CN104335898A - Method for in vitro intermediate propagation of skimmia reeuesiana - Google Patents

Method for in vitro intermediate propagation of skimmia reeuesiana Download PDF

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CN104335898A
CN104335898A CN201410050810.8A CN201410050810A CN104335898A CN 104335898 A CN104335898 A CN 104335898A CN 201410050810 A CN201410050810 A CN 201410050810A CN 104335898 A CN104335898 A CN 104335898A
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illumination
seed
skimmia
medium
rubella
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CN104335898B (en
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李其友
阳永学
曾红霞
肖康飞
祝菊红
程维舜
张娜
杨皓琼
毕秋荣
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WUHAN INSTITUTE OF AGRICULTURAL SCIENCE
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Abstract

The invention discloses a method for in vitro intermediate propagation of skimmia reeuesiana. The method comprises the following steps: (1) selecting and disinfecting an explant: by using a mature skimmia reeuesiana seed the explant, disinfecting by using 70% alcohol and then disinfecting by using 0.1% HgCl2 and washing by using sterile water; (2) seed germination: inoculating the disinfected seed into germination culture for culturing, wherein the seed germinates to enable cotyledon and euphylla to emerge; (3) calluses induction and proliferation: slicing a terminal bud with the euphylla, inoculating to a proliferation medium for subculture, wherein calluses are continuously proliferated; (4) rooting culture: inoculating a test-tube plantlet obtained by expanding propagation to a rooting medium and culturing the seedling to differentiate a white adventitious root; and (5) domestication and transplanting. The method is simple, convenient and feasible, great in propagation coefficient, low in production cost and short in period. The average propagation coefficient reaches 4.5, the rooting percentage is 90% and the survival rate of transplanting is 95%. The method is simple and convenient to operate and great in propagation coefficient and lays a foundation for industrial production of skimmia reeuesiana sprouts.

