CN106305416A - Efficient regeneration method with zenia insignis leaves being explants - Google Patents
Efficient regeneration method with zenia insignis leaves being explants Download PDFInfo
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- CN106305416A CN106305416A CN201610662934.0A CN201610662934A CN106305416A CN 106305416 A CN106305416 A CN 106305416A CN 201610662934 A CN201610662934 A CN 201610662934A CN 106305416 A CN106305416 A CN 106305416A
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- Prior art keywords
- blade
- outer implant
- appoint
- regeneration method
- bud
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses an efficient regeneration method with zenia insignis leaves being explants and belongs to the technical field of plant tissue culturing. With the leaves being materials, a zenia insignis regeneration system is researched for improvement in the regeneration capacity of zenia insignis, and an efficient regeneration system is established and used for providing effective reference value for genetic resource conservation and genetic engineering research later. The efficient tissue culturing and plant regeneration method can be free of influences of external conditions, and robust, neat and uniform zenia insignis tissue culture seedlings are produced on a large scale in an industrialized mode. The zenia insignis leaves serve as the drawn materials, and compared with the reported mode that hypocotyls and annual branches serve as explants to construct a regeneration system, the efficient regeneration method has the advantages of being convenient to operate, wide in range of drawn materials, high in repeatability and efficiency and the like. The method not only can meet the requirement for seedlings of the market, but also can modify and innovate zenia insignis genetic resources through transgene research to lay a foundation for sustainable utilization of the zenia insignis genetic resources.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to a kind of with appoint bean blade be outer implant efficiently the most again
Generation method.
Background technology
Appointing bean (Zenia insignis Chun) to have another name called Common zenia, Ren Shu, tree of choping off the head, for Caesalpiniaceae, Common zenia belongs to, speed
Raw fallen leaves megaphanerophyte.Appointing bean is China's rare tree species, and single genus plants, and belongs to China's second class protection wild tree species.Appoint Caragana arborescens kind main
Being distributed in Vietnam of Malaysia of Tropical Asia India (or South East Asia Mainland) to south China (or southwest) region, China mainly exists
Hunan, Guangdong, osmanthus, Yunnan, Guizhou Province etc. save distribution.Appointing bean Sprout reproduction ability strong, growth rapidly, and is grown into forest and is become a useful person early, its timber material
Of fine quality good, can be used as firewood；Its well developed root system, resistance, flourishing with flowers and leaves, wide adaptability, is excellent green tree species；Its leaf rich in
Abundant protein, perishable, feedstuff fertilizer can be made, it is possible to improvement soil, conserve water and soil.Visible, appointing bean is a kind of multipurpose
Seeds, are Fuel-plantation Trees, are again water and soil conservation seeds.Particularly in China south China and southwest, large area is distributed
In Limestone Area.In Limestone Area owing to rock is exposed, poor water retention property and the shallow feature such as barren of soil layer cause this area
Vegetation sparse, the environmental problem such as severe water and soil erosion is day by day serious, therefore, appoints these seeds of bean to show certain development space.
Appointing bean breeding potential the highest, result has dividing of biennial bearing, and difficult with former bean cuttage root-taking, the rate that is propagated from cuttings is the highest.Mesh
Before, although appointing bean seed seedling-raising and introduction planting technology to become ripe, wood comprehensive research on utilization also obtains certain progress, but
Appointing bean not yet to set up ripe efficient regenerating system, the work of the aspects such as fine-variety breeding is the most not yet carried out, its physiological and ecological characteristic
Studying the fewest, there is not been reported for molecular biology level, and hereditary information is deficient.
Set up by tissue culture and appoint a whole set of ripe efficient regenerating system of bean plant, big except obtaining in a short time
The vegetable material that scale is the most consistent, it is simple to carry out Germ-plasma resources protection and expanding propagation, for later Fast-propagation nursery stock, meets and produces need
Effective way to be provided；Can also future to appoint bean or pea for genetic test, the research of low temperature stress and utilize genetic engineering
Improve etc. biotechnology and appoint the tolerance to cold etc. of bean breeding to study offer basis.
