CN105900845A - Somatic embryo rapid propagation seedling raising method for camellia chekiangoleosa - Google Patents
Somatic embryo rapid propagation seedling raising method for camellia chekiangoleosa 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 invention discloses a rapid propagation and seedling raising method for somatic embryos of camellia chekiangoleosa, which comprises the steps of 1) explant pretreatment, 2) sterilization of camellia chekiangoleosa fruits and establishment of an aseptic system, 3) inoculation and callus induction, 4) somatic embryo induction, 5) secondary embryo induction and 6) secondary embryo differentiation into strong seedlings. The quick somatic embryo propagation seedling raising method for camellia chekiangkiang, provided by the invention, adopts N6+ NAA 1.0mg/L +6-BA 1.0mg/L +2, 4-D1.0 mg/L as a culture medium to induce embryogenic callus, and after inoculation and culture for about 7 days, cotyledon blocks on the culture medium begin to expand, and a large amount of yellowish, compact and compact embryogenic callus with smooth and glossy surface begins to appear around 15 days of cotyledon blocks. After the embryogenic callus is transferred to a somatic embryo induction culture medium, somatic embryos begin to appear about 20 days. After the proliferation and differentiation culture of secondary embryos, buds and roots are differentiated within about 15 days, and strong seedlings are finally formed. The invention establishes an efficient somatic embryogenesis system for the Zhejiang camellia oleifera and provides a large number of seedlings for the Zhejiang camellia oleifera.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to a kind of body embryo rapid seedling cultivation method of Camellia chekiangoleosa.
Background technology
Camellia chekiangoleosa, another name: C.chekiangoleosa.Formal name used at school:Camellia
chekiangoleosa.Theaceae, Camellia, is the distinctive seeds of China.Be distributed in Southern Zhejiang, east, Jiangxi, and Zhejiang, Fujian, San Sheng border, Anhui high mountain on.Camellia chekiangoleosa has to be viewed and admired and two kinds of purposes of oil.Plant forms is graceful, leaf color jade green, has beautiful very large Flos Carthami, beautiful in colour, fruit shape such as Fructus Mali pumilae, completely sets Fructus Pyracanthae summer and autumn, cold by force, for the Parents of Flos Camelliae Japonicae resistance breeding.Its tea core oil yield of these seeds is high, and oil is good, and Oleum Camelliae quality and oil yield are superior to C. olelfera, is a kind of well edible oil materials source, has good economic outlook, and market development potential is huge.Camellia chekiangoleosa resource distribution is in wild or semi-wild state mostly, and it is serious, close in imminent danger that natural habitat is destroyed by anthropic factor.
The natural propagation rate of oil tea is low, and tissue culture rapid propagating technology therefore can be used to solve, and at present both at home and abroad the most not yet Camellia chekiangoleosa is correlated with tissue culture technology report.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, it is an object of the invention to provide a kind of body embryo rapid seedling cultivation method of Camellia chekiangoleosa, setting up out efficient Camellia chekiangoleosa body embryo generation system, the short time can be that Camellia chekiangoleosa provides a large amount of seedling.
Technical scheme: in order to realize foregoing invention purpose, the technical solution used in the present invention is:
A kind of body embryo rapid seedling cultivation method of Camellia chekiangoleosa, step is as follows:
1) using Camellia chekiangoleosa seed as material, decapsidate, standby after sterilizing；
2) go to have eliminated the plumule of the seed of bacterium, plumular axis, radicle, leave behind cotyledon, cotyledon tangent plane is attached to embryonic callus induction media surface and inoculates；Temperature and humidity control, first carries out light culture, afterwards illumination cultivation, until inducing callus；Embryonic callus induction culture medium prescription is: N6+
1.0mg/L NAA+1.0-2.0 mg/L 6-BA+1.0 mg/L 2,4-D；
3) embryo callus is taken, stripping and slicing, it is inoculated in body embryonal induction culture medium, carries out body embryonal induction cultivation；Body embryonal induction culture medium prescription is: N6+
1.0mg/L NAA+2.0-3.0 mg/L 6-BA；
4) method using step 3), carries out the induction of Secondary embryos on Secondary embryos inducing culture；Secondary embryos inducing culture based formulas: N6+
0.5mg/L NAA+1.0-2.0mg/L ZT+0-
0.001mg/L TDZ；
5) being transferred to Secondary embryos in N6 culture medium cultivate, after 10d, differentiation is sprouted and root, ultimately forms strong sprout.
