CN103371103A - Rapid propagation method for tissue culture of Rhododendron delavayi Franch - Google Patents
Rapid propagation method for tissue culture of Rhododendron delavayi Franch Download PDFInfo
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- 241001617585 Rhododendron delavayi Species 0.000 title abstract 5
- 230000001954 sterilising effect Effects 0.000 claims abstract description 27
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 27
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 241000208422 Rhododendron Species 0.000 claims description 32
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 29
- 229930006000 Sucrose Natural products 0.000 claims description 29
- 239000002609 medium Substances 0.000 claims description 29
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
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- 239000010455 vermiculite Substances 0.000 claims description 4
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- 229910052902 vermiculite Inorganic materials 0.000 claims description 4
- 238000011161 development Methods 0.000 claims description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
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Abstract
The invention relates to a rapid propagation method for tissue culture of Rhododendron delavayi Franch. The method comprises the following steps: 1) selection of explants; 2) sterilization of the explants; 3) induction culture of new branches; 4) proliferation culture; 5) strong seedling culture; and 6) rooting ex vitro. The invention has the following beneficial effects: (1) the rapid propagation method for tissue culture of Rhododendron delavayi Franch by using bloomed tender stem segments as explants is established, so as to successfully solve the technical problems of hard sterilization and easy browning of explants; and (2) the Rhododendron delavayi Franch bred by the rapid propagation method for tissue culture provided by the invention has growth coefficient of 100-150 times and ex vitro rooting rate of 85-90%within a year and a half, so as to greatly increase propagation coefficient of the Rhododendron delavayi Franch and provide effective and reliable propagation method for the preservation and industrialization of the species.
Description
Technical field
The present invention relates to field of plant tissue culture technique, particularly relate to method for quickly breeding and the medium thereof of delavay rhododendron in the Ericaceae Rhododendron.
Background technology
Delavay rhododendron (
Rhododendron delavayiFranch), have another name called lantana, another name horse nose tassel, ox blood flower, the dog spattered drops of blood, Camellia, close bucket flower are the tree-like cuckoo subgroup of Ericaceae Rhododendron monkeyhead rhododendron leaf group, and its trunk is obvious, and corolla is bell, redness, the spongy fine hair of Ye Mi back of the body canescence.Delavay rhododendron is generally dungarunga, large flower and brilliant color, and tree performance is graceful, ornamental value is high, mainly be distributed in the southwest such as Yunnan, Guizhou, Sichuan, Tibet than the high altitude localities, be one of easy primary cuckoo kind of taming, in the afforestation exploitation, have using value.
At present, the propagation method of delavay rhododendron mainly comprises by taking wild resource, but the method transplanting survival rate is not high, and wild resource is destroyed serious.Cottage method is sturdy and be subjected to seasonal effect because of branch, can not realize large-scale production.Planting seed method reproduction coefficient is high, but seedling bloom need to be more than at least 6 years, blooming cycle is long, can not satisfy the extensive use of afforestation.Therefore adopting tissue culture technique is the important channel of realizing the delavay rhododendron large-scale breeding, can reduce the destruction to wild resource, accelerates the domestication process of primary cuckoo and the application on the gardens.
In recent years, to the existing more report of the Study on tissue culture of delavay rhododendron.(the Agriculture of Anhui science such as Zhou Yan, 2007(35): 9213-9214), (Plant Physiology Communications such as Cheng Xuemei, 2008(44): 297-298), (Southwestern University's journal (natural science edition) 2012 such as Hong Yi, 34(8): 61-66) in succession delavay rhododendron has been carried out group training research, all obtained group training seedling, but the stem section that above-mentioned research all is based on behind the seed sprouting is explant, and the group training seedling that obtains still will be through just blooming behind the cultivating and growing for many years.In addition, a little less than the group training seedling growth of the fast traditional font system that sets up by the seed approach, the stem section is sturdy not, and growth rate is slow after transplanting, so there is certain defective in its group training system.The present invention is directed to the delavay rhododendron botanical character, utilize the maternal plant stem section of having bloomed to be explant, set up the efficient tissue-culturing rapid propagation system of delavay rhododendron, growth coefficient is controlled at 5-6 doubly, and the tissue culture seeding stem segment of cultivation is sturdy, and has realized the outside sprout-cultivating-bottle technology.
