CN106922536A - A kind of method that fast energy-saving cultivates bletilla seedling - Google Patents
A kind of method that fast energy-saving cultivates bletilla seedling Download PDFInfo
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- CN106922536A CN106922536A CN201710240730.2A CN201710240730A CN106922536A CN 106922536 A CN106922536 A CN 106922536A CN 201710240730 A CN201710240730 A CN 201710240730A CN 106922536 A CN106922536 A CN 106922536A
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- seedling
- bletilla
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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
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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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
Abstract
The present invention relates to bletilla seedling breeding field, the method that more particularly to a kind of fast energy-saving cultivates bletilla seedling is comprised the following steps：By the sowing of aseptic bletilla seed, sprouting culture is carried out, be 1 2cm to the bletilla height of seedling degree for sprouting culture, be transplanted in seedlings nursing plate, cover preservative film, be positioned over the transition culture of slow seedling room；It is carried in greenhouse after the completion of the culture of slow seedling room, root pseudobulb a diameter of 1 2cm long to 16 20cm to bletilla seedling, carries out slow seedling 10 15 days before Field planting, then directly carries out Field planting；Sprouting the medium component used by culture is：Contain the 20mM of potassium ion 15, the 62g/L of sucrose 58,6 BA 0.8, the 1.5 2.5mg/L and 4.7g/L of agar powder 4.3 of 1.2mg/L, NAA in 1/2MS culture mediums.The method is two step bletilla tissue-culturing rapid propagation methods, energy saving consumption and toxigenic capacity, and cultivation period is short.
Description
Technical field
The present invention relates to bletilla seedling breeding field, a kind of method that bletilla seedling is cultivated in particular to fast energy-saving.
Background technology
Bletilla (splendid achnatherum) category (Bleatta Reichb.f.) is the raw herbaceous plant in orchid family (Orchidaceae) ground, with higher
Medical value and ornamental value.Its pseudobulb is a kind of traditional Chinese medicine, with hold back gas, ooze phlegm, stop blooding, the work(such as the carbuncle that disappears
Effect, bletilla category is distributed widely in Asian countries, and it has four kinds in China, is respectively bletilla (Bletilla striata
(Thunb.) Reichb.F.), magnificent bletilla (Bletilla kinesis (Rolfe) Schltr.), chrysanthemum bletilla (Bletilla
Ochracea Schltr.) and small bletilla (Bletilla formosana (Hayata) Schltr.).Bletilla is mainly grown in me
The ground such as state's distribution East China, Central-South, southwestern and Gansu, Shaanxi.It is maximum with Guizhou yield, it is best in quality.
The stem tuber hyoscine of bletilla, can astringing to arrest bleeding, detumescence and promoting granulation；Take orally and controlling pulmonary tuberculosis hemoptysis, bronchiectasis is coughed up
Blood, gastric ulcer haematemesis, hematuria, the disease aspect such as have blood in stool have extraordinary effect；Traumatism and bleeding, burn and scald, rhagadia manus et pedis are controlled in external application
Also there is effect Deng disease.Therefore, bletilla has at aspects such as biological medicine, health food, textile printing and dyeing, sapecial coating and daily-use chemical industries
Huge commercial exploitation.With the exploitation of bletilla food and dyeing industry, the demand of beauty culture, bletilla consumption increases increasingly
It is many, list in《Endangered animal and plant kind international trade pact》Protected.Its price is extremely sold well on Market of Chinese Materia Medica at home,
Guizhou bletilla such as in 2016 domestic Market of Chinese Materia Medica selects goods in 1200 yuans/kg or so, and gradeless and uniformly-priced goods is in 800 yuan of people
Coin/kg or so, but still it is on the rise, and show as the situation of selling well situation that supply falls short of demand.
The natural germination rate of bletilla seed is low, and the mankind are to the destruction in habitat and excessive excavation in addition, and bletilla plant is wild
Resource is seriously damaged, and the protection to its wild resource is extremely urgent.
