CN107750949B - Utilize the method for stem-tip tissue breeding hybrid cymbidium seedling - Google Patents
Utilize the method for stem-tip tissue breeding hybrid cymbidium seedling Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The present invention provides the methods using stem-tip tissue breeding hybrid cymbidium seedling, and the method includes the selections of explant；Lateral bud disinfection；Protocorm Fiber differentiation；Protocorm Multiplication culture；Protocorm differentiation culture and Rooting and hardening-off culture.Induction period of the present invention improves protocorm inductivity；Multiplicative stage reduces protocorm aberration rate and glass rate；Differential period reduces bud aberration rate, and the development of strong plantlets and rootage stage seedling early growth is more healthy and stronger, and new root entangling ratio reduces, and root system more disperses and stretches, while seedling aberration rate reduces.
Description
Present patent application be the applying date be on July 22nd, 2015, application No. is 2015104339758, entitled one
Kind efficiently breeds the divisional application of the Chinese patent application of the method for hybrid cymbidium seedling using stem-tip tissue.
Technical field
The present invention relates to a kind of method using stem-tip tissue breeding hybrid cymbidium seedling, in particular to one kind can make to plant
Seedling growth and development is more healthy and stronger, and new root entangling ratio reduces, the method for tissue culture that seedling aberration rate reduces.
Background technique
Hybrid cymbidium (Cymbidium hubridum) is to grow nonparasitically upon another plant to plant as parent through excessive generation with big flower pattern some in Cymbidium
Big, bright in luster, robust growth the excellent variety group of the flower pattern that cross selection cultivates.Hybrid cymbidium posture is graceful, and leaf is long
Dark green, flower appearance is rough, and the florescence up to 2-3 months, is epochmaking decorative indoor plant, is world-renowned " orchid nova ",
Have both state orchid delicate fragrance elegance and cattleya it is rich and varied, it is in very great demand in International Flower market, it is deep by people's all over the world
Like.
Currently, mainly passing through protocorm and Multiple Buds using the stem apex of virus-free healthy and strong maternal plant lateral bud as explant in production
Approach mass propagation hybrid cymbidium seedling.The factorial production hybrid cymbidium seedling is primarily present following four problem:
First is that, if stem-tip tissue is too big, disinfection is often not thorough, sterilizes successfully when the stem apex using lateral bud is bred
Rate is extremely low, while stem-tip tissue absorption nutriment is slower, and browning is easy during culture.If stem-tip tissue is too small, viability is big
Big to reduce, late stage of culture is easily dead, causes protocorm inductivity lower.
Second is that hybrid cymbidium multiplicative stage protocorm easily makes a variation, vitrification phenomenon is more serious, and this protocorm later period is difficult
With Bud Differentiation.
Third is that hybrid cymbidium differential period a part of protocorm can gradually ossify, it is mainly shown as that base portion pseudobulb can be different
It is often loose, undifferentiated leaf, or i.e. enabled differentiation leaf, but blade is smaller, is unable to further growth development, leads to protocorm differentiation
Bud mutation it is different, generate largely rigid seedling.
Fourth is that hybrid cymbidium strong plantlets and rootage stage seedling aberration rate is higher, seedling is not healthy and strong enough, can generate more inhomogeneity
Too long bamboo pole seedling between the variation seedling of type, such as blade alternate or the cross seedling for arrangement of growing thickly, stipes.In addition, seedling is new
Root entangling ratio is high, is not easy to separate, and is unfavorable for the transplanting domestication of later period greenhouse.
Summary of the invention
The present invention is directed to disadvantage mentioned above, carries out large-scale technological innovation to it, provides a kind of utilization stem-tip tissue breeding
The method of hybrid cymbidium seedling, optimizes explant sterilization method and explant selects cutting technique, designs efficient induction, increases
It grows, break up, the culture medium prescription in strong plantlets and rootage stage, improving stem-tip tissue disinfection success rate, protocorm inductivity to reach；
Reduce protocorm aberration rate and glass rate；Reduce Bud Differentiation aberration rate；So that seedling early growth develops more healthy and stronger, new root entangling
Ratio reduces, and root system more disperses and stretches, while the purpose that seedling aberration rate reduces.
