CN105850737A - Rhododendron Delavayi 'Cosmopolitan' chromosome doubling method - Google Patents

Rhododendron Delavayi 'Cosmopolitan' chromosome doubling method Download PDF

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Publication number
CN105850737A
CN105850737A CN201610218315.2A CN201610218315A CN105850737A CN 105850737 A CN105850737 A CN 105850737A CN 201610218315 A CN201610218315 A CN 201610218315A CN 105850737 A CN105850737 A CN 105850737A
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culture
chromosome doubling
cosmopolitan
callus
alpine rose
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CN201610218315.2A
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CN105850737B (en
Inventor
蒋淑磊
李志斌
白霄霞
李国松
赵玉芬
李振勤
徐立军
边光亚
李燕
赵儒丹
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SHIJIAZHUANG SHENZHOU FLOWER RESEARCH INSTITUTE CO., LTD.
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Shijiazhuang Academy of Agriculture and Forestry Sciences
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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
    • A01H4/008—Methods for regeneration to complete plants
    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A01H1/08—Methods for producing changes in chromosome number
    • 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

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

Abstract

The invention belongs to the field of biological breeding, is specially used for Rhododendron Delavayi 'Cosmopolitan' chromosome polyploidy breeding and particularly relates to a Rhododendron Delavayi 'Cosmopolitan' chromosome doubling method. The method comprises the following steps: inducing Rhododendron Delavayi 'Cosmopolitan' explants to generate calluses, and treating the calluses with a chemical reagent colchicine to double chromosomes; transferring the calluses into a differential medium for culturing and inducing adventitious buds to generate; transferring the adventitious buds into a propagation culture medium for propagation culture; inoculating strong seedlings into a rooting culture medium and carrying out rooting culture and acclimatization and transplanting; observing the chromosomes under a microscope; and screening doubled plant germchit. The method for doubling chromosomes of an ornamental plant, Rhododendron Delavayi 'Cosmopolitan', is provided in the invention for the first time. According to the polyploidy Rhododendron Delavayi 'Cosmopolitan' cultured by the method, the ornamental value of Rhododendron Delavayi 'Cosmopolitan' is effectively improved.

