CN107873517A - A kind of method by Chinese herbaceous peony cotyledon evoked callus regenerated adventitious bud - Google Patents

A kind of method by Chinese herbaceous peony cotyledon evoked callus regenerated adventitious bud Download PDF

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Publication number
CN107873517A
CN107873517A CN201711122690.8A CN201711122690A CN107873517A CN 107873517 A CN107873517 A CN 107873517A CN 201711122690 A CN201711122690 A CN 201711122690A CN 107873517 A CN107873517 A CN 107873517A
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China
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callus
culture
herbaceous peony
chinese herbaceous
cotyledon
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孙晓梅
潘庆
杨宏光
张丽杰
姚希诺
李雪婷
费日雯
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Shenyang Agricultural University
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Shenyang Agricultural University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The present invention relates to a kind of method by Chinese herbaceous peony cotyledon evoked callus regenerated adventitious bud, pass through Chinese herbaceous peony embryo Primary culture, it will be cut after seed disinfection, it is inoculated into using MS solid mediums as minimal medium, incubation time 15d obtains embryo seedling, and 20~30d of strong seedling culture obtains the tissue-cultured seedling of robust growth；45~60d of induction of callus time, obtain full bud green, hard-packed callus lines；Callus lines are accessed into adventitious buds differentiation culture medium, obtain the vigorous adventitious bud of growing way.Convenient material drawing of the present invention, the tissue-cultured seedling by the use of seed as explant culture, is not subject to seasonal restrictions, and can draw materials in the anniversary, and primary embryo culture pollution rate is far below other explants；Callus induction rate and Differentiation ration of adventitious buds is greatly improved；Callus induction and adventitious buds differentiation stage, anti-browning agent is added, effectively suppress browning problem, effectively solve the problems, such as high with Chinese herbaceous peony explant evoked callus pollution rate.

