CN103960130A - Adventitious bud induction method for Juglans mandshurica Maxim. - Google Patents
Adventitious bud induction method for Juglans mandshurica Maxim. Download PDFInfo
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- CN103960130A CN103960130A CN201410195477.XA CN201410195477A CN103960130A CN 103960130 A CN103960130 A CN 103960130A CN 201410195477 A CN201410195477 A CN 201410195477A CN 103960130 A CN103960130 A CN 103960130A
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- juglans mandshurica
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- zygotic embryo
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Abstract
The invention relates to an adventitious bud induction method for Juglans mandshurica Maxim.. The method comprises the following steps: (1) selecting an explant; (2) disinfecting a zygotic embryo; (3) pre-culturing the zygotic embryo; (4) selecting an adventitious bud explant; and (5) inducing and differentiating. The method is simple, convenient, rapid and effective, a large batch of tissue culture seedlings can be bred in a short time, a foundation can be laid for future culture of excellent clone plants, endangered trees in a crisis state in China can be protected and bred effectively, and a good foundation is laid for future further protection and utilization of the trees.
Description
Technical field
The present invention relates to juglans mandshurica quick reproduction technique production technology in forestry biotechnology.
Background technology
At present, juglans mandshurica (Juglans mandshurica Max im.) is under the jurisdiction of Juglandaceae Juglans, one of Northeast Forest Areas three large hard wealthy seeds, its material hard and compact, texture is perfectly straight attractive in appearance, prematurity exocarp, root (branch) skin, shell and blade all can be used as medicine, and of many uses, economic worth is high.Due to long-term over-exploitation, cause its resource closely in imminent danger, can not meet the needs in the fields such as domestic and international market economic development and medical science far away.Under normal condition, juglans mandshurica seed belongs to dormancy, and reproduction speed is slow, and efficiency is low, and the cycle is long.
Cultivate about the tissue of juglans mandshurica, because just inoculation pollution rate is high, it is more difficult that explant is sprouted, and brownization of isolated culture, degenerates seriously, and growth coefficient is low etc., and problem fails to obtain fine solution, not extensive use on producing so far always.At present, as carrying out somatic embryo generation, explant successfully induces somatic embryo (Wang Yanqing etc. 2000 taking the immature embryo of juglans mandshurica; Zhang Jianying etc. 2010), have no whole plant.Carry out vegetative propagation (Zhou Yu etc. 2001, Zhang Jianying etc. 2011) with juglans mandshurica resting shoot, vegetative propagation effect does not become effective.
Summary of the invention
The object of the invention is to overcome the weak point existing in above-mentioned technology, provide a kind of by selecting suitable explant, again through plant growth regulator treatment, under suitable condition, cultivate, and inducing juglans mandshurica Multiple Buds, Multiple Buds is inoculated on inducing culture and cultivates and obtain a large amount of indefinite buds after plant division.
In order to achieve the above object, the technical solution used in the present invention is:
(1) selection of explant
Take every year ripe juglans mandshurica seed 8-10 month, through clear water washing, juglans mandshurica exocuticle is got rid of in airing in the sun, dry under laying temperature 20-25 DEG C condition, ventilation is for subsequent use, shell is smashed with hammer, get the zygotic embryo of a small amount of cotyledon of that end band of juglans mandshurica seed tip
(2) zygotic embryo sterilization
First infiltrate 30 seconds 2 times with 70% ethanol, then use 0.1% mercuric chloride HgCl
2solution disinfection 15 minutes, uses aseptic water washing 5-7 time, then in sterile water, soaks 2-4 hour, for subsequent use,
(3) zygotic embryo preculture
In sterile water, soak after 2-7 hour, juglans mandshurica zygotic embryo exocuticle after sterilization is removed, be inoculated in the MS medium that adds hormone heteroauxin IBA0.01mg/L and cultivate, sucrose 30g/L, agar 6g/L, cultivation temperature 24-26 DEG C, light application time 16h/d, intensity of illumination 20001x, cultivates 25-30 days
(4) selection of indefinite bud explant
Juglans mandshurica zygotic embryo base portion cotyledon after cultivating opens, develop into after the plantlet with different number axillalry buds, the cotyledon that juglans mandshurica plant base portion is existed gets rid of, by tweezers clamping plants stems section, juglans mandshurica plant is started to be cut into the respectively stem section with different axillalry buds of about 1.0-2.0cm from base portion by sterile scissors, as the explant of adventitious bud inducing differentiation
(5) Induction and differentiation
Explant is seeded in adventitious bud induction culture base, medium is additional hormone 6-benzyl aminoadenine (6-BA) 2.0mg/L of DKW medium and IBA0.01mg/L, adjective gland purine AD10mg/L simultaneously, sucrose 30g/L, agar 6g/L, cultivation temperature is 25-27 DEG C, light application time 16h/d, intensity of illumination 1000-2000l x, cultivate explant base portion incision edge after 7-8 days and occur green bud point, cultivate and within 15-20 days, obtain indefinite bud.