Description

A kind of alpine rose " young beauty " chromosome doubling method
Technical field
The invention belongs to Biology Breeding field, be exclusively used in alpine rose " young beauty " ploidy breeding, special Do not relate to a kind of alpine rose " young beauty " chromosome doubling method.
Background technology
Alpine rose " young beauty " (Rhododendron Delavayi " Cosmopolitan ") Cuculidae Cuculus polioephalus Belong to, originate in China central and west regions, evergreen shrubs and dungarunga, the secondary color kind of outer pink words spoken by an actor from offstage, plant Dense, plant type is unfolded, leaf green light.Polyploid breeding is a kind of effective breeding technique, is also that plant enters One of important channel changed." epimegetic " is polyploid the most significant formalness feature, show stem sturdy, Blade is generous, blade face is coarse, leaf color is deepened, corolla is big and abundant, petal increases, color is rich and gaudy, additionally, Also there is the delay advantage such as the florescence of some flowers kind, resistance enhancing, improve ornamental value and the business of flowers Product are worth.Along with this variety requirement is got more and more by market, need further to alpine rose plant germplasm resource Carry out innovating and rearing new variety.
Summary of the invention
For above-mentioned technical problem, it is an object of the invention to provide a kind of alpine rose " young beauty " chromosome doubling Technical method, for realizing the purpose of invention, the present invention provides techniques below scheme:
A kind of alpine rose " young beauty " chromosome doubling method, comprises the following steps:
(1) it is transferred in callus culture base train after alpine rose " young beauty " outer implant being sterilized Supporting, induction produces callus；
(2) chemical reagent Colchicine is used to process callus, it is achieved chromosome doubling；
(3) callus will be processed through chemical reagent Colchicine to be transferred in Adventitious bud culture base train Support；
(4) it is transferred in proliferated culture medium carry out enrichment culture by the adventitious bud differentiated；
(5) the healthy and strong good tissue culture plant inoculation through enrichment culture carries out root culture in root media；
(6) Seedling of taking root selecting root system to grow to 2-3cm carries out seedling exercising, with mixing that turfy soil and perlite are made into Close substrate；
(7) Chromosome Number Observation is carried out after acclimatization and transplants under the microscope, after filtering out chromosome doubling Plant seedling.
The method of the outer implant sterilization described in step (1): by the outer implant of the ethanol postincubation of mass concentration 75% 30S, with aseptic water washing 4-5 time, then processes 1 minute with the mercuric chloride of mass concentration 0.1%；
Described callus culture base, is prepared the WPM minimal medium of gained: 230ml by following methods Middle interpolation sucrose 30g, agar powder 6.5g, 2-ip 6mg, be settled to 1 liter with pure water, adjusts pH value 5.4, Cultivation temperature 22 ± 2 DEG C, light culture, incubation time 30 days.
Step (2) detailed process is: callus processes 24h through the Colchicine of mass concentration 0.05%, With aseptic water washing 4 times；
Adventitious bud culture base described in step (3), is prepared the WPM of gained: 230ml by following methods Minimal medium interpolation sucrose 30g, agar 6.5g, TDZ 0.1mg, 2-ip 1mg, constant volume water to 1 liter, Adjust pH value 5.4, cultivation temperature 22 ± 2 DEG C, intensity of illumination 3500Iux, incubation time 45 days.
Proliferated culture medium described in step (4), is prepared the WPM base of gained: 230ml by following methods Basal culture medium adds sucrose 30g, agar powder 6.5g, 2-ip 3mg, NAA 0.5mg, uses pure water constant volume To 1 liter, adjust pH value 5.4, cultivation temperature 22 ± 2 DEG C, intensity of illumination 3500Iux, incubation time 60 days.
Step (5) root media, is prepared in the WPM culture medium of gained: 230ml big by following methods Secondary element subtracts half, wherein NH in WPM culture medium4NO3、(NH4)2SO4、MgSO4.7H2O、 KH2PO4、KNO3、K2SO4Amount reduce half, add sucrose 20g, agar powder 6.5g, activated carbon 1g, IBA0.8mg, is settled to 1 liter with pure water, adjusts pH value 5.4, cultivation temperature 22 ± 2 DEG C, intensity of illumination 3500Iux, incubation time 90 days.
Mixed-matrix described in step (6), including the turfy soil that weight ratio is 5:1 and perlite.