Description

A kind of method of Skimmia japonica Rubella Vitro Quick Reproduction
Technical field
The present invention relates to plant biotechnology field, more specifically relate to a kind of method of Skimmia japonica Rubella Vitro Quick Reproduction.
Background technology
Skimmia japonica Rubella (Skimmia japonica Rubella), has another name called carbuncle pearl, belongs to Rutaceae mattress taro platymiscium, is one of in recent years rather well received high-grade ornamental flower.The emerald green light of Skimmia japonica Rubella blade, the bronzing bud of early summer is exquisite lovely, and the fragrance of flowers is strong; The full branch of autumn and winter season Red Star, bright-coloured wish is dripped, and the phase of bearing fruit is half a year more than, is sight leaf, sees flower, sees all good excellent flower variety of fruit (Wu Difei, the good grass one Red Star bacterium taro of year night sight fruit.Chinese flower potted landscape, 2007, (2): 5-5).
Skimmia japonica Rubella can be sowed and breed with cuttage, and adopt seminal propagation, offspring there will be separation, can not keep the good characteristic of maternal plant, and longer to the required cycle of blooming from sowing, produces now and above seldom adopts.Cottage propagation survival rate less than 20%, and needs a large amount of female parents, can not meet Production requirement far away, and deterioration of variety phenomenon easily appears in long-term nutrition breeding.If from imported from Holland, the every strain of high 10cm seedling more than 10 yuan (Meng Yanqiong, Li Renjie, Yi Xingkai, Mei Jun, Li Jingya, the in vitro pedicel axillary buds induction of Skimmia japonica Rubella and adventitious buds proliferation research.China's agronomy circular, 2007,23(11): 81-85), seedling is expensive, causes enterprise's production cost to increase.Adopt plant tissue culture technique, factorial praluction Skimmia japonica Rubella seedling, not only can overcome variet complexity, problem such as kind sexual involution etc. that sowing and cuttage bring, and can not by the impact growth whole year high quality seedling of physioclimate, breeding research for Skimmia japonica Rubella provides material and technical support, significant to the fast development promoting Skimmia japonica Rubella industry.
At present, tissue-culturing rapid propagation about Skimmia japonica Rubella only has one section of report, and Meng Yanqiong etc. (2007) adopt the bennet of Skimmia japonica Rubella to be explant, at MS+2,4-D0.3mg/L+IAA0.2mg/L+BA3.0mg/L+KT2.0mg/L medium induction Axillary Buds Sprouting, inductivity can reach 91.1%.Shoot proliferation on MS+NAA0.8mg/L+BA3.0mg/L+KT2.0mg/L, growth coefficient can reach 8.73(Meng Yan fine jade, Li Renjie, Yi Xingkai, Mei Jun, Li Jingya, the in vitro pedicel axillary buds induction of Skimmia japonica Rubella and adventitious buds proliferation research.China's agronomy circular, 2007,23(11): 81-85).Because Skimmia japonica Rubella is bloomed by time restriction, adopt bennet to draw materials limited, and do not carry out the test of culture of rootage aspect in experiment, be restricted in the nursery of actual factory.
Summary of the invention
The object of the invention is a kind of method that there are provided Skimmia japonica Rubella Vitro Quick Reproduction, the method is simple and easy to do, and reproduction coefficient is large, production cost is low, and the cycle is short, and average reproduction coefficient reaches 4.5, breeding cycle 20 ~ 25d, rooting rate about 90%, transplanting survival rate 95%.Adopt the bud seedling of seed asepsis sprouting, carry out the factorial praluction of plantlet in vitro under artificial controlled condition, not by the impact of natural conditions, easy and simple to handle, reproduction coefficient is high, and production cost is low, and the cycle is short, produces lay a good foundation for Skimmia japonica Rubella Industrialization of seeds and seedlings.
To achieve the above object, the present invention adopts following technical measures:
(1) explant is selected and sterilization:
With Skimmia japonica Rubella mature seed for explant, under flowing water, rinse 30min, used 70%(volume ratio) after alcohol disinfecting, then be 0.1%(mass volume ratio by concentration) mercury chloride (HgCl 2) sterilization 8 ~ 10min, then use aseptic water washing 4 times.
(2) seed germination:
By in the seed access germination medium that disinfects, temperature be 24-26 DEG C, humidity is 35-45%, intensity of illumination 3000 ~ 5000 μm of ol m -2s -1, illumination every day 14 ~ 16h, cultivate through 25 ~ 30d, seed germination grows cotyledon, after 15 ~ 20d, grows 1 ~ 2 true leaf.
Described seed germination medium comprises MS minimal medium+0.5mg/L GA 3+ sucrose 30g/L, pH value is 5.8 ~ 6.0.
(3) clump bud inducement and propagation:
Cut 1 ~ 2 true leaf and above terminal bud, be inoculated on clump bud inducement medium, temperature be 24-26 DEG C, humidity is 35-45%, intensity of illumination 3000 ~ 5000 μm of ol m -2s -1, illumination every day 14 ~ 16h, cultivates through 25 ~ 30d, obtains Multiple Buds.Select the stem eye that grows thickly of normal development, cut into 1-2 stem eye carry out squamous subculture every 20 ~ 25d, average each switchable 4-5 bottle of subculture 1 bottle, makes clump bud constantly breed.
Described clump bud inducement and proliferated culture medium comprise MS minimal medium+1.0 ~ 1.5mg/L6-BA+0.1 ~ 0.3mg/L NAA+0.5mg/L GA 3+ sucrose 30g/L, pH value is 5.8 ~ 6.0.
(4) culture of rootage:
By test-tube plantlet long for plant height 3 ~ 5cm, be inoculated in root media, temperature be 24-26 DEG C, humidity is 35-45%, intensity of illumination 3000 ~ 5000 μm of ol m -2s -1, illumination every day 14-16h, will differentiate 4 ~ 6 white adventive root through 14-16d seedling.
Described root media comprises 1/2MS+0.5 ~ 1.0mg/L IBA+ sucrose 30g/L, and pH value is 5.8 ~ 6.0.
(5) rooting culture:
In greenhouse, after culture of rootage 25-30d, by robust growth, the Skimmia japonica Rubella plantlet in vitro with more than 3 new roots carries out rooting culture, and the mixed-matrix formula of transplanting is by peat: perlite: vermiculite=3:1:1(V/V).After transplanting, with sprayer water spray, make root system and matrix close contact, under moving into full exposure after then sheltering from heat or light one week.Greenhouse experiment is 20 DEG C ~ 25 DEG C, illumination 14 ~ 16h, and intensity of illumination is 2000 μm of ol m -2s -1.
The present invention for explant, sets up Skimmia japonica Rubella tissue-culturing rapid propagation system with the terminal bud of seed asepsis sprouting, appreciation rate higher (4 ~ 5 times), propagation is stable, root system is healthy and strong, and transplanting survival rate is higher, in Skimmia japonica Rubella seedling plant modification, have important using value.
The present invention compared with prior art, has the following advantages and effect:
1, the present invention compared with prior art, and seed is drawn materials simple and convenient, and quantity is large, easily sprouts.
2, to obtain clump bud reproduction rate high in the present invention, and every subculture once expands 4 ~ 5 times.
3, the present invention sets up Skimmia japonica Rubella group culturation rapid propagating technology, for good basis is laid in follow-up Skimmia japonica Rubella genetic breeding and transgenosis work.
Accompanying drawing explanation
Fig. 1 is the excellent potted plant schematic diagram of a kind of Skimmia japonica Rubella.
Mature seed after erythrocarpus peeling is used for experiment, and white arrow indication is erythrocarpus.
Fig. 2 is that a kind of seed germination grows cotyledon schematic diagram.
Fig. 3 is that a kind of seed germination grows schematic diagram after true leaf.
Seedling grows true leaf, for the terminal bud of breeding.
Fig. 4 is the Multiple Buds schematic diagram after a kind of terminal bud propagation.
Fig. 5 is that a kind of rooting of vitro seedling cultivates schematic diagram.
Fig. 6 is a kind of test-tube plantlet schematic diagram of normally taking root.
Fig. 7 is the Skimmia japonica Rubella seedling schematic diagram of normal growth after a kind of rooting and transplant.
Specific implementation method
In order to understand the present invention better, further illustrate essence of the present invention below in conjunction with specific embodiments and the drawings, but content of the present invention is not limited thereto.