To achieve the above object, the present invention takes following technical proposals to realize:
A method of hybrid cymbidium seedling is bred using stem-tip tissue, comprising the following steps:
Step a, the selection of explant cut the healthy and strong lateral bud for the 6-12cm that hybrid cymbidium maternal plant base portion is grown；
Step b, lateral bud disinfection, carries out disinfection to the lateral bud cut, cuts explant of the stem-tip tissue as culture；
Step c, protocorm Fiber differentiation, explant is inoculated into protocorm induction medium, is cultivated 30-60 days, training
Supporting temperature is 25-28 DEG C, light application time 8-14h/d, intensity of illumination 1000-2000lux, and induction obtains protocorm；It is described
The ingredient of induced medium includes: to spend treasured No. 3 1-3g/L, caseinhydrolysate 2-4g/L, adenine 0.5-1.5g/L, active carbon
0.5-1.5g/L, agar 6-8g/L, sucrose 15-24g/L；
The protocorm group of induced synthesis is cut into fritter by step d, Protocorm Multiplication culture, and every piece contains 3-5 protocorm
Stem is inoculated into proliferated culture medium, gradually forms new protocorm, repeatedly cutting proliferation, and every 30-40 days subcultures switching is primary；
The ingredient of the proliferated culture medium is identical as Fiber differentiation based component；
The protocorm group that multiplicative stage is formed is cut into fritter by step e, protocorm differentiation culture, and every piece containing 5-10
Protocorm is inoculated into differential medium and carries out differentiation culture, and protocorm differentiation goes out young leaves new root；The differential medium at
Dividing includes: to spend treasured No. 3 1-3g/L, caseinhydrolysate 2-4g/L, adenine 0.4-1.0g/L, TDZ (Thidiazuron) 0.05-
0.15mg/L, active carbon 0.5-1.5g/L, agar 5-9g/L, sucrose 13-26g/L.
Step f, Rooting and hardening-off culture, cut differentiation culture after high 5-6cm bud, be inoculated into Rooting and hardening-off culture base into
Row culture, obtains the seedling of robust growth, wherein the ingredient of Rooting and hardening-off culture base includes: to spend No. 1 1-4g/L of treasured, potassium nitrate
0.7-2.1g/L, ferrous sulfate heptahydrate 18-32mg/L, two water disodium ethylene diamine tetraacetate 30-43g/L, caseinhydrolysate 2-4g/
L, adenine 0.5-1.5g/L, mashed potatoes 12-28g/L, banana puree 35-65g/L, coconut milk 50-150mL, active carbon 0.5-
1.5g/L, agar 6-8g/L, sucrose 14-27g/L.
Further, in step b, the method that carry out disinfection to lateral bud the following steps are included:
The lateral bud cut is rinsed well with flowing water, peels off lateral bud outer blade, cutting side bastem portion lignifying group layer by layer
It knits, until being left 6-8 piece leaf, continues to be rinsed well with flowing water, then sterilize 15min with 12% calcium hypochlorite, use blotting paper
Surface moisture is blotted, takes and is further sterilized on aseptic working platform；
Continue to peel off stem apex outer blade, until being left 3-5 piece leaf, then sterilizes 12min, nothing with 5% calcium hypochlorite
Bacterium water rinses 3-5 times；
Continue to shell outer blade, until being left 1-2 piece leaf, then sterilizes 6min, aseptic water washing 3- with 1% calcium hypochlorite
5 times, surface moisture is blotted with blotting paper；
The spire of growing point outer layer is carefully peelled off in face under the microscope, until exposing growing point；
Cut the stem-tip tissue containing growing point, about 2-3mm size, the explant as culture.