To appointing, bean Study on tissue culture is domestic have been reported at present, is concentrated mainly on and utilizes the hypocotyl appointing bean aseptic seedling,
Seedling tender stem and the inducing culture of annotinous branch, for appointing other positions and each clonal tissue of bean aseptic seedling
Cultivate and not yet studies have reported that.Prior art is outer implant with axillalry bud and the bean seedling tender stem of a bean, whole root induction
Process time is 1 year；With appointing the cutting of the terminal bud of bean aseptic seedling, hypocotylar upper, stage casing and annotinous branch as outer planting
Body, needs about 75d from callus induction to obtaining complete tissue cultured seedling, the inductivity 50%～75% of bud, the inductivity of root
85%.Being outer implant relative to plumular axis and tender stem, blade is as regeneration and the outer implant of genetic transformation, root induction process used time
Shorter, and inductivity raising, also there are draw materials convenience, processing ease, wide material sources, be conducive to regeneration and the repetition of genetic transformation
The advantage such as carry out.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, it is an object of the invention to provide a kind of to appoint bean blade as outer planting
The highly efficient regeneration method of body.The method is outer implant to appoint a bean blade, operate easier, draw materials more extensive, expanding propagation is more efficient.Profit
A bean seedling of a large amount of high-quality can be the most quickly obtained by the method.
The present invention appoints bean regenerating system with blade for investigation of materials, it is intended to improves and appoints bean regeneration capacity, sets up the most again
Raw system, provides effective reference value for plasm resource protection afterwards and genetic engineering research.
The purpose of the present invention is achieved through the following technical solutions:
A kind of highly efficient regeneration method to appoint bean blade to be outer implant, comprises the steps:
(1) induction of sorite and elongation: the blade choosing aseptic seedling is outer implant, is inoculated into outer implant bud inducement and cultivates
Evoking adventive bud in base, during inoculation, blade abaxial side is upward；Cultivate to blade, begin with the appearance of bud point, outer implant is transferred to
Evoking adventive bud elongation in Elongation of adventitious bud culture medium；Adventitious bud induction frequency is up to more than 78%；
(2) root culture: when Elongation of adventitious bud to 3～4cm, cut adventitious bud, is inoculated in root media induction
Take root.Rooting rate is up to more than 85%.
The condition of culture of the induction described in step (1) is temperature 25 ± 2 DEG C, intensity of illumination 1500～2500lx, illumination
Time 12～15h.
Bud inducement culture medium described in step (1) is MS+6-BA 2.0～4.0mg/L+2,4-D 0.01～0.03mg/
L+IBA 0.01～0.02mg/L+ sucrose 28～35g/L+ agar 4.5～5.5g/L；PH is 5.8～6.0.
Elongation of adventitious bud culture medium described in step (1) be MS+6-BA 0.5～1.6mg/L+IBA 0.01～
0.03mg/L+ sucrose 28～35g/L+ agar 4.5～5.5g/L；PH is 5.8～6.0.
The condition of culture of the induction described in step (2) is temperature 25 ± 2 DEG C, intensity of illumination 1500～2500lx, illumination
Time 12～15h.
Root media described in step (2) is MS+NAA 0.01～0.02mg/L+ sucrose 28～35g/L+ agar
4.5～5.5g/L；PH is 5.8～6.0.
The blade of the aseptic seedling described in step (1), is made by the steps and obtains:
A, the acquisition of aseptic seedling: by seed water-bath soak, after seed tiling have on the culture dish of moistening filter paper；Place 20
～after 25h, select health and the seed that expands carries out disinfection；By planting seed in the MS of blank (without any hormone) after sterilization
Culture medium is cultivated；Seed germination rate is to more than 95%；
B, appoint the acquisition of bean blade: seed culture is unfolded to aseptic seedling leaf and come, and cuts blade as outer implant.
