CN108077067A - A kind of tissue culture and rapid propagation method of cotton rose - Google Patents

A kind of tissue culture and rapid propagation method of cotton rose Download PDF

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Publication number
CN108077067A
CN108077067A CN201611022683.6A CN201611022683A CN108077067A CN 108077067 A CN108077067 A CN 108077067A CN 201611022683 A CN201611022683 A CN 201611022683A CN 108077067 A CN108077067 A CN 108077067A
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China
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culture
cotton rose
bud
seedling
propagation method
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CN108077067B (en
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栗丹
曾俊
刘少奎
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Sichuan Qicai Forestry Industry Development Co Ltd
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Sichuan Qicai Forestry Industry Development Co Ltd
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001Culture apparatus for tissue culture
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008Methods for regeneration to complete plants

Abstract

The present invention discloses a kind of tissue culture and rapid propagation method of cotton rose, including:(1) bud inducement cultivation:Stem section of the cotton rose with bud point is cut as explant, is inoculated into after sterilization treatment in inducing culture, is cultivated, until axillary bud sprouting and growing to certain length;(2) culture of rootage:Step (1) described axillary bud is cut, is inoculated into root media, is cultivated, forms rooted seedling;(3) Multiplying culture:The stem section or stem apex of the band simple bud of step (2) described rooted seedling are cut, is inoculated into proliferated culture medium, is cultivated, forms cotton rose seedling;(4) acclimatization and transplants:Step (3) the cotton rose seedling is moved into outdoor, closes bottle hardening after 2-3 days, is transplanted to seedbed, overlay film covers sunshade net culture 8-12 days, until the cotton rose seedling grows new root, then striping, goes sunshade net culture.The method can promote to grow, and the seedling percent of culture is high, and rooting rate is up to 100%, and incubation time is short, can realize the quick breeding of cotton rose.

Description

A kind of tissue culture and rapid propagation method of cotton rose
Technical field
The present invention relates to plant tissue culture fast breeding technique fields, and in particular to a kind of tissue culture and rapid propagation method of cotton rose.
Background technology
Cotton rose originates in China Yellow River basin to South China, is distributed in provinces such as Hunan, Sichuan, Yunnan, Guangdong. There is the cultivation history of nearly 3,000 years in China, be mainly planted in the ground such as Hunan, Sichuan at present, Flos Hibisci Mutabilis is the city in Chengdu again Flower, people mostly plant lotus in pond side.In addition to as watching, flower, the leaf of cotton rose can be used as medicine, and have clearing heat and detoxicating, detumescence And other effects.Existing cotton rose breeding and culturing technology is usually cuttage or grafting method, and repoductive time is long, and survival rate is relatively low, obtains Less than popularization.
The content of the invention
In view of this, the present invention provides a kind of tissue culture and rapid propagation method of cotton rose, when the seedling that the method obtains is cultivated Between it is short, survival rate is high, and rooting rate is up to 100%, and seedling is neat, and convenient for the later stage, unified transplanting and Cultivate administration are, it can be achieved that wood The quick breeding of lotus.
For solution more than technical problem, technical solution provided by the invention is a kind of tissue culture and rapid propagation method of cotton rose, institute Tissue culture and rapid propagation method is stated to comprise the following steps:
(1) bud inducement cultivation:Stem section of the cotton rose with bud point is cut as explant, induction training is inoculated into after sterilization treatment It supports in base, up to axillary bud sprouting on the explant and grows to certain length in 2-3 week of culture;
(2) culture of rootage:Step (1) described axillary bud is cut, is inoculated into root media, 3-6 week of culture, until armpit Bastem portion takes root, and root growth forms rooted seedling to certain length;
(3) Multiplying culture:The stem section or stem apex of the band simple bud of step (2) described rooted seedling are cut, is inoculated into Multiplying culture In base, in 2-3 week of culture, until the stem section or stem apex are taken root, and the simple bud grows to certain length, forms cotton rose children Seedling;
(4) acclimatization and transplants:Step (3) the cotton rose seedling is moved into outdoor, bottle hardening is closed after 2-3 days, transplants to seedling Bed, overlay film covers sunshade net culture 8-12 days, until the cotton rose seedling grows new root, then striping, goes sunshade net culture.