Further, the stem-tip tissue containing growing point is uniformly cut into 4 fritters (cross cutting method) or 2 fritters (one from center
It is divided into two methods), every piece is individually inoculated into induced medium.
Further, the ingredient of the induced medium and proliferated culture medium includes: to spend treasured No. 3 2g/L, caseinhydrolysate 3g/
L, adenine 1g/L, active carbon 1g/L, agar 7g/L, sucrose 20g/L.
Further, the ingredient of the differential medium includes: to spend treasured No. 3 2g/L, caseinhydrolysate 3g/L, adenine 1g/
L, TDZ0.1mg/L, active carbon 1g/L, agar 7g/L, sucrose 20g/L.
Further, the ingredient of Rooting and hardening-off culture base includes: to spend No. 1 1-4g/L of treasured, potassium nitrate 0.7-2.1g/L, seven water sulphur
Sour ferrous iron 18-32mg/L, two water disodium ethylene diamine tetraacetate 30-43mg/L, caseinhydrolysate 2-4g/L, adenine 0.5-
1.5g/L, mashed potatoes 12-28g/L, banana puree 35-65g/L, coconut milk 100ml/L, active carbon 0.5-1.5g/L, agar 6-8g/
L, sucrose 14-27g/L.
Preferably, the ingredient of Rooting and hardening-off culture base includes: to spend No. 1 2g/L of treasured, potassium nitrate 1.5g/L, ferrous sulfate heptahydrate
27.8mg/L, two water disodium ethylene diamine tetraacetate 37.3mg/L, caseinhydrolysate 3g/L, adenine 1g/L, mashed potatoes 20g/L,
Banana puree 50g/L, coconut milk 100ml/L, active carbon 1g/L, agar 7g/L, sucrose 20g/L.
In short, the invention has the benefit that
Cymbidium grandiforium cultural method provided by the invention, firstly, multiplicative stage, the culture prepared using the present invention
Base can significantly reduce protocorm aberration rate, glass rate.
Differential period, the culture medium prepared using the present invention can significantly reduce the aberration rate of Bud Differentiation, reduce the seedling number that ossifys
Amount.
Strong plantlets and rootage stage, the culture medium prepared using the present invention can make seedling early growth develop more healthy and stronger, new root entangling
Ratio reduces, and root system more disperses and stretches, while reducing seedling aberration rate.
Further, using sterilization method of the invention and explant selection cutting technique can be improved stem-tip tissue disinfection at
Power, while increasing stem-tip tissue and culture medium contact area, promote absorption of nutrient ingredients, reduce melting brown rate, improves protocorm
Inductivity.
Specific embodiment
The present invention will be described in detail combined with specific embodiments below.
Embodiment 1
The ingredient of induced medium and proliferated culture medium is as follows:
Spend treasured No. 3 2g/L, caseinhydrolysate 3g/L, adenine 1g/L, active carbon 1g/L, agar 7g/L, sucrose 20g/L.
Embodiment 2
The ingredient of induced medium and proliferated culture medium includes:
Spend treasured No. 3 1g/L, caseinhydrolysate 2g/L, adenine 0.5g/L, active carbon 0.5g/L, agar 6g/L, sucrose
15g/L。
Embodiment 3
The ingredient of induced medium and proliferated culture medium includes:
Spend treasured No. 3 3g/L, caseinhydrolysate 4g/L, adenine 1.5g/L, active carbon 1.5g/L, agar 8g/L, sucrose
24g/L。
Embodiment 4
The ingredient of differential medium includes:
Treasured No. 3 2g/L, caseinhydrolysate 3g/L, adenine 1g/L, TDZ0.1mg/L, active carbon 1g/L, agar 7g/L are spent,
Sucrose 20g/L.
Embodiment 5
The ingredient of differential medium includes:
Spend No. 3 1g/L of treasured, caseinhydrolysate 2g/L, adenine 0.4g/L, TDZ0.05mg/L, active carbon 0.5g/L, agar
5g/L, sucrose 13g/L.
