CN104782502B - Method for rapidly obtaining regenerated plants of fiber hemps - Google Patents
Method for rapidly obtaining regenerated plants of fiber hemps Download PDFInfo
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- CN104782502B CN104782502B CN201510238469.3A CN201510238469A CN104782502B CN 104782502 B CN104782502 B CN 104782502B CN 201510238469 A CN201510238469 A CN 201510238469A CN 104782502 B CN104782502 B CN 104782502B
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Abstract
The invention provides a method for rapidly obtaining regenerated plants of fiber hemps. The method comprises the following steps: (1) carrying out sterile treatment on seeds of the fiber hemps and sowing seeds to obtain sterile seedlings; (2) tearing cotyledons from the sterile seedlings with the seedling ages of 2-3 days; cutting off leaf tips and putting into a bud induction culture medium containing thidiazuron and alpha-naphthylacetic acid to be cultured; (3) after culturing by the step (2) for 18-24 days, cutting off adventitious buds when the adventitious buds stretch to be 1cm-2cm high; transferring the adventitious buds into a rooting culture medium containing indolebutyric acid to be cultured and rooted, wherein the culture time is 35-42 days; and obtaining rooted seedlings. With the adoption of the method, explants are convenient and rapid to take, the period is short, the induction regeneration rate is relatively high and cotyledon explants can be obtained by only 3 days; and strong regenerated cluster buds are obtained by only 18-24 days; and seeds can be sowed at any time to obtain the explants, and the process is not limited by factors including material taking seasons, a disinfection process and the like, so that the method is suitable for hemp genetic transformation researches.
Description
Technical field
The present invention relates to a kind of method quickly obtaining fine Fructus Cannabiss regeneration plant.
Background technology
Fructus Cannabiss (cannabis sativaL.) it is Cannabaceae (cannabinaceace) Cannabis (cannabis) 1 year
Raw herbaceous plant, has the plant utilization history of thousands of years in China.Fructus Cannabiss are precious from head to foot, can produce the textile fabric of high-quality,
Also huge applications are had to be worth at aspects such as food, medicinal and building materials industries.Fructus Cannabiss are divided into according to its tetrahydrocannabinol (thc) content
Marijuana hemp, osculant hemp and fibre use three kinds of Fructus Cannabiss, and China is that fibre plants big country with Fructus Cannabiss, and cultivated area accounts for the 30% of the world
Left and right.
Due to last century, much country forbids planting all Fructus Cannabiss, leads to many preciousness germ plasm resources to be lost, to Fructus Cannabiss
Research also lags far behind other industrial crops.And, Fructus Cannabiss are dioecious Cross Pollinateds, germplasm heterozygosity
High.Carry out breed breeding using conventional line breeding method and Improvement advance is slow.Technique for gene engineering is in plant breeding
Commonly used method, but the transgenic plant of objective trait improvement will be obtained, setting up an efficient regenerating system is premise.
In the research of Fructus Cannabiss regenerating system, the selection of explant is particularly significant.Prior art is largely determined by explant
Select, the explant currently used for Fructus Cannabiss regeneration has stem apex, stem-segment with node, hypocotyls, a blade, these materials draw materials the cycle and
Convenience is restricted.The stem apex of the seedling plucked from land for growing field crops, stem section etc., as explant, by the season of growth and will be disappeared
The loaded down with trivial details restriction of malicious program (referring to Yin Pinxun, Yang Ming, Guo Hongyan, Liu Zhenbo, Hu Xueli, Chen Yu, the isolated culture of Fructus Cannabiss with fast
Speed breeding [j]. southwest agricultural journal. 2005 (04)).Sow the aseptic seedling producing from (not removing kind of a skin) after seed disinfection
Stem apex, hypocotyls, stem-segment with node, as explant, are grown up to suitably drawing materials period from being seeded into aseptic seedling, are needed 15-20 days,
Impact experimental period compactness (referring to Zhang Liguo, Song Xianyou, Fang Yuyan, Zheng Nan, Wu Guangwen. Fructus Cannabiss new varieties dragon Fructus Cannabiss one
Number regenerating system pre-test [j]. China's fiber crops industry science. 2012 (03)).The Chinese patent Shen of Application No. 200610088353.7
Please, it is inoculated in culture medium by after seed disinfection, stem apex explant after 20 days, could be obtained, after 30 days, obtain lateral bud, cultivation cycle
Longer.
