CN106069748B - The commercial tissue culture of sprout mating system of American red-maple splendidness in October - Google Patents
The commercial tissue culture of sprout mating system of American red-maple splendidness in October Download PDFInfo
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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
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Abstract
The present invention provides a kind of commercial tissue culture of sprout mating systems of American red-maple " October is brilliant ", include the following steps：(1) nursery stock individual tender tip is selected, removes blade and long petiole, disinfection cuts off apoptotic fraction；(2) it is inoculated in primary culture medium and cultivates；(3) by the axillary bud of sprouting, single bud is slit longitudinally into along stem section, is inoculated in Multiplying culture in solid multiplication culture medium；(4) the group's bud that will be proliferated out, is transferred in strong seedling culture base after segmentation and carries out strong seedling culture；(5) by the tissue-cultured seedling Jing Guo strong seedling culture, bottom stem section is cut off, stem section is inserted into root induction culture in root media；(6) tissue-cultured seedling of culture of rootage rooting culture in the greenhouse will be carried out, obtains commodity seedling.The present invention for a kind of economically feasible, character is stable, seedling is neat, the woody plant tissure cultural method that can be applied to large-scale commercial production, disclosure satisfy that the demand of commercialization nursery stock production.
Description
Technical field
The present invention relates to the seedling breeding methods of the extensive tissue cultures of American red-maple " October is brilliant " business.
Background technology
American red-maple " October is brilliant ", Acer rubrum'October Glory' are a kind of Aceraceae Acer L red maples
The xylophyta plant of wooden class is one kind in American red-maple improvement gardening kind.It is fallen leaves megaphanerophyte, grows most fast, adaptability
It is extremely strong, cold-resistant, drought-enduring, moisture-proof.Acidity to neutral soil makes autumn scenery more gorgeous.To pernicious gas resistance, especially to chlorine
Absorbability is strong.It is widely used in park, cell, street plant, not only with Landscape but also can does shade tree, it is deep by people
Like, be beautification, one of the preferable rare tree species in green planted city gardens introduced in recent years.And it can uniquely be used as trade
The Colored- leaf Plants of tree.At present, American red-maple " October is brilliant " seedling can be obtained by cutting propagation and seed propagation approach, with
Afforestation in recent years and the development of urban landscape are used as depositing dust plant action in urban landscape and become increasingly conspicuous, and nursery stock makes
Dosage is increasing.
It was to be introduced by each Scientific Research in University mechanism of China from foreign countries that plant tissue culture technique is introduced earliest, by nearly 50 years
Scientific research, particularly since nearly 20 years, with the raising to seed seedling quality requirement, particularly in ornamental herb
Field is widelyd popularize, and ready commercial, and China produces tissue culture seedling more than 3,000,000,000 plants of (bulb) (grain) per year at present, but main
In terms of concentrating on the draft industrial crops such as draft fancy horticulture plant, potato, strawberry.
Due to the longer physiology time limit of xylophyta, more chromosome numbers, height cortex and xylem's differentiation and
The factors such as scientific research and education system, the lot of research that numerous research units of China carry out depart from the economically feasible of commodity production
Property, the indices such as property stability, seedling regularity can not be applied to large-scale commercial production.Utilize woody plant tissure
Culture technique carries out the business activity of seedling breeding only in recent years with the business seedling that market is neat to batch, character is consistent
Wooden demand, which continues to increase, just to be started.
The content of the invention
It is existing to overcome the object of the present invention is to provide a kind of tissue culture of sprout mating system of splendidness in American red-maple October
The defects of with the presence of technology, meets the needs of business seedling breeding field development.