Embodiment 6
The ingredient of differential medium includes:
Spend No. 3 3g/L of treasured, caseinhydrolysate 4g/L, adenine 1.0g/L, TDZ0.15mg/L, active carbon 1.5g/L, agar
9g/L, sucrose 26g/L.
Embodiment 7
The ingredient of Rooting and hardening-off culture base includes:
Spend No. 1 2g/L of treasured, potassium nitrate 1.5g/L, ferrous sulfate heptahydrate 27.8mg/L, two water disodium ethylene diamine tetraacetates
37.3g/L, caseinhydrolysate 3g/L, adenine 1g/L, mashed potatoes 20g/L, banana puree 50g/L, coconut milk 100ml/L, activity
Charcoal 1g/L, agar 7g/L, sucrose 20g/L.
Embodiment 8
The ingredient of Rooting and hardening-off culture base includes:
Spend No. 1 1g/L of treasured, potassium nitrate 0.7g/L, ferrous sulfate heptahydrate 18mg/L, two water disodium ethylene diamine tetraacetate 30mg/
L, caseinhydrolysate 2g/L, adenine 0.5g/L, mashed potatoes 12g/L, banana puree 35g/L, coconut milk 50ml/L, active carbon
0.5g/L, agar 6g/L, sucrose 14g/L.
Embodiment 9
The ingredient of Rooting and hardening-off culture base includes:
Spend No. 1 4g/L of treasured, potassium nitrate 2.1g/L, ferrous sulfate heptahydrate 32mg/L, two water disodium ethylene diamine tetraacetate 43mg/
L, caseinhydrolysate 4g/L, adenine 1.5g/L, mashed potatoes 28g/L, banana puree 65g/L, coconut milk 150ml/L, active carbon
1.5g/L, agar 8g/L, sucrose 27g/L.
Embodiment 10
First, cut the healthy and strong lateral bud for the 6-12cm that hybrid cymbidium maternal plant base portion is grown.
Second, the lateral bud cut is rinsed well with flowing water, peels off lateral bud outer blade layer by layer, cutting side bastem portion is wooden
Change tissue, until being left 6 or so leaves, continues to be rinsed well with flowing water, then sterilize 15min with 12% calcium hypochlorite, use
Blotting paper blots surface moisture, takes and further sterilizes on aseptic working platform；
Continue to peel off stem apex outer blade, until being left 3 or so leaves, then sterilize 12min with 5% calcium hypochlorite,
Aseptic water washing 3-5 times；
Continue to peel off outer blade, until remaining 1-2 piece or so leaf, then sterilizes 6min, sterile water with 1% calcium hypochlorite
It rinses 3-5 times, blots surface moisture with blotting paper；
The spire of growing point outer layer is carefully peelled off in face under the microscope, until exposing growing point；
The stem-tip tissue containing growing point is cut, the stem-tip tissue containing growing point is uniformly cut by about 2-3mm size from center
4 fritters (cross cutting method), every piece is individually inoculated into induced medium as explant.
Stem-tip tissue disinfection success rate can be improved using sterilization method of the invention.Meanwhile explant activity is kept, no
Lead to death as disinfecting time is too long.
Disinfection success rate can be made to reach 85% or more using sterilization method of the invention, be sterilized in preferred the present embodiment
Success rate can achieve 90% or more.
And the stem-tip tissue containing growing point is uniformly cut into 4 fritters from center after sterilizing, if being inhaled because explant is too big
It is slower to receive nutriment, browning is easy during culture.If explant is too small, late stage of culture is easily dead, leads to protocorm inductivity
It is lower.