The condition that water-bath described in step A is soaked is that 85～90 DEG C of water-baths soak 8～12min；
The step of the sterilization described in step A is as follows: aseptically, with 75% ethanol sterilizing 25～30s, sterilized water
Rinse 1 time, 0.1～0.2% mercuric chloride sterilizing 5min～10min, sterilized water cleaning down 5 times.
The time of the placement described in step A is preferably 24h；
The condition of the cultivation described in step A is temperature 25 ± 2 DEG C, intensity of illumination 1500～2500lx, light application time 10
～15h.
Described 6-BA refers to 6-benzyl aminoadenine；2,4-D refer to 2, and 4-dichlorphenoxyacetic acid, is a kind of auxins
Like thing；IBA refers to indolebutyric acid；NAA refers to a-naphthalene acetic acid.
The present invention, relative to prior art, has such advantages as and effect:
The high-efficiency tissue of the present invention is cultivated and plant regeneration method, can break away from the impact of external condition, scale and factory
Metaplasia output is healthy and strong, the bean group of appointing of neat and consistent trains seedling.Seeds preprocess before tissue culture's operation and sterilization method mesh
Before not yet report, the method processes while seed can make seed germination unaffected, it is thus achieved that the date of seed Seedling shortens, and accelerates
Test rate.Appoint bean leaf as material of drawing materials, with it has been reported that plumular axis, annotinous branch build regeneration body as outer implant
System compares, and has the advantages such as easy to operate, extensive, the repeatable strong and efficiency of drawing materials is high.The method can not only meet market pair
The demand of seedling, and can be by transgenic research to appointing bean germ plasm resource improve and innovate, for appointing bean germ plasm resource
Sustainable use lays the foundation.
Accompanying drawing explanation
Fig. 1 is that 17d appoints bean aseptic seed Seedling, is used for cutting blade.
Fig. 2 is at MS+6-BA 2.0～4.0mg/L+2,4-D 0.01～0.03mg/L+IBA 0.01～0.02mg/L+
Sucrose 28～35g/L+ agar 4.5～5.5g/L, pH is on the upper leaf explant cultivating 40d of bud inducement cultivation of 5.8～6.0
Sorite.
Fig. 3 is at MS+6-BA 0.5～1.6mg/L+IBA 0.01～0.03mg/L+ sucrose 28～35g/L+ agar 4.5
～in the Elongation of adventitious bud culture medium that 5.5g/L, pH are 5.8～6.0, cultivate the adventitious bud of 15d elongation.
Fig. 4 is at MS+NAA 0.01～0.02mg/L+ sucrose 28～35g/L+ agar 4.5～5.5g/L, pH be 5.8～
The root induced on the root media of 6.0.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention do not limit
In this.
Embodiment 1
Step one: Seeds preprocess.Seed is placed in water-bath, soaks 12min with 85 DEG C of water-baths.Afterwards seed is tiled
Having on the culture dish of moistening filter paper, the moment keeps filter paper humidity.After seed places 24h in culture dish, select health and substantially
The seed expanded carries out next step test operation.
Step 2: seed disinfection sterilizing.On aseptic super-clean bench, with 75% ethanol sterilizing 28s, sterilized water rinses 1 time；
0.15% mercuric chloride sterilizing 8min, sterilized water cleaning down 5 times.By planting seed in the MS culture medium of blank (without any hormone)
Middle cultivation, cultivation temperature 25 ± 2 DEG C, intensity of illumination 1500lx, light application time 15h.Observing seed germination situation, germination rate reaches
96.54%.
Step 3: tissue culture's material obtains.After 7d, find that seed starts to sprout, stretch out plumular axis from cotyledon.Appoint Macroptilium
Leaf is unearthed, and about 10d appoints bean seed Seedling true leaf to unfold and come, it is thus achieved that complete aseptic seedling, using the true leaf of aseptic seedling as outer implant
Test.
