CN102197785A - Medium for rapid breeding and propagation of fraxinus americana tissue culture and application thereof - Google Patents
Medium for rapid breeding and propagation of fraxinus americana tissue culture and application thereof Download PDFInfo
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- CN102197785A CN102197785A CN 201010131281 CN201010131281A CN102197785A CN 102197785 A CN102197785 A CN 102197785A CN 201010131281 CN201010131281 CN 201010131281 CN 201010131281 A CN201010131281 A CN 201010131281A CN 102197785 A CN102197785 A CN 102197785A
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Abstract
The invention relates to forest tissue culture, and specifically relates to a medium for rapid breeding and propagation of fraxinus americana tissue culture and application thereof. The medium employs an MS or a WPM medium as a minimal medium, and the medium is added with growth augmentors to a final concentration of: CPPU 0.05-0.5mg/ L, NAA or IBA 0.1-0.5mg/L, and white granulated sugar 20-40g/L, and a pH of the medium is adjusted to 5.8-5.9. In the invention, plant-growth regulators of CPPU and NAA or IBA are added to the two phase solidified MS or WPM medium, so that the propagation effect on fraxinus americana embryo inducing aseptic seedling stems is substantial. The invention also provides application of the medium in rapid breeding and propagation of fraxinus americana tissue culture, so as to provide a feasible scheme for factory production of fraxinus americana in the future.
Description
Technical field
The present invention relates to the forest tissue culture, specifically a kind of U.S. Chinese wax tissue-culturing quick-propagation proliferated culture medium and application thereof.
Background technology
U.S.'s Chinese wax (Fraxinus americana) has another name called the Bai Ash, and the Oleaceae deciduous tree originates in the North America.U.S.'s Chinese wax is drought-enduring, cold-resistant, waterlogging, salt tolerant alkali, is the fine tree species of coastal wetland afforestation.Its growth is fast, and afforestation effect is good, the ecological benefits height, and material is good, quality is hard, and texture is perfectly straight, and structure is carefully even, and corrosion resistant power is strong, high resilience and gloss, easily processing, have " Manchurian ash " laudatory title (document 1. Dong must be intelligent, Su Guoxing. biological property of American ash and economic use.The Jiangsu forestry science and technology, 2003,30, (1): 32-34).Chinese wax is for a long time based on seminal propagation, because its juvenile stage is long, and tool tangible solid interval, have a strong impact on the production and the breeding of seed.Therefore, the application on Chinese wax of vegetative propagation and biotechnology has very big potentiality.Simultaneously, to those individualities through selection, breed improvement and genetic manipulation, carry out vegetative propagation and can quicken the stock breeding process of Chinese wax (2. hole winter of document plum, Tan Yanshuan, Shen Hailong. tissue culture of Fraxinus plant and plant regeneration. Plant Physiology Communications, 2003,39 (6): 677-680).Different seeds, that different explants is cultivated desired minimal medium is all variant, the suitable culture base, can effectively improve growth coefficient (3. man of virtue and ability girls of document. compound leaf maple tissue culture regenerating system is set up and the genetic transformation Primary Study. Chinese doctorate paper, 2005,05:1-99).Among the various compositions of medium, plant growth regulating substance forms for the organ in the tissue culture, play important and tangible regulating action (document 4. Wang Hui plums. the research of camplotheca acuminata tissue culture and genetic transformation. Chinese doctorate paper, 2004,06:1-91).Largeleaf Chinese Ash seedling rate of increase on MS+6-BA 0.2+KT 0.8+IBA 0.2 medium is 2.5 (document 6. Hong Yuan models, Shen Yujia, Hong Qing, Li Shunpeng.The cultured in vitro of Largeleaf Chinese Ash and breeding fast. Plant Physiology Communications, 2006,42 (6): 1139); Fine hair Chinese wax is explant with the stem section, and on MS+BA4.0～6.0mg/L+NAA2.0mg/L medium, average growth coefficient is 3.59 (document 6. Wang Lei, Li Shujuan, Xu Yungang.Zhan Ya light fine hair Chinese wax mature embryo and stem section are cultivated the foundation of breeding system. forestry science and technology, 2008,33 (3): 1-3).Reached 6-7 and utilize medium of the present invention, U.S.'s Chinese wax to expand numerous growth coefficient.Therefore, research U.S. Chinese wax tissue-culturing rapid propagation proliferated culture medium for the batch production production of U.S.'s Chinese wax from now on provides feasible program, is significant to exploitation and the popularization of this seeds.