In step 1), select the seed of Wuyi Mountain, Fujian Province Camellia chekiangoleosa as material, carefully remove seed hull, it is to avoid seed is caused damage；With clear water, seed is soaked 1.5h；Afterwards, seed coat is removed；It is carried out with detergent, and flowing water rinses 2h.
In step 1), by superclean bench by the alcohol wipe of 75%, open uviol lamp sterilizing 20min；The seed tweezers rinsed by flowing water load in aseptic bottle, pour the ethanol of 75% into, make seed be completely immersed in ethanol, stir sterilizing 30S, pour out ethanol aseptic water washing 1 time；Pour 0.1% mercuric chloride agitation sterilizing into, stir sterilizing 8min, pour out mercuric chloride, with aseptic water washing 4-5 time.
Step 2) in, the culture bottle inoculated being placed in artificial climate constant incubator, first carries out light culture 3d, afterwards illumination 24h/d, intensity of illumination 2000lx, temperature controls at 25 DEG C, and relative humidity is maintained at 40-50%.
Step 2) in, through the cultivation of 7d, the cotyledon piece being inoculated in culture medium starts to expand, and the periphery of 15d cotyledon piece starts substantial amounts of embryo callus occur.
Step 2) in, embryonic callus induction culture medium prescription: N6+
1.0mg/L NAA+1.0 mg/L 6-BA+1.0 mg/L 2,4-D。
Step 2) in, embryonic callus induction culture medium prescription: N6+
1.0mg/L NAA+2.0 mg/L 6-BA+1.0 mg/L 2,4-D。
In step 3), body embryonal induction culture medium prescription: N6+
1.0mg/L NAA+3.0 mg/L 6-BA。
In step 4), Secondary embryos inducing culture based formulas: N6+ 0.5mg/L NAA+1.0mg/L ZT+ 0.001mg/L TDZ.
In step 4), Secondary embryos inducing culture based formulas: N6+
0.5mg/L NAA+1.0mg/L ZT。
Beneficial effect: compared with prior art, the body embryo rapid seedling cultivation method of the Camellia chekiangoleosa of the present invention, with N6+NAA 1.0mg/L+6-BA 1.0mg/L+2,4-D 1.0mg/L is culture medium induced embryonic callus, after inoculation, cultivating through about 7d, the cotyledon piece in culture medium starts to expand, and the periphery of about 15d cotyledon piece starts consolidation the most faint yellow, fine and close, the glossiness embryo callus of smooth surface occur.After being transferred to by embryo callus in body embryonal induction culture medium, about 20d begins to somatic embryo occur.Through propagation and the differentiation culture of Secondary embryos, about 15d differentiation is sprouted and root, ultimately forms strong sprout.The present invention sets up out efficient Camellia chekiangoleosa body embryo generation system, and the short time can be that Camellia chekiangoleosa provides a large amount of seedling, has good practicality.
Accompanying drawing explanation
Fig. 1 is 1 week figure after Camellia chekiangoleosa outer implant inoculation；
Fig. 2 is the callus agglomerate figure that Camellia chekiangoleosa contains each stage of development
Fig. 3 is the Secondary embryos figure of Camellia chekiangoleosa；
Fig. 4 is Secondary embryos seedling differentiation figure.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further.
Embodiment 1
A kind of body embryo rapid seedling cultivation method of Camellia chekiangoleosa, step is as follows:
1) outer implant pretreatment
Select the seed that Wuyi Mountain, Fujian Province Camellia chekiangoleosa is gathered in the crops in October, 2014 as material, carefully remove seed hull, it is to avoid seed is caused damage.With clear water, seed is soaked 1.5h.Afterwards, seed coat is removed.It is carried out with detergent, and flowing water rinses 2h.