Explant through the invention selects cutting technique, increases the contact area of explant and culture medium, nutrients
Matter assimilation effect is more preferable, can reduce explant browning rate, improves protocorm inductivity.Third, protocorm Fiber differentiation will be cut
Explant be inoculated into protocorm induction medium after, 10d or so starts to expand, and surface, which gradually induces, generates many light green colors
Dotted particle, 25d or so develops into green protocorm.Protocorm full grains, surface are covered with white fluff.The induction training
Support the culture medium in base selection example 1.Cultivation cycle is 30-60d, and cultivation temperature is 25-28 DEG C, light application time 8-14h/
D, intensity of illumination 1000-2000lux.Fiber differentiation based component and cultural method of the invention are sampled, protocorm inductivity is big
In 95%.
4th, the protocorm group of induced synthesis is cut into fritter by Protocorm Multiplication culture, and every piece contains 3-5 protocorm,
It is inoculated into proliferated culture medium, gradually forms new protocorm, repeatedly cutting proliferation；Every 30-40d subculture is transferred 1 time, is formed big
The protocorm of amount；The Multiplying culture based component is identical as Fiber differentiation based component, the culture medium in same selection example 1.
Condition of culture is identical as the Fiber differentiation stage.
Multiplicative stage normotrophic protocorm is light green, and rough and have tiny protrusion, short white is covered on surface
Villus, protocorm later period easy Bud Differentiation.
And the protocorm to make a variation is bottle green or blackish green, surface is smooth and glossy.Meanwhile glass occurs for some protocorms
Glass phenomenon, is transparent.The protocorm surface of variation is without white fluff, although the speed of growth is quickly, vigor is very low, the later period
Bud Differentiation ratio is low, or even is unable to Bud Differentiation.
And protocorm aberration rate, vitrifying can be significantly reduced using Multiplying culture based component and cultural method of the invention
Rate.Growth coefficient is between 4-5, and aberration rate is less than 3%, and glass rate is less than 1%, and aberration rate is less than in preferred the present embodiment
2%.
5th, the protocorm group that the multiplicative stage is formed is cut into fritter by protocorm differentiation culture, and every piece former containing 5-10
Bulb is inoculated into differential medium and carries out differentiation culture, and protocorm differentiation goes out young leaves new root；The differential medium selection is real
Apply the culture medium of example 4.Cultivation cycle is 40-60d, and cultivation temperature is 25-28 DEG C, light application time 8-14h/d, and intensity of illumination is
2000-3000lux。
Differential period, a part of protocorm can gradually ossify, show as base portion pseudobulb can aberrant mast, undifferentiated leaf, or
The i.e. enabled differentiation leaf of person, but blade is smaller, is unable to further growth development, causes the bud mutation of protocorm differentiation different, generates a large amount of
Rigid seedling.This seedling leaf is harder, surface is as dehydration, and the later period normally can not take root and develop.
Rigid seedling ratio can greatly be reduced using differential medium ingredient of the invention and cultural method, ossify seedling ratio
Between 2%-4%, it is preferred that less than 2%.
6th, the bud of plant height 5-6cm after differentiation culture is chosen, is inoculated into Rooting and hardening-off culture base and is cultivated, wherein
Rooting and hardening-off culture base selection example 7.Cultivation cycle is 60-80d, and cultivation temperature is 25-28 DEG C, light application time 8-14h/
D, intensity of illumination 2500-3000lux.
The hybrid cymbidium strong plantlets and rootage stage, seedling aberration rate was higher, and seedling is not healthy and strong enough, and a part of kind of seedling leaf is loosely not
It is straight and upright, fragile, plant is more slim and frahile.In addition, the strong plantlets and rootage stage can also generate two kinds of variation seedling, such as blade alternate
Or too long bamboo pole seedling between the cross seedling of arrangement of growing thickly, stipes.Using Rooting and hardening-off culture based component of the invention and culture
Method can greatly reduce the ratio of the variating seedlings such as cross seedling, bamboo pole seedling, and variating seedling rate is less than 3%.Moreover, kind seedling leaf is straight
Vertical, stem thickness is strengthened, plant type is tall and straight.Average plant height is 8-10cm, and the number of blade is greater than 4, and radical is greater than 4.
