CN103371103B - Rapid propagation method for tissue culture of Rhododendron delavayi Franch - Google Patents

Rapid propagation method for tissue culture of Rhododendron delavayi Franch Download PDF

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CN103371103B
CN103371103B CN201310324529.4A CN201310324529A CN103371103B CN 103371103 B CN103371103 B CN 103371103B CN 201310324529 A CN201310324529 A CN 201310324529A CN 103371103 B CN103371103 B CN 103371103B
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rhododendron
agar
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CN103371103A (en
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朱春艳
顾宏辉
余金良
朱丹华
盛小光
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HANGZHOU BOTANICAL GARDEN
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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

A kind of rapid propagation method for tissue culture of Rhododendron delavayi Franch
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 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, be the tree-like cuckoo subgroup of Ericaceae Rhododendron monkeyhead rhododendron leaf group, its trunk is obvious, and corolla is bell, red, and Ye Mi carries on the back the spongy fine hair of 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 comparatively high altitude localitiess, southwest such as Yunnan, Guizhou, Sichuan, Tibet, be one of comparatively easy primary cuckoo kind of taming, in 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 destroys serious to wild resource.Cottage method because of branch sturdy and by seasonal effect, can not large-scale production be realized.Planting seed method reproduction coefficient is high, and the needs more than at least 6 years but seedling is bloomed, blooming cycle is long, can not meet the extensive use of afforestation.Therefore adopt tissue culture technique to be realize important channel to delavay rhododendron large-scale breeding, the destruction to wild resource can be reduced, accelerate the application on the domestication process of primary cuckoo and gardens.
In recent years, more report is had to 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 carry out organizing training research to delavay rhododendron, all obtain plantlet in vitro, but above-mentioned research be all based on seed sprouting after stem section be explant, the plantlet in vitro obtained still just can will be bloomed after cultivating and growing for many years.In addition, the plantlet in vitro growth of the rapid propagation system set up by seed approach is weak, and stem section is sturdy not, and after transplanting, growth rate is slow, and therefore its group training system exists certain defect.The present invention is directed to delavay rhododendron botanical character, utilize the maternal plant stem section of having bloomed to be explant, establish the efficient tissue-culturing rapid propagation system of delavay rhododendron, growth coefficient controls at 5-6 doubly, and the tissue culture seeding stem segment of cultivation is sturdy, and achieves 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 and having bloomed plant for explant, provide a kind of medium and the cultural method that improve delavay rhododendron group training efficiency, for the plant modification of delavay rhododendron nursery stock provides technical support.Another object of the present invention is to provide a kind of delavay rhododendron tissue-culturing quick-propagation medium.
In order to realize the first above-mentioned object, present invention employs following technical scheme:
A kind of rapid propagation method for tissue culture of Rhododendron delavayi Franch, the method is carried out as follows:
1) selection of explant: spring or autumn are from the then hair generating branch of clip the delavay rhododendron maternal plant that blooms with 4-6 resting bud;
2) explant sterilization: cut off branch blade, first soaks with running water and rinses 30-60 minute, blots after washing 1-2 time with liquid detergent; Then, with 75% alcohol disinfecting 15-30 second on desinfection chamber super-clean bench, with aseptic water washing 3-5 time, then with 0.1% mercury chloride sterilization 12-15 minute, finally with sterile distilled water rinse 3-5 all over after blot;
3) a new branch Fiber differentiation: by the branch of bacterium of having gone out excision stem apex, remaining branch is cut into the long stem section of 2-3cm, and every length of tape has 1-2 axillalry bud, and oblique cutting enters in inducing culture; In 23-26 DEG C of light 16 hours, within black 8 hours, cultivate after 25-35 days, the stem section resting bud development growth branch that makes new advances is long to 2-4cm; Described inducing culture is made up 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) Multiplying culture: the branch that clip newly grows, inserts after removing terminal bud in proliferated culture medium, and in 23-26 DEG C of light 16 hours, black 8 hours, cultivate after 45-60 days, stem segment length went out Multiple Buds; This stage can repeat; Described proliferated culture medium is made up 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 to the long Multiple Buds of 1-2cm and shear and move in strong seedling culture base, in 23-26 DEG C of light 16 hours/black 8 hours, cultivates 25-35 days, moves in matrix, carry out outside sprout-cultivating-bottle after growing to 3-5cm; Described strong seedling culture base is made up 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, to the high bottle seedling of 3-5 cm be grown to outdoor placement 1 week, moving into containing containing peat after washing away the agar on branch: perlite: in the matrix of vermiculite volume ratio 2:1:1, grows under moisture-heat preservation condition, taking root after 1-2 month.
In order to realize the second above-mentioned object, present invention employs following technical scheme:
A kind of this medium of delavay rhododendron tissue-culturing quick-propagation medium is made up 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 up 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 up 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 up 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) establish utilization to have bloomed the delavay rhododendron tissue culture and rapid propagation method that tender stem section is explant, successfully solve the technical barrier of the difficult sterilizing of explant, easy brownization;
(2) delavay rhododendron bred by tissue culture and rapid propagation method of the present invention internal breeding half a year coefficient be 100-150 doubly, outside sprout-cultivating-bottle rate reaches 85-90%, greatly improve the reproduction coefficient of delavay rhododendron, for the preservation of these species and industrialization provide reliable and effective propagation method.
Embodiment
By following examples, the present invention is described in further detail, but content of the present invention is not limited thereto.
embodiment 1
Rapid propagation method for tissue culture of Rhododendron delavayi Franch 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, rapid propagation method for tissue culture of Rhododendron delavayi Franch
(1) selection of explant: spring is about the new of 5-10 cm from clip delavay rhododendron maternal plant and sends out branch, with 4-6 resting bud;
(2) explant sterilization: branch cuts off blade, first uses tap water 30 minutes, blots after washing 1 time with liquid detergent; Then, with 75% alcohol disinfecting 15 seconds on desinfection chamber super-clean bench, with aseptic water washing 3 times, then sterilize 12 minutes with 0.1% mercury chloride, blot after finally rinsing 3 times with sterile distilled water;
(3) bud inducement is cultivated: by the branch of bacterium of having gone out excision stem apex, remaining branch is cut into the long stem section (every length of tape has 1-2 axillalry bud) of 2 cm, and oblique cutting enters in bud inducement medium; After 24 DEG C of light (16 hours)/black (8 hours) cultivate 30 days, it is long that stem segment with axillary buds grows to 2-4cm;
(4) Multiplying culture: the branch that clip newly grows, inserts after removing terminal bud in proliferated culture medium, and after 24 DEG C of light (16 hours)/black (8 hours) cultivate 50 days, stem segment length goes out Multiple Buds; This stage can repeat;
(5) strong seedling culture: shearing growing to the long Multiple Buds of 2cm in implantation strong seedling culture base, cultivating 30 days in 24 DEG C of light (16 hours)/black (8 hours);
(6) outside sprout-cultivating-bottle: in annual November, to the high bottle seedling of 3-5 cm be grown to outdoor hardening 1 week, move into containing containing peat after washing away the agar on branch: perlite: in the matrix of vermiculite volume ratio 2:1:1, grows under moisture-heat preservation condition, takes root after 1 month.
embodiment 2
Rapid propagation method for tissue culture of Rhododendron delavayi Franch, the method is carried 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, rapid propagation method for tissue culture of Rhododendron delavayi Franch
(1) selection of explant: autumn is about the new of 5-10 cm from clip delavay rhododendron maternal plant and sends out autumn growth, with 4-6 resting bud;
(2) explant sterilization: branch cuts off blade, first uses tap water 60 minutes, blots after washing 2 times with liquid detergent; Then, with 75% alcohol disinfecting 30 seconds on desinfection chamber super-clean bench, with aseptic water washing 5 times, then sterilize 15 minutes with 0.1% mercury chloride, blot after finally rinsing 5 times with sterile distilled water;
(3) bud inducement is cultivated: by the branch of bacterium of having gone out excision stem apex, remaining branch is cut into the long stem section (every length of tape has 1-2 axillalry bud) of 2 cm, and oblique cutting enters in bud inducement medium; After 25 DEG C of light (16 hours)/black (8 hours) cultivate 35 days, it is long that stem segment with axillary buds grows to 2-4cm;
(4) Multiplying culture: the branch that clip newly grows, inserts after removing terminal bud in proliferated culture medium, and after 25 DEG C of light (16 hours)/black (8 hours) cultivate 60 days, stem segment length goes out Multiple Buds; This stage can repeat;
(5) strong seedling culture: shearing growing to the long Multiple Buds of 2cm in implantation strong seedling culture base, cultivating 35 days in 25 DEG C of light (16 hours)/black (8 hours);
(6) outside sprout-cultivating-bottle: in the annual 2-3 month, to the high bottle seedling of 3-5 cm be grown to outdoor hardening 1 week, move into containing containing peat after washing away the agar on branch: perlite: in the matrix of vermiculite volume ratio 2:1:1, grows under moisture-heat preservation condition, takes 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, pH 4.8, high-temperature sterilization; Spring, from clip edible tender branch delavay rhododendron maternal plant, wipes out stem apex after sterilizing, and remaining branch segment is retreaded and inserted in bud inducement medium, cultivates 35 days in 26 DEG C of light (16 hours)/black (8 hours); The axillalry bud newly grown is gone push up in 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, 26 DEG C of light (16 hours)/black (8 hours) are cultivated 60 days; Shear in immigration strong seedling culture base by growing to the long Multiple Buds of 1-2cm: 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, cultivates 35 days in 26 DEG C of light (16 hours)/black (8 hours); In the annual 12-1 month, by growing to the high bottle seedling of 3-5 cm outdoor hardening 1 week, growing under moisture-heat preservation condition, taking root after 2 months; All the other process are all same as embodiment 1.