Step 4: the induction of sorite and elongation.On aseptic operating platform, cut leaf as outer implant by sterile razor blade,
True leaf one is divided into four, is inoculated into bud inducement culture medium (MS+6-BA 4.0mg/L+2,4-D 0.01mg/L+IBA 0.01mg/L+
Sucrose 28g/L+ agar 5g/L；PH is 5.8) in evoking adventive bud, during inoculation, blade abaxial side is upward；After cultivating 30d, on blade
Begin with bud point to occur, outer implant is transferred to Elongation of adventitious bud culture medium (MS+6-BA 0.5mg/L+IBA 0.01mg/L+ sugarcane
Sugar 28g/L+ agar 5g/L；PH is 6.0) in evoking adventive bud elongation.Condition of culture is temperature 25 ± 2 DEG C, intensity of illumination
2000lx, light application time 15h.Adventitious bud induction frequency is up to 82%.
Step 5: root culture.When observing Elongation of adventitious bud to 3～4cm, cut adventitious bud, be inoculated in root culture
Root induction in base (MS+NAA 0.015mg/L+ sucrose 35g/L+ agar 4.5g/L, pH is 5.9), condition of culture is temperature 25
± 2 DEG C, intensity of illumination 2000lx, light application time 13h.About 7d, has observed in root media that Article 1 root length goes out.After 5d
Obtain complete tissue cultured seedling.Rooting rate is up to 85.6%.
Embodiment 2
Step one: Seeds preprocess.Seed is placed in water-bath, soaks 8min with 90 DEG C of water-baths.Afterwards seed is tiled
Having on the culture dish of moistening filter paper, the moment keeps filter paper humidity.After seed places 24h in culture dish, select health and substantially
The seed expanded carries out next step test operation.
Step 2: seed disinfection sterilizing.On aseptic super-clean bench, with 75% ethanol sterilizing 30s, sterilized water rinses 1 time；
0.1% mercuric chloride sterilizing 10min, sterilized water cleaning down 5 times.By planting seed in the MS culture medium of blank (without any hormone)
Middle cultivation, cultivation temperature 25 ± 2 DEG C, intensity of illumination 2000lx, light application time 12h.Observing seed germination situation, germination rate reaches
98%.
Step 3: tissue culture's material obtains.After 7d, find that seed starts to sprout, stretch out plumular axis from cotyledon.Appoint Macroptilium
Leaf is unearthed, and about 10d appoints bean seed Seedling true leaf to unfold and come, it is thus achieved that complete aseptic seedling, using the true leaf of aseptic seedling as outer implant
Test.
Step 4: the induction of sorite and elongation.On aseptic operating platform, cut leaf as outer implant by sterile razor blade,
True leaf one is divided into four, is inoculated into bud inducement culture medium (MS+6-BA 3.0mg/L+2,4-D 0.03mg/L+IBA 0.015mg/L+
Sucrose 35g/L+ agar 5.5g/L；PH is 5.9) in evoking adventive bud, during inoculation, blade abaxial side is upward；After cultivating 30d, blade
On begin with bud point occur, outer implant is transferred to Elongation of adventitious bud culture medium (MS+6-BA 1.0mg/L+IBA 0.02mg/L+
Sucrose 35g/L+ agar 5.5g/L；PH is 5.8) in evoking adventive bud elongation.Condition of culture is temperature 25 ± 2 DEG C, intensity of illumination
2500lx, light application time 12h.Adventitious bud induction frequency can 79%.
Step 5: root culture.When observing Elongation of adventitious bud to 3～4cm, cut adventitious bud, be inoculated in root culture
Root induction in base (MS+NAA0.02mg/L+ sucrose 28g/L+ agar 5.5g/L, pH6.0), condition of culture is temperature 25 ± 2
DEG C, intensity of illumination 2500lx, light application time 15h.About 7d, has observed in root media that Article 1 root length goes out.Obtain after 5d
Obtain complete tissue cultured seedling.Rooting rate is up to 85%.