The length of axillary bud growth is 2-3cm wherein in step (1);The length of root growth is 5-7cm in step (2);Step Suddenly the length that simple bud grows in (3) is 4-6cm.
Preferably, the composition of the inducing culture described in step (1) is:MS minimal mediums supplement 0.1-0.5mg/L 6-BA, 0.05-0.15mg/L NAA, 10-30g/L sucrose, 4-6g/L agar, the concentration ratio of the 6-BA and the NAA are big In 1.
It is more highly preferred to, the composition of the inducing culture described in step (1) is:MS minimal mediums supplement 0.2mg/L 6-BA, 0.1mg/L NAA, 20g/L sucrose, 6g/L agar.
Preferably, the sterilization treatment of step (1) described explant is:After the explant is cleaned up, first with 75% 25-35s of ethanol postincubation, sterile water wash 1-2 times, then with 0.1% mercuric chloride handle 8-10min, sterile water wash 3-5 It is secondary, finally moisture is sucked with aseptic filter paper.
Preferably, the composition of step (2) root media and the proliferated culture medium described in step (3) is:1/2MS Minimal medium, supplement 0.1-0.5mg/L NAA, 0.05-0.15mg/L 6-BA, 10-30g/L sucrose, 4-6g/L fine jades Fat, the concentration ratio of the NAA and 6-BA are more than 1.
It is more highly preferred to, the composition of the proliferated culture medium described in step (2) root media and step (3) is: 1/2MS minimal mediums, supplement 0.2mg/L NAA, 0.1mg/L 6-BA, 20g/L sucrose, 6g/L agar.
Preferably, the bud inducement cultivation, culture of rootage, the temperature of Multiplying culture are 23-27 DEG C, and intensity of illumination is 1500-2000Lx, photoperiod 14/10h.
Preferably, the matrix in step (4) described seedbed is 1 by volume ratio:Perlite and the vermiculite composition of (0.5-1).
It is more highly preferred to, the matrix in step (4) described seedbed is 1 by volume ratio:1 perlite and vermiculite composition.
Preferably, the matrix carries out sterilization treatment before cotton rose seedling replanting with Bravo.
Preferably, step (4) overlay film, cover the temperature of sunshade net culture for 23-27 DEG C, relative humidity for 90- 99%, the striping, going the temperature of sunshade net culture, relative humidity is 70-80% for 23-27 DEG C.
It is more highly preferred to, step (4) overlay film covers the temperature of sunshade net culture for 25 DEG C, relative humidity 95%, institute It states striping, go the temperature of sunshade net culture as 25 DEG C, relative humidity 75%.
Preferably, the shading rate of the sunshade net is 75%.
Preferably, step (4) the cotton rose seedling is poured after the completion of transplanting with 1000 times of dilute solution sprayings of carbendazim Water.
MS minimal mediums described herein have higher inorganic salt concentration, can ensure the ore deposit needed for tissue growth Matter nutrition is more stable ionic equilibrium solution, its nitrate content is high, and the quantity and ratio of nutrient are suitable, can meet The nutrition and physiological requirements of plant cell, thus the scope of application is wider, most plants tissue-culturing quick-propagation uses it as The minimal medium of culture medium.
The 1/2MS minimal mediums are in MS minimal mediums, and a great number of elements halves, the culture of remaining constant formation Base.
The 6-BA is 6- benzyl aminoadenines, is a kind of artificial synthesized plant cytokinin, main function The formation for promoting bud, can also evoked callus occur, promote cell division, promote the differentiation of undifferentiated tissue, promote life The accumulation of object substance in vivo, promotes lateral bud, prevents aging, is usually used in plant tissue, cell culture.
The NAA is methyl α-naphthyl acetate, is the auxin analog in a plant growth regulators, and cuttage is used in plant Method uses when breeding, it can also be used to which Plant Tissue Breeding, promoting cell division, induced synthesis adventitious root increases seat with expanding Fruit prevents shedding, changes female, male flower ratio etc., can be entered through tender epidermis, the seed of blade, branch in plant, with nutrition stream Transporting is to complete stool.
