CN103416303A - Inducing method for spring dendrobium protoemm-like body - Google Patents
Inducing method for spring dendrobium protoemm-like body Download PDFInfo
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- CN103416303A CN103416303A CN2013102900804A CN201310290080A CN103416303A CN 103416303 A CN103416303 A CN 103416303A CN 2013102900804 A CN2013102900804 A CN 2013102900804A CN 201310290080 A CN201310290080 A CN 201310290080A CN 103416303 A CN103416303 A CN 103416303A
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Abstract
The invention discloses an inducing method for spring dendrobium protoemm-like body, which comprises the following steps: taking a tender tissue of a spring dendrobium plant as an explant, and carrying out inducing to obtain a germ-free seedling; inoculating a non-bud stem of the caudex of the germ-free seedling into an inducing culture medium after treatment of ultrasonic waves, and carrying out inducing cultivation to obtain the spring dendrobium protoemm-like body. As the non-bud stem of the caudex of the germ-free seedling is taken as an inducing material, and the generation of the protoemm-like body is induced under the assistance of ultrasonic wave treatment, a new important way is provided for the spring dendrobium tissue culture and fast propagation technology; the protoemm-like body obtained according to the method not only retains the favorable property of an original variety, but also is high in pullulation efficiency which is 2 to 3 times of that of cluster buds, and the application value in production is high; as the protoemm-like body can serve as an acceptor material of spring dendrobium transgenic breeding while cluster buds cannot, so that more transgenic spring dendrobium varieties can be obtained through the protoemm-like body.
Description
Technical field
The present invention relates to field of plant tissue culture technique, be specifically related to a kind of abductive approach of spring stem of noble dendrobium protocorms.
Background technology
The spring stem of noble dendrobium has another name called the U.S. flower stem of noble dendrobium, because blooming spring, gaining the name, is this plant of the perennial epiphytic orchid of the orchid family Dendrobium, with the common autumn stem of noble dendrobium, compares, have strain shape compact, spend many, the characteristics such as look gorgeous, savory and extensive management, be domestic and international comparatively popular high-grade pot flowers at present.
The training of mainly employing group of spring stem of noble dendrobium seedling and cottage propagation, because tissue culture propagation speed is fast, reproduction coefficient is high, is specially adapted to detoxification and new varieties and promotes, therefore occupy an leading position in the production of spring dendrobium stem good quality seedling.
The existing clonal quick breeding method for tissue culture of spring dendrobe cultivation kind of viewing and admiring is mostly that to take the propagation of Multiple Buds be starting point, and its growth rate is about 1.5～2 times/month, and proliferate efficiency is lower.Part spring dendrobe cultivation kind adopts the sowing aseptic seed to be bred, the rataria imbibition of seed, the blastocyte division growth at middle part, break through chalazal end kind skin and form spherical or oval-shaped protocorm, the growth rate of protocorm is about 4～5 times/month, but the offspring that seed propagation obtains, its genotype is separated very large with cultivated character.
It is reported and carry out the dendrobium tissue culture expanding propagation by protocorms (protocorm-like bodies, PLBs) approach, its breeding rate is 2～3 times of Multiple Buds far above the proliferate efficiency of bud propagation protocorms, on producing, has great using value.Protocorms is a kind of particular tissues structure that orchid is organized the excessive form-protocorm of similar seed germination produced in the training process: during the training of hybrid cymbidium seedling stem apex detoxify group, can induce the institutional framework that forms similar its seed protocorm by shoot apical meristem (rather than seed).Can not cause qualitative variation by the breeding of protocorms approach, be kept variety stability.
Torrid zone orchid has all been realized (comprising Moth orchid, dendrobium, hybrid cymbidium, Wen Xinlan and Bowring cattleya etc.) plant regeneration system of protocorms approach at present.The Chinese patent literature that is CN101180951B as publication number discloses a kind of tissue cultivation quick breeding method of orchid, and it comprises:
(1) induce cultivation: the seed of orchid or young shoot are seeded on inducing culture, induce the initial protocorm of sesame seed size, described inducing culture is to improve the semisolid culturemedium that MS is minimal medium additional other compositions, and concrete composition is: KNO
3, NH
4NO
3, KH
2PO
4, MgSO
4* 7H
2O, CaCl
2, KI, H
3BO
3, MnSO
4* 4H
2O, ZnSO
4* 7H
2O, Na
2MoO
4* 2H
2O, CuSO
4* 5H
2O, CoCl
2* 6H
2O, Na
2* EDTA, FeSO
4* 7H
2O, C
6H
12O
6* 2H
2O, NH
3CH
2COOH, C
12H
17ClN
4OS*HCl, C
8H
11O
3N*HCl, NC
5H
4COOH, sucrose, 6-BA, active carbon, agarose; The described condition of culture of cultivation of inducing is: prior to dark training under 20～25 ℃ 15～20 days, and then forward under 20～25 ℃, 1000～1500Lux and induce and cultivate 2～3 months;
(2) propagation and subculture are cultivated: initial protocorm is transferred on proliferated culture medium and breeds cultivation, every surrounding, by after the initial protocorm after propagation or protocorms cutting, carrying out the subculture cultivation, make protocorm or protocorms fast breeding.