Description

A kind of method by Chinese herbaceous peony cotyledon evoked callus regenerated adventitious bud
Technical field
The invention belongs to the field of tissue culture of plant, and in particular to a kind of to be regenerated by Chinese herbaceous peony cotyledon evoked callus The method of adventitious bud.
Background technology
Chinese herbaceous peony（Paeonia lactiflora）For Paeoniaceae Paeonia perennial root flowers, have and view and admire and medicinal valency Value, it is traditional famous flower of China, cultivation history is long, is known as the title of " flower phase ", is that Landscape often uses plant.
At present, the rearing new variety work of Chinese herbaceous peony is more using tradition such as artificial orientation's pollination, induced mutations and natural mutations Breeding method, but stamen valve or anandrous problem, and natural hybridization pollination setting percentage be present in some kinds of Chinese herbaceous peony Low, the factor such as reproduction isolation between kind limits the application of traditional breeding way.These reasons make the Chinese herbaceous peony traditional breeding method time Length, efficiency of selection is low, renewal speed is slow, causes Chinese herbaceous peony Breeding Situation to be difficult to the demand for meeting growing flowers market.Group It is currently used efficient breeding practice to knit culture technique, and the fast development of transgenic technology in recent years, for the product of Chinese herbaceous peony Kind improvement is advantageously.Rely on Chinese herbaceous peony tissue culture technique and establish regenerative system, the genetic transformation of Chinese herbaceous peony gene is realized, so as to change Become the biological characteristics of Chinese herbaceous peony, seek a high yield for Chinese herbaceous peony, the breeding road of stabilization is the joint research mesh of domestic and foreign scholars Mark.
In most plants, blade is generally acknowledged evoked callus optimal material, and when cotyledon is development of plants First leaf, stores substantial amounts of nutrient, and cell division is active.Forefathers have not to the callus induction of Chinese herbaceous peony, propagation, differentiation With the achievement in research of degree, most of research terminates in callus proliferation, though small part research has the differentiation of adventitious bud, but All it is using the explant that open country grows naturally as material, explant has the problem of pollution is unmanageable, and early stage disinfects Waste substantial amounts of manpower and materials, and also influenceed by season, it is impossible to the anniversary is studied, constrain Chinese herbaceous peony rapid breeding and Renewal speed, callus differentiation cycle length in addition, easy browning, Differentiation ration of adventitious buds is only 10%-20%, and bud growing way compared with It is weak.
The content of the invention
In order to make up above-mentioned the deficiencies in the prior art, the invention provides one kind by Chinese herbaceous peony cotyledon evoked callus again The method of raw adventitious bud, can effectively solve the problems, such as, raising adventitious bud point high with Chinese herbaceous peony explant evoked callus pollution rate Rate, strengthens the growing way of bud, and is not influenceed by season, can be cultivated with the anniversary.
Technical scheme is as follows：
A kind of method by Chinese herbaceous peony cotyledon evoked callus regenerated adventitious bud, it has main steps that：
1) Chinese herbaceous peony embryo Primary culture：It will be cut, be inoculated into using MS solid mediums as basic culture after seed disinfection Base, addition gibberellin 0.5mg L-1, cellar culture is carried out, incubation time 15d obtains embryo seedling；
2) Chinese herbaceous peony tissue-cultured seedling strong seedling culture：Embryo seedling is transferred into using MS solid mediums as minimal medium, adds gibberellin 0.5mg· L-1, the mgL of 6-benzyl aminopurine 0.5-1, kinetin 0.5mg L-1, it is strong that cellar culture 20-30 d obtain growth Strong tissue-cultured seedling；
3) callus induction：The cotyledon of tissue-cultured seedling is cut from root, is inoculated into callus tissue culture base, callus training Base is supported using the 1/2MS solid mediums improved as minimal medium, adds Thidiazuron 0.5mgL-1, 2,4- dichlorphenoxyacetic acids 0.2 mg·L-1, polyvinylpyrrolidone 1gL-1, incubation time 45-60 d, obtain full bud green, hard-packed callus Tissue block；
4) adventitious buds differentiation culture：By callus lines access adventitious buds differentiation culture medium, adventitious bud culture base with improve 1/ 2MS solid mediums are minimal medium, addition Thidiazuron 0.2 mgL-1, the mgL of methyl α-naphthyl acetate 0.5-1, polyvinyl pyrrole Alkanone 1gL-1, incubation time 45-60 d.
In such scheme, the formula of described improvement 1/2MS solid mediums is：A great number of elements improvement is NH4NO3 412.5 mg·L-1、KNO3 475 mg·L-1、CaCl2·2H2O 220 mg·L-1、MgSO4·7H2O 185 mg·L-1、 KH2PO4 350 mg·L-1, other trace elements, molysite, organic component content are constant.
In such scheme, the specific method of described seed disinfection is：Paeonia lactiflora seed is placed under flowing water and rinsed well, is shelled Except kind of a skin, once, with sterilizing filter paper the surface of the seed water is blotted with 75% alcohol-pickled 30 s, aseptic water washing in superclean bench Point, then use 0.1%HgCl2 5min is soaked, aseptic water washing 3~5 times, sterilizing filter paper blots the surface of the seed moisture, is sterilized Seed afterwards.
In such scheme, the female parent of described Paeonia lactiflora seed is powder jade slave, male parent is Fen Yu buildings.
In such scheme, described conventional culture conditions are：25 ± 2 DEG C of temperature, light application time 12hd-1, intensity of illumination 1200±100lx。
