uneconomical, and cost is high, and difficulty is also large.Compare somatic embryo system and indefinite bud system, adventitious shoot regeneration system has and obviously has superiority, if same plant can use somatic embryo mode and adventitious shoot regeneration mode to carry out transgenosis simultaneously, generally all can adopt the mode of adventitious shoot regeneration.The direct evoking adventive bud time cycle is short, and about 3 week of general induction, indefinite bud starts to occur, then grows and within about 4 weeks, can move bottle and take root, and the whole cycle was at about 3 months; And adopting the mode of somatic embryos, step is various, and the time is long, from dip-dye, need 9 months to 1 year to obtaining plant.Adopt the mode of adventitious shoot regeneration, lopsided seedling is few, and regrowth growing way is prosperous, and adopts the mode of somatic embryo to have more lopsided seedling.
The scholars such as Wang Yong disclose induction and the regeneration plant of Folium Mori indefinite bud, be that acceptor is carried out the research of mulberry tree gene engineering and prepared with Folium Mori, but its basic element of cell division is lower with auxin concentration, and be the method for taking once to induce, do not consider the difference of indefinite bud initiation and development to hormone concentration requirement, so cotyledon adventitious bud induction frequency is very low; In addition, inorganizable of the method and anatomical method prove that the bud of inducing is indefinite bud or plumule source.
Summary of the invention
The technical problem solved: for the deficiencies in the prior art, the invention provides a kind of abductive approach of mulberry tree cotyledon indefinite bud, according to difference degree of the passing induction that indefinite bud initiation and development requires hormone concentration, the startup that evoking adventive bud occurs under high concentration of cytokinin (6-BA), proceed to after indefinite bud starts in the low concentration basic element of cell division (6-BA) and promote uncertain buds growth, method is stablized, and inductivity is high.
Technical scheme: the abductive approach of mulberry tree cotyledon indefinite bud provided by the invention, comprises the following steps:
(1) Seed imbibition and cultivation
Mulberry Seeds clear water is soaked after 12h, uses running water 1-2h, secondly with 7% liquor natrii hypochloritis or 0.1% mercuric chloride solution sterilizing 15 minutes, then with sterile purified water flushing 3-4 time.
(2) cotyledon is separated
Under the Mulberry Seeds that imbibition sterilizing is good is placed in anatomical lens, tear seed coat off with sharp mouth tweezer, by two panels cotyledon separately, with scalpel, cotyledon is excised from base portion.
(3) adventitious bud inducing
Separation obtained on cotyledon access adventitious bud induction culture base 1, to be placed in illumination box 26 DEG C, photoperiod 16L/8D Fiber differentiation, proceeds to after cultivating 3 weeks in adventitious bud induction culture base 2.
Described adventitious bud induction culture base 1 is: MS medium+30g/L sucrose+7g/L agar powder+K8P vitamin+10% coconut milk+3mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), and pH value is 5.8-6.0.
Described adventitious bud induction culture base 2 is: MS medium+30g/L sucrose+7g/L agar powder+K8P vitamin+10% coconut milk+2mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), and pH value is 5.8-6.0.
(4) indefinite bud growth
After adventitious bud formation, move in mulberry tree tissue cultures subculture medium when adventitious bud formation 3-4 sheet blade, subculture medium is: MS medium+30g/L sucrose+7g/L agar powder+B family vitamin+0.5mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), and pH value is 5.8-6.0.
(5) adventitious bud rooting
When indefinite bud grows to 1 centimetre, move into root media and form whole plant, root media is: 1/2MS medium+30g/L sucrose+7g/L agar powder+B family vitamin+0.1mg/L indolebutyric acid (IBA), and pH value is 5.8-6.0.
beneficial effect: the present invention is that Mulberry transgenic provides an effective acceptor regenerative system, and the time cycle is short, and adventitious bud inducing is effective, result is stablized, repeating effect is good, for extensive transgenosis mulberry tree creates possibility, realizes mulberry tree kind matter and carries out germplasm innovation and deposit by transgenic approach.
Accompanying drawing explanation
Fig. 1 is cotyledon separation graph; Fig. 2 is the indefinite bud that induction cotyledon produces;
Fig. 3 is mulberry tree cotyledon indefinite bud stereoscan photograph; Fig. 4 is mulberry tree cotyledon indefinite bud generation histologic section.
Embodiment
Mode below by embodiment is described in further details the present invention, and these embodiments are only used for the present invention is described, do not limit the scope of the invention.
Embodiment 1
(1) Seed imbibition and cultivation
Get 200 first cross Sang Feng to speed mulberry seed, put into beaker and add clear water and soak 12 hours, then beaker is placed on tap down-flow water and rinses 2 hours; To burn water in the cup afterwards falls dry, and add 75% alcohol and rock 10 minutes, after the essence of falling dry wine, clear water rinses 2 times; Add 7% liquor natrii hypochloritis's surface disinfection 15 minutes, constantly rock therebetween, move in superclean bench, rinse 3 times with sterilizing aquae destillata.