Advantage of the present invention is:
The present invention is simple and convenient, effective fast; can breed at short notice large quantities of group training seedlings; can carry out basis for cultivate choiceness plant later; effectively protect and breed the crisis state that national endangered species exists, for further protecting from now on and utilize these species to establish good basis.
The invention provides the method for juglans mandshurica evoking adventive bud, the method efficiency is high, and reproduction speed is fast, and average bud ratio is higher, and adventitious bud induction frequency reaches 90.6%, and propagation multiple is in 7.5 left and right.The present invention confirms to carry out the fast numerous of juglans mandshurica seedling and can pass through seed seedling-raising method and grafting, thereby has saved the time, has removed time restriction and reproductive efficiency improves greatly, can be fast for the construction of juglans mandshurica economic forest provides sufficient seedling; Meanwhile, the molecular improvement that this efficient asexual reproduction method is juglans mandshurica particularly transgenic technology provides good basis.
Embodiment
Below in conjunction with embodiments of the invention are described in further detail.
Embodiment 1,
(1) selection of explant
Take ripe juglans mandshurica seed annual August, through clear water washing, juglans mandshurica exocuticle is got rid of in airing in the sun, dry under 20 DEG C of conditions of laying temperature, ventilation is for subsequent use, shell is smashed with hammer, get the zygotic embryo of a small amount of cotyledon of that end band of juglans mandshurica seed tip
(2) zygotic embryo sterilization
First infiltrate 30 seconds 2 times with 70% ethanol, then use 0.1% mercuric chloride HgCl
2solution disinfection 15 minutes, uses aseptic water washing 5 times, then in sterile water, soaks 2 hours, for subsequent use,
(3) zygotic embryo preculture
In sterile water, soak after 2 hours, the juglans mandshurica zygotic embryo exocuticle after sterilization is removed, be inoculated in the MS medium that adds hormone heteroauxin IBA0.01mg/L and cultivate, sucrose 30g/L, agar 6g/L, 24 DEG C of cultivation temperature, light application time 16h/d, intensity of illumination 20001x, cultivates 25 days
(4) selection of indefinite bud explant
Juglans mandshurica zygotic embryo base portion cotyledon after cultivating opens, develop into after the plantlet with different number axillalry buds, the cotyledon that juglans mandshurica plant base portion is existed gets rid of, by tweezers clamping plants stems section, juglans mandshurica plant is started to be cut into the respectively stem section with different axillalry buds of about 1.0cm from base portion by sterile scissors, as the explant of adventitious bud inducing differentiation
(5) Induction and differentiation
Explant is seeded in adventitious bud induction culture base, medium is additional hormone 6-benzyl aminoadenine (6-BA) 2.0mg/L of DKW medium and IBA0.01mg/L, adjective gland purine AD10mg/L simultaneously, sucrose 30g/L, agar 6g/L, cultivation temperature is 25 DEG C, light application time 16h/d, intensity of illumination 10001x, cultivate explant base portion incision edge after 7-8 days and occur green bud point, cultivate and within 15 days, obtain induced bud
Embodiment 2,
(1) selection of explant
Take ripe juglans mandshurica seed annual September, through clear water washing, juglans mandshurica exocuticle is got rid of in airing in the sun, dry under 23 DEG C of conditions of laying temperature, ventilation is for subsequent use, shell is smashed with hammer, get the zygotic embryo of a small amount of cotyledon of that end band of juglans mandshurica seed tip
