CN104885934B - Method adopting pseudobulb to induce regeneration and propagation of malaxis microtatantha plants - Google Patents
Method adopting pseudobulb to induce regeneration and propagation of malaxis microtatantha plants Download PDFInfo
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- CN104885934B CN104885934B CN201510234271.8A CN201510234271A CN104885934B CN 104885934 B CN104885934 B CN 104885934B CN 201510234271 A CN201510234271 A CN 201510234271A CN 104885934 B CN104885934 B CN 104885934B
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Abstract
The invention belongs to a rapid propagation method for important plant materials in rare and endangered wild plants, and particularly relates to a wild orchid tissue culture method. The method comprises material selection and disinfection, induction culture, propagation culture, root induction and transplant. The invention aims to provide a method by adopting pseudobulb to induce regeneration and propagation of malaxis microtatantha plants, and to provide technical support for promoting the study and application of rare orchid species. The method is high in propagation coefficient and high in rooting percentage; plants are short in growth time, good in growth and tidy; the seedling cultivation process is greatly shortened, and the cost is significantly lowered. The method also provides technical support for malaxis microtatantha protection as well as large-scale and industrialized production, popularization and application.
Description
Technical field
The invention belongs to important vegetable material method for quickly breeding in wild plant in imminent danger, more particularly to wild orchid
Method for tissue culture.
Background technology
Little natural pond is bluem.microtatantha (schlechter) szlachetko, orchid family, Chinese unique wheat, country
Level protection plant (cites ii level), herbelet is given birth on ground, and pseudobulb is little, and avette or subsphaeroidal, 1 piece of leaf, close to floor file, ovum
, to width egg shape, scape is upright for shape, and very thin, Chang Zise slightly flattens, the very narrow wing of both sides tool；12 centimetres of raceme length, generally
Have 10 20 flowers, flower very little, yellow, April at florescence, be distributed in In Central Jiangxi (east is solid), Fujian (Wuyi Mountain, Yongfu, Liaanjiang county)
With east of Taiwan (Taroko Gorge).It is born on sylvan life or the rock at dark and damp place, 200 600 meters of height above sea level.With liver moss symbiosis, to environment
Condition require rather harsh, typically uninhabited, air quality is good, vegetation is luxuriant, the environment had abundant water resources just is distributed,
There is certain indicative significance for environmental condition.In view of this plant, build is less, such as non-be in the florescence it is more difficult to identification, and also exist
Naturally procreation ability, Spreading and diffusion is indifferent, the problems such as natural renovation is difficult, causes good seed rare.Little natural pond orchid plant
Regeneration contributes to solving scarcity of resources, alleviates the pressure of wild resource subject to severe risks of damage, limited wild effectively to protect
Resource is to promote the research of rare cymbidium variety, utilize the research of the rare cymbidium variety of promotion, using offer technical support, induction
Little natural pond orchid bulb plant regeneration has no report.
Content of the invention
It is an object of the present invention to provide a kind of method of employing pseudobulb induction little natural pond orchid plant regeneration and breeding.Precious for promoting
The research of dilute cymbidium variety, utilization provide technical support.
The method of the present invention is achieved in that
A kind of employing pseudobulb induction little natural pond orchid plant regeneration and the method for breeding, specifically comprise the following steps that
1) material selection and sterilization: the band leaf pseudobulb plant that the same day is taken is wrapped with wet gauze, takes back rearmounted flowing water
Under rinse well, then disinfection, for inoculating.
2) Fiber differentiation: rear material will be disinfected, blot through filter paper to keep flat after surface moisture and be inoculated in through high temperature, height
In the bulb bud inducement cultivation base of pressure sterilizing；Condition of culture: culture room temperature is 23 ± 2 DEG C, light application time 5h/d, intensity of illumination
500～1000lx；Blade engender white and gradually death, pseudobulb gradually greening, grow up；
3) Multiplying culture: will be through above-mentioned Fiber differentiation, obtained well-grown diameter grows into 0.1-0.3 cm's
Pseudobulb, is seeded in proliferated culture medium, light application time 5h/d, intensity of illumination 1000～1300lx；
4) root induction: the Multiplying culture pseudobulb unrooted seedling individual plant with vanelets out is transferred to and taken root
In culture medium；Light application time 6h/d, intensity of illumination 1300～1500lx；
5) transplant: when root induction culture test tube seedling grows to root 1～3, during 1, leaf, test tube seedling is placed on natural light
Lower refining seedling 5～7 days, opens bottle cap hardening 1-2 days；Then take out cultivating seedling, wash away the culture medium being attached on root system, move into water
In the grass and fertile soil mass ratio mixed-matrix for 1:2, moisturizing is sheltered from heat or light, and (at 15～25 DEG C, humidity should be maintained at temperature control
75%～85%), obtain the complete regenerated plant of robust growth.