The method of the present invention, includes the following steps：
(1) explant is chosen：It is limited to the non-lignifying that spring of 3~4 years nursery stock individuals sprouts naturally growth selection year
Tender tip, tender tip materials length is 10~15cm, has 3~4 internodes；
Remove blade and long petiole, disinfection；
Preferably, the sterilization method, includes the following steps：
Explant surface dirt is wiped with alcohol swab, is subsequently placed in 1：2.5~3.5, preferably 1：(commodity float 3 bleaching water
Plain boiled water：Sterile water, volume ratio) in rinsing 10~20 minutes, preferably 15 minutes, during which jiggle 3~5 times, be subsequently placed in nothing
It is rinsed 2~4 times in bacterium water, stands 1~3 minute, then with surface Ming Shui is blotted on aseptic filter paper, cut off incision in petiole and stem section
Mouth apoptotic fraction, retains morphology 0.8~1.2cm of upper end, preferably 1cm, preferably 1.0~2.0cm of lower end, 1.5cm；
Alternatively, sterilization method includes the following steps：
Using hydrogen peroxide or hypochlorite disinfectant, conditions for sterilization is：Hydrogen peroxide concentration is 5%~15%, and disinfecting time is
10~30 minutes, sodium hypochlorite weight concentration was 2%~15%, and disinfecting time is 5~30 minutes, then is blotted on aseptic filter paper
Surface Ming Shui；
(2) Primary culture：The explant disinfected is inoculated in primary culture medium, and is transferred in culturing room, 20~
30 DEG C, preferably 25 DEG C, when half-light culture 40~60 is small, preferably 48 it is small when, then give illumination 1200~1800LUX, preferably
1500LUX, preferably 12~20/8h of photoperiod, 16/8h, carry out Primary culture 2~4 weeks, and preferably 3 weeks, dormancy axillary bud sprouting opened
Dynamic culture is completed；
The primary culture medium using water as solvent, contains following component：
PH value 5.50~6.00, preferably 5.80；
The formula can quickly break axillary bud dormancy, promote axillary bud sprouting, while can effectively avoid the production of callus
It is raw.
The WPM derives from commercially available prefabricated WPM finished products, containing various chemotrophy ingredients and trace element, but not
Containing sucrose and agar；1/2WPM refers to weighing the commercially available WPM finished products of 1/2 mass；
The TDZ is from commercially available import packing TDZ (Chinese name " Thidiazuron " drug, analysis are pure), strong cell point
Split plain class substance；
The GA3GA is dispensed from commercially available import3(Chinese name gibberellin), is one kind of gibberellin class substance, plant
One kind of growth regulator is mainly used for breaking plant dormancy.
(3) Multiplying culture：By the axillary bud that Primary culture is sprouted, single bud is slit longitudinally into along stem section, is inoculated in solid multiplication
In culture medium, 20~30 DEG C, preferably 25 DEG C, when light culture 40~60 is small, preferably 48 it is small when, then to illumination, 1200~
1800LUX, preferably 1500LUX, preferably 12~20/8h of photoperiod, 16/8h, preferably Multiplying culture 3~5 weeks, 4 weeks are proliferated multiplying power
Up to 3 times, the group's bud being proliferated out is obtained, it is preferred that the group's bud being proliferated out is divided into single bud, and is re-seeded into increasing within every 4 weeks
It grows in culture medium, expansion amount multiplication；
The solid multiplication culture medium using water as solvent, contains following component：
PH value is 5.50~6.00, preferably 5.80；
The IAA dispenses IAA drugs from commercially available import, and the entitled heteroauxin of Chinese is analyzed pure；
The KT dispenses KT drugs from commercially available import, and the entitled kinetin of Chinese is analyzed pure；
The formula can ensure that shoot proliferation is artificially controllable, and multiplication multiplying power is 3--4 times, and sprout is healthy and strong, and glass rate is low
In 0.1%；
(4) strong seedling culture：The group's bud that will be proliferated out in proliferated culture medium, is transferred in strong seedling culture base, light after segmentation
According to 2000~3000LUX, preferably 2500LUX, preferably 12~20/8h of photoperiod, 16/8h, preferably 20~30 DEG C, 25 DEG C, culture 5
~7 weeks, preferably 6 weeks, single bud robust growth, and growing height reaches 3cm, obtains the tissue-cultured seedling of strong seedling culture；
The strong seedling culture base using water as solvent, contains following component：
1~2g/L of WPM, preferably 1.35g/L
20~30g/L of sucrose
5.0~7.0g/L of agar
PH value is 5.50~6.00, preferably 5.80；
(5) culture of rootage：By the tissue-cultured seedling Jing Guo strong seedling culture, the stem section of excision stem section 3~5mm of bottom, stem section insertion
8~10mm of root media, is inserted vertically into, 2000~3000LUX of illumination, preferably 2500LUX, 12~20/8h of photoperiod, preferably
16/8h, preferably 20~30 DEG C, 25 DEG C are cultivated 2~5 weeks, and preferably 3 weeks, every plant of plant had effectively healthy and strong root 2~5, root long
20~40mm, no lateral root obtain culture of rootage tissue-cultured seedling；
The root media, using water as solvent, including the component matched as follows：
PH value is 5.50~6.00, preferably 5.80；
The IBA dispenses IBA drugs from commercially available import, and the entitled indolebutyric acid of Chinese is analyzed pure；
AC dispenses AC drugs, the entitled activated carbon of Chinese, superfines, bamboo charcoal from commercially available import；
The root that the formula can promote plant to generate is connected with plant structure, avoids generating from callus false
Root, and the root grown it is sturdy, without lateral root, damage can be reduced in transplanting, is quickly survived.
