CN102972291A - Tissue culture and propagation method and inductive culture mediums for Chongzuo camellia nitidissima - Google Patents
Tissue culture and propagation method and inductive culture mediums for Chongzuo camellia nitidissima Download PDFInfo
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- CN102972291A CN102972291A CN2012104828341A CN201210482834A CN102972291A CN 102972291 A CN102972291 A CN 102972291A CN 2012104828341 A CN2012104828341 A CN 2012104828341A CN 201210482834 A CN201210482834 A CN 201210482834A CN 102972291 A CN102972291 A CN 102972291A
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Abstract
The invention discloses a tissue culture and propagation method and inductive culture mediums for Chongzuo camellia nitidissima. According to the method, the improved MS, 6-benayl aminopurine (6-BA) and 3-indoleacetic acid (IAA) are used as an inductive culture medium for the cluster buds of Chongzuo camellia nitidissima; and the combination of the two growth hormones can be used for activating acting enzymes in the body cells of Chongzuo camellia nitidissima, and promoting the germination and the growth of the explants of Chongzuo camellia nitidissima in a proper concentration and a weakly acidic environment; the thick and strong cluster buds can be formed after the culturing for 55 days, and the propagation rate is increased by 20 times. According to the method disclosed by the invention, 1/2 improved MS and indolebutyric acid (IBA) or 1/2 improved MS and napthaleneacetic acid (NAA) are further used as an inductive culture medium for rooting, and capable of effectively promoting the single seedlings of Chongzuo camellia nitidissima to root to form new plants. Via the tissue culture and propagation method applying the three inductive culture mediums for Chongzuo camellia nitidissima, Chongzuo camellia nitidissima can be rapidly and effectively propagated, thus solving the problems that wild resources are diminished gradually and the natural propagation rate of Chongzuo camellia nitidissima is low, and effectively protecting the precious plant genetic resource which is rare in the world.
Description
Technical field
The present invention relates to Plant Tissue Breeding breeding field, especially a kind of Chongzuo Camellia nitidissima tissue culture propagation method and inducing culture thereof.
Background technology
Chongzuo Camellia nitidissima (C.chungtsoensis S.Y.Liang et L.D.Huang), evergreen shrubs, it is the rare kind of nearest newfound Camellia nitidissima, after camellia azalea, second everblooming Camellia nitidissima kind finding in the world, it is the cadmium yellow of color in Camellia nitidissima family not only, and constantly blooms the whole year.The Chongzuo Camellia nitidissima only has a small amount of distribution at the local limestone low mountainous region of Chongzuo, Guangxi.This kind is approximate with the lemon yellow Camellia nitidissima, and difference is to spend large buff, petal 13-16 lobe, perpetual bloom.Chongzuo Camellia nitidissima flower is evenly distributed, and branch is dense, but normal growth under the full exposure condition has very high ornamental value, is having broad application prospects aspect family potted plant, landscaping and the Camellia Breeding.Narrow, the limited amount of the natural range of Chongzuo Camellia nitidissima, because branch is thin and delicate, the cuttage root-taking rate is low, the vegetative propagation difficulty is difficult to satisfy the landscape flower market demand.Up to now, though some are arranged about the report that the Camellia Section Chrysantha tissue is cultivated both at home and abroad, have no the group training research report about the Chongzuo Camellia nitidissima.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of Chongzuo Camellia nitidissima tissue culture propagation method, effectively to utilize existing resource Fast-propagation Chongzuo Camellia nitidissima, protects this in the world rare rare species germ plasm resource.
Another technical problem that the present invention will solve provides inducing clumping bud medium and the root induction medium for above-mentioned Chongzuo Camellia nitidissima tissue culture propagation method.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: Chongzuo Camellia nitidissima tissue culture propagation method, the explant of Chongzuo Camellia nitidissima is inoculated in the inducing clumping bud medium cultivates, when treating that Multiple Buds grows to 3cm, Multiple Buds is divided into Xiao Cong or single seedling is inoculated in the root induction medium and cultivates;
Above-mentioned inducing clumping bud medium contains the material of following concentration take modified MS medium as the basis:
6-benzyl aminopurine 2～4mg/L 3-indolyl acetic acid 0.6～0.8mg/L
Its pH value is 4.5～5.8;
Above-mentioned root induction medium contains the material of following concentration take 1/2 modified MS medium as the basis:
Indolebutyric acid 0.6～1.2mg/L or naa 0.6～1.2mg/L.