However, obtaining the most common method of bletilla field planting using the predominance of bletilla Fruit pod explant.It is main
There are direct seeding method (live seedling) and tissue culture method (tissue-cultured seedling).Wherein direct sowing method (patent publication No.：
CN104126397A, bletilla direct sowing and seedling method, patent publication No.：CN106068790A, a kind of bletilla seed sowing nursery
Method) technical problem that is primarily present is rooting rate not high, the production cycle is long, seedling quality differs and sprouts, and medicine (hormone) makes
With many etc., but it also has the advantage of its own, and such as consumed energy is low and Field planting survival rate is high.Tissue culture method (patent
Number：CN105145352A, a kind of efficient group culturation rapid propagating technology of bletilla seedling；CN104719152A, bletilla factorial seedling-culturing method)
It is primarily present toxigenic capacity too high, the consumption energy (electric power) is high, direct planting survival rates are low and domestication seedling also needs to greenhouse etc. no
Foot.
In view of this, it is special to propose the present invention.
The content of the invention
The present invention provides a kind of quick (the 9-11 month) method of bletilla industrial seedling rearing, and its low cost obtains seedling quality
Height, (tissue culture room 2 months+slow seedling room (15 days)+greenhouse culture 6~8) with short production cycle individual month), successfully solve current bletilla
Industrial seedling rearing high cost (energy cost of cost of labor+culturing room) and production cycle (6~8 months+greenhouse in tissue culture room white silk long
Seedling culture 6 months) the problems such as, field planting survival rate more than 98%.
In order to realize above-mentioned purpose of the invention, spy uses following technical scheme：
A kind of method that fast energy-saving cultivates bletilla seedling, comprises the following steps：
(a), by the sowing of aseptic bletilla seed, sprouting culture is carried out, be 1-2cm to the bletilla height of seedling degree for sprouting culture, will
It is transplanted in seedlings nursing plate, covers preservative film, is positioned over the transition culture of slow seedling room；
It is carried in greenhouse after the completion of the culture of (b), slow seedling room, long to 16-20cm to bletilla seedling, root pseudobulb is a diameter of
1-2cm, carries out slow seedling 10-15 days before Field planting, then directly carries out Field planting；
In step (a), the medium component used by the sprouting culture is：Contain potassium ion 15- in 1/2MS culture mediums
20mM, sucrose 58-62g/L, 6-BA 0.8-1.2mg/L, NAA1.5-2.5mg/L and agar powder 4.3-4.7g/L.
For the former live and tissue culture method production bletilla seedling cycle it is long, human cost is high, the consumption energy is high and takes soil
Many etc. the deficiencies of ground area.The present invention carries out aseptic seeding tissue culture from bletilla seed, by traditional " four steps (stage) bletilla group
The fast numerous method (sprouting culture, strong seedling culture, culture of rootage and greenhouse domestication culture) of training " is improved to " two step bletilla tissue-culturing rapid propagation methods
(sprouting culture of rootage and the culture of seedlings nursing plate greenhouse) ", whole process is more simple and easy to do, and required labour is greatly decreased, not only
Energy saving is consumed and toxigenic capacity, and the time cycle that industrial fast breeding produces bletilla seedling is shorten to 9-11 month, whole
It is individual production finished product cycle time, field-transplanting survival rate be more than 98%, than existing field-transplanting survival rate lifting 8% with
On.The bletilla seed provided using the present invention is planted, and produces the finished product cycle at 3~4 years or so, identified, the bletilla quality for obtaining
It is superior, hence it is evident that higher than bletilla quality of the prior art.
Specifically, the present invention is specific defines the medium component sprouted used by culture, and the composition is according to bletilla initial
Growth phase it is different to the demand of nutriment, improve the germination medium of bletilla tissue culture, especially, it is determined that different potassium
The 1/2MS culture mediums dialogue and the growth effect of early stage of ion concentration.Eventually passing through culture medium of the invention carries out sprouting culture,
The sprouting of root can be effectively facilitated, and growth conditions are good, and good basis is provided into seedlings nursing plate to transplant.There is provided using the present invention
Germination medium culture after, it is no longer necessary to single strong seedling culture, culture of rootage before, and coordinate other steps, by tradition
" four steps (stage) bletilla tissue-culturing rapid propagation method " be improved to " two step bletilla tissue-culturing rapid propagation methods ", when substantially reducing bletilla tissue culture
Between, whole step is easier, reduces the amount of labour used and energy cost, and can also obtain the bletilla seedling of high-quality, is conducive to extensive
The quick tissue culture of batch production.