2) sterilizing of safflower oil tea fruit and the foundation of sterile system
By superclean bench by the alcohol wipe of 75%, open uviol lamp sterilizing 20min.The seed tweezers rinsed by flowing water load in aseptic bottle, pour the ethanol of 75% into, make seed be completely immersed in ethanol, stir sterilizing 30S, pour out ethanol aseptic water washing 1 time.Pour 0.1% mercuric chloride agitation sterilizing into, stir sterilizing 8min, pour out mercuric chloride, with aseptic water washing 4-5 time.
3) inoculation and the induction of callus
The seed of bacterium of having gone out is placed in sterile petri dish by grain, prolongs seed cotyledons seam vertical direction with sterile scalpel and be cut in half, be carefully removed plumule, plumular axis, radicle, leave behind cotyledon, tangent plane is attached to embryonic callus induction media surface and inoculates.Being placed in artificial climate constant incubator by the culture bottle inoculated, first carry out light culture 3d, afterwards illumination 24h/d, intensity of illumination 2000lx, temperature controls at 25 DEG C, and relative humidity is maintained at 40-50%.
Through the cultivation of about 7d, as it is shown in figure 1, the cotyledon piece being inoculated in culture medium starts to expand, the periphery of about 15d cotyledon piece starts substantial amounts of embryo callus occur.These embryo callus grow fine, and present consolidation faint yellow, fine and close, smooth surface is glossy, amount is many state.
Using N6 culture medium as minimal medium, configure the embryonic callus induction culture medium of different formulations, carry out organizing parallel contrast more and cultivate, find that different culture media is very big on inductivity impact, specific as follows:
N6+
1.0mg/L NAA+1.0 mg/L 6-BA+1.0 mg/L 2,4-D, inductivity 68.67%；
N6+
1.0mg/L NAA+2.0 mg/L 6-BA+1.0 mg/L 2,4-D, inductivity 57.65%；
N6+
1.0mg/L NAA+2.0 mg/L 6-BA+0.5mg/L 2,4-D, inductivity 37%；
N6+
1.0mg/L NAA+3.0 mg/L 6-BA+0.5 ~ 2.0 mg/L 2,4-D, inductivity ＜ 30%.
4) induction of somatic embryo
Open culture dish, clamp embryo callus with tweezers gently, and (Flos Carthami oil tea embryo callus has the habit grown of uniting, the size of stripping and slicing to be controlled well to be cut into some pieces with cutter, to ensure embryo callus energy continued growth), build culture dish.With tweezers, the embryo callus cut is put in the body embryonal induction culture medium being cooled to solid, the culture bottle inoculated is placed in artificial climate constant incubator and cultivates, illumination 24h/d, intensity of illumination 2000lx, temperature controls at 25 DEG C, and relative humidity is maintained at 40-50%.
Embryo callus is transferred in body embryonal induction culture medium, as shown in Figure 2, figure a, d are embryo callus, it is apparent that globular embryo, heart-shape embryo in figure b, figure e, f have been differentiated to form plumelet, it can be seen that the most sturdy root in figure c, g, h, i, about 20d starts somatic embryo occur.Further, it is observed that the somatic embryo development early forms of different development stage on same embryo callus, globular embryo, heart-shape embryo and cotyledon shape embryo etc. are included.
Using N6 culture medium as minimal medium, configure the body embryonal induction culture medium of different formulations, carry out organizing parallel contrast more and cultivate, find that different culture media is very big on inductivity impact, specific as follows:
N6+
0.5mg/L NAA+1.0-4.0 mg/L 6-BA, inductivity ＜ 10% or be 0；
N6+
1.5mg/L NAA+1.0-4.0 mg/L 6-BA, inductivity ＜ 30%；
N6+
1.0mg/L NAA+1.0 mg/L 6-BA, inductivity 5.08%；
N6+
1.0mg/L NAA+2.0 mg/L 6-BA, inductivity 35%；
N6+
1.0mg/L NAA+3.0 mg/L 6-BA, inductivity 53.33%；
N6+
1.0mg/L NAA+4.0 mg/L 6-BA, inductivity 19.6%.
5) induction of Secondary embryos
Use body embryonal induction method, Secondary embryos inducing culture carries out the induction of Secondary embryos.Through the cultivation of about 20d, as shown in Figure 3, it is thus achieved that substantial amounts of Secondary embryos, afford ample material for carrying out of subsequent experimental.
