CN104488708A - Method for tissue culture of buddha pedestal hoop - Google Patents

Method for tissue culture of buddha pedestal hoop Download PDF

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Publication number
CN104488708A
CN104488708A CN201410727351.2A CN201410727351A CN104488708A CN 104488708 A CN104488708 A CN 104488708A CN 201410727351 A CN201410727351 A CN 201410727351A CN 104488708 A CN104488708 A CN 104488708A
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callus
adventitious bud
naa
induction
culture medium
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CN201410727351.2A
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吕永平
陈志�
汪一婷
牟豪杰
周迪江
陈剑平
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Zhejiang Academy of Agricultural Sciences
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Zhejiang Academy of Agricultural Sciences
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Abstract

The invention relates to a method for tissue culture of a buddha pedestal hoop, and belongs to the field of plant tissue culture. The method comprises the following steps: preparing a culture medium, selecting and disinfecting an explant, carrying out callus induction, inducing adventitious buds, multiplying the adventitious buds, culturing sound seedlings, inducing rootage of the adventitious buds, and domesticating and transplanting tissue culture seedlings, wherein a basic culture medium selects an MS culture medium, the used amount of sucrose is 20-40g/L, a coagulating agent is agar powder with the used amount of 8-10g/L, the pH value of the culture medium is adjusted to 5.6-5.8 before sub-package; a callus inducing culture medium comprises 3.0-5.0mg/L MS+6-BA and 0.05-0.2mg/L NAA; an adventitious bud inducing culture medium comprises 1.0-3.0mg/L MS+6-BA and 0.05-0.2mg/L NAA; an adventitious bud multiplying culture medium comprises 0.5-1.0mg/L MS+6-BA and 0.05-0.2mg/L NAA; and a rootage culture medium is 0.01-0.5mg/L MS+IBA or 0.01-0.5mg/L 1/2 MS+IBA. The method provided by the invention has the advantage that an established tissue culture rapid propagation technology system of the buddha pedestal hoop has a multiplication coefficient of 2-2.5, a rooting rate of 100% and a transplantation survival rate of 100%.