Tissue culture is to carry out cultured in vitro with part tissue of plant or organ on aseptic synthetic medium, to obtain the asexual reproduction method of whole plant.Because breaking away from the plant parent, in test tube, cultivates culture, so also claim cultured in vitro.Plant Tissue Breeding is to be the emerging technology that base growth is got up with plant physiology at the beginning of 20th century, and this technology is used widely in research and production.This technology has the acceleration breeding, shortens reproductive process, and the improvement quality is saved the space, reduces work, and the anniversary experiment is produced, and be not subjected to characteristics such as natural conditions restriction, and the volume of tissue cultivating seedling is little, is easy to carry to exchange with resource.At present, rapid propagation in vitro and batch production are to be most widely used, to develop the most ripe modern biotechnology in forestry, having produced important effect aspect raising forest output and the quality, have brought great economic benefit, social benefit and ecological benefits.
Medium is the important component part of Plant Tissue Breeding, is the support that tissue cultivating seedling grows.Because of the required nutrient difference of growth and development of plants, so the plant of different cultivars needs different types of medium, only select optimum medium, could farthest obtain regeneration plant, effectively improve growth coefficient.
Plant growth regulator is the key substance in the medium, though amount is few, plays very important and tangible regulating and controlling effect in Plant Tissue Breeding.The plant growth regulator that is usually used in tissue culture has two big classes: i.e. cytokinin and auxins.The main effect of cytokinin is division and the expansion that promotes cell, makes stem increase slightly the differentiation of induced bud; It is the elongation growth that promotes cell that auxins mainly acts on, hestening rooting.In Plant Tissue Breeding, phytocytomine commonly used has forchlorfenuron (CPPU), 6-benzyl aminopurine (6-BA), kinetin (KT) and zeatin (ZT), plant growing commonly used have methyl (NAA), indolebutyric acid (IBA) and 2,4 dichlorophenoxyacetic acid (2,4-D).Different types of plant growth regulator mechanism of action difference, the splitting ability of phytocytomine, auximone promote the ability of cell elongation that power is arranged, in order to obtain higher growth coefficient, need the plant growth regulator of screening suitable kind and concentration.
Carbon source is the indispensable material of Plant Tissue Breeding, and it not only can provide energy to explant, and can keep certain osmotic pressure, and the concentration of carbohydrate not only influences multiplication rate, but also the quality of influence propagation bud.Carbon source commonly used has fructose, glucose, sucrose, maltose, sorbierite etc., and wherein sucrose is the most frequently used, and sucrose can be supported the vigorous growth of most plant isolated cultures, is used as the standard carbon source of Plant Tissue Breeding and extensive use always.Recently, many sucrose that studies show that might not be optimum carbon sources, and different plants are incomplete same to the reaction of different carbohydrates, are necessary to explore alternative carbon source, to improve proliferate efficiency and to save production cost.
Forchlorfenuron (CPPU) white crystal is insoluble in water, is dissolved in organic solutions such as ethanol, methyl alcohol, acetone, stable storage under the normal temperature, low toxicity.Forchlorfenuron is a kind of phenylurea class plant growth regulator with cytokine activity, and its biologically active is but high 10～100 times than 6-benzyl aminopurine (6-BA), can quicken cell mitogen, promote plant growing, is widely used in farming, forestry.
Methyl (NAA) claim the a-methyl again, and pure product are white tasteless crystal, are soluble in organic solvents such as acetone, ether, benzene, ethanol and chloroform, and low toxicity can be earlier with a small amount of alcohol dissolving, thin up again during preparation.Methyl is the broad spectrum type plant growth regulator, can promote cell elongation, promotes plant establishment.
Indolebutyric acid (IBA) has another name called the 3-indolebutyric acid, and pure product are that white is to faint yellow crystalline solid.Main application: promote the growth of plant main root, improve germination rate, survival rate.
Summary of the invention
Low for overcoming existing Fraxinus tree tissue culture multiplication rate, the propagation switching cycle is long, agar, sugar consumption amount are big, the culture medium cost height, the needs that are unfavorable for large-scale industrialized production, the purpose of this invention is to provide a kind of suitable U.S. Chinese wax ripe kind embryonal induction quick proliferated culture medium of aseptic seedling stem sections tissue culture and application thereof, make Fraxinus tree tissue culture multiplication rate fast, the propagation switching cycle shortens, the agar consumption is little, culture medium cost reduces, and is beneficial to the needs of large-scale industrialized production.