Summary of the invention
In order to solve above-mentioned technical problem, an object of the present invention is to utilize the plant that bloomed to be explant, a kind of medium and cultural method that improves delavay rhododendron group training efficient is provided, breeding for the batch production of delavay rhododendron nursery stock provides technical support.Another object of the present invention provides a kind of delavay rhododendron tissue-culturing quick-propagation medium.
In order to realize the first above-mentioned purpose, the present invention has adopted following technical scheme:
A kind of delavay rhododendron quick breeding method for tissue culture, the method is carried out as follows:
1) selection of explant: spring or autumn from the delavay rhododendron maternal plant that blooms clip with the then hair generating branch of 4-6 resting bud;
2) explant sterilization: cut off the branch blade, soak with running water first and flushing 30-60 minute, blot with after liquid detergent washing 1-2 time; Then,, with aseptic water washing 3-5 time, again with 0.1% mercury chloride sterilization 12-15 minute, blot with after sterile distilled water flushing 3-5 time at last with 75% alcohol disinfecting 15-30 second at the desinfection chamber super-clean bench;
3) new branch is induced cultivation: the branch of the bacterium of will having gone out excision stem apex, and remaining branch is cut into the long stem section of 2-3cm, and every section with 1-2 axillalry bud, and oblique cutting enters in the inducing culture; In 23-26 ℃ of light 16 hours, to cultivate after 25-35 days in black 8 hours, the stem section resting bud development growth branch that makes new advances is long to 2-4cm; Described inducing culture is made of following component: WPM minimal medium+ZT 3.0-4.0 mg/L+NAA0.2-0.4 mg/L+20 g/L sucrose or white sugar+6-10 g/L agar, pH4.8-5.2, high-temperature sterilization;
4) propagation is cultivated: the branch that clip newly grows, go to insert in the proliferated culture medium behind the terminal bud, and in 23-26 ℃ of light 16 hours, black 8 hours, cultivate after 45-60 days, the stem segment length goes out Multiple Buds; This stage can repeat; Described proliferated culture medium is made of following component: WPM minimal medium+ZT 1.0-2.0 mg/L+NAA 0.1-0.2 mg/L+20g/L sucrose or white sugar+6-10 g/L agar, pH 4.8-5.2, high-temperature sterilization;
5) strong seedling culture: will grow the long Multiple Buds of 1-2cm and shear and move in the strong seedling culture base, and in 23-26 ℃ of light 16 hours/black 8 hours, cultivate 25-35 days, and in the immigration matrix, carry out outside sprout-cultivating-bottle until long behind the 3-5cm; Described strong seedling culture base is made of following component: WPM minimal medium+ZT 0.5-1.0 mg/L+NAA0.05-0.1 mg/L+20g/L sucrose or white sugar+6-10 g/L agar, pH 4.8-5.2, high-temperature sterilization;
6) outside sprout-cultivating-bottle: in annual March 10 to next year, length is arrived the high bottle seedling of 3-5 cm in outdoor 1 week of placement, move into behind the agar on the flush away branch and contain peat: perlite: in the matrix of vermiculite volume ratio 2:1:1, under the moisture-heat preservation condition, grow, take root after 1-2 month.
In order to realize the second above-mentioned purpose, the present invention has adopted following technical scheme:
A kind of this medium of delavay rhododendron tissue-culturing quick-propagation medium is made of following component: WPM minimal medium+ZT 0.5-4.0 mg/L+NAA0.05-0.4 mg/L+20 g/L sucrose or white sugar+6-10 g/L agar, pH4.8-5.2.
As preferably, this medium is made of following component: WPM minimal medium+ZT 3.0-4.0 mg/L+NAA0.2-0.4 mg/L+20 g/L sucrose or white sugar+6-10 g/L agar, pH4.8-5.2.