Embodiment 3
Step one: Seeds preprocess.Seed is placed in water-bath, soaks 10min with 88 DEG C of water-baths.Afterwards seed is tiled
Having on the culture dish of moistening filter paper, the moment keeps filter paper humidity.After seed places 24h in culture dish, select health and substantially
The seed expanded carries out next step test operation.
Step 2: seed disinfection sterilizing.On aseptic super-clean bench, with 75% ethanol sterilizing 25s, sterilized water rinses 1 time；
0.2% mercuric chloride sterilizing 5min, sterilized water cleaning down 5 times.By planting seed in the MS culture medium of blank (without any hormone)
Middle cultivation, cultivation temperature 25 ± 2 DEG C, intensity of illumination 2500lx, light application time 10h.Observing seed germination situation, germination rate reaches
95%.
Step 3: tissue culture's material obtains.After 7d, find that seed starts to sprout, stretch out plumular axis from cotyledon.Appoint Macroptilium
Leaf is unearthed, and about 10d appoints bean seed Seedling true leaf to unfold and come, it is thus achieved that complete aseptic seedling, using the true leaf of aseptic seedling as outer implant
Test.
Step 4: the induction of sorite and elongation.On aseptic operating platform, cut leaf as outer implant by sterile razor blade,
True leaf one is divided into four, is inoculated into bud inducement culture medium (MS+6-BA 2.0mg/L+2,4-D 0.02mg/L+IBA 0.02mg/L+
Sucrose 30g/L+ agar 4.5g/L；PH is 6.0) in evoking adventive bud, during inoculation, blade abaxial side is upward；After cultivating 30d, blade
On begin with bud point occur, outer implant is transferred to Elongation of adventitious bud culture medium (MS+6-BA1.6mg/L+IBA 0.03mg/L+
Sucrose 30g/L+ agar 4.5g/L；PH is 5.9) in evoking adventive bud elongation.Condition of culture is temperature 25 ± 2 DEG C, intensity of illumination
1500lx, light application time 14h.Adventitious bud induction frequency is up to 78%.
Step 5: root culture.When observing Elongation of adventitious bud to 3～4cm, cut adventitious bud, be inoculated in root culture
Root induction in base (MS+NAA 0.01mg/L+ sucrose 32g/L+ agar 5.2g/L, pH is 6.0), condition of culture be temperature 25 ±
2 DEG C, intensity of illumination 1500lx, light application time 12h.About 7d, has observed in root media that Article 1 root length goes out.Obtain after 5d
Obtain complete tissue cultured seedling.Rooting rate is up to 90%.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (8)
1. one kind is the highly efficient regeneration method of outer implant to appoint bean blade, it is characterised in that comprise the steps:
(1) induction of sorite and elongation: the blade choosing aseptic seedling is outer implant, outer implant is inoculated in bud inducement culture medium
Evoking adventive bud, during inoculation, blade abaxial side is upward；Cultivating and begin with the appearance of bud point to blade, it is indefinite outer implant to be transferred to
Evoking adventive bud elongation in bud elongation medium；
(2) root culture: when Elongation of adventitious bud to 3～4cm, cut adventitious bud, be inoculated in root induction in root media；
Bud inducement culture medium described in step (1) is MS+6-BA 2.0～4.0mg/L+2,4-D 0.01～0.03mg/L+
IBA 0.01～0.02mg/L+ sucrose 28～35g/L+ agar 4.5～5.5g/L；PH is 5.8～6.0；
Elongation of adventitious bud culture medium described in step (1) is MS+6-BA 0.5～1.6mg/L+IBA 0.01～0.03mg/L+
Sucrose 28～35g/L+ agar 4.5～5.5g/L；PH is 5.8～6.0.