The inducing culture complicated component that the method adopts, the condition of inducing cultivation is also comparatively loaded down with trivial details and the cycle is long, key be the method resulting be protocorm, do not possess the germplasm feature consistency that clonal reproduction possesses.Up to now, the spring stem of noble dendrobium induces the research of protocorms regeneration to there is not yet successful report by the plant material.
Summary of the invention
The invention provides a kind of abductive approach of spring stem of noble dendrobium protocorms, the spring stem of noble dendrobium protocorms proliferate efficiency that this abductive approach obtains is high, can reach 4.57 times/month.
A kind of abductive approach of spring stem of noble dendrobium protocorms comprises:
(1) tender tissue of getting spring stem of noble dendrobium plant, as explant, is induced and is obtained aseptic seedling;
(2) get the aseptic seedling stem foot without the leaf leaf stem section, be inoculated in inducing culture and induced cultivation after ultrasonic is processed, obtain described spring stem of noble dendrobium protocorms.
What the present invention chose the aseptic seedling stem foot is induced material without the leaf leaf stem section, and adopts ultrasonic to process the generation of helper-inducer protocorms.The aseptic seedling stem foot is low without leaf leaf stem section maturity, is easy to form callus and further is divided into protocorms.
Protocorms abductive approach of the present invention is to take the tender tissue of spring dendrobe cultivation kind Dendrobium Sanya to build as starting material, the tender tissue maturity is low, be easy to dedifferentiation and be regenerated as aseptic seedling, described tender tissue can be selected stem apex or axillalry bud.
Select the aseptic seedling stem foot not select stem apex or axillalry bud without the leaf leaf stem section be induced material, because of the material without the bud growing point under certain condition, easier dedifferentiation forms callus and further is divided into protocorms, and stem apex or axillalry bud material easily directly form new plant from the bud growing point.
As preferably, the described length without the leaf leaf stem section is 2.5～3.5mm.As further preferred, the described length without the leaf leaf stem section is 3mm.Under the sterile water soaking state, carry out the ultrasonic processing without the leaf leaf stem section.Not adopting under the ultrasonic treatment conditions, all do not obtain protocorms.
When ultrasonic is processed, the small complex in water is activated under hyperacoustic effect, and these complexs become cavitation bubble; Cavitation bubble concussion, growth, shrink, collapse, produce HTHP and torrent make membrane perforation in the moment of collapse, cell is caused to damage to a certain degree, thereby the wound responsing reaction of activated cell obtains protocorms.
As preferably, the intensity that described ultrasonic is processed is 40KHz, and the time is 12min.Processing time is long easily causes excessive injury to induced material, affects the inductivity of protocorms.
After ultrasonic is finished dealing with, induced material is inoculated in inducing culture and is induced cultivation.As preferably, described inducing culture is for containing 0.3～0.4mg/L6-benzyl aminoadenine (6-BA), 0.04～0.06mg/L phenyl thiadiazolyl group urea (TDZ), the solid 1/2MS medium of 145～155mL/L Coconut Juice.As further preferably, described inducing culture is for containing 0.2mg/L6-benzyl aminoadenine, 0.05mg/L phenyl thiadiazolyl group urea, the solid 1/2MS medium of 150mL/L Coconut Juice.
The described condition of cultivation of inducing is: cultivation temperature 25-28 ℃, intensity of illumination 6000-12000lx incubation time is 1～2 month.Cultivate on described inducing culture after 1～2 month and can obtain described protocorms.The protocorms obtained can be used for the acceptor material of transgenic breeding, also can directly through propagation and subculture, cultivate acquisition spring stem of noble dendrobium plant.
The present invention also provides a kind of spring stem of noble dendrobium quick breeding method for tissue culture, comprises inducing cultivation, propagation to cultivate and subculture is cultivated, and described method of inducing cultivation is the abductive approach of described spring stem of noble dendrobium protocorms.