Using N6 culture medium as minimal medium, configure the Secondary embryos inducing culture of different formulations, carry out organizing parallel contrast more and cultivate, find that different culture media is very big on inductivity impact, specific as follows:
N6+
0.5mg/L NAA+1.0mg/L ZT+
0.001mg/L TDZ, inductivity 80.82%；
N6+
0.5mg/L NAA+1.0mg/L ZT, inductivity 75.95%；
N6+
0.5mg/L NAA+2.0mg/L ZT+0-
0.001mg/L TDZ, inductivity ＜ 75%；
N6+
1.0mg/L NAA+1.0-2.0mg/L ZT+
0-0.001mg/L TDZ, inductivity ＜ 32%；
N6+
1.5mg/L NAA+1.0-2.0mg/L ZT+
0-0.001mg/L TDZ, inductivity ＜ 22%；
6) Secondary embryos is divided into strong sprout
With N6 culture medium for Secondary embryos division culture medium, additive method is with the induction of Secondary embryos.After Secondary embryos is transferred in N6 culture medium, as shown in Figure 4, little by little turing green, about 15d differentiation is sprouted and root, ultimately forms strong sprout.
Above all kinds of culture medium are both needed to add 100g/L bananas juice, 30g/L sucrose, 5.8g/L agar, regulate pH to 5.8-6.0.
Claims (10)
1. the body embryo rapid seedling cultivation method of a Camellia chekiangoleosa, it is characterised in that step is as follows:
1) using Camellia chekiangoleosa seed as material, decapsidate, standby after sterilizing；
2) go to have eliminated the plumule of the seed of bacterium, plumular axis, radicle, leave behind cotyledon, cotyledon tangent plane is attached to embryonic callus induction media surface and inoculates；Temperature and humidity control, first carries out light culture, afterwards illumination cultivation, until inducing callus；Embryonic callus induction culture medium prescription is: N6+ 1.0mg/L NAA+1.0-2.0 mg/L 6-BA+1.0 mg/L 2,4-D；
3) embryo callus is taken, stripping and slicing, it is inoculated in body embryonal induction culture medium, carries out body embryonal induction cultivation；Body embryonal induction culture medium prescription is: N6+ 1.0mg/L NAA+2.0-3.0 mg/L 6-BA；
4) method using step 3), carries out the induction of Secondary embryos on Secondary embryos inducing culture；Secondary embryos inducing culture based formulas: N6+ 0.5mg/L NAA+1.0-2.0mg/L ZT+0-0.001mg/L TDZ；
5) being transferred to Secondary embryos in N6 culture medium cultivate, after 10d, differentiation is sprouted and root, ultimately forms strong sprout.
The body embryo rapid seedling cultivation method of Camellia chekiangoleosa the most according to claim 1, it is characterised in that in step 1), selects the seed of Wuyi Mountain, Fujian Province Camellia chekiangoleosa as material, carefully removes seed hull, it is to avoid seed is caused damage；With clear water, seed is soaked 1.5h；Afterwards, seed coat is removed；It is carried out with detergent, and flowing water rinses 2h.
The body embryo rapid seedling cultivation method of Camellia chekiangoleosa the most according to claim 1, it is characterised in that in step 1), by superclean bench by the alcohol wipe of 75%, opens uviol lamp sterilizing 20min；The seed tweezers rinsed by flowing water load in aseptic bottle, pour the ethanol of 75% into, make seed be completely immersed in ethanol, stir sterilizing 30S, pour out ethanol aseptic water washing 1 time；Pour 0.1% mercuric chloride agitation sterilizing into, stir sterilizing 8min, pour out mercuric chloride, with aseptic water washing 4-5 time.
The body embryo rapid seedling cultivation method of Camellia chekiangoleosa the most according to claim 1, it is characterized in that, step 2) in, the culture bottle inoculated is placed in artificial climate constant incubator, first carry out light culture 3d, afterwards illumination 24h/d, intensity of illumination 2000lx, temperature controls at 25 DEG C, and relative humidity is maintained at 40-50%.
The body embryo rapid seedling cultivation method of Camellia chekiangoleosa the most according to claim 1, it is characterised in that step 2) in, through the cultivation of 7d, the cotyledon piece being inoculated in culture medium starts to expand, and the periphery of 15d cotyledon piece starts substantial amounts of embryo callus occur.