Highly efficient regeneration method to appoint bean blade to be outer implant the most according to claim 1, it is characterised in that: step (1)
Described in the condition of culture of induction be temperature 25 ± 2 DEG C, intensity of illumination 1500～2500lx, light application time 12～15h.
Highly efficient regeneration method to appoint bean blade to be outer implant the most according to claim 1, it is characterised in that: step (2)
Described in the condition of culture of induction be temperature 25 ± 2 DEG C, intensity of illumination 1500～2500lx, light application time 12～15h.
Highly efficient regeneration method to appoint bean blade to be outer implant the most according to claim 1, it is characterised in that: step (2)
Described in root media be MS+NAA0.01～0.02mg/L+ sucrose 28～35g/L+ agar 4.5～5.5g/L；PH is
5.8～6.0.
Highly efficient regeneration method to appoint bean blade to be outer implant the most according to claim 1, it is characterised in that: step (1)
Described in the blade of aseptic seedling, be made by the steps and obtain:
A, the acquisition of aseptic seedling: by seed water-bath soak, after seed tiling have on the culture dish of moistening filter paper；Place 20～
After 25h, select health and the seed that expands carries out disinfection；After sterilization, planting seed is cultivated in blank MS culture medium；
B, appoint the acquisition of bean blade: seed culture is unfolded to aseptic seedling leaf and come, and cuts blade as outer implant.
Highly efficient regeneration method to appoint bean blade to be outer implant the most according to claim 5, it is characterised in that: in step A
The condition that described water-bath is soaked is that 85～90 DEG C of water-baths soak 8～12min.
Highly efficient regeneration method to appoint bean blade to be outer implant the most according to claim 5, it is characterised in that: in step A
The step of described sterilization is as follows: aseptically, with 75% ethanol sterilizing 25～30s, aseptic water washing 1 time, 0.1～
0.2% mercuric chloride sterilizing 5min～10min, sterilized water cleaning down 5 times.
Highly efficient regeneration method to appoint bean blade to be outer implant the most according to claim 5, it is characterised in that: in step A
The condition of described cultivation is temperature 25 ± 2 DEG C, intensity of illumination 1500～2500lx, light application time 10～15h.
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| CN201610662934.0A CN106305416B (en) | 2016-08-12 | 2016-08-12 | It is a kind of using appoint beans blade as the highly efficient regeneration method of explant |
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| CN201610662934.0A CN106305416B (en) | 2016-08-12 | 2016-08-12 | It is a kind of using appoint beans blade as the highly efficient regeneration method of explant |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102823414A (en) * | 2012-09-18 | 2012-12-19 | 广西壮族自治区林业科学研究院 | Non-tube cutting seedling method of tissue subcultured buds of zenia |
| CN103392491A (en) * | 2013-08-14 | 2013-11-20 | 广西壮族自治区林业科学研究院 | Method for cutting seedling of Zenia insignis through adopting quarter sawing process |
| CN104145793A (en) * | 2014-08-08 | 2014-11-19 | 广西壮族自治区林业科学研究院 | Root-cutting seedling method of zenia insignis |
-
2016
- 2016-08-12 CN CN201610662934.0A patent/CN106305416B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102823414A (en) * | 2012-09-18 | 2012-12-19 | 广西壮族自治区林业科学研究院 | Non-tube cutting seedling method of tissue subcultured buds of zenia |
| CN103392491A (en) * | 2013-08-14 | 2013-11-20 | 广西壮族自治区林业科学研究院 | Method for cutting seedling of Zenia insignis through adopting quarter sawing process |
| CN104145793A (en) * | 2014-08-08 | 2014-11-19 | 广西壮族自治区林业科学研究院 | Root-cutting seedling method of zenia insignis |
Non-Patent Citations (2)
| Title |
|---|
| 张天宇、姚湘明: "翅荚木愈伤组织的诱导研究", 《林业勘察设计(福建)》 * |
| 邱运亮: "翅荚木试管苗快速繁殖技术的研究", 《林业科技开发》 * |
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