As preferably, this medium is made of following component: WPM minimal medium+ZT 1.0-2.0 mg/L+NAA 0.1-0.2 mg/L+20g/L sucrose or white sugar+6-10 g/L agar, pH 4.8-5.2.
As preferably, this medium is made of following component: WPM minimal medium+ZT 0.5-1.0 mg/L+NAA0.05-0.1 mg/L+20g/L sucrose or white sugar+6-10 g/L agar, pH 4.8-5.2.
The invention has the beneficial effects as follows:
(1) set up and utilize the tender stem section of having bloomed to be the delavay rhododendron tissue culture and rapid propagation method of explant, successfully solved the technical barrier of the difficult sterilization of explant, easy brownization;
(2) delavay rhododendron that breeds by tissue culture and rapid propagation method of the present invention is 100-150 times at internal breeding half a year coefficient, the outside sprout-cultivating-bottle rate reaches 85-90%, greatly improved the reproduction coefficient of delavay rhododendron, for preservation and the industrialization of these species provides reliable and effective propagation method.
Embodiment
The present invention is described in further detail by following examples, but content of the present invention is not limited to this.
Embodiment 1
Delavay rhododendron quick breeding method for tissue culture the method, carry out as follows:
1, the preparation of medium:
(1) inducing culture: WPM minimal medium+ZT 3.0 mg/L+NAA 0.2mg/L+20 g/L sucrose+8 g/L agar, pH 5.0, high-temperature sterilization;
(2) proliferated culture medium: WPM minimal medium+ZT 2.0 mg/L+NAA 0.2 mg/L+20g/L sucrose+8 g/L agar, pH 5.0, high-temperature sterilization;
(3) strong seedling culture base: WPM minimal medium+ZT 0.5 mg/L+NAA 0.05 mg/L+20g/L sucrose+8 g/L agar, pH 5.0, high-temperature sterilization.
2, delavay rhododendron quick breeding method for tissue culture
(1) selection of explant: spring, clip from the delavay rhododendron maternal plant was about the new branch of 5-10 cm, with 4-6 resting bud;
(2) explant sterilization: branch cuts off blade, first with running water flushing 30 minutes, blots with after the liquid detergent washing 1 time; Then,, with aseptic water washing 3 times, again with 0.1% mercury chloride sterilization 12 minutes, blot after washing 3 times with sterile distilled water at last with 75% alcohol disinfecting 15 seconds at the desinfection chamber super-clean bench;
(3) bud is induced cultivation: the branch excision stem apex of the bacterium of will having gone out, and remaining branch is cut into the long stem section (every section with 1-2 axillalry bud) of 2 cm, and oblique cutting enters in the bud inducing culture; In 24 ℃ of light (16 hours)/black (8 hours) cultivation is after 30 days, it is long that stem segment with axillary buds grows to 2-4cm;
(4) propagation is cultivated: the branch that clip newly grows, go to insert in the proliferated culture medium behind the terminal bud, and in 24 ℃ of light (16 hours)/black (8 hours) cultivation is after 50 days, the stem segment length goes out Multiple Buds; This stage can repeat;
(5) strong seedling culture: will grow the long Multiple Buds of 2cm and shear in the implantation strong seedling culture base, and cultivate 30 days in 24 ℃ of light (16 hours)/black (8 hours);
(6) outside sprout-cultivating-bottle: in annual November, length is arrived the high bottle seedling of 3-5 cm in outdoor 1 week of hardening, move into behind the agar on the flush away branch and contain peat: perlite: in the matrix of vermiculite volume ratio 2:1:1, under the moisture-heat preservation condition, grow, take root after 1 month.
Embodiment 2
Delavay rhododendron quick breeding method for tissue culture, the method carry out as follows:
1, the preparation of medium:
(1) inducing culture: WPM minimal medium+ZT 4.0 mg/L+NAA 0.2mg/L+20 g/L sucrose+6 g/L agar, pH 5.2, high-temperature sterilization;
(2) proliferated culture medium: WPM minimal medium+ZT 2.0 mg/L+NAA 0.1 mg/L+20g/L sucrose+6 g/L agar, pH 5.2, high-temperature sterilization;
(3) strong seedling culture base: WPM minimal medium+ZT 1.0 mg/L+NAA 0.1 mg/L+20g/L sucrose+6 g/L agar, pH 5.2, high-temperature sterilization.