(2) zygotic embryo sterilization
First infiltrate 30 seconds 2 times with 70% ethanol, then use 0.1% mercuric chloride HgCl
2solution disinfection 15 minutes, uses aseptic water washing 6 times, then in sterile water, soaks 3 hours, for subsequent use,
(3) zygotic embryo preculture
In sterile water, soak after 4 hours, the juglans mandshurica zygotic embryo exocuticle after sterilization is removed, be inoculated in the MS medium that adds hormone heteroauxin IBA0.01mg/L and cultivate, sucrose 30g/L, agar 6g/L, 25 DEG C of cultivation temperature, light application time 16h/d, intensity of illumination 20001x, cultivates 27 days
(4) selection of indefinite bud explant
Juglans mandshurica zygotic embryo base portion cotyledon after cultivating opens, develop into after the plantlet with different number axillalry buds, the cotyledon that juglans mandshurica plant base portion is existed gets rid of, by tweezers clamping plants stems section, juglans mandshurica plant is started to be cut into the respectively stem section with different axillalry buds of about 1.5cm from base portion by sterile scissors, as the explant of adventitious bud inducing differentiation
(5) Induction and differentiation
Explant is seeded in adventitious bud induction culture base, medium is additional hormone 6-benzyl aminoadenine (6-BA) 2.0mg/L of DKW medium and IBA0.01mg/L, adjective gland purine AD10mg/L simultaneously, sucrose 30g/L, agar 6g/L, cultivation temperature is 26 DEG C, light application time 16h/d, intensity of illumination 15001x, cultivate explant base portion incision edge after 8 days and occur green bud point, cultivate and within 15-20 days, obtain induced bud.
Embodiment 3,
(1) selection of explant
Take ripe juglans mandshurica seed annual October, through clear water washing, juglans mandshurica exocuticle is got rid of in airing in the sun, dry under 25 DEG C of conditions of laying temperature, ventilation is for subsequent use, shell is smashed with hammer, get the zygotic embryo of a small amount of cotyledon of that end band of juglans mandshurica seed tip
(2) zygotic embryo sterilization
First infiltrate 30 seconds 2 times with 70% ethanol, then use 0.1% mercuric chloride HgCl
2solution disinfection 15 minutes, uses aseptic water washing 7 times, then in sterile water, soaks 4 hours, for subsequent use,
(3) zygotic embryo preculture
In sterile water, soak after 7 hours, the juglans mandshurica zygotic embryo exocuticle after sterilization is removed, be inoculated in the MS medium that adds hormone heteroauxin IBA0.01mg/L and cultivate, sucrose 30g/L, agar 6g/L, 26 DEG C of cultivation temperature, light application time 16h/d, intensity of illumination 20001x, cultivates 30 days
(4) selection of indefinite bud explant
Juglans mandshurica zygotic embryo base portion cotyledon after cultivating opens, develop into after the plantlet with different number axillalry buds, the cotyledon that juglans mandshurica plant base portion is existed gets rid of, by tweezers clamping plants stems section, juglans mandshurica plant is started to be cut into the respectively stem section with different axillalry buds of about 2.0cm from base portion by sterile scissors, as the explant of adventitious bud inducing differentiation
(5) Induction and differentiation
Explant is seeded in adventitious bud induction culture base, medium is additional hormone 6-benzyl aminoadenine (6-BA) 2.0mg/L of DKW medium and IBA0.01mg/L, adjective gland purine AD10mg/L simultaneously, sucrose 30g/L, agar 6g/L, cultivation temperature is 27 DEG C, light application time 16h/d, intensity of illumination 20001x, cultivate explant base portion incision edge after 8 days and occur green bud point, cultivate and within 20 days, obtain induced bud.