Content of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, provides a kind of cycle shorter, easy to operate
The method quickly obtaining fine Fructus Cannabiss regeneration plant.
The present invention solves its technical problem and employed technical scheme comprise that, a kind of side quickly obtaining fine Fructus Cannabiss regeneration plant
Method, comprises the steps:
(1) fibre cannabis seeds are obtained aseptic seedling through aseptic process, sowing；
Referring to Cheng Chaohua, Li Yujun, Zhao Lining, Tang dragonfly, a surname consolidates, and triple factures obtain cannabis seeds aseptic seedling research
[j]. China's fiber crops industry science. 2011 (01)；
(2) on superclean bench, cotyledon is torn from the aseptic seedling of 2 days seedling ages or 3 days seedling ages (should not cut, no
Regeneration frequency then can be affected), cut blade tip, be just placed on the bud inducement cultivation base containing Thidiazuron (tdz) and α-naphthaleneacetic acid (naa)
Turn out adventitious bud；Described bud inducement cultivation base is ms basal medium, and Thidiazuron concentration is 0.1-0.2mg/l, α-naphthaleneacetic acid
Concentration is 0.01-0.05mg/l, additional saccharose 30g/l, agar (6-7) g/l, ph5.5-6.0, intensity of illumination 2200-
2600lx, light application time 14-16h/d, 24 ± 2 DEG C of temperature, incubation time is 18-24 days；
(3), after cultivating 18-24 days through step (2), when Elongation of adventitious bud is to 1-2 centimetre high, adventitious bud is cut, goes to
On root media containing indolebutyric acid (iba), culture is taken root, and described root media is that ms nutrient substance subtracts for 1/2ms(
Half) basal medium, wherein indolebutyric acid (iba) concentration are 0.3-0.5mg/l, additional saccharose 30g/l, agar (6-7) g/l,
Ph5.5-6.0, intensity of illumination 2200-2600lx, light application time 14-16h/d, 24 ± 2 DEG C of temperature；Incubation time is 35-42 days,
Obtain seedling of taking root.
Obtained seedling implanting and cultivating according to a conventional method of taking root.
Further, in step (2), described go ace's leaf be preferably 1/2 cotyledon.
Indication " Fructus Cannabiss " of the present invention, is fibre and uses Fructus Cannabiss, its tetrahydrocannabinol (thc) mass content < 0.3%.
The method of the present invention particularly suitable fibre Hemp Varieties, regeneration plant inductivity is 46%-48%.
The sterilization based on seed for the present invention, peeling are processed, and first time proposition cotyledon sets up regenerating system as explant,
Can convenient, be quickly obtained regeneration plant.The aseptic seedling cotyledon that 2 day seedling ages or 3 day seedling age are used as explant, outside these
Implant is aseptic condition, and growth potential is homogeneous, can obtain a large amount of regeneration buds in 28 days, reduces 7- experimental period than original method
14 days, this can provide very big facility for the genetic transformation work in future.
The present invention adopts the cotyledon of 2 days seedling ages or 3 days seedling age aseptic seedling as explant, obtains cotyledon and only needs from being seeded into
Take 3 day time, regeneration of cotyledons frequency reaches 46%-48%, can quickly and conveniently obtain regeneration plant.
Using the present invention, explant is drawn materials convenient, fast, obtains cotyledon explant it is only necessary to 3 days, obtain healthy and strong again
Raw clump bud it is only necessary to 18-24 days, cycle is short, and can sow at any time, obtain explant, season of not drawn materials, disinfecting process
Etc. the restriction of factor, it is highly suitable for Study on Genetic Transformation.