(6) tame：It will carry out the culture of rootage tissue-cultured seedling of 3 weeks to be transferred in greenhouse, cover 85% shading rate sunshade net, cover
Two layers of lid is kept for 5~10 days, preferably 7 days, then covers 1 layer of 85% shading rate sunshade net 5~10 days, preferably 7 days, you can promote
The tissue-cultured seedling of American red-maple " October is brilliant " is made to adapt to alternating temperature and natural lighting environment, you can obtain seedling；
Preferably, step (7) transplanting is further included：
2~4 days before transplanting, preferably 3 days, ventilation, it was 13~35 DEG C, preferably 18--26 DEG C to keep warm indoor temperature；
Tissue culture of sprout of the step (6) by domestication is transplanted in 72 cave forest pallets, transplanting medium is configured to treasure
Zhu Yan：Vermiculite is 1：1 volume ratio；
After the completion of transplanting, 550~650 mesh, preferably 600 mesh nozzles sprinkle profoundly water immediately, plastic covering film and 95% sunshade
Net 2~4 days, preferably 3 days, remove plastic film, and covering 90% sunshade net of individual layer is further cultured for 3~7 days, preferably 5 days；
Then sunshade net is removed, 550~650 mesh, preferably 600 mesh nozzle supplementary fertilizer water carry out foliage dressing.Foliar fertilizer 20-
20-20 quick-acting fertilizers, EC values are 0.6ms/cm, and 10 points of fine day every morning sprays once, normally conserved after two weeks.
Quick-acting fertilizer described in 20-20-20 quick-acting fertilizers is the water-soluble balanced fertilizer of U.S. peters brands 20-20-20
The beneficial effects of the invention are as follows：For a kind of economically feasible, property stability, seedling it is neat, can be applied to it is big
The woody plant tissure cultural method of scale commercial production, disclosure satisfy that the demand of commercialization nursery stock production.
Specific embodiment
Embodiment 1
(1) explant is chosen：Selection is limited to the non-lignifying sprouted naturally in the spring of 4 years raw nursery stock individuals greenhouse-grown year
Tender tip, tender tip materials length be 15cm, have 4 internodes；Remove blade and long petiole, disinfection；Sterilization method is as follows：
Explant surface dirt is wiped with alcohol swab, is subsequently placed in 1：3 bleaching water (commodity bleaching water：Sterile water, volume
Than) in rinsing 15 minutes, during which jiggle 4 times, be subsequently placed in sterile water and rinse 3 times, standing 2 minutes, then with sterile filter
Surface Ming Shui is blotted on paper, cuts off lower cut apoptotic fraction in petiole and stem section, retains morphology upper end 1cm, lower end 1.5cm；
(2) Primary culture：The explant disinfected is inoculated in primary culture medium, and is transferred in culturing room, 25 DEG C,
When half-light culture 48 is small, illumination 1500LUX, photoperiod 16/8h are then given, carries out Primary culture 3 weeks, dormancy axillary bud sprouting opens
Dynamic culture is completed.