As preferably, it is the liquid MS medium that contains 0.1～0.3mg/L6-benzyl aminoadenine that described propagation is cultivated the proliferated culture medium adopted.As further preferred, described proliferated culture medium is the liquid MS medium that contains 0.2mg/L6-benzyl aminoadenine.Breed cultivation and can obtain the ready-made protocorms rate of increase on this proliferated culture medium, be about 4.57 times/month.
When carrying out large-scale production, also can adopt the solid 1/2MS medium that contains 0.2mg/L6-benzyl aminoadenine, proliferate efficiency is higher (4.35 times/month) and cost-saving also.
Adopt solid 1/2MS medium containing 0.8～1.2mg/L6-benzyl aminoadenine, 0.1～0.3mg/L methyl α-naphthyl acetate (NAA) to promote protocorms to change into minute to sprout and carry out the propagation of bud thereafter, and then adopt the solid 1/2MS medium that contains 1.8～2.2mg/L6-benzyl aminoadenine, 0.1～0.2mg/L methyl α-naphthyl acetate (NAA) to organize taking root and strong seedling culture of training seedling, finally organize hardening and the transplant survival of training seedling, complete whole spring stem of noble dendrobium tissue-culturing quick-propagation process.
Compared with prior art, beneficial effect of the present invention is embodied in:
(1) to take spring stem of noble dendrobium aseptic seedling stem foot be induced material without the leaf leaf stem section in the present invention, and adopt ultrasonic to process the helper-inducer protocorms to produce, for spring dendrobe tissue culture fast breeding technique provides new important channel;
(2) protocorms obtained has not only kept the merit of former kind, and proliferate efficiency is high, is 2～3 times of adventitious buds proliferation efficiency, on producing, has great using value;
(3) protocorms can be used as the acceptor material of spring stem of noble dendrobium transgenic breeding, and Multiple Buds can not, thereby can obtain the transformed variety of more spring stems of noble dendrobium by protocorms.
The accompanying drawing explanation
Fig. 1 is the aseptic seedling of growing on minimal medium;
Fig. 2 is the protocorms of growing on inducing culture;
Fig. 3 is the protocorms of breeding on liquid MS medium;
Fig. 4 is the protocorms of breeding on solid 1/2MS medium;
Fig. 5 is the group training seedling obtained by the protocorms differentiation;
Fig. 6 is the spring stem of noble dendrobium plantlet that the group training seedling growth in Fig. 5 obtains.
Embodiment
Embodiment 1
A kind of spring stem of noble dendrobium quick breeding method for tissue culture comprises:
(1) select the stem apex of spring dendrobe cultivation kind Dendrobium Sanya plant or axillalry bud as explant, induce and obtain aseptic seedling;
By stem apex or with the stem section of axillalry bud after flowing water rinses, process 7～10min with 70% Ethanol Treatment 1min, 0.1% mercuric chloride, in the access minimal medium, induce under 25-28 ℃ of illumination (6000-12000lx) condition and cultivate approximately 30 days, obtain aseptic seedling (as shown in Figure 1);
Minimal medium is the solid 1/2MS medium containing 1.0mg/L6-BA, 0.2mg/L NAA;
(2) get the aseptic seedling stem foot without the leaf leaf stem section, after ultrasonic is processed, be inoculated in inducing culture, induce under 25-28 ℃ of illumination (6000-12000lx) condition and cultivate 30-60 days, obtain protocorms (as shown in Figure 2);
What cut 3mm aseptic seedling stem foot is induced material without the leaf leaf stem section, under the sterile water soaking state, through the 40KHz ultrasonic, processes 12min, is connected in inducing culture, induces and obtains protocorms;
Inducing culture is the solid 1/2MS medium containing 0.2mg/L6-BA, 0.05mg/L TDZ;
(3) protocorms is proceeded in proliferated culture medium, under 25-28 ℃ of illumination (6000-12000lx) condition, propagation is cultivated 30 days;
Proliferated culture medium is the liquid MS medium containing 0.2mg/L6-BA, or contains the solid 1/2MS medium of 0.2mg/L6-BA;
While by the liquid MS medium containing 0.2mg/L6-BA, breeding cultivation, the rate of increase of protocorms is 4.57 times/month (as shown in Figure 3); While by the solid 1/2MS medium containing 0.2mg/L6-BA, breeding cultivation, the rate of increase of protocorms is 4.35 times/month (as shown in Figure 4);
(4) after propagation has been cultivated, protocorms is proceeded to mitogenetic medium, under 25-28 ℃ of illumination (l6000-12000lx) condition, cultivation is 25 days, impels protocorms to change into minute to sprout and carry out the propagation of bud;
Mitogenetic medium is the solid 1/2MS medium containing 1.0mg/L6-BA, 0.2mg/L NAA;
(5) after the indefinite bud of protocorms grows up to the group training seedling (as shown in Figure 5) of high about 10cm, it is transferred in root media, 25-28 ℃ of illumination (6000-12000lx) is lower cultivates 30 days, is taken root and strong seedling culture;
Root media is the solid 1/2MS medium containing 2.0mg/L6-BA, 0.1mg/L NAA;
(6) will organize the training transplantation of seedlings to land for growing field crops after hardening, obtain spring stem of noble dendrobium Dendrobium Sanya plantlet (as shown in Figure 6).