Claims (1)
1. a method for juglans mandshurica evoking adventive bud, is characterized in that:
(1) selection of explant
Take every year ripe juglans mandshurica seed 8-10 month, through clear water washing, juglans mandshurica exocuticle is got rid of in airing in the sun, dry under laying temperature 20-25 DEG C condition, ventilation is for subsequent use, shell is smashed with hammer, get the zygotic embryo of a small amount of cotyledon of that end band of juglans mandshurica seed tip
(2) zygotic embryo sterilization
First infiltrate 30 seconds 2 times with 70% ethanol, then use 0.1% mercuric chloride HgCl
2solution disinfection 15 minutes, uses aseptic water washing 5-7 time, then in sterile water, soaks 2-4 hour, for subsequent use,
(3) zygotic embryo preculture
In sterile water, soak after 2-7 hour, juglans mandshurica zygotic embryo exocuticle after sterilization is removed, be inoculated in the MS medium that adds hormone heteroauxin IBA0.01mg/L and cultivate, sucrose 30g/L, agar 6g/L, cultivation temperature 24-26 DEG C, light application time 16h/d, intensity of illumination 20001x, cultivates 25-30 days
(4) selection of indefinite bud explant
Juglans mandshurica zygotic embryo base portion cotyledon after cultivating opens, develop into after the plantlet with different number axillalry buds, the cotyledon that juglans mandshurica plant base portion is existed gets rid of, by tweezers clamping plants stems section, juglans mandshurica plant is started to be cut into the respectively stem section with different axillalry buds of about 1.0-2.0cm from base portion by sterile scissors, as the explant of adventitious bud inducing differentiation
(5) Induction and differentiation
Explant is seeded in adventitious bud induction culture base, medium is additional hormone 6-benzyl aminoadenine (6-BA) 2.0mg/L of DKW medium and IBA0.01mg/L, adjective gland purine AD10mg/L simultaneously, sucrose 30g/L, agar 6g/L, cultivation temperature is 25-27 DEG C, light application time 16h/d, intensity of illumination 1000-2000l x, cultivate explant base portion incision edge after 7-8 days and occur green bud point, cultivate 15-20 days acquisition induced bundles and sprout, Multiple Buds is inoculated on medium and obtains a large amount of indefinite buds after cultivation after plant division.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106472317A (en) * | 2016-10-19 | 2017-03-08 | 黑龙江省林业科学研究所 | The method of the isolated culture adventive bud evoked plant regeneration of Juglans mandshurica |
| CN109729977A (en) * | 2019-03-04 | 2019-05-10 | 沈阳农业大学 | A method of inhibiting explant body pollution brown stain during juglans mandshurica stem section culture |
| CN112273234A (en) * | 2020-11-10 | 2021-01-29 | 上海市农业科学院 | Method for transforming deformed seedlings into normal plants in peach embryo rescue process |
| CN113598059A (en) * | 2021-09-23 | 2021-11-05 | 沈阳农业大学 | Method for rapidly regenerating plants by inducing adventitious buds of mature embryos of juglans mandshurica maxim |
-
2014
- 2014-05-09 CN CN201410195477.XA patent/CN103960130A/en active Pending
Non-Patent Citations (3)
| Title |
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| 刘喜星等: ""绿岭"核桃种胚组培苗腋芽快繁体系的初步研究", 《农业科学》 * |
| 张建瑛等: "胡桃楸胚性愈伤组织诱导与体细胞胚胎发生", 《植物研究》 * |
| 樊靖等: "核桃离体培养和植株再生", 《园艺学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106472317A (en) * | 2016-10-19 | 2017-03-08 | 黑龙江省林业科学研究所 | The method of the isolated culture adventive bud evoked plant regeneration of Juglans mandshurica |
| CN106472317B (en) * | 2016-10-19 | 2018-09-25 | 黑龙江省林业科学研究所 | The method of the isolated culture adventive bud evoked plant regeneration of Juglans mandshurica |
| CN109729977A (en) * | 2019-03-04 | 2019-05-10 | 沈阳农业大学 | A method of inhibiting explant body pollution brown stain during juglans mandshurica stem section culture |
| CN109729977B (en) * | 2019-03-04 | 2022-09-06 | 沈阳农业大学 | Method for inhibiting browning of explant pollution in process of cultivating juglans mandshurica stem segments |
| CN112273234A (en) * | 2020-11-10 | 2021-01-29 | 上海市农业科学院 | Method for transforming deformed seedlings into normal plants in peach embryo rescue process |
| CN112273234B (en) * | 2020-11-10 | 2022-03-01 | 上海市农业科学院 | Method for transforming deformed seedlings into normal plants in peach embryo rescue process |
| CN113598059A (en) * | 2021-09-23 | 2021-11-05 | 沈阳农业大学 | Method for rapidly regenerating plants by inducing adventitious buds of mature embryos of juglans mandshurica maxim |
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Application publication date: 20140806 |