With the ripe aseptic seedling stem-segment with single bud of planting embryonal induction of U.S.'s Chinese wax is material, is inoculated in the proliferated culture medium.The U.S.'s quick proliferated culture medium of Chinese wax tissue culture consists of:
It is to be minimal medium with MS or WPM medium, adds growth and urges into thing in minimal medium, adds each thing to final concentration and be in minimal medium: forchlorfenuron 0.05～0.5mgL
-1, methyl or indolebutyric acid 0.1～0.5mgL
-1, white sugar 20～40gL
-1, regulate medium pH value to 5.8～5.9.
Described proliferated culture medium is to be a kind of two solidifying medium of being in of minimal medium allotment between solid-state and liquid with MS or WPM medium, promptly also is added with final concentration and is in minimal medium: 3.0～4.0gL
-1Agar powder.
Described forchlorfenuron concentration is 0.1～0.3mgL
-1, described methyl or indolebutyric acid concentration are 0.1～0.3mgL
-1, described white sugar concentration is 25～35gL
-1
Wherein the MS medium adds forchlorfenuron 0.15mgL
-1, methyl or indolebutyric acid 0.2mgL
-1With white sugar 30gL
-1Best results.
Described proliferated culture medium can be used as the proliferated culture medium of U.S.'s Chinese wax kind embryonal induction aseptic seedling stem sections fast breeding.
U.S.'s Chinese wax tissue-culturing quick-propagation process is: with U.S.'s Chinese wax mature and plump seed is explant, divest kind of a shell, behind flushing with clean water 2～3h, go up with volumetric concentration 68-72% alcohol at superclean bench (being aseptic workbench) successively and soak into 30-50s, aseptic water washing 2～3 times, mass concentration 0.09～0.11% mercuric chloride (HgCl
2) sterilization 6～10min, aseptic water washing 3～4 times cuts embryo, is inoculated in MS or the WPM medium, when height of seedling 3～4cm is high, cuts the aseptic seedling stem-segment with single bud, is inoculated in the described proliferated culture medium of claim 1.
The present invention has following advantage:
1, saves production cost.The used agar amount of two solidifying medium is half of solid culture medium approximately, and the white sugar price is about 16% of a homemade sucrose.Agar and sucrose account for 95% of culture medium cost, therefore, utilize the two solidifying medium of inventing to carry out U.S.'s Chinese wax tissue-culturing quick-propagation and can save the medium production cost greatly.
2, growth coefficient height.Fraxinus tree tissue culture growth coefficient was about 3 in the past, and the medium growth coefficient that utilizes invention is 6～7, and multiplication rate has improved 2 times.
3, switching cycle is short.Two solidifying medium contact more tight with culture, help cultivating the absorption of seedling nutrition, and can effective support live culture, solid culture medium propagation U.S. Chinese wax optimal period is in 5～6 weeks, utilizing the two solidifying medium propagation U.S. Chinese wax optimal period of invention is about 4 weeks, switching cycle reduces more than 25% than solid culture, and therefore, growth rate is fast.
4, the sprout after the propagation is sturdy, and it is vigorous to breed the seedling growth that, and the average bud length after 4 weeks can reach 2.1cm, and the cauline leaf growth is neat healthy and strong.
Embodiment
With the ripe embryo of U.S.'s Chinese wax is explant, compare variety classes medium and plant growth regulator, reach the influence of plant modifying agent concentration and white sugar concentration, filter out best enrichment culture based formulas U.S.'s Chinese wax kind embryonal induction aseptic seedling stem-segment with single bud propagation.
The standard recipe of the standard recipe WPM medium of MS medium
Embodiment 1
1) acquisition of the preparation of inducing culture, sterilization and sterilizable material
MS is a minimal medium, adds agar 6.0gL
-1, white sugar 30gL
-1, regulate medium pH value 5.8～5.9, be sub-packed in the glass triangle flask of 100ml every bottle of about 40ml.Medium and the damp and hot sterilization of sterilization employing high pressure of cultivating vessel.The medium that branch is installed places in the high-pressure sterilizing pot with experiment apparatus such as the culture vessel that bandages, tweezers, scalpel, and 121 ℃, 1.1kg.cm
-3Sterilized 20 minutes.
Choose full U.S. Chinese wax seed, divest kind of a shell, behind flushing with clean water 2～3h, on superclean bench, soak into 30s, aseptic water washing 2～3 times, 0.1%HgCl with 70% alcohol
2Sterilization 8min, aseptic water washing 3～4 times cuts embryo, is inoculated in the MS medium, is positioned over culturing room.