Explant is young tender seed.
The tender seed of children is first through cleaning and use 0.1%HgCl
2Solution disinfection is processed 20～30min, uses 0.2%HgCl again
2Solution disinfection is processed 15～20min.
Cultivate all at 25 ± 2 ℃ of temperature, light application time 12～14h.d
-1, intensity of illumination 2000lx condition under carry out.
Chongzuo Camellia nitidissima inducing clumping bud medium, take modified MS medium as the basis, contain the material of following concentration:
6-benzyl aminopurine 2～4mg/L 3-indolyl acetic acid 0.6～0.8mg/L.
The pH value of Chongzuo Camellia nitidissima inducing clumping bud medium is 4.5～5.8.
Above-mentioned modified MS medium contains composition: Ca (NO
3)
24H
2O 300mg/L; MgSO
47H
2O 370mg/L; KNO
31900mg/L; NH
4NO
31650mg/L; KH
2PO
4340mg/L; Na
2EDTA 37.3mg/L; FeSO
47H
2O 27.8mg/L; KI0.83mg/L; H
3BO
30.63mg/L; MnSO
44H
2O 22.3mg/L; ZnSO
47H
2O 8.6mg/L; Na
2Mo4H
2O 0.025mg/L; CuSO
45H
2O 0.025mg/L; CoCl
26H
2O 0.003mg/L; Inositol 50mg/L; Nicotinic acid 0.5mg/L; Hydrochloric acid pyridoxic acid 0.5mg/L; Thiamine hydrochloride 0.5mg/L; Glycine 2mg/L.
Chongzuo golden flower root induction medium, take 1/2 modified MS medium as the basis, contain the material of following concentration:
Indolebutyric acid 0.6～1.2mg/L or naa 0.6～1.2mg/L.
Above-mentioned 1/2 modified MS medium contains composition: Ca (NO
3)
24H
2O 150mg/L; MgSO
47H
2O 185mg/L; KNO
3950mg/L; NH
4NO
3825mg/L; KH
2PO
4170mg/L; Na
2EDTA 37.3mg/L; FeSO
47H
2O 27.8mg/L; KI0.83mg/L; H
3BO
30.63mg/L; MnSO
44H
2O 22.3mg/L; ZnSO
47H
2O 8.6mg/L; Na
2Mo4H
2O 0.025mg/L; CuSO
45H
2O 0.025mg/L; CoCl
26H
2O 0.003mg/L; Inositol 50mg/L; Nicotinic acid 0.5mg/L; Hydrochloric acid pyridoxic acid 0.5mg/L; Thiamine hydrochloride 0.5mg/L; Glycine 2mg/L.
The present invention adopts improvement MS+6-benayl aminopurine (6-BA)+3-indolyl acetic acid (IAA) as Chongzuo Camellia nitidissima inducing clumping bud medium, these two kinds of somatotropin are combined under suitable concentration and the weakly acidic environment, can activate the effect enzyme in the Camellia nitidissima somatic cell of Chongzuo, promote sprouting and the growth of Chongzuo Camellia nitidissima explant, cultivate and just can form sturdy Multiple Buds in 55 days, expand and numerously reach 20 times.The present invention also adopts 1/2 improvement MS+ indolebutyric acid (IBA) or 1/2 improvement MS+ naa (NAA) as the root induction medium, can effectively promote the single seedling rooting of Chongzuo Camellia nitidissima to form new plant.Use the Chongzuo Camellia nitidissima tissue culture propagation method of above-mentioned three kinds of inducing cultures; can fast and effeciently breed the Chongzuo Camellia nitidissima; solve its wild resource and day by day reduced the problem low with the natural propagation rate, effectively protected this in the world rare rare species germ plasm resource.
Embodiment
Embodiment 1
(1) inducing clumping bud
Get the tender seed of Chongzuo camellia chrysantha young, use first liquid detergent clean surface dirt, then flowing water flushing 30min, then at superclean bench with 75% Ethanol Treatment 30s, aseptic water washing 2 times is used 0.1HgCl again
2Sterilization 20min, aseptic water washing 5 times; Use again 0.2%HgCl
2Sterilization 15min, aseptic water washing 6 times; Use the aseptic filter paper suck dry moisture, choose the milky embryo and plant in adding 6-BA 4mg/L, IAA 0.6mg/L with improvement MS, the pH value is adjusted on 5.0 the medium, is 25 ± 1 ℃ in temperature, and light application time is 12h.d
-1, under the condition of intensity of illumination 2000lx, cultivated 65 days, on average obtain 15.2 times Multiple Buds, Multiple Buds is more sturdy, green, it is slightly slow to grow.