(2) cotyledon is separated
Seed after surface disinfection process is placed in superclean bench on anatomical lens, with scalpel at hilum place side cut-away seed coat breach, gently presses seed seed coat breach offside with tweezers, complete embryo is extruded gently; With dissecting forceps, two panels cotyledon in embryo is separated gently, with scalpel, cotyledon is excised from base portion.
(3) adventitious bud inducing
Separation is obtained cotyledon and be seeded in (MS medium+30g/L sucrose+7g/L agar powder+K8P vitamin+10% coconut milk+3mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA) in adventitious bud induction culture base 1, pH value 6.0), to be placed in illumination box 26 DEG C, photoperiod 16L/8D Fiber differentiation, cultivate and move in adventitious bud induction culture base 2 (MS medium+30g/L sucrose+7g/L agar powder+K8P vitamin+10% coconut milk+2mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), pH value is 6.0) for 3 weeks.
(4) indefinite bud growth
Indefinite bud shifts out to during 3-4 sheet blade by uncertain buds growth, cut off lower blade, move into indefinite bud subculture medium (MS medium+30g/L sucrose+7g/L agar powder+B family vitamin+0.5mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), pH value is 6.0).
(5) adventitious bud rooting
Treat that indefinite bud grows to 1 centimetre, top stem section is cut and moves into root media (1/2MS medium+30g/L sucrose+7g/L agar powder+B family vitamin+0.1mg/L indolebutyric acid (IBA), pH value is 6.0); Plant is taken out after forming whole plant by adventitious bud rooting, washes away root agar, moves in indoor growing matrix and grows hardening.
Fig. 1 is that embodiment 1 indefinite bud is separated, and as can be seen from Figure 1, is separated the not subsidiary any embryonic tissue of the cotyledon obtained; Fig. 2 is the indefinite bud that induction cotyledon produces, and be a bud as can be seen from Figure 2, neither one bud occupies advantage and plays main bud effect; Fig. 3 is indefinite bud scanning electron microscope (SEM) photograph, and as can be seen from Figure 3 on fine structure, indefinite bud stacks mutually, does not have dividing of main bud lateral bud; Fig. 4 is indefinite bud histologic section, is attached thereto when indefinite bud occurs as can be seen from Figure 4 without any vascular tissue.
Table 1 embodiment 1 cotyledon adventitious bud inducing situation (after Fiber differentiation investigation in 1 month)
Embodiment 2
(1) Seed imbibition and cultivation
Get 200 mulberry seeds that grow directly from seeds, put into beaker and add clear water immersion 12h, then beaker is placed on tap down-flow water and rinses 2 hours; Then drained by burning water in the cup, add 0.1% mercuric chloride solution surface disinfection 15 minutes, constantly rock therebetween, move in superclean bench, after being poured out by mercuric chloride solution, aqua sterilisa rinses 3 times.
By good for surface disinfection seed access germination medium (1/2MS medium+15g/L sucrose+7g/L agar powder, pH value 6.0), cultivate and be separated for cotyledon two days later.
(2) cotyledon is separated
Seed after cultivation is placed in superclean bench on anatomical lens, with scalpel at hilum place side cut-away seed coat breach, gently tears seed coat indentation, there with two tweezers, strip out complete for embryo.With dissecting forceps, two panels cotyledon in embryo is separated gently, with scalpel, cotyledon is excised from base portion.
(3) adventitious bud inducing
(MS medium+30g/L sucrose+7g/L agar powder+K8P vitamin+10% coconut milk+3mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA) in adventitious bud induction culture base 1 is seeded in by being separated the cotyledon obtained, pH value is 6.0), to be placed in illumination box 26 DEG C, photoperiod 16L/8D Fiber differentiation, cultivate and move in adventitious bud induction culture base 2 (MS medium+30g/L sucrose+7g/L agar powder+K8P vitamin+10% coconut milk+2mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), pH value is 6.0) for 3 weeks.
(4) indefinite bud growth
Indefinite bud shifts out to during 3-4 sheet blade by uncertain buds growth, cut off lower blade, move into indefinite bud subculture medium (MS medium+30g/L sucrose+7g/L agar powder+B family vitamin+0.5mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), pH value is 6.0).
(5) adventitious bud rooting
Treat that indefinite bud grows to 1 centimetre, top stem section is cut and moves into root media (1/2MS medium+30g/L sucrose+7g/L agar powder+B family vitamin+0.1mg/L indolebutyric acid (IBA), pH value is 6.0).Form whole plant after adventitious bud rooting, plant is taken out, washes away root agar, move in indoor growing matrix and grow hardening.
Table 2 embodiment 2 adventitious bud inducing situation
Embodiment 3
(1) Seed imbibition and cultivation
Get 200 Guangdong producing hybrid mulberries, put into beaker and add a little clear water immersion 12h, then beaker is placed on tap down-flow water and rinses 2 hours.Drained by burning water in the cup, add 0.1% mercuric chloride solution surface disinfection 15 minutes, constantly rock therebetween, move in superclean bench, after being poured out by mercuric chloride solution, aqua sterilisa rinses 3 times.