The process that step (7) is concrete is: selects stem apex and the tip of a root of plant, carries out section, under the microscope Observe chromosome doubling situation.
A kind of alpine rose " young beauty " the chromosome doubling method that the present invention provides, uses chemical substance autumn waters--limid eyes Celestial element processes alpine rose " young beauty " callus thus induction polyploid, provides alpine rose plant germplasm Source is innovated, and for cultivating new varieties further, improves ornamental value.
Detailed description of the invention
Being described in conjunction with the embodiments technical scheme, applied chemistry material Colchicine processes alpine rose " young beauty " callus thus induction polyploid.
Wherein, 1 liter of WPM minimal medium composition is:
A great number of elements: NH4NO3400mg；(NH4)2SO4132mg；MgSO4.7H2O 370mg；KH2PO4 170mg；KNO3400mg；K2SO4900mg；
Calcium salt: CaCl296mg；Ca(NO3)2·4H2O 556mg；
Trace element: H3BO36.2mg；MnSO4.H2O 22.3mg；Na2MoO4·2H2O 0.25mg； CuSO4.5H2O 0.025mg；ZnSO4·7H2O 8.6mg；
Iron salt: Na2-EDTA 37.3mg；FeSO4.7H2O 27.8mg；
Vitamin: inositol 100mg；Nicotinic acid (vitamin PP) 0.5mg；Benadon 0.5mg；Hydrochloric acid Thiamine 0.5mg；Glycine 2mg.
Concrete grammar is as follows:
(1) outer implant is gathered
In tender tip growth in spring actively, in the fine morning, " Hydrargyri Oxydum Rubrum is good for the good alpine rose of growth selection gesture People " plant, the tender tissue on clip top and terminal bud, take outer implant 30, peel off the coated squama of terminal bud part Sheet, prepares to disinfect.
(2) disinfect
The most each outer implant toothbrush dips in dish detergent diluent and scrubs one time, then rinses 30 points with tap water Clock, with 75% ethanol postincubation 30S on superclean bench, with aseptic water washing 4-5 time, the mercuric chloride of 0.1% Process 1 minute, then with aseptic water washing 4-5 time.
(3) outer implant callus induction
Outer implant after sterilization is inoculated in callus culture base, cultivates for basic with the WPM of 230ml Base adds sucrose 30g, agar powder 6.5g, 2-ip 2mg, NAA 0.5mg, pure water constant volume to 1 liter, adjusts Whole pH value to 5.4, cultivation temperature 22 ± 2 DEG C, light culture, incubation time 30 days.
(4) Colchicine processes callus
In an aseptic environment, the Colchicine that callus is immersed in 0.05% processes 24h, rushes with sterilized water Wash 4 times.
(5) callus is through breaking up again
Selection quality is in granular form, the callus of rubicundity color, is transferred in redifferential medium.230ml Culture medium be WPM minimal medium add sucrose 30g, agar 6.5g, TDZ 0.1mg, 2-ip 1mg, Constant volume water, to 1 liter, adjusts pH value 5.4, cultivation temperature 22 ± 2 DEG C, intensity of illumination 3500Iux, incubation time 45 days.
(6) enrichment culture
By callus through the bud of differential growth again, be transferred to proliferated culture medium, the culture medium of 230ml with WPM is that minimal medium adds sucrose 30g, and agar 6.5g, NAA 0.5mg, 2-ip 3mg, constant volume water is extremely 1 liter, adjust pH value 5.4, cultivation temperature 22 ± 2 DEG C, intensity of illumination 3500Iux, incubation time 60 days.
(7) root culture
After enrichment culture, the sprout of 2-3cm length is selected to be transferred in root media, the cultivation of 230ml Base, wherein NH in WPM culture medium4NO3、(NH4)2SO4、MgSO4.7H2O、KH2PO4、KNO3、 K2SO4Amount minimizing half, interpolation sucrose 20g, agar 6.5g, activated carbon 1g, IBA 0.8mg, IAA 0.3mg, It is settled to 1 liter with pure water, adjusts pH value 5.4, cultivation temperature 22 ± 2 DEG C, intensity of illumination 3500Iux, training Support 90 days time.
(8) acclimatization and transplants
When root length is to 2-3cm in root media, get final product acclimatization and transplants.First bottle Seedling is placed in greenhouse before transplanting Middle domestication about 7 days, opens bottle cap and places 3 days, taken out gently by Seedling with tweezers, use warm water cleaning culture medium Soaking 4 minutes with the carbendazim of 1% afterwards, plant in seedling culture hole plate, transplanting tissue cultured seedling matrix ratios is turfy soil: Perlite was 5:1, waters permeable, with plastic sheeting sunshade 3-5 days.
(9) chromosome detection
Choose stem apex and the tip of a root processing alpine rose " young beauty " plant through Colchicine, by micro- Sem observation chromosome number, the plant that screening doubles.