It should be noted that the medium component that the present invention is provided is：In 1/2MS culture mediums containing potassium ion 15-20mM,
Sucrose 58-62g/L, 6-BA 0.8-1.2mg/L, NAA 1.5-2.5mg/L and agar powder 4.3-4.7g/L, potassium ion therein
Concentration is that the potassium concentration in 1/2MS culture mediums is increased into concentration of the invention, can such as add potassium chloride, potassium sulfate,
Other compositions sucrose, 6-BA, NAA and agar powder are also the composition of extra addition.
Preferably, in step (a), the aseptic bletilla seed is obtained by following steps：
The bletilla Fruit pod of full maturity and cracking is cut off, the bletilla seed for obtaining soaks 4-6min with 75% ethanol,
Then 75% ethanol is removed.
Further, the aseptic bletilla seed is obtained by following steps：
The crust of the full maturity of collection and uncracked bletilla Fruit pod is cleaned up, is then aseptically used
75% ethanol soaks 4-6min, with aseptic water washing 3-4 time, is burnt 3-5 times with alcolhol burner flame envelope afterwards, is placed in suction on aseptic paper
Solid carbon dioxide point, cuts off and obtains aseptic bletilla seed.
In its natural state, bletilla is bloomed in or so annual mid-April, about in September part or so maturation after pollination, but
It is to want full maturity until cracking is about in or so November.Therefore, selection is collected full maturity but is not opened in or so October
The bletilla Fruit pod split, is placed on 4 DEG C of refrigerator cold-storages standby.When using, bletilla Fruit pod is taken out, by uncracked bletilla capsule with washing
Clothing powder solution is cleaned, and 5~10min is rinsed under running water, and the crust of bletilla Fruit pod is cleaned up.
The present invention is carried out disinfection using 75% alcohol to bletilla Fruit pod and seed first, it is to avoid sterilized using mercuric chloride in the past
Healthy to tissue culture personnel and environmental pollution potential hazard influence.
Uncracked bletilla seed is directly sowed on the culture medium of the 900mm diameter Petri dishes for having configured, each Fruit pod
Sow culture dish and process 10~12, about 200~300, the seed per culture dish culture medium, then with sealed membrane by culture medium
Mouth seal.
And ftracture bletilla Fruit pod Seed sterilization sterilization after, with toothpick by seed point on culture medium, after dibbling is finished, will
Culture medium is sealed with sealed membrane.
Preferably, in step (a), the condition cultivated of sprouting is：Temperature is 22-24 DEG C, and light intensity is 1500-
2500LX, light application time is 11-13h/d.
Further, the time for sprouting culture is 55-65 days.
The present invention coordinates above-mentioned condition culture 55-65 days by specific medium component, sprouts the bletilla height of seedling of culture
It is 1-2cm to spend, and growth is vigorous, effectively facilitates the sprouting of root, and good basis is provided into seedlings nursing plate to transplant.
Preferably, the composition in the seedlings nursing plate is：It is the 1/ of 15-20mM that 6-BA and potassium concentration are filled in fertile soil
2MS nutrient solutions, containing 6-BA 0.8-1.2mg/L and potassium concentration are the 1/2MS nutrient solutions of 15-20mM in the fertile soil
0.8-1.2mg/L.The 1/2MS nutrient solutions added i.e. in seedlings nursing plate are increased to the 1/2MS trainings of 15-20mM configurations for potassium concentration
Nutrient solution, the rising of potassium ion can add potassium chloride, potassium sulfate etc. in 1/2MS nutrient solutions.