By good for surface disinfection seed access germination medium (1/2MS medium+15g/L sucrose+7g/L agar powder, pH value 6.0), find that there is the seed be contaminated by bacterial therebetween, uncontaminated seed is moved in new germination medium.
(2) cotyledon is separated
The cultivation seed of 3 days is placed in superclean bench on anatomical lens, with scalpel at hilum place side cut-away seed coat breach, tears seed coat indentation, there gently with two tweezers, strip out complete for embryo.With dissecting forceps, two panels cotyledon in embryo is separated, with scalpel, cotyledon is excised from base portion.
(3) adventitious bud inducing
Separation is obtained cotyledon and be seeded in (MS medium+30g/L sucrose+7g/L agar powder+K8P vitamin+10% coconut milk+3mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA) in adventitious bud induction culture base 1, pH value is 6.0), to be placed in illumination box 26 DEG C, photoperiod 16L/8D Fiber differentiation, cultivate and move in adventitious bud induction culture base 2 (MS medium+30g/L sucrose+7g/L agar powder+K8P vitamin+10% coconut milk+2mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), pH value is 6.0) for 3 weeks.
(4) indefinite bud growth
Indefinite bud shifts out to during 3-4 sheet blade by uncertain buds growth, cut off lower blade, move into indefinite bud subculture medium (MS medium+30g/L sucrose+7g/L agar powder+B family vitamin+0.5mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), pH value is 6.0).
(5) adventitious bud rooting
Treat that indefinite bud grows to 1 centimetre, top stem section is cut and moves into root media (1/2MS medium+30g/L sucrose+7g/L agar powder+B family vitamin+0.1mg/L indolebutyric acid (IBA), pH value is 6.0).Form whole plant after adventitious bud rooting, plant is taken out, washes away root agar, move in indoor growing matrix and grow hardening.
Table 3 embodiment 3 adventitious bud inducing situation
Claims (1)
1. an abductive approach for mulberry tree cotyledon indefinite bud, is characterized in that comprising the following steps:
(1) Mulberry Seeds clear water is soaked after 12h, use running water 1-2h, then use 7% liquor natrii hypochloritis or 0.1% mercuric chloride solution sterilizing 15 minutes, then rinse 3-4 time with sterile purified water;
(2) under anatomical lens, peel off seed coat in gnotobasis, be separated cotyledon;
(3) separation obtained on cotyledon access adventitious bud induction culture base 1, to be placed in illumination box 26 DEG C, photoperiod 16L/8D Fiber differentiation, proceeds in adventitious bud induction culture base 2 after cultivating 3 weeks;
Described adventitious bud induction culture base 1 is: MS medium+30g/L sucrose+7g/L agar powder+K8P+10% coconut milk+3mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), and pH value is 5.8-6.0;
Described adventitious bud induction culture base 2 is: MS medium+30g/L sucrose+7g/L agar powder+K8P+10% coconut milk+2mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), and pH value is 5.8-6.0;
(4) move into when adventitious bud formation 3-4 sheet blade in mulberry tree tissue cultures subculture medium;
Described subculture medium is: MS medium+30g/L sucrose+7g/L agar powder+B family vitamin+0.5mg/L6-benzyl aminoadenine (6-BA)+0.1mg/L methyl α-naphthyl acetate (NAA), and pH value is 5.8-6.0;
(5) when indefinite bud grows to 1 centimetre, move into root media and form whole plant;
Described root media is: 1/2MS medium+30g/L sucrose+7g/L agar powder+B family vitamin+0.1mg/L indolebutyric acid (IBA), and pH value is 5.8-6.0.
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| CN108575757A (en) * | 2018-05-18 | 2018-09-28 | 安徽圣桑林业科技发展有限公司 | One seed pod mulberry tissue cultures |
| CN109329054A (en) * | 2018-10-18 | 2019-02-15 | 广东海洋大学 | A kind of vitrified method of reduction mulberries adventitious bud |
| CN111053030A (en) * | 2019-11-15 | 2020-04-24 | 湖北省农业科学院经济作物研究所 | Culture medium for obtaining mulberry test-tube plantlet and preparation method and application thereof |
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| CN103168676A (en) * | 2013-03-08 | 2013-06-26 | 湖北大学 | New Broussonetia papyrifera mulberry tree hybrid distant hybridization and polyploidization breeding method |
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Patent Citations (3)
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|---|---|---|---|---|
| CN103125377A (en) * | 2011-11-24 | 2013-06-05 | 赵新红 | H.rosa-sinensis immature embryo isolated culture and special culture medium thereof |
| CN103168676A (en) * | 2013-03-08 | 2013-06-26 | 湖北大学 | New Broussonetia papyrifera mulberry tree hybrid distant hybridization and polyploidization breeding method |
| CN103270952A (en) * | 2013-06-18 | 2013-09-04 | 西南大学 | Sinkiang drug mulberry tissue culture and rapid propagation culture medium |
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| Effect of nutrients on in vitro culture of Morus alba L. (White mulberry);Ankit Pradhan，et al.;《Bioresearch bulletin》;20100622;第1卷;摘要，第10页左栏第1段 * |
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