The technical program mainly utilizes the principle of micro cuttage, carries out tissue culture expanding propagation to cotton rose, 6-BA is primarily to facilitate The growth of bud, NAA are mainly used for the growth of adventitious root.In bud inducement cultivation, 6-BA/NAA (concentration ratio) > 1, induction of armpit The sprouting of bud;It is taking root and in Multiplying culture, is being with the addition of NAA and 6-BA, and NAA/6-BA (concentration ratio) > 1, induction of root Generation, while promote the development of bud.
Compared with forefathers' fast breeding technique is studied, the advantages of technical scheme, is that the breeding cycle can be obviously shortened, side It multiplication in case and takes root for same culture medium, which can meet and take root, eliminate hardening while bud development is promoted Before the step of taking root.
In addition, matrix treatments, water management, disinfectant measure, humidity control during preceding and transplanting are transplanted in the technical solution Method processed may be such that transplanting survival rate reaches 100%, higher than state of the art.
Technical scheme carries out bud inducement cultivation using stem section of the cotton rose with bud point as explant, sprouts armpit Bud, then axillary bud is subjected to culture of rootage and forms rooted seedling, rooted seedling is cut into the stem section with simple bud or stem apex carries out Multiplying culture, Cotton rose seedling is formed, then carries out acclimatization and transplants, the quick breeding of cotton rose is realized by above method, shortens growth cycle. By the screening to inducing culture, root media, proliferated culture medium, optimal nutrient media components and proportioning are obtained, is used The culture medium of afore-mentioned plants growth regulator proportioning, obtained seedling incubation time is short, can promote to grow, and survival rate is high, Rooting rate is up to 100%, and seedling is neat, and unified transplanting and Cultivate administration convenient for the later stage realize the quick breeding of cotton rose.
Specific embodiment
In order to which those skilled in the art is made to more fully understand technical scheme, with reference to specific embodiment pair The present invention is described in further detail.
Embodiment 1
A kind of tissue culture and rapid propagation method of cotton rose described in the present embodiment, comprises the following steps:
(1) bud inducement cultivation:Stem section of the cotton rose with bud point is cut as explant, after cleaning up, first with 75% Ethanol postincubation 30s, sterile water wash 2 times, then handle 9min, sterile water wash 4 times, finally with sterile filter with 0.1% mercuric chloride Paper sucks moisture, is inoculated into the inducing culture after adjusting pH to 6.0 and sterilization treatment and cultivates, until armpit on the explant Bud is sprouted and grows to certain length, and the composition of wherein inducing culture is:MS minimal mediums, supplement 0.2mg/L 6-BA, 0.1mg/L NAA, 20g/L sucrose, 6g/L agar;
(2) culture of rootage:Step (1) described axillary bud is cut, is inoculated into root media and cultivates, until axillary bud base portion is given birth to Root, and root growth forms rooted seedling, the composition of wherein root media is to certain length:1/2MS minimal mediums, supplement 0.2mg/L NAA, 0.1mg/L 6-BA, 20g/L sucrose, 6g/L agar;
(3) Multiplying culture:The stem section or stem apex of the band simple bud of step (2) described rooted seedling are cut, is inoculated into Multiplying culture It is cultivated in base, until the stem section or stem apex are taken root, and the simple bud grows to certain length, forms cotton rose seedling, wherein The composition of proliferated culture medium is:1/2MS minimal mediums, supplement 0.2mg/L NAA, 0.1mg/L 6-BA, 20g/L sucrose, 6g/ L agar;
(4) acclimatization and transplants:Step (3) the cotton rose seedling is moved into outdoor, bottle hardening is closed after 2-3 days, transplants to seedling Bed, the wherein matrix in seedbed are 1 by volume ratio:1 perlite and vermiculite composition, and sterilization treatment is first carried out using Bravo, After the completion of transplanting, with the spraying watering of 1000 times of solution of carbendazim, with covered rearing with plastic film moisturizing, with the sunshade that shading rate is 75% Net shades, and controlled at 25 DEG C, relative humidity 95% is cultivated 8-12 days, until the cotton rose seedling grows newly Root, then striping, go sunshade net culture, temperature is 25 DEG C, relative humidity 75%.