The present invention by seedlings nursing plate add 6-BA and improved 1/2MS nutrient solutions, further promote root growth and
More preferable nutrition is provided, upgrowth situation is substantially better than being not added with 6-BA and improvement 1/2MS nutrient solutions fertile soil.
Further, in the seedlings nursing plate, the spacing in the rows of bletilla seedling is 28-33mm, and line-spacing is 33-38mm.Closed by setting
The spacing in the rows and line-spacing of reason, promote bletilla seedling preferably to grow.
Bletilla seedling covers preservative film, mainly plays moisture-keeping function, it is preferable that the preservative film is apart from bletilla seedling top 450-
550mm；
The time of the transition culture is 14-16 days.
By the transition culture of a period of time, for greenhouse culture provides condition.
Further, the transition culture is：
The condition of culture of preceding 9-10d is：22~24 DEG C of temperature, 1000~1500LX of light intensity, light application time is 11-13h/
d；；
The condition of culture of 5-6d is afterwards：When 20~26 DEG C of temperature, light intensity are natural light, daily light irradiation time is natural lighting
It is long.
Further, the greenhouse is duplicature greenhouse；Duplicature greenhouse can prevent high temperature and high light from being caused to bletilla seedling
Injury.
Further, in greenhouse culture, seedlings nursing plate is placed on culturing rack, throws off the preservative film covered on seedlings nursing plate, training
Support 6~8 months.
The present invention first combines bletilla tissue culture and greenhouse shelf seedlings nursing plate nursery, improves greenhouse culture bletilla seedling
Utilization rate, solve bletilla nursery land used it is excessive the problems such as.
Further, insect prevention and diseases prevention are noted in greenhouse incubation, wherein to carry out sterilization one in canopy every other month
It is secondary；Growing state according to seedling provides foliar fertilizer.
Further, during Field planting, every mu of 6000~8000 plants of seedling in ground.
In addition, the formula of the 1/2MS culture mediums in the present invention is as shown in table 1.
The specific composition of table 1MS culture mediums
Compared with prior art, beneficial effects of the present invention are：
(1) it is fast it is numerous during, bletilla Fruit pod or seed are carried out disinfection using 75% alcohol first, it is to avoid profit in the past
With mercuric chloride sterilization influence healthy to tissue culture personnel and environmental pollution potential hazard.
(2) while improving bletilla different phase tissue culture medium component, by traditional, " four steps (stage) bletilla tissue culture is fast
Numerous method " is improved to " two step bletilla tissue-culturing rapid propagation methods ", substantially reduces the bletilla tissue culture time, by it is original reduce the amount of labour used and
Energy cost, and the bletilla seedling of high-quality can also be obtained, be conducive to large-scale industrialized quickly tissue culture nursery.
(3) it is different to the demand of nutriment in initial growth phase according to bletilla first, it is determined that different potassium ions
The 1/2MS culture mediums dialogue and the growth effect of early stage of content concn, improve the germination medium of bletilla tissue culture, can effectively promote
Enter the sprouting of root, and growth conditions are good, and good basis is provided into seedlings nursing plate to transplant.
(4) bletilla tissue culture and greenhouse shelf seedlings nursing plate nursery are combined first, improves greenhouse culture bletilla seedling
Utilization rate, solve bletilla nursery land used it is excessive the problems such as.
(5) slow seedling transition chamber is utilized first, bletilla is solved and is transplanted seedlings field planting to the survival rate of greenhouse, and then be field planting
Bletilla survival rate is provided and ensured.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described.
Fig. 1 is bletilla germination and growth situation map of 20 days on different culture media；
Fig. 2 is the growing state figure of method acquisition bletilla seedling when cultivating 4 months of different cultivation bletilla seedlings.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
A kind of method that fast energy-saving cultivates bletilla seedling, including following key step：
Configuration culture medium：
By each composition configuration 1/2MS culture mediums in table 1, while configuration, addition potassium chloride causes the potassium in culture medium
Ion concentration is 15mM, obtains improved 1/2MS culture mediums, is subsequently adding sucrose, 6-BA, NAA and agar powder so that culture medium
The concentration of this several composition of middle addition is sucrose 58g/L, 6-BA 0.8mg/L, NAA 1.5mg/L and agar powder 4.7g/L.