The primary culture medium, with aqueous solvent, including the component matched as follows：
PH value 5.80.
(3) Multiplying culture：By the axillary bud that Primary culture is sprouted, single bud is slit longitudinally into along stem section, is inoculated in solid multiplication
In culture medium, 25 DEG C, when light culture 48 is small, illumination 1500LUX, photoperiod 16/8h are then given, Multiplying culture 4 weeks is proliferated
The group's bud being proliferated out is divided into single bud in every 4 weeks by the group's bud gone out, and is re-seeded into proliferated culture medium, and expansion amount increases
It grows.
The solid multiplication culture medium, with aqueous solvent, including the component matched as follows：
PH value is 5.80.
(4) strong seedling culture：The group's bud that will be proliferated out in proliferated culture medium, is transferred in strong seedling culture base, light after segmentation
According to 2500LUX, photoperiod 16/8h, 25 DEG C, cultivate 6 weeks, single bud robust growth, and growing height reaches 3cm, is strengthened
The tissue-cultured seedling of seedling culture.
The strong seedling culture base, using water as solvent, including the component matched as follows：
WPM 1.35g/L
Sucrose 30g/L
Agar 6.5g/L
PH value is 5.80.
(5) culture of rootage：By the tissue-cultured seedling Jing Guo strong seedling culture, the stem section of excision stem section bottom 3mm, stem section insertion is taken root
Culture medium 8--10mm, is inserted vertically into, illumination 2500LUX, photoperiod 16/8h, are cultivated 6 weeks, every plant of plant has effective by 25 DEG C
Healthy and strong root 2, root long 10--30mm, no lateral root obtain culture of rootage tissue-cultured seedling.
The root media, using water as solvent, including the component matched as follows：
PH value is 5.80.
(6) tame：It will carry out the culture of rootage tissue-cultured seedling of 6 weeks to be transferred in greenhouse, cover 85% shading rate sunshade net, cover
Two layers of lid is kept for 7 days, then covers 1 layer of 85% shading rate sunshade net 7 days, you can promotes the group of American red-maple " October is brilliant "
It trains seedling and adapts to alternating temperature and natural lighting environment.
(7) transplant：
3 days before transplanting, ventilation, it is 18--26 DEG C to keep warm indoor temperature；
Tissue culture of sprout of the step (6) by domestication is transplanted in 72 cave forest pallets, transplanting medium is configured to treasure
Zhu Yan：Vermiculite is 1：1 volume ratio；
After the completion of transplanting, 600 mesh nozzles sprinkle profoundly water immediately, and it is thin to remove plastics for plastic covering film and 95% sunshade net 3 days
Film, covering 90% sunshade net of individual layer are further cultured for 4 days；
Then sunshade net is removed, 600 mesh nozzle supplementary fertilizer water carry out foliage dressing.Foliar fertilizer is with 20：20：20 quick-acting fertilizers, EC
It is worth for 0.6ms/cm, 10 points of fine day every morning sprays once, normally conserved after two weeks.
Embodiment 2
(1) explant is chosen：It is limited to the tender of the non-lignifying that spring of 3 years nursery stock individuals sprouts naturally growth selection year
The tip, tender tip materials length is 10cm, has 3 internodes；
Except blade and long petiole, disinfection；Sterilization method is as follows：
With dioxygen water sterilization, conditions for sterilization is：Hydrogen peroxide concentration is 10%, and disinfecting time is 20 minutes, then with sterile filter
Surface Ming Shui is blotted on paper；
(2) Primary culture：The explant disinfected is inoculated in primary culture medium, and is transferred in culturing room, 25 DEG C,
When half-light culture 48 is small, illumination 1500LUX, photoperiod 16/8h are then given, carries out Primary culture 3 weeks, dormancy axillary bud sprouting opens
Dynamic culture is completed.
The primary culture medium, using water as solvent, including the component matched as follows：
PH value 5.80.