Embodiment 2
Utilize the method identical with embodiment 1, but step (2) is processed without ultrasonic.Do not induce the acquisition protocorms.
Embodiment 3
Utilize the method identical with embodiment 1, but step (2) adopts the 40KHz ultrasonic to process 15min.In one week, material is almost all dead, does not obtain protocorms.
Embodiment 4
Utilize the method identical with embodiment 1, but step (2) adopts the 40KHz ultrasonic to process 5min.Material is all brown stains in 40d, do not obtain protocorms.
Embodiment 5
Utilize the method identical with embodiment 1, but using the stem apex of the long spring dendrobe cultivation kind Dendrobium Sanya plant of 3mm or with the stem section of axillalry bud as induced material, carry out inducing of protocorms.Result has no equally protocorms and generates, and material all grows up to new plantlet.
Claims (9)
1. the abductive approach of a spring stem of noble dendrobium protocorms comprises:
(1) tender tissue of getting spring stem of noble dendrobium plant, as explant, is induced and is obtained aseptic seedling;
(2) get the aseptic seedling stem foot without the leaf leaf stem section, be inoculated in inducing culture and induced cultivation after ultrasonic is processed, obtain described spring stem of noble dendrobium protocorms.
2. abductive approach as claimed in claim 1, is characterized in that, described tender tissue is stem apex or axillalry bud.
3. abductive approach as claimed in claim 1, is characterized in that, the described length without the leaf leaf stem section is 2.5～3.5mm.
4. abductive approach as claimed in claim 1, is characterized in that, the intensity that described ultrasonic is processed is 40KHz, and the time is 12min.
5. abductive approach as claimed in claim 1, is characterized in that, described inducing culture is for containing 0.3～0.4mg/L6-benzyl aminoadenine, 0.04～0.06mg/L phenyl thiadiazolyl group urea, the solid 1/2MS medium of 145～155mL/L Coconut Juice.
6. abductive approach as claimed in claim 5, is characterized in that, described inducing culture is for containing 0.2mg/L6-benzyl aminoadenine, 0.05mg/L phenyl thiadiazolyl group urea, the solid 1/2MS medium of 150mL/L Coconut Juice.
7. abductive approach as claimed in claim 1, is characterized in that, the described condition of cultivation of inducing is: cultivation temperature 25-28 ℃, and intensity of illumination 6000-12000lx, incubation time is 1～2 month.
8. a spring stem of noble dendrobium quick breeding method for tissue culture, comprise and induce cultivation, propagation to cultivate and subculture is cultivated, and it is characterized in that, the abductive approach that described method of inducing cultivation is the arbitrary described spring stem of noble dendrobium protocorms of claim 1～7.
9. spring stem of noble dendrobium quick breeding method for tissue culture as claimed in claim 8, is characterized in that, it is the liquid MS medium that contains 0.1～0.3mg/L6-benzyl aminoadenine that described propagation is cultivated the proliferated culture medium adopted.
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Cited By (3)
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| CN104351053A (en) * | 2014-11-12 | 2015-02-18 | 柳州市天姿园艺有限公司 | Seed seedling method of dendrobium officinale using stem tips as explants to breed |
| CN111955331A (en) * | 2020-07-31 | 2020-11-20 | 深圳市仙湖植物园管理处（深圳市园林研究中心） | Culture method and induction culture medium for artificially inducing dragonfly dendrobium to bloom |
| CN115530075A (en) * | 2022-10-24 | 2022-12-30 | 吉首大学 | Artificial efficient propagation method of dendrobium nobile |
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Application publication date: 20131204 |