Prepare seedlings nursing plate：
Fertile soil is taken, 6-BA and improved 1/2MS nutrient solutions is added, makes to contain 6-BA 0.8mg/L in fertile soil, contained
Improved 1/2MS nutrient solutions 0.8mg/L.
1) bletilla Fruit pod and seed disinfection.The bletilla Fruit pod of collection is taken, is cleaned with washing powder solution, rinsed under running water
5~10min, then uses 75% ethanol soaking disinfection 4min, then with aseptic water washing 3-4 times, be placed on superclean bench
Suck dry moisture on the filter paper of sterilizing.
2) culture is sprouted in sowing.The bletilla Fruit pod that will be disinfected with the scissors of sterilization is cut off from middle part, takes out
Seed, is inoculated into rapidly on the culture medium of the 900mm diameter Petri dishes for having configured；Each Fruit pod sows 10~12, culture dish,
Per about 200~300, the seed of culture dish culture medium, the mouth of culture medium is sealed with sealed membrane then, condition of culture is temperature
22~24 DEG C, light intensity 1500LX, the culture 2 months of daily illumination 12h and dark 12h.
3) transplant seedlings field planting.Deng bletilla seedling germination and growth on culture dish culture medium to 1~2cm or so (i.e. 2 months when
Between), it is transplanted to tweezers and is placed with the seedlings nursing plate of fertile soil, 6-BA and improved 1/2MS culture mediums mixing, every two young plant
Transplanting field planting spacing in the rows be 28mm, line-spacing is 33mm, 300 plants or so of a seedlings nursing plate co-transplantation, survival rate 99%, and bletilla
Seedling has no pollution in seedling raising process.
4) slow seedling room transition culture.The seedlings nursing plate that completion will be colonized covers preservative film and (support jack-up is used, apart from seedling
500mm or so), it is then placed into the culture medium slow seedling transition culture of slow seedling room；The condition of culture of preceding 10d is：Temperature 22~24
DEG C, light intensity 1000LX, daily light irradiation time be 12h illumination and 12h dark；The condition of culture of 5d is afterwards：20~26 DEG C of temperature, light
By force for natural light, daily light irradiation time be natural lighting duration.
5) greenhouse training orientation.The nursery disk cover for being completed in slow seedling room slow seedling is transported in greenhouse, its training is placed
Support on frame, throw off the preservative film covered on seedlings nursing plate, cultivate 6 months；Condition of culture is duplicature greenhouse, and the period in summer (prevents
High temperature and high light are damaged to bletilla seedling) plastic house sunshade net is given, other conditions are greenhouse natural conditions；Incubation
It is middle to note insect prevention and diseases prevention, wherein to carry out sterilization in canopy every other month once, will also provide one according to the growing state of seedling
Determine foliar fertilizer.
6) completely natural slow seedling before field planting.When the seedling of greenhouse culture grows to 16~18cm, root pseudobulb diameter
During for 1~2cm, the ventilating opening of culturing room's greenhouse is opened, slow seedling 15d before Field planting is carried out to the cultivating seedling in greenhouse.
7) Field planting.The bletilla seedling of nature slow seedling will be complete together with seedlings nursing plate, be transported to what is put in order
Crop field is colonized, and, at 6000~8000 plants or so, survival rate is more than 98% after field planting for every mu of ground seedling.
Embodiment 2
A kind of method that fast energy-saving cultivates bletilla seedling, including following key step：
Configuration culture medium：
By each composition configuration 1/2MS culture mediums in table 1, while configuration, addition potassium chloride causes the potassium in culture medium
Ion concentration is 20mM, obtains improved 1/2MS culture mediums, is subsequently adding sucrose, 6-BA, NAA and agar powder so that culture medium
The concentration of this several composition of middle addition is sucrose 60g/L, 6-BA 1mg/L, NAA 2mg/L and agar powder 4.5g/L.