2. culture medium:
(1) bulb bud inducement cultivation base vw+1.5 mg/l 6-ba+1.0 mg/l kt+0.3mg/l naa+ 1.5g/l
Peptone+25g/l su+6.5g/l ag+1.5 g/l ac+10 g/l banana+60 g/l potato, ph value is 5.6；
(2) proliferated culture medium vw+2.0mg/l 6-ba+1.0 mg/l kt+ 0.5mg/l naa+2g/l peptone+
1g/l vb6+ 20 g/l banana+70 g/l potato+25g/l su+6.5g/l ag+1.5 g/l ac, ph value is 5.6；
(3) root media 1/2vw+1.0 mg/l iba+0.1 mg/l naa+1.5 g/l vb6+25g/l su+
6.5g/l ag+1.5 g/l ac, ph value is 5.6；
Above is referred to: 1, ag is (abbreviation of agar culture medium solidifying agent and holder agar powder)
2nd, su is (abbreviation that sugar provides plant carbon source and maintains osmotic pressure sucrose)
3rd, maturity is 80% banana (without various process of accelerating the ripening)
4th, potato (for commercially available fresh food potato, the poor kind of preferred starch).
Present invention is disclosed a kind of little natural pond orchid bulb plant regeneration and quick breeding technology, through 20-30d Fiber differentiation,
50-70d Multiplying culture, 25-35d root induction, moisturizing is sheltered from heat or light, and survival rate is up to more than 95%.Plant adaptability after 1-2 week
Gradually strengthen, obtain the complete regenerated plant of robust growth.
The remarkable advantage of the present invention:
One: existing resource can be applied to greatest extent by the inventive method, by animal nutrition in the short time
The interior application demand solving scarce resource；
Two: contribute to solving scarcity of resources, the pressure of alleviation wild resource subject to severe risks of damage, effectively protect limited
Wild resource, for promoting the research of rare cymbidium variety, using offer technical support.
Three: relevant induction little natural pond orchid bulb plant regeneration and propagation method have no report.
Four: little natural pond orchid pseudobulb cultivate on bulb bud inducement cultivation base after gradually greening, grow up, growth coefficient is high, give birth to
Root rate is high, and the demand of plant growth time is short, grows fine, and neat and consistent substantially reduces nursery stock incubation, has saved a large amount of
Cost.For the protection that little natural pond is blue, and scale, industrialization production popularization and application provide technical support.
Five: strong innovation of the present invention, with high content of technology, public welfare is strong, for the research, the profit that promote rare cymbidium variety
With, have good prospect and and its significance, provide theoretical foundation and technical support for developing this kind of resource.
Specific embodiment
Embodiment 1
A kind of employing pseudobulb induction little natural pond orchid plant regeneration and the method for breeding, specifically comprise the following steps that
1) material selection and sterilization: band pseudobulb plant 1, will be taken the same day and wrapped with wet gauze, and take back laboratory rearmounted
Under flowing water, dust on plant and plant attachment are rinsed well；2nd, plant is disinfected: leaf pseudobulb will be carried after rinsing to plant
Strain is put in saturation bleaching powder supernatant and is soaked 10min, and is carefully pressed from both sides except close bulb base portion impurity and be carried out with little son of taking the photograph
Afterwards, lower punching 20 min of running water, distilled water dashes 2 times, puts in superclean bench and removes alcohol with after 75% alcohol disinfecting 30s,
Add 0.1% mercuric chloride (hgcl2) process 8min, mercury solution is poured into useless mercury bottle, sterilized water dashes 3 times, you can with inoculation.
2) Fiber differentiation: material will be disinfected, blot the band leaf pseudobulb plant after surface moisture through filter paper and entirely put down
Put and be inoculated in through in high temperature, autoclaved bulb bud inducement cultivation base；Condition of culture: culture room temperature is 23 ± 2 DEG C, illumination
Time 5h/d, intensity of illumination 800lx, incubation time 20d；Blade engenders white gradually death, and pseudobulb gradually adapts to
Culture medium that special design is prepared and after cultivating on this culture medium gradually greening, grow up；
3) Multiplying culture: will be through above-mentioned Fiber differentiation, obtained well-grown diameter grows into the pseudobulb of 0.2 cm,
It is seeded in respectively in proliferated culture medium, light application time 5h/d, intensity of illumination 1200lx, Multiplying culture time 55d；
4) root induction: the subculture Fiber differentiation pseudobulb unrooted seedling individual plant tap with vanelets out turns
Move on in root media；Light application time 6h/d, intensity of illumination 1400lx, root induction time 25d；
5) test tube seedling culture completes: when root induction culture test tube seedling grows to root 1, completes to induce little natural pond during 1, leaf
Blue bulb plant regeneration simultaneously can carry out Periodical Reproduction culture；
6) culture of rootage test tube seedling will be completed and be placed on natural light lower refining seedling 6 days, open bottle cap hardening 1 day, to strengthen test tube
The adaptability to outdoor environment for the seedling；Then take out cultivating seedling with taking the photograph son from blake bottle, wash away the culture being attached on root system
Base, moves in the matrix that pasture and water and fertile soil (1:2) mix, moisturizing is sheltered from heat or light (temperature control is at 20 DEG C), and humidity is maintained at 80%,
Avoid direct sunlight, survival rate is up to more than 96%.After 1 week, plant adaptability gradually strengthens, obtain robust growth complete again
Raw plant.