Description

A kind of method of Buddhist seat hoop tissue cultures
Technical field
The present invention relates to plant tissue culture technique, be specifically related to a kind of method of Buddhist seat hoop tissue cultures.
Background technology
Buddhist seat hoop (Bulbine mesembryanthoides subsp namaquensis) originates in South Africa, for hyacinthaceae squama apium perennial bulb class meat herbaceous plant, Buddhist seat hoop plant type is small and exquisite, peculiar, plant tool meat semicircle or olive-type blade, there is translucent window, there is certain ornamental value, main with little potted plant training at present, be applicable to balcony, windowsill and desk to intersperse, view and admire, there are larger market prospects.
Under nature, Buddhist seat hoop lateral bud is less, and meat blade cuttage survival rate is low, although strain Post flowering can self-pollination, seed is minimum and quantity is few, difficultly collects, and annual reproductive number is limited, is unfavorable for the popularization of Buddhist seat hoop.Plant tissue culture technique is considered to the most efficient plant seedling fast breeding technique means at present, but at present, domestic there is not yet utilizes plant tissue culture technology to carry out the bibliographical information of Fast-propagation to Buddhist seat hoop.
Summary of the invention
The object of the invention is to the deficiency overcoming prior art existence, and a kind of method of Buddhist seat hoop tissue cultures is provided, set up good Buddhist seat hoop tissue culture regeneration system, for the production of Buddhist seat hoop Industrialization of seeds and seedlings, new varieties initiative and quality genetic improvement etc. carry out technical support.
The object of the invention is to have come by following technical solution.The method of this Buddhist seat hoop tissue cultures, the method comprises the steps:
1) the choosing and processing of explant: choose Buddhist seat hoop spike as starting material, the Buddhist seat hoop spike collected peels off small ear base portion sepal, more whole inflorescence is cut into the segment of 0.5cm length, as group training explant;
2) callus of induce: on superclean bench, explant through surface sterilization is being transferred in aseptic inoculation dish, explant surface moisture is sucked with aseptic filter paper, be inoculated in calli induction media MS+6-BA 3.0 ~ 5.0mg/L+NAA 0.05 ~ 0.2mg/L and carry out callus of induce cultivation, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1;
3) adventitious bud inducing: by Buddhist seat hoop callus material transfer good for the propagation of acquisition in callus induction medium MS+6-BA 1.0 ~ 3.0mg/L+NAA 0.05 ~ 0.2mg/L, carry out indefinite bud, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1;
4) adventitious bud proliferation: on aseptic working platform, the indefinite bud 2 ~ 3 one clump that the 0.5 ~ 1.0cm obtained by callus of induce is high, utilize aseptic scalpel that base portion is caused some wounds, be inoculated in adventitious bud proliferation medium and carry out Multiplying culture, adventitious bud proliferation medium is MS+6-BA 0.5 ~ 1.0mg/L+NAA 0.05 ~ 0.2mg/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1;
5) adventitious bud rooting induction: Seedling height is separated into single indefinite bud to the indefinite bud of growing thickly of 2 ~ 4cm from base portion, the induction of adventive root is carried out in access root media, root media is MS+IBA 0.01 ~ 0.5mg/L or 1/2MS+IBA0.01 ~ 0.5mg/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1;
6) plantlet in vitro domestication and transplanting: after indefinite bud base portion grows the adventive root of 3 ~ 51 ~ 2cm, group training is moved to greenhouse, scattered light opens bottle cap after cultivating 5d, then cultivates 1 ~ 2d, completes before plantlet in vitro is transplanted and tames, then plantlet in vitro is taken out from blake bottle, clean plantlet in vitro surface agar, dry and slightly fold to plantlet in vitro surface, Buddhist seat hoop plantlet in vitro is planted in matrix, 15d is cultivated in moisturizing more than 90%, then normally cultivates in greenhouse;
Callus of induce, adventitious bud inducing and propagation, adventitious bud rooting induction minimal medium are MS medium, and dosage of sucrose is 20 ~ 40g/L, and coagulating agent is agar powder, and consumption is 8 ~ 10g/L, is adjusted to 5.6 ~ 5.8 before medium pH packing; Calli induction media is MS+6-BA 3.0 ~ 5.0mg/L+NAA 0.05 ~ 0.2mg/L; Callus induction medium is MS+6-BA 1.0 ~ 3.0mg/L+NAA 0.05 ~ 0.2mg/L; Adventitious bud proliferation medium is MS+6-BA 0.5 ~ 1.0mg/L+NAA 0.05 ~ 0.2mg/L; Root media is MS+IBA 0.01 ~ 1.0mg/L or 1/2MS+IBA 0.01 ~ 1.0mg/L; Callus of induce, adventitious bud inducing and propagation, adventitious bud rooting condition of culture are that cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1.
Before the selection of initial explant and process, callus of induce, adventitious bud inducing, adventitious bud proliferation, root induction, transplanting domestication process and after transplanting moisturizing more than 90% cultivate 15d.
Beneficial effect of the present invention is:
1, Buddhist seat hoop quick reproduction technique is carried out by tissue culture technique, produce by the restriction of area, season, weather and maternal plant growth year, be convenient to factorial seedling growth, can produce according to order, production time and scale controlled, for applying of Buddhist seat hoop provides sufficient high quality seedling guarantee.
2, reproduction coefficient reaches 2 ~ 2.5, rooting rate 100%, transplanting survival rate 100%, reaches the requirement that seedling factorial seedling growth is produced.
3, plantlet in vitro offspring seedling genetic background is consistent, keeps maternal merit to greatest extent.
4, be conducive to this genus other plant kind tissue-culturing rapid propagation Establishing to use for reference, promote.
5, set up good tissue-culturing rapid propagation system, for utilizing Plant Biotechnology from now on, the research work such as rearing new variety, genetic improvement is carried out to Buddhist seat hoop and lay the foundation.
Embodiment
Below by embodiment, the present invention is further elaborated, and help is understood the present invention by embodiment better, but the present invention is not limited only to following embodiment.
The method of this Buddhist seat hoop tissue cultures, the method comprises the steps:
1, medium and condition of culture: minimal medium selects MS medium, dosage of sucrose is 20 ~ 40g/L, and coagulating agent is agar powder, and consumption is 8 ~ 10g/L, is adjusted to 5.6 ~ 5.8 before medium pH packing; Calli induction media is MS+6-BA 3.0 ~ 5.0mg/L+NAA 0.05 ~ 0.2mg/L; Callus induction medium is MS+6-BA 1.0 ~ 3.0mg/L+NAA0.05 ~ 0.2mg/L; Adventitious bud proliferation medium is MS+6-BA 0.5 ~ 1.0mg/L+NAA 0.05 ~ 0.2mg/L; Root media is MS+IBA 0.01 ~ 1.0mg/L or 1/2MS+IBA 0.01 ~ 1.0mg/L; Callus of induce, adventitious bud inducing and propagation, adventitious bud rooting condition of culture are that cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1.
2, the process of explant and sterilization: choose Buddhist seat hoop spike as starting material, the Buddhist seat hoop spike collected peels off small ear base portion sepal, again whole inflorescence is cut into the segment of about 0.5cm length, as explant, the explant liquid detergent solution cleaning 30min handled well, period constantly stirs gently, then explant material 30 ~ 60min of cleaning through liquid detergent of running water, on the superclean bench of sterilization, the Buddhist seat hoop explant cleaned through flowing water is being transferred in aseptic conical flask, with 75% alcohol-pickled 30 ~ 40s, period constantly rocks conical flask gently, removal is attached to Buddhist seat hoop explant blibbing, pour out 75% alcohol, use 0.1%HgCl2 solution soak-out material 8 ~ 10min again, or be 1%NaClO solution soak-out material 10 ~ 15min with effective chlorine density, constantly conical flask is rocked gently between soak period, removal is attached to Buddhist seat hoop explant blibbing, pour out 0.1%HgCl 2solution or 1%NaClO solution, by sterile water wash through the explant material of disinfecting 4 ~ 5 times, the explant sterile water after process soaks, for subsequent use.
3, callus of induce: on superclean bench, Buddhist seat hoop explant through surface sterilization is transferred in aseptic inoculation dish, explant surface moisture is sucked with aseptic filter paper, be inoculated in calli induction media MS+6-BA 3.0 ~ 5.0mg/L+NAA0.05 ~ 0.2mg/L and carry out callus of induce, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1.
4, adventitious bud inducing: by Buddhist seat hoop callus material transfer good for the propagation of acquisition in callus induction medium MS+6-BA 1.0 ~ 3.0mg/L+NAA 0.05 ~ 0.2mg/L, carry out differentiation adventitious buds induction, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1.
5, adventitious bud proliferation: on aseptic working platform, the indefinite bud 2 ~ 3 one clump that the 0.5 ~ 1.0cm obtained by callus induction is high, utilize aseptic scalpel that base portion is caused some wounds gently, be inoculated in adventitious bud proliferation medium and carry out Multiplying culture, adventitious bud proliferation medium is MS+6-BA 0.5 ~ 1.0mg/L+NAA 0.05 ~ 0.2mg/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1.
6, adventitious bud rooting induction: Seedling height is split into single to the indefinite bud of growing thickly of 2 ~ 4cm from base section, the induction of adventive root is carried out in access root media, root media is MS+IBA 0.01 ~ 1.0mg/L or 1/2MS+IBA 0.01 ~ 1.0mg/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1.
7, plantlet in vitro domestication and transplanting: after indefinite bud base portion grows the meat adventive root of 3 ~ 52 ~ 3cm, seedling of taking root moves to greenhouse, scattered light opens bottle cap after cultivating 5d, then cultivates 1 ~ 2d, and the plant that taken root by Buddhist seat hoop is carefully taken out from blake bottle, the medium of plantlet in vitro base portion is cleaned with running water, then place 2 ~ 3 days in ventilating and cooling place, dry and slightly fold to plantlet in vitro surface, Buddhist seat hoop plantlet in vitro is planted in matrix, 15d is cultivated in moisturizing more than 90%, then normally cultivates in greenhouse.