In addition, strong plantlets and rootage stage ideal seedling, root system is more dispersed, root system stretching, extension is good, and bottle outlet is easy to clean, no
Easily cause damage.
And the seedling that developmental condition is bad, root system are entangled, are not easy to separate, some root system tips can expand balling
Shape is unfavorable for the transplanting domestication of later period greenhouse.
Root system entanglement ratio can be significantly reduced using Rooting and hardening-off culture based component of the invention and cultural method, tangle
Ratio is less than 3%.
Although the invention has been described by way of example and in terms of the preferred embodiments, but it is not for limiting the present invention, any this field
Technical staff without departing from the spirit and scope of the present invention, may be by the methods and technical content of the disclosure above to this hair
Bright technical solution makes possible variation and modification, therefore, anything that does not depart from the technical scheme of the invention, and according to the present invention
Technical spirit any simple modifications, equivalents, and modifications made to the above embodiment, belong to technical solution of the present invention
Protection scope.
Claims (4)
1. a kind of method using stem-tip tissue breeding hybrid cymbidium seedling, which is characterized in that
It the described method comprises the following steps:
Step a, the selection of explant cut the healthy and strong lateral bud for the 6-12cm that hybrid cymbidium maternal plant base portion is grown；
Step b, lateral bud disinfection, carries out disinfection to the lateral bud cut, cuts explant of the stem-tip tissue as culture；
Step c, protocorm Fiber differentiation, explant is inoculated into protocorm induction medium, is cultivated 30-60 days, culture temperature
Degree is 25-28 DEG C, light application time 8-14h/d, and intensity of illumination is that 1000-2000lux induces to obtain protocorm；The induction training
Feeding base consists of the following compositions: Hua Bao No. 3 2g/L, caseinhydrolysate 3g/L, adenine 1g/L, active carbon 1g/L, agar 7g/L,
Sucrose 20g/L；
The protocorm group of induced synthesis is cut into fritter by step d, Protocorm Multiplication culture, and every piece contains 3-5 protocorm, connects
Kind gradually forms new protocorm into proliferated culture medium, repeatedly cutting proliferation, and every 30-40 days subcultures switching is primary；The increasing
The ingredient for growing culture medium is identical as Fiber differentiation based component；
The protocorm group that proliferation obtains is cut into fritter by step e, protocorm differentiation culture, and every piece contains 5-10 protocorm, connects
Kind carries out differentiation culture into differential medium, and protocorm differentiation goes out young leaves new root；Differential medium consists of the following compositions: flower
Treasured No. 3 2g/L, caseinhydrolysate 3g/L, adenine 1g/L, TDZ0.1 mg/L, active carbon 1g/L, agar 7g/L, sucrose 20g/
L；
Step f, Rooting and hardening-off culture cut the bud of high 5-6cm after differentiation culture, are inoculated into Rooting and hardening-off culture base and are trained
It supports, obtains the seedling of robust growth, wherein Rooting and hardening-off culture base consists of the following compositions: No. 1 1-4g/L of Hua Bao, potassium nitrate
0.7-2.1g/L, ferrous sulfate heptahydrate 18-32mg/L, two water disodium ethylene diamine tetraacetate 30-43g/L, caseinhydrolysate 2-4g/
L, adenine 0.5-1.5g/L, mashed potatoes 12-28g/L, banana puree 35-65g/L, coconut milk 50-150mL, active carbon 0.5-
1.5g/L, agar 6-8g/L, sucrose 14-27g/L.