(2) root induction
When treating that Multiple Buds grows to 3～4cm, Multiple Buds is divided into Xiao Cong or single seedling is inoculated into the root induction medium, the root induction culture medium prescription is: on 1/2 improvement MS, add IBA 0.6mg/L and make; Be 25 ± 1 ℃ in temperature, light application time is 12h.d
-1, under the condition of intensity of illumination 2000lx, cultivate the adventive root that began to grow white in 30 days, grew 1～2 in 50 days, sturdy, the adventive root of the about 1cm of length.
Embodiment 2
(1) inducing clumping bud
Get the tender seed of Chongzuo camellia chrysantha young, use first liquid detergent flush away surface dirt, then flowing water flushing 30min, then at superclean bench with 75% Ethanol Treatment 40s, aseptic water washing 2 times is used 0.1HgCl again
2Sterilization 25min, aseptic water washing 5 times; Use again 0.2%HgCl
2Sterilization 15min, aseptic water washing 6 times; Blot explant with aseptic filter paper, choose the milky embryo and plant in adding 6-BA 3mg/L, IAA 0.70mg/L with improvement MS, the pH value is adjusted on 5.5 the medium, is 25 ± 1 ℃ in temperature, and light application time is 12h.d
-1, under the condition of intensity of illumination 2000lx, cultivated 60 days, on average obtain 18 times Multiple Buds, Multiple Buds is sturdy, leaf strong green, plumpness, growing way is good.
(2) root induction
When treating that Multiple Buds grows to 3～4cm, Multiple Buds is divided into Xiao Cong or single seedling is inoculated into the root induction medium, the root induction culture medium prescription is: on 1/2 improvement MS, add IBA 1.2mg/L and make; Be 25 ± 1 ℃ in temperature, light application time is 12h.d
-1, under the condition of intensity of illumination 2000lx, cultivate the adventive root that began to bear white in 30 days, grew 2～3 in 50 days, sturdy, the adventive root of the about 2cm of length.
Embodiment 3
(1) inducing clumping bud
Get the tender seed of Chongzuo camellia chrysantha young, use first liquid detergent flush away surface dirt, then flowing water flushing 30min, then at superclean bench with 75% Ethanol Treatment 40s, aseptic water washing 2 times is used 0.1HgCl again
2Sterilization 30min, aseptic water washing 5 times; Use again 0.2%HgCl
2Sterilization 20min, aseptic water washing 6 times; Blot explant with aseptic filter paper, plant in adding 6-BA 2mg/L, IAA 0.8mg/L with improvement MS, the pH value is adjusted on 5.8 the medium, is 25 ± 1 ℃ in temperature, and light application time is 12h.d
-1, under the condition of intensity of illumination 2000lx, cultivated 55 days, on average obtain 20 times Multiple Buds, Multiple Buds is more sturdy, the leaf strong green, growth is very fast.
(2) root induction
When treating that Multiple Buds grows to 3～4cm, Multiple Buds is divided into Xiao Cong or single seedling is inoculated into the root induction medium, the root induction culture medium prescription is: on 1/2 improvement MS, add NAA 0.6mg/L and make; Be 25 ± 1 ℃ in temperature, light application time is 12h.d
-1, under the condition of intensity of illumination 2000lx, cultivate the adventive root that began to grow white in 30 days, grew 2 in 48 days, sturdy, the adventive root of the about 2cm of length.
Among the above embodiment, modified MS medium contains composition and is: Ca (NO
3)
24H
2O 300mg/L; MgSO
47H
2O 370mg/L; KNO
31900mg/L; NH
4NO
31650mg/L; KH
2PO
4340mg/L; Na
2EDTA 37.3mg/L; FeSO
47H
2O27.8mg/L; KI 0.83mg/L; H
3BO
30.63mg/L; MnSO
44H
2O 22.3mg/L; ZnSO
47H
2O 8.6mg/L; Na
2Mo4H
2O 0.025mg/L; CuSO
45H
2O 0.025mg/L; CoCl
26H
2O 0.003mg/L; Inositol 50mg/L; Nicotinic acid 0.5mg/L; Hydrochloric acid pyridoxic acid 0.5mg/L; Thiamine hydrochloride 0.5mg/L; Glycine 2mg/L.