(3) Multiplying culture：By the axillary bud that Primary culture is sprouted, single bud is slit longitudinally into along stem section, is inoculated in solid multiplication
In culture medium, 25 DEG C, when light culture 48 is small, illumination 1500LUX, photoperiod 16/8h are then given, Multiplying culture 4 weeks is proliferated
The group's bud being proliferated out is divided into single bud in every 4 weeks by the group's bud gone out, and is re-seeded into proliferated culture medium, and expansion amount increases
It grows.
The solid multiplication culture medium, using water as solvent, including the component matched as follows：
PH value is 5.80.
(4) strong seedling culture：The group's bud that will be proliferated out in proliferated culture medium, is transferred in strong seedling culture base, light after segmentation
According to 2500LUX, photoperiod 16/8h, 25 DEG C, cultivate 6 weeks, single bud robust growth, and growing height reaches 3cm, is strengthened
The tissue-cultured seedling of seedling culture.
The strong seedling culture base, using water as solvent, including the component matched as follows：
WPM 1.35g/L
Sucrose 30g/L
Agar 6.5g/L
PH value is 5.80.
(5) culture of rootage：By the tissue-cultured seedling Jing Guo strong seedling culture, the stem section of excision stem section bottom 3mm, stem section insertion is taken root
Culture medium 8--10mm, is inserted vertically into, illumination 2500LUX, photoperiod 16/8h, are cultivated 6 weeks, every plant of plant has effective by 25 DEG C
Healthy and strong root 2, root long 10--30mm, no lateral root obtain culture of rootage tissue-cultured seedling.
The root media, using water as solvent, including the component matched as follows：
PH value is 5.80.
(6) tame：It will carry out the culture of rootage tissue-cultured seedling of 6 weeks to be transferred in greenhouse, cover 85% shading rate sunshade net, cover
Two layers of lid is kept for 7 days, then covers 1 layer of 85% shading rate sunshade net 7 days, you can promotes the group of American red-maple " October is brilliant "
It trains seedling and adapts to alternating temperature and natural lighting environment.
(7) transplant：
3 days before transplanting, ventilation, it is 18--26 DEG C to keep warm indoor temperature；
Tissue culture of sprout of the step (6) by domestication is transplanted in 72 cave forest pallets, transplanting medium is configured to treasure
Zhu Yan：Vermiculite is 1：1 volume ratio；
After the completion of transplanting, 600 mesh nozzles sprinkle profoundly water immediately, and it is thin to remove plastics for plastic covering film and 95% sunshade net 3 days
Film, covering 90% sunshade net of individual layer are further cultured for 4 days；
Then sunshade net is removed, 600 mesh nozzle supplementary fertilizer water carry out foliage dressing.Foliar fertilizer 20-20-20 quick-acting fertilizers, EC
It is worth for 0.6ms/cm, 10 points of fine day every morning sprays once, normally conserved after two weeks.
Claims (3)
1. the commercial tissue culture of sprout mating system of American red-maple " October is brilliant ", which is characterized in that include the following steps：
(1) it is limited to the nursery stock individual tender tip for the non-lignifying that spring of 3~4 years nursery stock individuals sprouts naturally growth selection year, it is tender
Tip materials length is 10~15cm, has 3~4 internodes；Blade and long petiole are removed, after disinfection, is cut off above and below petiole and stem section
Notch apoptotic fraction retains morphology 0.8~1.2cm of upper end, 1.0~2.0cm of lower end；
(2) explant disinfected is inoculated in primary culture medium and cultivated；
Cultural method is as follows：When 20~30 DEG C of half-light cultures 40~60 are small, then give illumination 1200~1800LUX, the photoperiod 12
~20/8h is cultivated 2~4 weeks, dormancy axillary bud sprouting；
The primary culture medium using water as solvent, contains the component matched as follows：
PH value is 5.50~6.00；
(3) axillary bud for sprouting Primary culture, is slit longitudinally into single bud along stem section, is inoculated in solid multiplication culture medium and cultivates,
Obtain the group's bud being proliferated out；