2) proliferated culture medium preparation and sterilization
MS is a minimal medium, adds agar 3.0gL respectively
-1(MS two solidifying medium of the present invention) and 6.0gL
-1(MS solid culture medium) makes up two types of medium, adds white sugar 20gL again
-1, forchlorfenuron 0.1mgL
-1, methyl 0.1mgL
-1, regulate medium pH value 5.8～5.9, be sub-packed in the glass triangle flask of 100ml every bottle of about 40ml.The same step of sterilizing methods " 1 "
3) treat that aseptic seedling grows to 3～4cm, cut stem-segment with single bud, be inoculated in the proliferated culture medium, 4 week the back observe growing states.Drawn by table 1, up to 6.2, average bud is up to 2.34cm to stem-segment with single bud at MS two solidifying medium growth coefficients, and propagation seedling robust growth.
Culturing room's condition: 25 ± 1 ℃ of temperature, humidity 60～70%, intensity of illumination 30～40umolm
-2S
-1(14hd
-1).
Table 1 different MS medium state is to the influence of bud propagation
Embodiment 2
The acquisition of this example materials is identical with embodiment 1 method with condition, and difference from Example 1 is:
2) proliferated culture medium preparation and sterilization MS are minimal medium, add 0.2mgL respectively
-1Forchlorfenuron, 0.2mgL
-16-benzyl aminoadenine (6-BA) and 0.2mgL
-1Thiadiazoles phenylurea (TDZ) is combined into 3 types of medium, adds agar 3.5gL again
-1, sucrose 30gL
-1, methyl 0.2mgL
-1, regulate medium pH value 5.8～5.9, be sub-packed in the glass triangle flask of 100ml every bottle of about 40ml.Sterilizing methods is with example 1 step " 1 ".
3) treat that aseptic seedling grows to about 3～4cm, cut stem-segment with single bud, be inoculated in the proliferated culture medium, 4 week the back observe growing states, adding 0.2mgL
-1In the medium of forchlorfenuron, growth coefficient is up to 6.4, the average high 2.16cm of bud, and propagation seedling robust growth.
The dissimilar basic elements of cell division of table 2 are to the influence of bud propagation
Embodiment 3
The acquisition of this example materials is identical with embodiment 1 method with condition, and difference from Example 1 is:
WPM is a minimal medium, adds agar 4.0gL
-1, white sugar 40gL
-1, forchlorfenuron 0.3mgL
-1, indolebutyric acid 0.3mgL
-1, regulate medium pH value 5.8～5.9, be sub-packed in the glass triangle bottle of 100ml every bottle of about 40ml.Sterilizing methods is with example 1 step " 1 ".
3) treat that aseptic seedling grows to 3～4cm, cut stem-segment with single bud, be inoculated in the proliferated culture medium, 4 week the back observe growing states, growth coefficient reaches 6.8, the average high 1.96cm of bud, and propagation seedling robust growth.
Proliferated culture medium of the present invention is effective to kind of embryonal induction aseptic seedling stem sections propagation, and wherein the MS medium adds forchlorfenuron 0.15mgL
-1, methyl 0.2mgL
-1, white sugar 30gL
-1Best results, growth coefficient reaches 6.9, and bud is up to 2.34cm.It is remarkable to U.S.'s Chinese wax kind embryonal induction aseptic seedling stem sections cultivation effect to show that two solidifying MS and WPM medium add plant growth regulator forchlorfenuron and methyl or indolebutyric acid.The invention provides the application of this medium in U.S.'s Chinese wax tissue-culturing quick-propagation, for the batch production production of U.S.'s Chinese wax from now on provides feasible program.
Claims (8)
1. U.S.'s Chinese wax tissue-culturing quick-propagation proliferated culture medium is characterized in that:
It is to be minimal medium with MS or WPM medium, adds growth and urges into thing in minimal medium, adds each thing to final concentration and be in minimal medium: forchlorfenuron 0.05～0.5mgL
-1, methyl or indolebutyric acid 0.1～0.5mgL
-1, white sugar 20～40gL
-1, regulate medium pH value to 5.8～5.9.
2. proliferated culture medium according to claim 1 is characterized in that:
Described proliferated culture medium is to be a kind of two solidifying medium of being in of minimal medium allotment between solid-state and liquid with MS or WPM medium, promptly also is added with final concentration and is in minimal medium: 3.0～4.0gL
-1Agar powder.