The body embryo rapid seedling cultivation method of Camellia chekiangoleosa the most according to claim 1, it is characterised in that step 2) in, embryonic callus induction culture medium prescription: N6+ 1.0mg/L NAA+1.0
mg/L 6-BA+1.0 mg/L 2,4-D。
The body embryo rapid seedling cultivation method of Camellia chekiangoleosa the most according to claim 1, it is characterised in that step 2) in, embryonic callus induction culture medium prescription: N6+ 1.0mg/L NAA+2.0 mg/L 6-BA+1.0 mg/L 2,4-D.
The body embryo rapid seedling cultivation method of Camellia chekiangoleosa the most according to claim 1, it is characterised in that in step 3), body embryonal induction culture medium prescription: N6+
1.0mg/L NAA+3.0 mg/L 6-BA。
The body embryo rapid seedling cultivation method of Camellia chekiangoleosa the most according to claim 1, it is characterised in that in step 4), Secondary embryos inducing culture based formulas: N6+
0.5mg/L NAA+1.0mg/L ZT+ 0.001mg/L TDZ。
The body embryo rapid seedling cultivation method of Camellia chekiangoleosa the most according to claim 1, it is characterised in that in step 4), Secondary embryos inducing culture based formulas: N6+
0.5mg/L NAA+1.0mg/L ZT。
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| CN107743870A (en) * | 2017-11-22 | 2018-03-02 | 中南林业科技大学 | The method that a kind of oil tea half is dehydrated the sterile regeneration plant of embryo |
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| CN111492973A (en) * | 2020-05-12 | 2020-08-07 | 黄冈师范学院 | Method for obtaining regeneration plants from common camellia oleifera through somatic embryogenesis |
| CN111771720A (en) * | 2020-07-16 | 2020-10-16 | 湖南大学 | Efficient white flower oil tea Jiangxiang non-2 somatic embryo induction method |
| CN111903525A (en) * | 2020-08-17 | 2020-11-10 | 重庆福林农业生物技术研究院有限公司 | Induction method of embryogenic callus of camellia oleifera abel with large fruit |
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| CN114027189A (en) * | 2021-11-03 | 2022-02-11 | 黄冈师范学院 | Method for obtaining regeneration plants from mature embryos of common camellia oleifera through somatic embryogenesis |
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| CN107743870A (en) * | 2017-11-22 | 2018-03-02 | 中南林业科技大学 | The method that a kind of oil tea half is dehydrated the sterile regeneration plant of embryo |
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| CN110604058A (en) * | 2019-10-17 | 2019-12-24 | 中国林业科学研究院亚热带林业研究所 | Tissue culture seedling raising method for camellia chekiangoleosa immature embryos |
| CN110604058B (en) * | 2019-10-17 | 2021-04-06 | 中国林业科学研究院亚热带林业研究所 | Tissue culture seedling raising method for camellia chekiangoleosa immature embryos |
| CN111492973A (en) * | 2020-05-12 | 2020-08-07 | 黄冈师范学院 | Method for obtaining regeneration plants from common camellia oleifera through somatic embryogenesis |
| CN111771720A (en) * | 2020-07-16 | 2020-10-16 | 湖南大学 | Efficient white flower oil tea Jiangxiang non-2 somatic embryo induction method |
| CN111771720B (en) * | 2020-07-16 | 2022-08-19 | 湖南大学 | Efficient induction method for Jiangxi non-2 somatic embryos of camellia oleifera |
| CN111903525A (en) * | 2020-08-17 | 2020-11-10 | 重庆福林农业生物技术研究院有限公司 | Induction method of embryogenic callus of camellia oleifera abel with large fruit |
| CN113331059A (en) * | 2021-07-21 | 2021-09-03 | 贵州大学 | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants |
| CN114027189A (en) * | 2021-11-03 | 2022-02-11 | 黄冈师范学院 | Method for obtaining regeneration plants from mature embryos of common camellia oleifera through somatic embryogenesis |
| CN114027189B (en) * | 2021-11-03 | 2022-11-18 | 黄冈师范学院 | Method for obtaining regeneration plants from mature embryos of common camellia oleifera through somatic embryogenesis |
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