Wherein, bud inducement cultivation, culture of rootage, the temperature of Multiplying culture are 25 DEG C, intensity of illumination 2000Lx, the photoperiod 14/10h。
In the present embodiment, step (1) bud inducement cultivation is after 2 weeks, and the axillary bud growth that is sprouted on explant to 2-3cm; Step (2) culture of rootage after 1 week, take root by axillary bud base portion, and root long is 2-4cm, rooting rate 100%, and top axillary bud after 3 weeks It is extending to 5cm, axillary bud is extending to 10cm after 6 weeks;Step (3) Multiplying culture after 1 week stem section or stem apex take root, 3 Zhou Houdan Bud length to 5cm, cotton rose seedling at this time can carry out acclimatization and transplants;And continue culture can be again up to the cotton rose seedling after 6 weeks Carry out shoot proliferation culture, growth coefficient average out to 5;The lotus seedling of 10 days grows new root, transplant survival after step (4) transplanting Rate is up to 100%.
Embodiment 2
A kind of tissue culture and rapid propagation method of cotton rose described in the present embodiment, with embodiment 1 difference lies in:
The composition of step (1) inducing culture is:MS minimal mediums, supplement 0.1mg/L 6-BA, 0.05mg/LNAA, 10g/L sucrose, 4g/L agar;
The composition of step (2) root media is:1/2MS minimal mediums, supplement 0.1mg/L NAA, 0.05mg/L6- BA, 10g/L sucrose, 4g/L agar;
The composition of step (3) proliferated culture medium is:1/2MS minimal mediums, supplement 0.1mg/L NAA, 0.05mg/L6- BA, 10g/L sucrose, 4g/L agar.
In the present embodiment, step (1) bud inducement cultivation is after 2 weeks, and the axillary bud growth that is sprouted on explant to 1-2cm; Step (2) culture of rootage after 10 days, take root by axillary bud base portion, and root long is 2-3cm, rooting rate 100%, and top axillary bud after 3 weeks It is extending to 4cm, axillary bud is extending to 8cm after 6 weeks;Step (3) Multiplying culture after 10 days stem section or stem apex take root, 4 Zhou Houdan Bud length to 5-6cm, cotton rose seedling at this time can carry out acclimatization and transplants;And continue culture can be again up to the cotton rose seedling after 7 weeks Secondary progress shoot proliferation culture, growth coefficient average out to 5;The lotus seedling of 12 days grows new root after step (4) transplanting, transplants into Motility rate is up to 95%.
Embodiment 3
A kind of tissue culture and rapid propagation method of cotton rose described in the present embodiment, with embodiment 1 difference lies in:
The composition of step (1) inducing culture is:MS minimal mediums, supplement 0.5mg/L 6-BA, 0.15mg/LNAA, 30g/L sucrose, 6g/L agar;
The composition of step (2) root media is:1/2MS minimal mediums, supplement 0.5mg/L NAA, 0.15mg/L6- BA, 30g/L sucrose, 6g/L agar;
The composition of step (3) proliferated culture medium is:1/2MS minimal mediums, supplement 0.5mg/L NAA, 0.15mg/L6- BA, 30g/L sucrose, 6g/L agar.
In the present embodiment, step (1) bud inducement cultivation is after 2 weeks, and the axillary bud growth that is sprouted on explant to 2cm;Step Suddenly (2) culture of rootage after 1 week, take root by axillary bud base portion, and root long is 2-4cm after 3 weeks, rooting rate 100%, and top axillary bud can Elongation is to 4cm, and axillary bud is extending to 9cm after 6 weeks;Step (3) Multiplying culture after 1 week stem section or stem apex take root, simple bud is long after 3 weeks To 4cm, cotton rose seedling at this time can carry out acclimatization and transplants;And continue culture up to the cotton rose seedling after 6-7 weeks can again into Row shoot proliferation culture, growth coefficient average out to 5;The lotus seedling of 11 days grows new root, transplanting survival rate after step (4) transplanting Up to 98%.