2, delavay rhododendron quick breeding method for tissue culture
(1) selection of explant: autumn, clip from the delavay rhododendron maternal plant was about the new autumn growth of 5-10 cm, with 4-6 resting bud;
(2) explant sterilization: branch cuts off blade, first with running water flushing 60 minutes, blots with after the liquid detergent washing 2 times; Then,, with aseptic water washing 5 times, again with 0.1% mercury chloride sterilization 15 minutes, blot after washing 5 times with sterile distilled water at last with 75% alcohol disinfecting 30 seconds at the desinfection chamber super-clean bench;
(3) bud is induced cultivation: the branch excision stem apex of the bacterium of will having gone out, and remaining branch is cut into the long stem section (every section with 1-2 axillalry bud) of 2 cm, and oblique cutting enters in the bud inducing culture; In 25 ℃ of light (16 hours)/black (8 hours) cultivation is after 35 days, it is long that stem segment with axillary buds grows to 2-4cm;
(4) propagation is cultivated: the branch that clip newly grows, go to insert in the proliferated culture medium behind the terminal bud, and in 25 ℃ of light (16 hours)/black (8 hours) cultivation is after 60 days, the stem segment length goes out Multiple Buds; This stage can repeat;
(5) strong seedling culture: will grow the long Multiple Buds of 2cm and shear in the implantation strong seedling culture base, and cultivate 35 days in 25 ℃ of light (16 hours)/black (8 hours);
(6) outside sprout-cultivating-bottle: in the annual 2-3 month, length is arrived the high bottle seedling of 3-5 cm in outdoor 1 week of hardening, move into behind the agar on the flush away branch and contain peat: perlite: in the matrix of vermiculite volume ratio 2:1:1, under the moisture-heat preservation condition, grow, take root after 1 month.
Embodiment 3
In the present embodiment, described inducing culture is: WPM minimal medium+ZT 3.0mg/L+NAA 0.3 mg/L+20 g/L white sugar+10 g/L agar, and pH 4.8, high-temperature sterilization; Spring is clip children shoot from the delavay rhododendron maternal plant, wipes out stem apex after the sterilization, and remaining branch segment is retreaded and inserted in the bud inducing culture, cultivates 35 days in 26 ℃ of light (16 hours)/black (8 hours); The axillalry bud that newly grows is gone to push up in the rear insertion proliferated culture medium: WPM minimal medium+ZT 1.0 mg/L+NAA 0.05 mg/L+20g/L white sugar+10 g/L agar, pH 4.8, high-temperature sterilization, cultivated 60 days 26 ℃ of light (16 hours)/black (8 hours); The long shearing to the long Multiple Buds of 1-2cm moved in the strong seedling culture base: WPM minimal medium+ZT 0.5 mg/L+NAA 0.05 mg/L+20g/L white sugar+10 g/L agar, pH 4.8, high-temperature sterilization was cultivated 35 days in 26 ℃ of light (16 hours)/black (8 hours); In the annual 12-1 month, the long high bottle seedling of 3-5 cm that arrives in outdoor 1 week of hardening, is grown under the moisture-heat preservation condition, take root after 2 months; All the other process all are same as embodiment 1.