Brief description
Fig. 1 is the Fructus Cannabiss aseptic seedling figure of 3 days seedling ages of the embodiment of the present invention 1 gained；
Fig. 2 is the 1/2 cotyledon figure that the embodiment of the present invention 1 is used as explant；
Fig. 3 is the Multiple Buds figure that the embodiment of the present invention 1 cotyledon base portion grows；
Fig. 4 is the embodiment of the present invention 1 Multiple Buds stretch scheme；
Fig. 5 is that the embodiment of the present invention 1 regeneration plant is taken root figure；
Fig. 6 is that the embodiment of the present invention 1 Transplantation of Regenerated Plantlets survives figure.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1
The method quickly obtaining fine Fructus Cannabiss regeneration plant of the present embodiment, comprises the steps:
(1) fibre cannabis seeds are obtained aseptic seedling through aseptic process, sowing；
Referring to Cheng Chaohua, Li Yujun, Zhao Lining, Tang dragonfly, a surname consolidates, and triple factures obtain cannabis seeds aseptic seedling research
[j]. China's fiber crops industry science. 2011 (01)；
(2) on superclean bench, cotyledon is torn from the aseptic seedling of 3 days seedling ages and (should not cut, otherwise can affect again
Raw frequency), cut blade tip, leave 1/2 cotyledon, be just placed in the bud inducement cultivation base containing Thidiazuron (tdz) and α-naphthaleneacetic acid (naa)
On turn out adventitious bud；Described bud inducement cultivation base is ms basal medium, and Thidiazuron concentration is 0.2mg/l, and α-naphthaleneacetic acid is dense
Spend for 0.05mg/l, additional saccharose 30g/l, agar 6.5g/l, ph5.8, intensity of illumination 2300lx, light application time 16h/d, temperature
24 ± 2 DEG C of degree, incubation time is 20 days；
(3), after cultivating 20 days through step (2), when Elongation of adventitious bud is to 1-2 centimetre high, adventitious bud is cut, goes to and contain
Culture on the root media of indolebutyric acid (iba) is had to take root, described root media is that ms nutrient substance halves for 1/2ms()
Basal medium, wherein indolebutyric acid (iba) concentration is 0.5mg/l, additional saccharose 30g/l, agar 6.5g/l, ph5.8, illumination
Intensity 2300lx, light application time 16h/d, 24 ± 2 DEG C of temperature；Incubation time is 35 days, obtains seedling of taking root.
Obtained seedling implanting and cultivating according to a conventional method of taking root.Fig. 1 is the big of 3 days seedling ages of the embodiment of the present invention 1 gained
Numb aseptic seedling figure；Fig. 2 is the 1/2 cotyledon figure that the embodiment of the present invention 1 is used as explant；Fig. 3 is the embodiment of the present invention 1 cotyledon base portion
Grow Multiple Buds figure；Fig. 4 is the embodiment of the present invention 1 Multiple Buds stretch scheme；Fig. 5 is that the embodiment of the present invention 1 regeneration plant is taken root figure；
Fig. 6 is that the embodiment of the present invention 1 Transplantation of Regenerated Plantlets survives figure.Illustrate to adopt the present invention, can smoothly obtain explant, produce
Regeneration plant, and rooting and transplant survives.
In the present embodiment, explant is drawn materials convenient, fast, obtains cotyledon explant it is only necessary to 3 days, obtain healthy and strong again
Raw clump bud it is only necessary to 20 days, cycle is short, and can sow at any time, obtain explant, season of not drawn materials, disinfecting process etc.
The restriction of factor, is highly suitable for Study on Genetic Transformation.
Embodiment 2
Choose 2 days, 4 days, 5 days, the aseptic seedling of 6 days seedling ages respectively, cotyledon torn and is processed, other process steps with
Embodiment 1.Regeneration frequency using 2-6 days different seedling age explants the results are shown in Table in 1.It can be seen that adopting 2 days, seedling age explant in 3 days
Body is more suitable for.