Embodiment 4
A kind of tissue culture and rapid propagation method of cotton rose described in the present embodiment, with embodiment 1 difference lies in:
In step (4) acclimatization and transplants, the matrix in seedbed is 1 by volume ratio:0.5 perlite and vermiculite composition.
In the present embodiment, the lotus seedling of 15 days grows new root after transplanting, and transplanting survival rate is up to 90%.
Embodiment 5
The influence of plant growth regulator ingredient and concentration to bud inducement cultivation in inducing culture
Stem section of several cotton roses with bud point is cut as explant, after cleaning up, first with 75% ethanol postincubation 30s, sterile water wash 2 times, then 9min is handled with 0.1% mercuric chloride, sterile water wash 4 times finally sucks water with aseptic filter paper Point, it is inoculated into inducing culture and cultivates, until sprouting axillary bud on the explant and growing to certain length, wherein inducing The composition of culture medium is:MS minimal mediums, supplement plant growth regulator, sucrose, agar, MS minimal mediums, sucrose, Under conditions of agar all same, it is grouped, is observed and recorded in culture medium according to the ingredient of plant growth regulator and concentration The culture situation of explant, specific grouping and cultivation results are shown in Table 1.
The influence result of plant growth regulator ingredient and concentration to bud inducement cultivation in 1 inducing culture of table
Grouping 6-BA(mg/L) NAA(mg/L) Explant number (a) Sprouting number (a)
1 0.1 -- 20 14
2 0.2 -- 20 16
3 0.5 -- 20 16
4 -- 0.05 20 13
5 -- 0.10 20 13
6 -- 0.15 20 12
7 0.2 0.05 20 18
8 0.2 0.10 20 20
9 0.2 0.15 20 17
As can be seen from the above table, 6-BA and NAA simultaneously using be applied alone any than the two when rudiment number it is high, and from 7-9 Group as can be seen that both simultaneously in use, 6-BA be 0.2mg/L, NAA be 0.1mg/L when, germination rate highest, be Fiber differentiation The most suitable plant growth regulator composition condition of base.
Embodiment 6
The influence of plant growth regulator ingredient and concentration to axillary bud culture of rootage and bud point number in root media
The axillary bud that it is formed after inducing is several, is inoculated into root media and cultivates, until axillary bud base portion is taken root, and root is given birth to Length forms rooted seedling, the composition of wherein root media is to certain length:1/2MS minimal mediums supplement plant growth tune Agent, sucrose, agar are saved, under conditions of 1/2MS minimal mediums, sucrose, agar all same, according to plant growth regulator Ingredient and concentration are grouped, and observe and record the culture situation of axillary bud in culture medium, and specific grouping and cultivation results are shown in Table 2.
The influence of plant growth regulator ingredient and concentration to axillary bud culture of rootage and bud point number in 2 root media of table
As can be seen from the above table, when NAA is used alone, control group IBA (existing common plant growth regulatings can be reached Agent, but IBA prices are far above NAA) same effect, rooting rate is basically identical, can meet need of rootedness, but average bud point quantity It is few, it is impossible to meet lower step multiplication demand.And NAA and 6-BA uses simultaneously than NAA is applied alone when bud point number it is high, and can from 7-9 groups To find out, the two uses simultaneously, and NAA 0.2mg/L, when 6-BA is 0.1mg/L, number of taking root, root long, bud point number are maximum, make a living The most suitable plant growth regulator composition condition of root and proliferated culture medium.
It the above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair The limitation of the present invention, protection scope of the present invention should be subject to claim limited range.For the art For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change Protection scope of the present invention is also should be regarded as into retouching.