Embodiment 1:
A method for Skimmia japonica Rubella Vitro Quick Reproduction, the steps include:
1, medium preparation:
1.1 minimal medium preparations:
Described medium is MS, gets macroelement mother liquor 100ml respectively; MS trace element mother liquor 10ml; The organic mother liquor 10ml of MS; MS molysite 10ml; Inositol 10ml; Mother liquor composition is as following table 1:
Table 1MS medium mother liquor formula
1.2 somatotropin preparations:
Basic element of cell division 6-BA(6-benzyladenine by required) growth hormone NAA(methyl α-naphthyl acetate), IBA(indolebutyric acid), GA 3(gibberellin) uses the NaOH of 0.1mol/L or alcohol to dissolve, then with being mixed with the mother liquor of 0.5mg/ml after aquae destillata dilution respectively, loading in volumetric flask, being placed in Refrigerator store.
1.3 medium preparations:
All medium all add the plant growth regulator of agar powder 7g/L and sucrose 30g/L and variable concentrations, by about the NaOH adjust pH to 6.0 of medium 0.1mol/L for preparing, except GA 3outside external demand filtration sterilization, other additives all adds before autoclaving, pressure 1.1kg/cm -2and in 121 DEG C time timing 20min.
2, plant seed is selected and sterilization:
Buy the excellent potted plant of Skimmia japonica Rubella (Fig. 1) December from Wei Erfu biotech inc, Wuhan, get red-coloured seeds, after peeling is cleaned, shell, rinse 30min under flowing water, used 70%(volume ratio) after alcohol disinfecting, then be 0.1%(mass volume ratio by concentration) HgCl 2sterilization 8 ~ 10min, with aseptic water washing 4 times.
3, seed germination:
By in the seed access germination medium that disinfects, temperature be 25 DEG C, humidity is 40%, intensity of illumination 3000 ~ 5000 μm of ol m -2s -1, illumination every day 14 ~ 16h, cultivate through 25 ~ 30d, seed germination grows cotyledon (Fig. 2), after 10 ~ 15d, grows 1 ~ 2 true leaf (Fig. 3).
4, clump bud inducement and propagation:
Cut 1 ~ 2 true leaf and above terminal bud, be inoculated on clump bud inducement medium, temperature be 25 DEG C, humidity is 40%, intensity of illumination 3000 ~ 5000 μm of ol m -2s -1, illumination every day 14 ~ 16h, cultivates through 25 ~ 30d, and obtain Multiple Buds (Fig. 4), growth coefficient is 4 ~ 6.Select the stem eye that grows thickly of normal development, cut into 1 ~ 2 stem eye carry out squamous subculture every 20 ~ 25d, average each switchable 4 ~ 5 bottles of subculture 1 bottle, makes clump bud constantly breed.
5, culture of rootage:
The test-tube plantlet long by plant height 2 ~ 3cm, being inoculated in root media, is 25 DEG C in temperature, and humidity is 40%, intensity of illumination 3000 ~ 5000 μm of ol m -2s -1, illumination every day 14 ~ 16h, 14 ~ 16d seedling starts to take root, and after 20d, each seedling can differentiate 4 ~ 6, and length is at the white adventive root of more than 3cm, and rooting rate is (Fig. 5, Fig. 6) more than 90%.
6, rooting culture:
When adventive root grows to more than 4cm, the Skimmia japonica Rubella test-tube plantlet with more than 3 new roots is uncapped in Indoor Natural illumination bottom and carries out hardening, transplant after 3d.After transplanting, with sprayer water spray, make root system and matrix close contact, under moving into full exposure after then sheltering from heat or light one week, plant strain growth normal (Fig. 7).The matrix of transplanting is peat: perlite: vermiculite=3:1:1(V/V) mixed-matrix.Temperature condition is 20 DEG C ~ 25 DEG C, illumination 14 ~ 16h, and intensity of illumination is 3000 ~ 5000 μm of ol m -2s -1.
4.5 are reached, breeding cycle 20 ~ 25d, rooting rate about 90%, transplanting survival rate 95% by the average reproduction coefficient of the present invention.Adopt the bud seedling of seed asepsis sprouting, carry out the factorial praluction of plantlet in vitro under artificial controlled condition, not by the impact of natural conditions, easy and simple to handle, reproduction coefficient is large, and production cost is low, and the cycle is short, produces lay a good foundation for Skimmia japonica Rubella Industrialization of seeds and seedlings.