2. culture medium:
Culture medium is prepared: culture medium thickness are 1.5cm；
(1) bulb bud inducement cultivation base vw+1.5 mg/l 6-ba+1.0 mg/l kt+0.3mg/l naa+ 1.5g/l
Peptone+25g/l su+6.5g/l ag+1.5 g/l ac+10 g/l banana+60 g/l potato, ph value is 5.6；
(2) proliferated culture medium vw+2.0mg/l 6-ba+1.0 mg/l kt+ 0.5mg/l naa+2g/l peptone+
1g/l vb6+ 20 g/l banana+70 g/l potato+25g/l su+6.5g/l ag+1.5 g/l ac, ph value is 5.6；
(3) root media 1/2vw+1.0 mg/l iba+0.1 mg/l naa+1.5 g/l vb6+25g/l su+
6.5g/l ag+1.5 g/l ac, ph value is 5.6；
Above is referred to:
1st, ag is (abbreviation of agar culture medium solidifying agent and holder agar powder)
2nd, su is (abbreviation that sugar provides plant carbon source and maintains osmotic pressure sucrose)
3rd, maturity is 80% banana (without various process of accelerating the ripening)
4th, potato (for commercially available fresh food potato)
5th, ac is activated carbon.
Claims (1)
1. a kind of employing pseudobulb induction little natural pond orchid plant regeneration and breeding method it is characterised in that: the method includes following
Step:
1) material selection and sterilization: the band leaf pseudobulb plant that the same day is taken is wrapped with wet gauze, takes back rearmounted flowing water undershoot
Wash clean, then disinfection, for inoculating；
2) Fiber differentiation: rear material will be disinfected, blot through filter paper to keep flat after surface moisture to be inoculated in and go out through high temperature, high pressure
In the bulb bud inducement cultivation base of bacterium；Condition of culture: culture room temperature is 23 ± 2 DEG C, light application time 5h/d, intensity of illumination 500
～1000lx；Blade engender white and gradually death, pseudobulb gradually greening, grow up；
3) Multiplying culture: will be through above-mentioned Fiber differentiation, obtained well-grown diameter grows into the false squama of 0.1-0.3 cm
Stem, is seeded in proliferated culture medium, light application time 5h/d, intensity of illumination 1000～1300lx；
4) root induction: the Multiplying culture pseudobulb unrooted seedling individual plant with vanelets out transfers to culture of rootage
In base；Light application time 6h/d, intensity of illumination 1300～1500lx；
5) transplant: when root induction culture test tube seedling grows to root 1～3, during 1, leaf, test tube seedling is placed on refining under natural light
Seedling 5～7 days, opens bottle cap hardening 1-2 days；Then take out cultivating seedling, wash away the culture medium being attached on root system, move into pasture and water and
Fertile soil mass ratio is that in the mixed-matrix of 1:2, moisturizing is sheltered from heat or light, temperature control at 15～25 DEG C, humidity should be maintained at 75%～
85%, obtain the complete regenerated plant of robust growth；
Described bulb bud inducement cultivation base: vw+1.5mg/l 6-ba+1.0mg/l kt+0.3mg/l naa+1.5g/l peptone
+ 25g/l su+6.5g/l ag+1.5g/l ac+10g/l banana+60 g/l potato, ph value is 5.6；
Described proliferated culture medium: vw+2.0mg/l 6-ba+1.0mg/l kt+0.5mg/l naa+2g/l peptone+1g/l vb6+
20g/l banana+70g/l potato+25g/l su+6.5g/l ag+1.5g/l ac, ph value is 5.6；
Described root media: 1/2vw+1.0mg/l iba+0.1mg/l naa+1.5g/l vb6+25g/l su+6.5g/l ag
+ 1.5g/l ac, ph value is 5.6；
Wherein, described ag is agar culture medium solidifying agent and the abbreviation of holder agar powder；Su for sugar provide plant carbon source and
Maintain the abbreviation of osmotic pressure sucrose；Ac is activated carbon.
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| CN104885934B true CN104885934B (en) | 2017-01-18 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102599063A (en) * | 2012-04-09 | 2012-07-25 | 向华 | Rapid propagation method of Bletilla striata |
| CN103299903A (en) * | 2013-05-23 | 2013-09-18 | 广西壮族自治区中国科学院广西植物研究所 | Efficient bletilla propagation method using pseudobulbs to induce pseudobulbs |
-
2015
- 2015-05-11 CN CN201510234271.8A patent/CN104885934B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102599063A (en) * | 2012-04-09 | 2012-07-25 | 向华 | Rapid propagation method of Bletilla striata |
| CN103299903A (en) * | 2013-05-23 | 2013-09-18 | 广西壮族自治区中国科学院广西植物研究所 | Efficient bletilla propagation method using pseudobulbs to induce pseudobulbs |
Non-Patent Citations (2)
| Title |
|---|
| 培养基与光照对沼兰种子非共生萌发的影响;王瑞霞等;《植物生态学报》;20101231;第34卷(第4期);第438-443页 * |
| 独花兰组织培养与快速繁殖（简报）;高丽等;《亚热带植物科学》;20101231;第39卷(第3期);第79页第2-6段 * |
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