Among the above embodiment, above-mentioned 1/2 modified MS medium contains composition and is: Ca (NO
3)
24H
2O 150mg/L; MgSO
47H
2O185mg/L; KNO
3950mg/L; NH
4NO
3825mg/L; KH
2PO
4170mg/L; Na
2EDTA 37.3mg/L; FeSO
47H
2O27.8mg/L; KI 0.83mg/L; H
3BO
30.63mg/L; MnSO
44H
2O 22.3mg/L; ZnSO
47H
2O 8.6mg/L; Na
2Mo4H
2O 0.025mg/L; CuSO
45H
2O 0.025mg/L; CoCl
26H
2O 0.003mg/L; Inositol 50mg/L; Nicotinic acid 0.5mg/L; Hydrochloric acid pyridoxic acid 0.5mg/L; Thiamine hydrochloride 0.5mg/L; Glycine 2mg/L.
Claims (9)
1. Chongzuo Camellia nitidissima tissue culture propagation method, it is characterized in that: the explant of Chongzuo Camellia nitidissima is inoculated in the inducing clumping bud medium cultivates, when treating that Multiple Buds grows to 3cm, Multiple Buds is divided into Xiao Cong or single seedling is inoculated in the root induction medium and cultivates;
Described inducing clumping bud medium contains the material of following concentration take modified MS medium as the basis:
6-benzyl aminopurine 2～4mg/L 3-indolyl acetic acid 0.6～0.8mg/L
Its pH value is 4.5～5.8;
Described root induction medium contains the material of following concentration take 1/2 modified MS medium as the basis:
Indolebutyric acid 0.6～1.2mg/L or naa 0.6～1.2mg/L.
2. Chongzuo according to claim 1 Camellia nitidissima tissue culture propagation method, it is characterized in that: described explant is young tender seed.
3. Chongzuo according to claim 2 Camellia nitidissima tissue culture propagation method, it is characterized in that: the tender seed of described children is first through cleaning and use 0.1%HgCl
2Solution disinfection is processed 20～30min, uses 0.2%HgCl again
2Solution disinfection is processed 15～20min.
4. Chongzuo according to claim 3 Camellia nitidissima tissue culture propagation method, it is characterized in that: described cultivation is all at 25 ± 2 ℃ of temperature, light application time 12～14h.d
-1, intensity of illumination 2000lx condition under carry out.
5. Chongzuo Camellia nitidissima inducing clumping bud medium is characterized in that containing the material of following concentration take modified MS medium as the basis:
6-benzyl aminopurine 2～4mg/L 3-indolyl acetic acid 0.6～0.8mg/L.
6. Chongzuo according to claim 5 Camellia nitidissima inducing clumping bud medium, the pH value that it is characterized in that this medium is 4.5～5.8.
7. according to claim 5 or 6 described inducing cultures, it is characterized in that: described modified MS medium contains composition and is: Ca (NO
3)
24H
2O 300mg/L; MgSO
47H
2O 370mg/L; KNO
31900mg/L; NH
4NO
31650mg/L; KH
2PO
4340mg/L; Na
2EDTA 37.3mg/L; FeSO
47H
2O 27.8mg/L; KI 0.83mg/L; H
3BO
30.63mg/L; MnSO
44H
2O 22.3mg/L; ZnSO
47H
2O 8.6mg/L; Na
2Mo4H
2O 0.025mg/L; CuSO
45H
2O0.025mg/L; CoCl
26H
2O 0.003mg/L; Inositol 50mg/L; Nicotinic acid 0.5mg/L; Hydrochloric acid pyridoxic acid 0.5mg/L; Thiamine hydrochloride 0.5mg/L; Glycine 2mg/L.
8. Chongzuo golden flower root induction medium is characterized in that containing the material of following concentration take 1/2 modified MS medium as the basis:
Indolebutyric acid 0.6～1.2mg/L or naa 0.6～1.2mg/L.