The regeneration frequency of the different seedling age explant of table 1
| Cotyledon seedling age (my god) | Regeneration frequency (%) |
| 2 | 47.8±5.1 |
| 3 | 46.7±5.7 |
| 4 | 36.7±4.9 |
| 5 | 22.2±3.5 |
| 6 | 11.9±3.7 |
Reference examples
Choose the aseptic seedling of two groups of 3 days seedling ages respectively, respectively cotyledon torn, cut process, other process steps are same
Embodiment 1.Be the results are shown in Table in 2 using the method for tearing and the regeneration frequency cutting method gained explant.It can be seen that using tearing more
It is suitable for.
The 3d seedling age explant regeneration frequency of table 2 different biopsy method method
| Method of drawing material | Regeneration frequency (%) |
| Tear | 47.3±4.8 |
| Cut | 6.2±3.7 |
Claims (2)
1. a kind of method quickly obtaining fine Fructus Cannabiss regeneration plant is it is characterised in that comprise the steps:
(1) fibre cannabis seeds are obtained aseptic seedling through aseptic process, sowing；
(2) on superclean bench, cotyledon is torn from the aseptic seedling of 2 days seedling ages or 3 days seedling ages, cuts blade tip, be just placed in
Adventitious bud is turned out on bud inducement cultivation base containing Thidiazuron and α-naphthaleneacetic acid；Described bud inducement cultivation base is the culture of ms basis
Base, Thidiazuron concentration is 0.1-0.2mg/l, and α-naphthaleneacetic acid concentration is 0.01-0.05mg/l, additional saccharose 30g/l, agar 6-
7g/l, ph5.5-6.0, intensity of illumination 2200-2600lx, light application time 14-16h/d, 24 ± 2 DEG C of temperature, incubation time is 18-
24 days；
(3) through step (2) cultivate 18-24 days after, when Elongation of adventitious bud is to 1-2 centimetre high, adventitious bud is cut, go to containing
On the root media of indolebutyric acid, culture is taken root, and described root media is 1/2ms basal medium, and wherein indolebutyric acid is dense
Spend for 0.3-0.5mg/l, additional saccharose 30g/l, agar 6-7g/l, ph5.5-6.0, intensity of illumination 2200-2600lx, during illumination
Between 14-16h/d, 24 ± 2 DEG C of temperature；Incubation time is 35-42 days, obtains seedling of taking root.
2. the method quickly obtaining fine Fructus Cannabiss regeneration plant according to claim 1 is it is characterised in that in step (2),
The described blade tip that cuts is to cut 1/2 cotyledon.
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| CN106605595B (en) * | 2016-12-12 | 2018-12-28 | 罗永志 | It is cultivated fine seed strains the method for fiery fiber crops by stem apex |
| CN107630034A (en) * | 2017-11-16 | 2018-01-26 | 中国农业科学院麻类研究所 | A kind of agriculture bacillus mediated hemp transgenic method |
| CN107912301B (en) * | 2017-11-29 | 2021-04-13 | 百色学院 | Method for inducing callus by using cannabis sativa seeds |
| CN108401902B (en) * | 2018-03-07 | 2021-05-25 | 黑龙江省科学院大庆分院 | Rapid propagation method for tissue culture of hemp stem tip |
| CN110419397A (en) * | 2019-09-06 | 2019-11-08 | 黑龙江汉美生工业大麻科技有限公司 | A kind of Cultivate administration method improving No. 5 CBD contents of imperial hemp |
| CN112602597A (en) * | 2020-12-31 | 2021-04-06 | 济南浩隆生物科技有限公司 | Method for obtaining industrial hemp full-female or full-male seedlings |
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| CN102934610A (en) * | 2012-10-23 | 2013-02-20 | 中国农业科学院油料作物研究所 | Breeding maintaining method of ricinus communist female plant |
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