Prepare seedlings nursing plate：
Take fertile soil, add 6-BA and improved 1/2MS nutrient solutions, make in fertile soil containing 6-BA 1mg/L, containing changing
The 1/2MS nutrient solutions 1mg/L for entering.
1) bletilla Fruit pod and seed disinfection.The bletilla Fruit pod of collection is taken, is cleaned with washing powder solution, rinsed under running water
5~10min, then uses 75% ethanol soaking disinfection 5min, then with aseptic water washing 3-4 times, be placed on superclean bench
Suck dry moisture on the filter paper of sterilizing.
2) culture is sprouted in sowing.The bletilla Fruit pod that will be disinfected with the scissors of sterilization is cut off from middle part, takes out
Seed, is inoculated into rapidly on the culture medium of the 900mm diameter Petri dishes for having configured；Each Fruit pod sows 10~12, culture dish,
Per about 200~300, the seed of culture dish culture medium, the mouth of culture medium is sealed with sealed membrane then, condition of culture is temperature
22~24 DEG C, light intensity 2000LX, the culture 2 months of daily illumination 13h and dark 11h.
3) transplant seedlings field planting.Deng bletilla seedling germination and growth on culture dish culture medium to 1~2cm or so (i.e. 2 months when
Between), it is transplanted to tweezers and is placed with the seedlings nursing plate of fertile soil, 6-BA and improved 1/2MS culture mediums mixing, every two young plant
Transplanting field planting spacing in the rows be 30mm, line-spacing is 35mm, 300 plants or so of a seedlings nursing plate co-transplantation, survival rate 100%, and in vain
And seedling has no pollution in seedling raising process.
4) slow seedling room transition culture.The seedlings nursing plate that completion will be colonized covers preservative film and (support jack-up is used, apart from seedling
500mm or so), it is then placed into the culture medium slow seedling transition culture of slow seedling room；The condition of culture of preceding 10d is：Temperature 22~24
DEG C, light intensity 1200LX, daily light irradiation time be 13h illumination and 11h dark；The condition of culture of 5d is afterwards：20~26 DEG C of temperature, light
By force for natural light, daily light irradiation time be natural lighting duration.
5) greenhouse training orientation.The nursery disk cover for being completed in slow seedling room slow seedling is transported in greenhouse, its training is placed
Support on frame, throw off the preservative film covered on seedlings nursing plate, cultivate 7 months；Condition of culture is duplicature greenhouse, and the period in summer (prevents
High temperature and high light are damaged to bletilla seedling) plastic house sunshade net is given, other conditions are greenhouse natural conditions；Incubation
It is middle to note insect prevention and diseases prevention, wherein to carry out sterilization in canopy every other month once, will also provide one according to the growing state of seedling
Determine foliar fertilizer.
6) completely natural slow seedling before field planting.When the seedling of greenhouse culture grows to 18~20cm, root pseudobulb diameter
During for 1~2cm, the ventilating opening of culturing room's greenhouse is opened, slow seedling 12d before Field planting is carried out to the cultivating seedling in greenhouse.
7) Field planting.The bletilla seedling of nature slow seedling will be complete together with seedlings nursing plate, be transported to what is put in order
Crop field is colonized, and, at 6000~8000 plants or so, survival rate is more than 98% after field planting for every mu of ground seedling.
Embodiment 3
A kind of method that fast energy-saving cultivates bletilla seedling, including following key step：
Configuration culture medium：
By each composition configuration 1/2MS culture mediums in table 1, while configuration, addition potassium chloride causes the potassium in culture medium
Ion concentration is 20mM, obtains improved 1/2MS culture mediums, is subsequently adding sucrose, 6-BA, NAA and agar powder so that culture medium
The concentration of this several composition of middle addition is sucrose 62g/L, 6-BA 1.2mg/L, NAA2.5mg/L and agar powder 4.7g/L.
Prepare seedlings nursing plate：
Take fertile soil, add 6-BA and improved 1/2MS nutrient solutions, make to contain 6-BA1.2mg/L in fertile soil, containing changing
The 1/2MS nutrient solutions 1.2mg/L for entering.