Claims (10)

1. a kind of tissue culture and rapid propagation method of cotton rose, it is characterised in that:The tissue culture and rapid propagation method comprises the following steps:
(1) bud inducement cultivation:Stem section of the cotton rose with bud point is cut as explant, inducing culture is inoculated into after sterilization treatment In, up to axillary bud sprouting on the explant and grow to certain length in 2-3 week of culture;
(2) culture of rootage:Step (1) described axillary bud is cut, is inoculated into root media, 3-6 week of culture, until axillary bud base Portion takes root, and root growth forms rooted seedling to certain length;
(3) Multiplying culture:The stem section or stem apex of the band simple bud of step (2) described rooted seedling are cut, is inoculated into proliferated culture medium, In 2-3 week of culture, until the stem section or stem apex are taken root, and the simple bud grows to certain length, forms cotton rose seedling;
(4) acclimatization and transplants:Step (3) the cotton rose seedling is moved into outdoor, bottle hardening is closed after 2-3 days, transplants to seedbed, Overlay film covers sunshade net culture 8-12 days, until the cotton rose seedling grows new root, then striping, goes sunshade net culture.
2. a kind of tissue culture and rapid propagation method of cotton rose according to claim 1, it is characterised in that:Luring described in step (1) The composition for leading culture medium is:MS minimal mediums, supplement 0.1-0.5mg/L 6-BA, 0.05-0.15mg/L NAA, 10- 30g/L sucrose, 4-6g/L agar, the concentration ratio of the 6-BA and NAA are more than 1.
3. a kind of tissue culture and rapid propagation method of cotton rose according to claim 2, it is characterised in that:Luring described in step (1) The composition for leading culture medium is:MS minimal mediums, supplement 0.2mg/L 6-BA, 0.1mg/L NAA, 20g/L sucrose, 6g/L fine jades Fat.
4. a kind of tissue culture and rapid propagation method of cotton rose according to claim 1, it is characterised in that:Step (1) described explant The sterilization treatment of body is:After the explant is cleaned up, first with 75% 25-35s of ethanol postincubation, sterile water wash 1- 2 times, then 8-10min is handled with 0.1% mercuric chloride, sterile water wash 3-5 times finally sucks moisture with aseptic filter paper.
5. a kind of tissue culture and rapid propagation method of cotton rose according to claim 1, it is characterised in that:Step (2) is described to take root The composition of proliferated culture medium described in culture medium and step (3) is:1/2MS minimal mediums supplement 0.1-0.5mg/L NAA, 0.05-0.15mg/L 6-BA, 10-30g/L sucrose, 4-6g/L agar, the concentration ratio of the NAA and the 6-BA are big In 1.
6. a kind of tissue culture and rapid propagation method of cotton rose according to claim 5, it is characterised in that:Step (2) is described to take root The composition of proliferated culture medium described in culture medium and step (3) is:1/2MS minimal mediums, supplement 0.2mg/L NAA, 0.1mg/L 6-BA, 20g/L sucrose, 6g/L agar.
7. a kind of tissue culture and rapid propagation method of cotton rose according to claim 1, it is characterised in that:The bud inducement cultivation, Culture of rootage, the temperature of Multiplying culture are 23-27 DEG C, and intensity of illumination is 1500-2000Lx, photoperiod 14/10h.
8. a kind of tissue culture and rapid propagation method of cotton rose according to claim 1, it is characterised in that:Step (4) described seedbed Matrix by volume ratio be 1:Perlite and the vermiculite composition of (0.5-1).
9. a kind of tissue culture and rapid propagation method of cotton rose according to claim 1, it is characterised in that:Step (4) overlay film, The temperature of sunshade net culture is covered for 23-27 DEG C, and relative humidity is 90-99%, and the striping, the temperature for going sunshade net culture are 23-27 DEG C, relative humidity is 70-80%.
10. a kind of tissue culture and rapid propagation method of cotton rose according to claim 1, it is characterised in that:Step (4) the wooden cottonrose hibiscus Rong seedling is after the completion of transplanting, with 1000 times of dilute solution spraying waterings of carbendazim.
CN201611022683.6A 2016-11-21 2016-11-21 Tissue culture and rapid propagation method of cotton rose Active CN108077067B (en)

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Publication number Priority date Publication date Assignee Title
CN109329062A (en) * 2018-11-27 2019-02-15 广西玉林市华睿茶业有限公司 A kind of tissue culture technique of cottonrose hibiscus leaf

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