Cultural method is as follows：When 20~30 DEG C of light cultures 40~60 are small, then to illumination, 1200~1800LUX, the photoperiod 12~
20/8h is cultivated 3~5 weeks, obtains the group's bud being proliferated out；
The group's bud being proliferated out is divided into single bud, and is re-seeded into proliferated culture medium within every 4 weeks, expansion amount multiplication；
The solid multiplication culture medium, using water as solvent, including the component matched as follows：
PH value is 5.50~6.00；
(4) the group's bud that will be proliferated out in proliferated culture medium, is transferred to after segmentation in strong seedling culture base, and culture obtains strong seedling culture
Tissue-cultured seedling；
Cultural method is as follows：2000~3000LUX of illumination, 12~20/8h of photoperiod, 20~30 DEG C are cultivated 5~7 weeks, are strengthened
The tissue-cultured seedling of seedling culture；
The strong seedling culture base, using water as solvent, including the component matched as follows：
1~2g/L of 1/2WPM
20~30g/L of sucrose
5.0~7.0g/L of agar
PH value is 5.50~6.00；
(5) by the tissue-cultured seedling Jing Guo strong seedling culture, the stem section of excision stem section 3~5mm of bottom, stem section is inserted vertically into root media
8~10mm, 2000~3000LUX of illumination, 12~20/8h of photoperiod are cultivated, 20~30 DEG C, cultivates 2~5 weeks, obtains training of taking root
Support tissue-cultured seedling；
The root media, using water as solvent, including the component matched as follows：
PH value is 5.50~6.00；
(6) tissue-cultured seedling for carrying out culture of rootage is tamed in the greenhouse, obtains commodity seedling；
Acclimation method is as follows, covers 85% shading rate sunshade net, covers two layers, is kept for 5~10 days, then covers 1 layer of 85% screening
Light rate sunshade net 5~10 days.
2. according to the method described in claim 1, it is characterized in that, in step (1), the sterilization method, including walking as follows
Suddenly：Explant surface dirt is wiped with alcohol swab, is subsequently placed in 1：2.5~3.5, middle rinsing 10~20 minutes is subsequently placed in nothing
It is rinsed 2~4 times in bacterium water, stands 1~3 minute, then blot surface Ming Shui；
Alternatively, sterilization method includes the following steps：
Using hydrogen peroxide or hypochlorite disinfectant, conditions for sterilization is：Hydrogen peroxide weight concentration is 5%~15%, and disinfecting time is
10~30 minutes, sodium hypochlorite weight concentration was 2%~15%, and disinfecting time is 5~30 minutes, blots surface Ming Shui.
3. method according to claim 1 or 2, which is characterized in that further include transplant step (7)：
2~4 days before transplanting, ventilation, it is 13~35 DEG C to keep warm indoor temperature；
Tissue culture of sprout of the step (6) by domestication is transplanted in forest pallet, transplanting medium is configured to perlite：Vermiculite
For 1：1 volume ratio；
After the completion of transplanting, 550~650 mesh, nozzle sprinkle profoundly water immediately, and plastic covering film and 95% sunshade net 2~4 days remove
Plastic film, covering 90% sunshade net of individual layer are further cultured for 3~7 days；
Then sunshade net is removed, 550~650 mesh, nozzle supplementary fertilizer water carries out foliage dressing, foliar fertilizer 20-20-20 quick-acting fertilizers,
EC values are 0.6ms/cm, and 10 points of fine day every morning sprays once, normally conserved after two weeks.
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| CN107094625B (en) * | 2017-05-05 | 2020-04-24 | 江苏东郁园林科技有限公司 | Tissue culture seedling breeding method for taxus mairei |
| CN110663553A (en) * | 2019-11-09 | 2020-01-10 | 江苏东郁植物科技有限公司 | Commercial tissue culture seedling breeding method for acer rubrum |
| CN110839528A (en) * | 2019-11-22 | 2020-02-28 | 江苏东郁植物科技有限公司 | Tissue culture seedling breeding method for acer rubrum |
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| WO2022260849A1 (en) * | 2021-06-09 | 2022-12-15 | Nant Holdings Ip, Llc | Methods and systems for producing a protein of interest in a plant |
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