1) bletilla Fruit pod and seed disinfection.The bletilla Fruit pod of the cracking of collection is taken, is cut off, obtain bletilla seed, Ran Houfang
Enter 75% ethanol soaking disinfection 5min, suctioned out seed and alcohol with micro-shifting device, be placed on filter paper dried up in super-clean bench it is standby.
2) culture is sprouted in sowing.With toothpick by the seed point after sterilization the 900mm diameter Petri dishes for having configured culture
On base, per about 200~300, the seed of culture dish culture medium, after dibbling is finished, the mouth of culture medium is sealed with sealed membrane, trained
The condition of supporting is the culture 2 months of 22~24 DEG C of temperature, light intensity 2500LX, daily illumination 11h and dark 13h.
3) transplant seedlings field planting.Deng bletilla seedling germination and growth on culture dish culture medium to 1~2cm or so (i.e. 2 months when
Between), it is transplanted to tweezers and is placed with the seedlings nursing plate of fertile soil, 6-BA and improved 1/2MS culture mediums mixing, every two young plant
Transplanting field planting spacing in the rows be 28mm, line-spacing is 38mm, 300 plants or so of a seedlings nursing plate co-transplantation, survival rate 100%, and in vain
And seedling has no pollution in seedling raising process.
4) slow seedling room transition culture.The seedlings nursing plate that completion will be colonized covers preservative film and (support jack-up is used, apart from seedling
500mm or so), it is then placed into the culture medium slow seedling transition culture of slow seedling room；The condition of culture of preceding 10d is：Temperature 22~24
DEG C, light intensity 1500LX, daily light irradiation time be 11h illumination and 13h dark；The condition of culture of 5d is afterwards：20~26 DEG C of temperature, light
By force for natural light, daily light irradiation time be natural lighting duration.
5) greenhouse training orientation.The nursery disk cover for being completed in slow seedling room slow seedling is transported in greenhouse, its training is placed
Support on frame, throw off the preservative film covered on seedlings nursing plate, cultivate 8 months；Condition of culture is duplicature greenhouse, and the period in summer (prevents
High temperature and high light are damaged to bletilla seedling) plastic house sunshade net is given, other conditions are greenhouse natural conditions；Incubation
It is middle to note insect prevention and diseases prevention, wherein to carry out sterilization in canopy every other month once, will also provide one according to the growing state of seedling
Determine foliar fertilizer.
6) completely natural slow seedling before field planting.When the seedling of greenhouse culture grows to 18~20cm, root pseudobulb diameter
During for 1~2cm, the ventilating opening of culturing room's greenhouse is opened, slow seedling 10d before Field planting is carried out to the cultivating seedling in greenhouse.
7) Field planting.The bletilla seedling of nature slow seedling will be complete together with seedlings nursing plate, be transported to what is put in order
Crop field is colonized, and, at 6000~8000 plants or so, survival rate is more than 98% after field planting for every mu of ground seedling.
Comparative example 1
Sprouting the culture medium added in culture medium and seedlings nursing plate used is：Contain potassium ion in 1/2MS culture mediums
10mM, sucrose 60g/L, 6-BA 1mg/L, NAA 2mg/L and agar powder 4.5g/L, other compositions and step are with embodiment 2.
Sprout 20 days, compare with embodiment 2, taken pictures under microscope (2 ×) under the same terms, the result for obtaining such as Fig. 1
It is shown.The figure on Fig. 1 left sides is the tissue that culture medium used in comparative example sprouts culture 20 days, and the figure on the right is the institute of embodiment 2
Culture medium sprouts the tissue that culture is obtained for 20 days.It is apparent that the bletilla seedling root that this comparative example sprouts culture sprouts weak, and group
Hair-weaving educates substantially small, and planting survival rates of subsequently transplanting seedlings are very low, less than 50%.And follow-up slow seedling transition culture and greenhouse culture
Upgrowth situation is poor, therefore, give up cultivation.