Claims (1)
1. delavay rhododendron quick breeding method for tissue culture is characterized in that the method carries out as follows:
1) selection of explant: spring or autumn from the delavay rhododendron maternal plant that blooms clip with the then hair generating branch of 4-6 resting bud;
2) explant sterilization: cut off the branch blade, soak with running water first and flushing 30-60 minute, blot with after liquid detergent washing 1-2 time; Then,, with aseptic water washing 3-5 time, again with 0.1% mercury chloride sterilization 12-15 minute, blot with after sterile distilled water flushing 3-5 time at last with 75% alcohol disinfecting 15-30 second at the desinfection chamber super-clean bench;
3) new branch is induced cultivation: the branch of the bacterium of will having gone out excision stem apex, and remaining branch is cut into the long stem section of 2-3cm, and every section with 1-2 axillalry bud, and oblique cutting enters in the inducing culture; In 23-26 ℃ of light 16 hours, to cultivate after 25-35 days in black 8 hours, the stem section resting bud development growth branch that makes new advances is long to 2-4cm; Described inducing culture is made of following component: WPM minimal medium+ZT 3.0-4.0 mg/L+NAA0.2-0.4 mg/L+20 g/L sucrose or white sugar+6-10 g/L agar, pH4.8-5.2, high-temperature sterilization;
4) propagation is cultivated: the branch that clip newly grows, go to insert in the proliferated culture medium behind the terminal bud, and in 23-26 ℃ of light 16 hours, black 8 hours, cultivate after 45-60 days, the stem segment length goes out Multiple Buds; This stage can repeat; Described proliferated culture medium is made of following component: WPM minimal medium+ZT 1.0-2.0 mg/L+NAA 0.1-0.2 mg/L+20g/L sucrose or white sugar+6-10 g/L agar, pH 4.8-5.2, high-temperature sterilization;
5) strong seedling culture: will grow the long Multiple Buds of 1-2cm and shear and move in the strong seedling culture base, and in 23-26 ℃ of light 16 hours/black 8 hours, cultivate 25-35 days, and in the immigration matrix, carry out outside sprout-cultivating-bottle until long behind the 3-5cm; Described strong seedling culture base is made of following component: WPM minimal medium+ZT 0.5-1.0 mg/L+NAA0.05-0.1 mg/L+20g/L sucrose or white sugar+6-10 g/L agar, pH 4.8-5.2, high-temperature sterilization;
6) outside sprout-cultivating-bottle: in annual March 10 to next year, length is arrived the high bottle seedling of 3-5 cm in outdoor 1 week of placement, move into behind the agar on the flush away branch and contain peat: perlite: in the matrix of vermiculite volume ratio 2:1:1, under the moisture-heat preservation condition, grow, take root after 1-2 month.
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| CN105475138A (en) * | 2015-12-18 | 2016-04-13 | 深圳职业技术学院 | Rapid propagation method of Rhododendron moulmainense Hook. f. |
| CN105660397A (en) * | 2016-01-13 | 2016-06-15 | 杭州市园林绿化股份有限公司 | High-frequency regeneration and rapid propagation method of Enkianthus chinensis tissue culture seedlings |
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| CN107079815A (en) * | 2017-05-25 | 2017-08-22 | 贵州师范大学 | A kind of method for inducing charming cuckoo and Rhododendron delavayi cenospecies callus |
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| CN105475138A (en) * | 2015-12-18 | 2016-04-13 | 深圳职业技术学院 | Rapid propagation method of Rhododendron moulmainense Hook. f. |
| CN105660397A (en) * | 2016-01-13 | 2016-06-15 | 杭州市园林绿化股份有限公司 | High-frequency regeneration and rapid propagation method of Enkianthus chinensis tissue culture seedlings |
| CN106258958A (en) * | 2016-08-08 | 2017-01-04 | 天水市果树研究所 | A kind of outside sprout-cultivating-bottle method of Fructus Pruni pseudocerasi tissue cultured seedling |
| CN107079815A (en) * | 2017-05-25 | 2017-08-22 | 贵州师范大学 | A kind of method for inducing charming cuckoo and Rhododendron delavayi cenospecies callus |
| CN107079815B (en) * | 2017-05-25 | 2019-06-11 | 贵州师范大学 | A method for inducing callus of Rhododendron charismatica and Rhododendron lantana hybrids |
| CN107494274A (en) * | 2017-10-12 | 2017-12-22 | 云南省农业科学院花卉研究所 | A kind of stripping bud sterilization method of alpine rose explant |
| CN114847162A (en) * | 2022-05-19 | 2022-08-05 | 福建农林大学 | Azalea in vitro culture and micro-bud grafting method |
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