2. the method according to claim 1, wherein including following to the method that lateral bud carries out disinfection in step b
Step:
The lateral bud cut is rinsed well with flowing water, peels off outer blade, cutting side bastem portion lignified tissue, until surplus layer by layer
Lower 6-8 piece leaf, 12% calcium hypochlorite sterilize 15min, then take on aseptic working platform and further sterilize；
Continue to peel off stem apex outer blade, until being left 3-5 piece leaf, 5% calcium hypochlorite sterilizes 12min；
Continue to shell outer blade, until being left 1-2 piece leaf, 1% calcium hypochlorite sterilizes 6min；Face under the microscope is carefully peelled off
The spire of growing point outer layer, until exposing growing point；The stem-tip tissue containing growing point is cut, the explant as culture.
3. according to the method described in claim 2, it is characterized in that, the stem-tip tissue containing growing point is uniformly cut into 4 from center
Fritter or 2 fritters, every piece is individually inoculated into induced medium.
4. the method according to claim 1, wherein Rooting and hardening-off culture base consists of the following compositions: Hua Bao 1
2g/L, potassium nitrate 1.5g/L, ferrous sulfate heptahydrate 27.8mg/L, two water disodium ethylene diamine tetraacetate 37.3g/L, caseinhydrolysate
3g/L, adenine 1g/L, mashed potatoes 20g/L, banana puree 50g/L, coconut milk 100ml/L, active carbon 1g/L, agar 7g/L, sugarcane
Sugared 20g/L.
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| CN109042342A (en) * | 2018-11-01 | 2018-12-21 | 翁源县天下泽雨农业科技有限公司 | A kind of method for tissue culture of Chunlan |
| CN109601386B (en) * | 2019-01-24 | 2020-08-25 | 北京农业职业学院 | Method for disinfecting bud cutting explant in stem tip tissue culture of cymbidium sinense |
| CN109804927B (en) * | 2019-03-05 | 2020-06-05 | 湖北红杏生态环境科技有限公司 | In-vitro rapid propagation method of cymbidium hybridum |
| CN111758561B (en) * | 2020-07-24 | 2022-04-15 | 广东省农业科学院环境园艺研究所 | Sterile sowing and breeding method of hybrid orchid seeds |
| CN113475396B (en) * | 2021-07-25 | 2023-04-28 | 杭州市农业科学研究院 | Hybrid orchid high-efficiency seedling method of spring sword and cymbidium |
| CN114532225B (en) * | 2022-02-28 | 2023-06-23 | 广西壮族自治区农业科学院 | Tissue culture rapid propagation and cultivation method for paphiopedilum delbrueckii |
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| CN1586166A (en) * | 2004-07-30 | 2005-03-02 | 中国科学院华南植物园 | Quick breeding method for cymbidium hyridus high quality sprout |
| CN101569287B (en) * | 2009-06-02 | 2011-08-10 | 中国科学院华南植物园 | Method for test tube propagation of seedlings of zygopetalum spp. |
| CN103371100B (en) * | 2012-04-17 | 2014-09-17 | 上海市农业科学院 | Tissue culture and rapid propagation method of nobile-type dendrobium seedlings |
| RU2523604C2 (en) * | 2012-10-25 | 2014-07-20 | Федеральное государственное бюджетное образоваельное учреждение высшего профессионального образования "Мордовский государственный университет им. Н.П. Огарёва" | METHOD OF PROPAGATION OF CYMBIDIUM in vitro |
| CN102972293A (en) * | 2012-12-04 | 2013-03-20 | 三亚柏盈热带兰花产业有限公司 | Rapid propagation method for cultivating tissue culture seedling by utilizing Mokara axillary bud tissue |
| CN105961199A (en) * | 2013-09-16 | 2016-09-28 | 周杰 | Tissue culture method of dendrobium officinale |
| KR20150057052A (en) * | 2013-11-18 | 2015-05-28 | 건국대학교 산학협력단 | A composition for improvement of regeneration system from protcorm-like body of Cymbidium plants and method therefor |
| KR101587707B1 (en) * | 2014-03-04 | 2016-02-02 | 고려대학교 산학협력단 | Producing method of orchid seedlings |
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| CN104938341B (en) | 2017-12-12 |
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