9. inducing culture according to claim 8, it is characterized in that: described 1/2 modified MS medium contains composition and is: Ca (NO
3)
24H
2O 150mg/L; MgSO
47H
2O 185mg/L; KNO
3950mg/L; NH
4NO
3825mg/L; KH
2PO
4170mg/L; Na
2EDTA 37.3mg/L; FeSO
47H
2O 27.8mg/L; KI 0.83mg/L; H
3BO
30.63mg/L; MnSO
44H
2O 22.3mg/L; ZnSO
47H
2O 8.6mg/L; Na
2Mo4H
2O 0.025mg/L; CuSO
45H
2O0.025mg/L; CoCl
26H
2O 0.003mg/L; Inositol 50mg/L; Nicotinic acid 0.5mg/L; Hydrochloric acid pyridoxic acid 0.5mg/L; Thiamine hydrochloride 0.5mg/L; Glycine 2mg/L.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104285813A (en) * | 2014-10-27 | 2015-01-21 | 南宁市金花茶公园 | Camellia chrysantha tissue culture propagation method |
| CN104488712A (en) * | 2014-12-11 | 2015-04-08 | 柳州博泽科技有限公司 | Camellia nitidissima sprout tissue culture and rapid propagation method |
| CN104488714A (en) * | 2014-12-11 | 2015-04-08 | 柳州博泽科技有限公司 | Camellia nitidissima tissue culture method |
| CN104521753A (en) * | 2014-12-11 | 2015-04-22 | 柳州博泽科技有限公司 | Rapid propagation method of camellia nitidissima |
| CN104642111A (en) * | 2015-02-09 | 2015-05-27 | 广西壮族自治区农业科学院花卉研究所 | Golden camellia seedling propagation method |
| CN104663433A (en) * | 2014-12-11 | 2015-06-03 | 柳州博泽科技有限公司 | Rapid tissue culture method for golden camellia |
| CN106069755A (en) * | 2016-06-17 | 2016-11-09 | 句容市下蜀窑业茶场 | A kind of strengthening seedling and rooting cultural method of tea-tree tissue culture seedling |
| CN107135948A (en) * | 2017-06-02 | 2017-09-08 | 广西壮族自治区南宁良凤江国家森林公园 | A kind of method that Camellia nitidissima spray cultivates tissue-cultured seedling under sunlight conditions |
| CN107155888A (en) * | 2017-06-02 | 2017-09-15 | 广西壮族自治区南宁良凤江国家森林公园 | A kind of method that Camellia nitidissima embryo tissue-cultured seedling is cultivated under sunlight conditions |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104285813A (en) * | 2014-10-27 | 2015-01-21 | 南宁市金花茶公园 | Camellia chrysantha tissue culture propagation method |
| CN104488712A (en) * | 2014-12-11 | 2015-04-08 | 柳州博泽科技有限公司 | Camellia nitidissima sprout tissue culture and rapid propagation method |
| CN104488714A (en) * | 2014-12-11 | 2015-04-08 | 柳州博泽科技有限公司 | Camellia nitidissima tissue culture method |
| CN104521753A (en) * | 2014-12-11 | 2015-04-22 | 柳州博泽科技有限公司 | Rapid propagation method of camellia nitidissima |
| CN104663433A (en) * | 2014-12-11 | 2015-06-03 | 柳州博泽科技有限公司 | Rapid tissue culture method for golden camellia |
| CN104488714B (en) * | 2014-12-11 | 2016-04-13 | 柳州博泽科技有限公司 | The tissue culture method of a kind of Camellia nitidissima |
| CN104488712B (en) * | 2014-12-11 | 2016-07-06 | 柳州博泽科技有限公司 | A kind of tissue culture and rapid propagation method of tender shoots Camellia nitidissima Chi |
| CN104642111A (en) * | 2015-02-09 | 2015-05-27 | 广西壮族自治区农业科学院花卉研究所 | Golden camellia seedling propagation method |
| CN106069755A (en) * | 2016-06-17 | 2016-11-09 | 句容市下蜀窑业茶场 | A kind of strengthening seedling and rooting cultural method of tea-tree tissue culture seedling |
| CN107135948A (en) * | 2017-06-02 | 2017-09-08 | 广西壮族自治区南宁良凤江国家森林公园 | A kind of method that Camellia nitidissima spray cultivates tissue-cultured seedling under sunlight conditions |
| CN107155888A (en) * | 2017-06-02 | 2017-09-15 | 广西壮族自治区南宁良凤江国家森林公园 | A kind of method that Camellia nitidissima embryo tissue-cultured seedling is cultivated under sunlight conditions |
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