Beneficial effects of the present invention are：Convenient material drawing, the tissue-cultured seedling by the use of seed as explant culture, is not limited by season System, can draw materials, and primary embryo culture pollution rate is far below other explants in the anniversary；Tissue-cultured seedling using seed as explant culture Material of the cotyledon as evoked callus, cell division is active, and growth is vigorous, and callus induction rate is high, up to 90% with On, Differentiation ration of adventitious buds is improved to more than 50%, and the growing way of adventitious bud is vigorous；Callus induction and adventitious buds differentiation stage, Anti- browning agent is added, effectively suppresses browning problem, improves operating efficiency, can effectively solve with Chinese herbaceous peony explant callus induction group Knit the problem of pollution rate is high.
Brief description of the drawings
Fig. 1 is Chinese herbaceous peony embryo germination.
Fig. 2 is the tissue-cultured seedling that embryo culture obtains.
Fig. 3 is that cotyledon starts to expand.
Fig. 4 is Callus formation.
Fig. 5 is callus differentiation adventitious bud.
Fig. 6 is adventitious bud seedling.
Embodiment
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
1. materials：The Chinese herbaceous peony hybrid seed that in late August, 2016 harvests in Agricultural University Of Shenyang Chinese herbaceous peony garden, the mother of seed This kind is powder jade slave, and male parent varieties are Fen Yu buildings, are the common kind of Chinese herbaceous peony.
2. seed disinfection and inoculation：
（1）Cleaning：Flowing water rinses 10 ~ 20min of seed, cleans surface dirt；
（2）Seed soaking：The big seed of full grain is selected, soaks 24 ~ 48h, makes kind of soft and soggyization, beneficial to a stripping kind skin；
（3）Remove kind of a skin：Cut with scalpel from the side of the seed, peel off kind of a skin, in materials due to Chinese herbaceous peony embryo it is tender it is small, be difficult to Completely strip, easily damage, a small amount of endosperm around embryo can be retained, the method is easy to operate, will not damage embryo, and embryo is sprouted Hair influences smaller；
（4）The Paeonia lactiflora seed for removing kind of skin is carried out disinfection in superclean bench, first with 75% alcohol-pickled 30s, aseptic water washing 1 time, sterilizing filter paper blots surface moisture, then is inhaled with 0.1% mercuric chloride soaking disinfection 5min, aseptic water washing 3 ~ 5 times, sterilizing filter paper Dry surface moisture；
（5）Seed after sterilization is inoculated into the culture medium for embryo culture, to prevent cross pollution, one kind of every bottle of inoculation Embryo, 200 bottles are inoculated with altogether.
3. tissue-cultured seedling culture：
（1）Embryo primary culture medium：MS minimal mediums, addition gibberellin 0.5mgL-1, additional saccharose 30gL-1, agar 6g·L-1, pH 5.8；
（2）Tissue-cultured seedling strong seedling culture base：MS minimal mediums, addition gibberellin 0.5mgL-1, 6-benzyl aminopurine 1.0mg L-1。
4. callus induction：
（1）Vegetable material：Tissue-cultured seedling cotyledon is cut from base portion, then is cut into the cotyledon piece of 0.5cm × 0.5cm sizes, every bottle connects 2 pieces of cotyledons of kind, are inoculated with 200 bottles altogether；
（2）Culture medium prescription：The 1/2MS solid mediums of improvement are minimal medium, specifically will be a large amount of in MS culture mediums Element improvement is NH4NO3 412.5 mg·L-1、KNO3 475 mg·L-1、CaCl2·2H2O 220 mg·L-1、MgSO4· 7H2O 185 mg·L-1、KH2PO4 350 mg·L-1, other trace elements, molysite, organic component content are constant；Add thiophene benzene Grand 0.5mgL-1, 2,4- dichlorphenoxyacetic acid 0.2mgL-1, polyvinylpyrrolidone 1gL-1, additional saccharose 30gL-1, Agar 6.0gL-1, pH 5.8；
（3）Condition of culture：25 ± 2 DEG C of temperature, light application time 12hd-1, 1200 ± 100lx of intensity of illumination, incubation time 60d.
5. adventitious buds differentiation：
（1）Full bud green, hard-packed callus lines access adventitious buds differentiation culture medium, every bottle of inoculation 2 will be derived Block callus, 200 bottles are inoculated with altogether；
（2）Culture medium prescription：The 1/2MS solid mediums of improvement are minimal medium, specifically will be a large amount of in MS culture mediums Element improvement is NH4NO3 412.5 mg·L-1、KNO3 475 mg·L-1、CaCl2·2H2O 220 mg·L-1、MgSO4· 7H2O 185 mg·L-1、KH2PO4 350 mg·L-1, other trace elements, molysite, organic component content are constant；Add thiophene benzene Grand 0.2 mgL-1, methyl α-naphthyl acetate 0.5mgL-1, polyvinylpyrrolidone 1gL-1, additional saccharose 30gL-1, agar 6.0g L-1, pH 5.8；
（3）Condition of culture：25 ± 2 DEG C of temperature, light application time 12hd-1, 1200 ± 100lx of intensity of illumination, incubation time 60 d。
6. statistical indicator and method：
（1）The embryo disinfected is accessed into embryo primary culture medium, germination rate and pollution rate are counted after cultivating 15d；
Embryo sum × 100% of embryo number/inoculation of germination rate=sprouting；
Embryo sum × 100% of embryo number/inoculation of pollution rate=pollution；
（2）Cotyledon is accessed in callus inducing medium, healing rate and melting brown rate are counted after cultivating 60d；
The cotyledon number of healing rate=formation callus/inoculation cotyledon sum × 100%；
The callus number of melting brown rate=browning/inoculation cotyledon sum × 100%；
（3）Callus is transferred and counts Differentiation ration of adventitious buds after cultivating 60d in adventitious buds differentiation culture medium；
The callus number of inductivity=differentiation budding/access callus sum × 100%.
7. result：Embryo is transferred in embryo primary culture medium, 7 d observe embryo germination, and 15 d kinds scutellums open （See Fig. 1）, investigation embryo germination rate is 74.5%, pollution rate 0.8%；Tissue-cultured seedling strong seedling culture base is transferred to, 30 d can be observed Training young plant leaf switchs to peak green, trains seedling rooting（See Fig. 2）；Cotyledon piece is inoculated in callus inducing medium, 30-60 d It is observed that cotyledon expands, callus is formed（See Fig. 3, Fig. 4）, investigation callus induction rate is 93.5%, and melting brown rate is 3.5%；It is transferred to callus differential medium, 30-60 d are observed that bud point, the adventitious bud that callus differentiates, no Normal bud seedling（See Fig. 5, Fig. 6）, Differentiation ration of adventitious buds 56.5%.