Claims (1)

1. a rapid propagation method for tissue culture of Rhododendron delavayi Franch, is characterized in that the method is carried out as follows:
1) selection of explant: spring or autumn are from the then hair generating branch of clip the delavay rhododendron maternal plant that blooms with 4-6 resting bud;
2) explant sterilization: cut off branch blade, first soaks with running water and rinses 30-60 minute, blots after washing 1-2 time with liquid detergent; Then, with 75% alcohol disinfecting 15-30 second on desinfection chamber super-clean bench, with aseptic water washing 3-5 time, then with 0.1% mercury chloride sterilization 12-15 minute, finally with sterile distilled water rinse 3-5 all over after blot;
3) a new branch Fiber differentiation: by the branch of bacterium of having gone out excision stem apex, remaining branch is cut into the long stem section of 2-3cm, and every length of tape has 1-2 axillalry bud, and oblique cutting enters in inducing culture; In 23-26 DEG C of illumination 16 hours, within dark 8 hours, cultivate after 25-35 days, stem section resting bud development growth makes new advances branch to 2-4cm length; Described inducing culture is made up 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) Multiplying culture: the branch that clip newly grows, inserts after removing terminal bud in proliferated culture medium, and in 23-26 DEG C of illumination 16 hours, dark 8 hours, cultivate after 45-60 days, stem segment length went out Multiple Buds; This stage repeats; Described proliferated culture medium is made up 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: shearing in immigration strong seedling culture base by growing to the long Multiple Buds of 1-2cm, in 23-26 DEG C of illumination 16 hours, dark 8 hours, cultivating 25-35 days, moving into after growing to 3-5cm in matrix, carry out outside sprout-cultivating-bottle; Described strong seedling culture base is made up 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, to the high bottle seedling of 3-5 cm be grown to outdoor placement 1 week, move into containing peat after washing away the agar on branch: perlite: in the matrix of vermiculite volume ratio 2:1:1, grows under moisture-heat preservation condition, 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.
CN105660397B (en) * 2016-01-13 2017-12-15 杭州市园林绿化股份有限公司 A kind of lantern tree tissue-cultured seedling high frequency regeneration rapid propagation method
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
CN107079815B (en) * 2017-05-25 2019-06-11 贵州师范大学 A method of inducing charming cuckoo and Rhododendron delavayi cenospecies callus
CN107494274B (en) * 2017-10-12 2019-10-22 云南省农业科学院花卉研究所 A kind of stripping bud sterilization method of alpine rose explant

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