3. proliferated culture medium according to claim 1 is characterized in that: described forchlorfenuron concentration is 0.1～0.3mgL
-1, described methyl or indolebutyric acid concentration are 0.1～0.3mgL
-1
4. proliferated culture medium according to claim 1 is characterized in that:
Described forchlorfenuron concentration is 0.15mgL
-1, described methyl or indolebutyric acid concentration are 0.2mgL
-1
5. proliferated culture medium according to claim 1 is characterized in that:
Described white sugar concentration is 30gL
-1
6. according to claim 4 or 5 described proliferated culture mediums, it is characterized in that: wherein the MS medium is a basal medium.
7. the application of the described proliferated culture medium of claim 1 is characterized in that: described proliferated culture medium can be used as the proliferated culture medium of U.S.'s Chinese wax kind embryonal induction aseptic seedling stem sections fast breeding.
8. the application of proliferated culture medium according to claim 7 is characterized in that:
U.S.'s Chinese wax tissue-culturing quick-propagation process is: with U.S.'s Chinese wax mature and plump seed is explant, divest kind of a shell, behind flushing with clean water 2～3h, on superclean bench, soak into 30～50s successively with volumetric concentration 68-72% alcohol, aseptic water washing 2～3 times, mass concentration 0.09～0.11% mercuric chloride (HgCl
2) sterilization 6～10min, aseptic water washing 3～4 times cuts embryo, is inoculated in MS or the WPM medium, when height of seedling 3～4cm, cuts the aseptic seedling stem-segment with single bud, is inoculated in the described proliferated culture medium of claim 1.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104381128A (en) * | 2014-10-15 | 2015-03-04 | 山东省林业科学研究院 | Method for improving tissue culture and rapid propagation proliferation rate of Fraxinus velutina |
| CN104396759A (en) * | 2014-12-10 | 2015-03-11 | 中国科学院遗传与发育生物学研究所 | Method for tissue culture and rapid breeding of Fraxinus chinensis |
| CN105532468A (en) * | 2016-01-11 | 2016-05-04 | 山东省林业科学研究院 | Method for acquiring regenerative plants through fraxinus velutina somatic embryo induction |
| CN106359084A (en) * | 2016-08-24 | 2017-02-01 | 盛世绿源科技有限公司 | Tissue culture method of American ash |
| CN108575188A (en) * | 2018-04-27 | 2018-09-28 | 鲁东大学 | A kind of method that Chinese wax percentage of seedgermination is improved under condition of tissue culture |
| CN109984038A (en) * | 2019-04-08 | 2019-07-09 | 鲁东大学 | Rapidly promote Chinese wax stem apex proliferation and the take root culture medium being completed at the same time and purposes |
-
2010
- 2010-03-24 CN CN2010101312816A patent/CN102197785B/en not_active Expired - Fee Related
Non-Patent Citations (2)
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| 《安徽农业科学》 20060130 陈之群等 "绒毛白蜡茎段的组织培养及植株再生" 第472-473页 1-8 第34卷, 第3期 * |
| 《植物生理学通讯》 20061231 洪源范等 "大叶白蜡的离体培养与快速繁殖" 第1139页 1-8 第42卷, 第6期 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104381128A (en) * | 2014-10-15 | 2015-03-04 | 山东省林业科学研究院 | Method for improving tissue culture and rapid propagation proliferation rate of Fraxinus velutina |
| CN104381128B (en) * | 2014-10-15 | 2016-03-02 | 山东省林业科学研究院 | A kind of method improving the Fraxinus velutina tissue-culturing rapid propagation rate of increase |
| CN104396759A (en) * | 2014-12-10 | 2015-03-11 | 中国科学院遗传与发育生物学研究所 | Method for tissue culture and rapid breeding of Fraxinus chinensis |
| CN104396759B (en) * | 2014-12-10 | 2016-04-06 | 中国科学院遗传与发育生物学研究所 | The method that ash tree tissue cultures is bred fast |
| CN105532468A (en) * | 2016-01-11 | 2016-05-04 | 山东省林业科学研究院 | Method for acquiring regenerative plants through fraxinus velutina somatic embryo induction |
| CN106359084A (en) * | 2016-08-24 | 2017-02-01 | 盛世绿源科技有限公司 | Tissue culture method of American ash |
| CN108575188A (en) * | 2018-04-27 | 2018-09-28 | 鲁东大学 | A kind of method that Chinese wax percentage of seedgermination is improved under condition of tissue culture |
| CN108575188B (en) * | 2018-04-27 | 2021-01-15 | 鲁东大学 | Method for improving germination rate of fraxinus chinensis seeds under tissue culture condition |
| CN109984038A (en) * | 2019-04-08 | 2019-07-09 | 鲁东大学 | Rapidly promote Chinese wax stem apex proliferation and the take root culture medium being completed at the same time and purposes |
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