Claims (2)

1. a method for Buddhist seat hoop tissue cultures, is characterized in that: the method comprises the steps:
1) the choosing and processing of explant: choose Buddhist seat hoop spike as starting material, the Buddhist seat hoop spike collected peels off small ear base portion sepal, more whole inflorescence is cut into the segment of 0.5cm length, as group training explant;
2) callus of induce: on superclean bench, explant through surface sterilization is being transferred in aseptic inoculation dish, explant surface moisture is sucked with aseptic filter paper, be inoculated in calli induction media MS+6-BA 3.0 ~ 5.0mg/L+NAA 0.05 ~ 0.2mg/L and carry out callus of induce cultivation, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1;
3) adventitious bud inducing: by Buddhist seat hoop callus material transfer good for the propagation of acquisition in callus induction medium MS+6-BA 1.0 ~ 3.0mg/L+NAA 0.05 ~ 0.2mg/L, carry out indefinite bud, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1;
4) adventitious bud proliferation: on aseptic working platform, the indefinite bud 2 ~ 3 one clump that the 0.5 ~ 1.0cm obtained by callus of induce is high, utilize aseptic scalpel that base portion is caused some wounds, be inoculated in adventitious bud proliferation medium and carry out Multiplying culture, adventitious bud proliferation medium is MS+6-BA 0.5 ~ 1.0mg/L+NAA 0.05 ~ 0.2mg/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1;
5) adventitious bud rooting induction: Seedling height is separated into single indefinite bud to the indefinite bud of growing thickly of 2 ~ 4cm from base portion, the induction of adventive root is carried out in access root media, root media is MS+IBA 0.01 ~ 0.5mg/L or 1/2MS+IBA0.01 ~ 0.5mg/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1;
6) plantlet in vitro domestication and transplanting: after indefinite bud base portion grows the adventive root of 3 ~ 51 ~ 2cm, group training is moved to greenhouse, scattered light opens bottle cap after cultivating 5d, then cultivates 1 ~ 2d, completes before plantlet in vitro is transplanted and tames, then plantlet in vitro is taken out from blake bottle, clean plantlet in vitro surface agar, dry and slightly fold to plantlet in vitro surface, Buddhist seat hoop plantlet in vitro is planted in matrix, 15d is cultivated in moisturizing more than 90%, then normally cultivates in greenhouse;
Callus of induce, adventitious bud inducing and propagation, adventitious bud rooting induction minimal medium are MS medium, and dosage of sucrose is 20 ~ 40g/L, and coagulating agent is agar powder, and consumption is 8 ~ 10g/L, is adjusted to 5.6 ~ 5.8 before medium pH packing; Calli induction media is MS+6-BA 3.0 ~ 5.0mg/L+NAA 0.05 ~ 0.2mg/L; Callus induction medium is MS+6-BA 1.0 ~ 3.0mg/L+NAA 0.05 ~ 0.2mg/L; Adventitious bud proliferation medium is MS+6-BA 0.5 ~ 1.0mg/L+NAA 0.05 ~ 0.2mg/L; Root media is MS+IBA 0.01 ~ 1.0mg/L or 1/2MS+IBA 0.01 ~ 1.0mg/L; Callus of induce, adventitious bud inducing and propagation, adventitious bud rooting condition of culture are that cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 14h/d, and intensity of illumination is 40 ~ 60 μm of olm -2s -1.
2. the method for Buddhist seat hoop tissue cultures according to claim 1, is characterized in that: before the selection of initial explant and process, callus of induce, adventitious bud inducing, adventitious bud proliferation, root induction, transplanting domestication process and after transplanting moisturizing more than 90% cultivate 15d.
CN201410727351.2A 2014-12-04 2014-12-04 Method for tissue culture of buddha pedestal hoop Pending CN104488708A (en)

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Publication number Priority date Publication date Assignee Title
CN102499090A (en) * 2011-11-07 2012-06-20 上海旭东园艺有限公司 Method for isolated culture of Haworthia succulent plants
CN103125394A (en) * 2013-03-13 2013-06-05 浙江省农业科学院 Method for establishing tissue culture regeneration system of tylecodon paniculatus
CN103141387A (en) * 2013-03-08 2013-06-12 浙江省农业科学院 Method for cultivating haworthia maughanii tissue
CN103270947A (en) * 2013-03-08 2013-09-04 浙江省农业科学院 Duvalia angustiloba tissue culturing method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102499090A (en) * 2011-11-07 2012-06-20 上海旭东园艺有限公司 Method for isolated culture of Haworthia succulent plants
CN103141387A (en) * 2013-03-08 2013-06-12 浙江省农业科学院 Method for cultivating haworthia maughanii tissue
CN103270947A (en) * 2013-03-08 2013-09-04 浙江省农业科学院 Duvalia angustiloba tissue culturing method
CN103125394A (en) * 2013-03-13 2013-06-05 浙江省农业科学院 Method for establishing tissue culture regeneration system of tylecodon paniculatus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
G. BRINGMANN等: "In vitro germination and establishment of tissue cultures of Bulbine caulescens and of two Kniphofia species (Asphodelaceae)", 《PLANT CELL REP》 *

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