Claims (7)

1. alpine rose " young beauty " chromosome doubling method, it is characterised in that comprise the following steps:
(1) it is transferred in callus culture base train after alpine rose " young beauty " outer implant being sterilized Supporting, induction produces callus；
(2) chemical reagent Colchicine is used to process callus, it is achieved chromosome doubling；
(3) callus will be processed through chemical reagent Colchicine to be transferred in Adventitious bud culture base train Support；
(4) it is transferred in proliferated culture medium carry out enrichment culture by the adventitious bud differentiated；
(5) the healthy and strong good tissue culture plant inoculation through enrichment culture carries out root culture in root media；
(6) Seedling of taking root selecting root system to grow to 2-3cm carries out seedling exercising, with mixing that turfy soil and perlite are made into Close substrate；
(7) Chromosome Number Observation is carried out after acclimatization and transplants under the microscope, after filtering out chromosome doubling Plant seedling.
2. according to a kind of alpine rose " young beauty " the chromosome doubling method described in claims 1, It is characterized in that, the method for the outer implant sterilization described in step (1): at the ethanol of mass concentration 75% Manage outer implant 30S, with aseptic water washing 4-5 time, then process 1 minute with the mercuric chloride of mass concentration 0.1%；
Described callus culture base, is prepared the WPM minimal medium of gained: 230ml by following methods Middle interpolation sucrose 30g, agar powder 6.5g, 2-ip 6mg, be settled to 1 liter with pure water, adjusts pH value 5.4, Cultivation temperature 22 ± 2 DEG C, light culture, incubation time 30 days.
3. according to a kind of alpine rose " young beauty " the chromosome doubling method described in claims 1, It is characterized in that, step (2) detailed process is: callus is through the Colchicine of mass concentration 0.05% Process 24h, with aseptic water washing 4 times；
Adventitious bud culture base described in step (3), is prepared the WPM of gained: 230ml by following methods Minimal medium interpolation sucrose 30g, agar 6.5g, TDZ 0.1mg, 2-ip 1mg, constant volume water to 1 liter, Adjust pH value 5.4, cultivation temperature 22 ± 2 DEG C, intensity of illumination 3500Iux, incubation time 45 days.
4. according to a kind of alpine rose " young beauty " the chromosome doubling method described in claims 1, It is characterized in that the proliferated culture medium described in step (4) is prepared gained by following methods: 230ml's WPM minimal medium adds sucrose 30g, agar powder 6.5g, 2-ip 3mg, NAA 0.5mg, with pure Water is settled to 1 liter, adjusts pH value 5.4, cultivation temperature 22 ± 2 DEG C, intensity of illumination 3500Iux, incubation time 60 days.
5. according to a kind of alpine rose " young beauty " the chromosome doubling method described in claims 1, It is characterized in that, step (5) root media, following methods prepare the WPM training of gained: 230ml Support in base, add sucrose 20g, agar powder 6.5g, activated carbon 1g, IBA 0.8mg, be settled to pure water 1 liter, adjust pH value 5.4；Wherein NH in WPM culture medium4NO3、(NH4)2SO4、MgSO4.7H2O、 KH2PO4、KNO3、K2SO4Amount reduces half；Cultivation temperature 22 ± 2 DEG C, intensity of illumination 3500Iux, training Support 90 days time.
6. according to a kind of alpine rose " young beauty " the chromosome doubling method described in claims 1, It is characterized in that, the mixed-matrix described in step (6), including the turfy soil that weight ratio is 5:1 and Margarita Rock.
7. according to a kind of alpine rose " young beauty " the chromosome doubling method described in claims 1, It is characterized in that, the process that step (7) is concrete is: selects stem apex and the tip of a root of plant, carries out section, Basis of microscopic observation chromosome doubling situation.
CN201610218315.2A 2016-04-08 2016-04-08 A kind of alpine rose " young beauty " chromosome doubling method Expired - Fee Related CN105850737B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107242139A (en) * 2017-08-15 2017-10-13 石家庄市农林科学研究院 A kind of abductive approach of alpine rose ' flour gold butterfly ' somatic embryo
CN108935087A (en) * 2018-09-11 2018-12-07 浙江农林大学 A kind of breeding method of Rhododendron fortuneilindl. polyploid

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CN103636492A (en) * 2013-11-14 2014-03-19 石家庄市农林科学研究院 Rhododendron hybrides cosmopolitan tissue culture rapid propagation method

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107242139A (en) * 2017-08-15 2017-10-13 石家庄市农林科学研究院 A kind of abductive approach of alpine rose ' flour gold butterfly ' somatic embryo
CN107242139B (en) * 2017-08-15 2024-04-19 石家庄市农林科学研究院 Induction method of rhododendron alpine 'Pink butterfly' somatic embryo
CN108935087A (en) * 2018-09-11 2018-12-07 浙江农林大学 A kind of breeding method of Rhododendron fortuneilindl. polyploid
CN108935087B (en) * 2018-09-11 2020-04-28 浙江农林大学 Method for cultivating polyploids of rhododendron micranthum

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