Claims (5)

  1. A kind of 1. method by Chinese herbaceous peony cotyledon evoked callus regenerated adventitious bud, it is characterised in that comprise the following steps：
    1) Chinese herbaceous peony embryo Primary culture：Cut, be inoculated into using MS solid mediums as basic training after Paeonia lactiflora seed is sterilized Support base, addition gibberellin 0.5mg L-1, cellar culture is carried out, incubation time 15d obtains embryo seedling；
    2) Chinese herbaceous peony tissue-cultured seedling strong seedling culture：Embryo seedling is transferred into using MS solid mediums as minimal medium, adds gibberellin 0.5mg· L-1, the mgL of 6-benzyl aminopurine 0.5-1, kinetin 0.5mg L-1, it is strong that cellar culture 20-30d obtains growth Strong tissue-cultured seedling；
    3) callus induction：The cotyledon of tissue-cultured seedling is cut from root, is inoculated into callus tissue culture base, callus training Base is supported using the 1/2MS solid mediums improved as minimal medium, the formula of described improvement 1/2MS solid mediums is：Greatly Secondary element improvement is NH4NO3 412.5 mg·L-1、KNO3 475 mg·L-1、CaCl2·2H2O 220 mg·L-1、MgSO4· 7H2O 185 mg·L-1、KH2PO4 350 mg·L-1, other trace elements, molysite, organic component content are constant, add thiophene benzene Grand 0.5mgL-1, the mgL of 2,4- dichlorphenoxyacetic acid 0.2-1, polyvinylpyrrolidone 1gL-1, incubation time 45-60d, Obtain full bud green, hard-packed callus lines；
    4) adventitious buds differentiation culture：By callus lines access adventitious buds differentiation culture medium, adventitious bud culture base with improve 1/ 2MS solid mediums are minimal medium, and the formula of described improvement 1/2MS solid mediums is：A great number of elements is improved NH4NO3 412.5 mg·L-1、KNO3 475 mg·L-1、CaCl2·2H2O 220 mg·L-1、MgSO4·7H2O 185 mg· L-1、KH2PO4 350 mg·L-1, other trace elements, molysite, organic component content are constant, addition Thidiazuron 0.2 mgL-1, The mgL of methyl α-naphthyl acetate 0.5-1, polyvinylpyrrolidone 1gL-1, incubation time 45-60d.
  2. 2. a kind of method by Chinese herbaceous peony cotyledon evoked callus regenerated adventitious bud according to claim 1, its feature It is：The specific method of described seed disinfection is：Paeonia lactiflora seed is placed under flowing water and rinsed well, kind of a skin is divested, ultra-clean 75% alcohol-pickled 30 s of workbench, aseptic water washing once, with sterilizing filter paper blot the surface of the seed moisture, then with 0.1% HgCl2 5min, aseptic water washing 3-5 times are soaked, sterilizing filter paper blots the surface of the seed moisture, the seed after being sterilized.
  3. 3. a kind of method by Chinese herbaceous peony cotyledon evoked callus regenerated adventitious bud, its feature exist according to claim 2 In：It is described divest kind of skin specific method be：Cut with scalpel from the side of the seed, retain a small amount of endosperm around embryo and avoid damage to Embryo.
  4. 4. a kind of method by Chinese herbaceous peony cotyledon evoked callus regenerated adventitious bud according to claim 1, its feature It is：The female parent of described Paeonia lactiflora seed is powder jade slave, male parent is Fen Yu buildings.
  5. 5. a kind of method by Chinese herbaceous peony cotyledon evoked callus regenerated adventitious bud according to claim 1, its feature It is：Described cellar culture is specifically：25 ± 2 DEG C of temperature, light application time 12hd-1, 1200 ± 100lx of intensity of illumination.
CN201711122690.8A 2017-11-14 2017-11-14 A kind of method by Chinese herbaceous peony cotyledon evoked callus regenerated adventitious bud Pending CN107873517A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109496493A (en) * 2018-12-10 2019-03-22 山东省林业科学研究院 A method of promote Chinese herbaceous peony bud to sprout
CN111602594A (en) * 2020-04-20 2020-09-01 四川农业大学 Method for reducing explant pollution in peony tissue culture
CN112913690A (en) * 2021-01-28 2021-06-08 四川农业大学 Peony in-vitro regeneration method

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CN104996304A (en) * 2015-08-24 2015-10-28 扬州大学 Culture medium and culture method for inducing callus differentiation through peony leaves

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CN104996304A (en) * 2015-08-24 2015-10-28 扬州大学 Culture medium and culture method for inducing callus differentiation through peony leaves

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109496493A (en) * 2018-12-10 2019-03-22 山东省林业科学研究院 A method of promote Chinese herbaceous peony bud to sprout
CN109496493B (en) * 2018-12-10 2021-05-07 山东省林业科学研究院 Method for promoting germination of peony buds
CN111602594A (en) * 2020-04-20 2020-09-01 四川农业大学 Method for reducing explant pollution in peony tissue culture
CN112913690A (en) * 2021-01-28 2021-06-08 四川农业大学 Peony in-vitro regeneration method

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Application publication date: 20180406