Claims (1)

1. a method for Skimmia japonica Rubella Vitro Quick Reproduction, the steps include:
(1) explant is selected and sterilization:
With Skimmia japonica Rubella mature seed for explant, under flowing water, rinse 30min, after being used 70% volume ratio alcohol disinfecting, then be 0.1% mass volume ratio mercury chloride sterilization 8 ~ 10min by concentration, then use aseptic water washing 4 times;
(2) seed germination:
By in the seed of sterilization access germination medium, temperature be 24-26 DEG C, humidity is 35-45%, intensity of illumination 3000 ~ 5000 μm of ol m -2s -1, illumination every day 14 ~ 16h, cultivate through 25 ~ 30d, seed germination grows cotyledon, after 15 ~ 20d, grows 1 ~ 2 true leaf;
Described seed germination medium comprises MS minimal medium+0.5mg/L GA 3+ sucrose 30g/L, pH value is 5.8 ~ 6.0;
(3) clump bud inducement and propagation:
Cut the terminal bud of 1 ~ 2 true leaf, be inoculated on clump bud inducement medium, temperature be 24-26 DEG C, humidity is 35-45%, intensity of illumination 3000 ~ 5000 μm of ol m -2s -1, illumination every day 14 ~ 16h, cultivates through 25 ~ 30d, obtains Multiple Buds, selects the stem eye that grows thickly, cut into 1-2 stem eye carry out squamous subculture every 20 ~ 25d, and average each subculture 1 bottle switching 4-5 bottle, makes clump bud constantly breed;
Described clump bud inducement and proliferated culture medium comprise MS minimal medium+1.0 ~ 1.5mg/L 6-BA+0.1 ~ 0.3 mg/L NAA+0.5mg/L GA 3+ sucrose 30g/L, pH value is 5.8 ~ 6.0;
(4) culture of rootage:
By test-tube plantlet long for plant height 3 ~ 5cm, be inoculated in root media, temperature be 24-26 DEG C, humidity is 35-45%, intensity of illumination 3000 ~ 5000 μm of ol m -2s -1, illumination every day 14-16h, will differentiate 4 ~ 6 white adventive root through 14-16d seedling;
Described root media comprises 1/2MS+0.5 ~ 1.0mg/L IBA+sucrose 30g/L, and pH value is 5.8 ~ 6.0;
(5) rooting culture:
In greenhouse, after culture of rootage 25-30d, the Skimmia japonica Rubella plantlet in vitro of more than 3 new roots is carried out rooting culture, and the mixed-matrix formula of transplanting is by peat: perlite: vermiculite=3:1:1V/V, after transplanting, spray water with sprayer, make root system and base contact, under moving into full exposure after then sheltering from heat or light one week, greenhouse experiment is 20 DEG C ~ 25 DEG C, illumination 14 ~ 16h, intensity of illumination is 2000 μm of ol m -2s -1.
CN201410050810.8A 2014-02-14 2014-02-14 A kind of method of Skimmia japonica Rubella Vitro Quick Reproduction Expired - Fee Related CN104335898B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106613192A (en) * 2016-12-09 2017-05-10 渠县金穗农业科技有限公司 Rapid propagation method for polygonum multiflorum
CN107182790A (en) * 2017-07-18 2017-09-22 上海市园林科学规划研究院 A kind of rapid propagation method of golden red mattress taro
CN109924073A (en) * 2019-03-26 2019-06-25 四川大学 A kind of nervate twayblade herb seed asepsis sprouting and sprouting and rooting method

Citations (1)

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Publication number Priority date Publication date Assignee Title
CN103444548A (en) * 2013-09-17 2013-12-18 南京通泽农业科技有限公司 Rapid skimmia propagation method

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
CN103444548A (en) * 2013-09-17 2013-12-18 南京通泽农业科技有限公司 Rapid skimmia propagation method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
孟艳琼等: ""红星茵芋离体花梗腋芽诱导及丛生芽增殖研究"", 《中国农学通报》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106613192A (en) * 2016-12-09 2017-05-10 渠县金穗农业科技有限公司 Rapid propagation method for polygonum multiflorum
CN107182790A (en) * 2017-07-18 2017-09-22 上海市园林科学规划研究院 A kind of rapid propagation method of golden red mattress taro
CN107182790B (en) * 2017-07-18 2019-03-15 上海市园林科学规划研究院 A kind of rapid propagation method of golden red mattress taro
CN109924073A (en) * 2019-03-26 2019-06-25 四川大学 A kind of nervate twayblade herb seed asepsis sprouting and sprouting and rooting method

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