Comparative example 2
Bletilla traditional four-step tissue culture method, step is as follows：
1) Seed Treatment before sowing：The bletilla Fruit pod that will be collected rinses 10min with running water, on superclean bench first
With 75% ethanol disinfection 30s, then 10min is soaked with 4% liquor natrii hypochloritis, Fruit pod is ceaselessly stirred during sterilization, it is disappeared
Malicious thorough, rinsed with sterile water 3 times is then stand-by with the moisture that aseptic filter paper blots Fruit pod surface.
2) sowing is sprouted：Sowing culture medium contains glucose 30g/L and agar powder 6.5g/L for 1/2MS culture mediums；In nothing
Under the conditions of bacterium, seed is uniformly seeded into media surface, then the blake bottle that will have been sowed is put into sterilized culturing room
Culture.
3) squamous subculture：After sprouting culture 1 month or so, start switching, 15-30 seedling of every bottle of switching, culture medium of transferring
To contain NAA 1.0mg/L, glucose 30g/L and agar powder 6.5g/L in 1/2MS culture mediums, switching culture medium is placed on culture
Cultivated in room.
4) culture of rootage：Squamous subculture 50d or so, starts switching, and 15 seedlings of every bottle of switching, switching culture medium is cultivated for MS
Base contains NAA 2.0mg/L, 6-BA 1.5mg/L, carbon dust 1 ‰, banana puree 8%, glucose 30g/L and agar powder 6.5g/L, will
The seedling transferred is cultivated in being placed on culturing room.
5) domestication canopy is built：The fertile sand soil of selection soil property builds domestication canopy, and installs automatic sprinkler, in canopy
The seedbed of 1.2-1.8m wide, 15-20cm high is made with seedling medium, it is the sunshade of 65-75% that degree of shading is laid outside domestication canopy
Net.
6) bottle outlet is transplanted：Selection is cultivated 4-6 month and has the tissue-cultured seedling of bulb, is placed on domestication canopy or outdoor shady place, 5-7
Tissue-cultured seedling is cleaned after it, the tissue-cultured seedling of wash clean is transplanted on the seedling bed of domestication canopy, spacing in the rows 5cm × 5cm, after transplanting in time
Pour root water.
7) water and fertilizer management：At 20-30 DEG C, soil moisture is maintained at 70-80% to temperature control in domestication canopy；Transplant latter
One time of nutrition liquid is poured within individual month, later every two weeks foliage-spray nutritious liquor 1 time.
8) seedling raising greenhouse.Domestication canopy culture 80-100 days, is transferred to seedling raising greenhouse culture 4 months.
9) Field planting.Field-transplanting survival rate is 90%, and the production finished product cycle was in 4~5 years.
In addition, embodiment 2, comparative example 2 are sprouted into culture nursery with direct sowing method comparing, sowed by bletilla seed based on starting
Calculate, bletilla seedling upgrowth situation is as shown in Figure 2 at 4 months.
In Fig. 2, control 1 is that direct sowing method sprouts culture；Control 2 is that the traditional tissue culture method in comparative example 2 sprouts culture；Treatment
Sprouted for the method for the embodiment of the present invention 2 and cultivated.
Figure it is seen that the bletilla seedling production method that the present invention is provided, the growing way and development feelings of the bletilla seedling of cultivation
Condition is significantly stronger than other groups.
Additionally, other embodiment cultivation bletilla seedling result is basically identical with embodiment 2.
Bletilla seedling production method disclosed by the invention, fast energy-saving successfully solves many of two kinds of nursery of current bletilla
The live seedling of problem, such as Fruit pod is slow-growing, the cycle is long, seedling quality it is not high it is high with tissue-cultured seedling production cost (it is artificial with soil into
Originally), consume energy (the mainly electricity cost of culturing room) high, the cycle is long and needs a series of problems, such as taming seedling greenhouse, carry significantly
The high speed of growth and efficiency that its seedling is produced using bletilla Fruit pod explant predominance, can at 9~11 months or so
To obtain Field planting seedling, compared to the production time that former method has saved 3~5 months, its Field planting survival rate exists
More than 98%.In addition, the present invention is only once manually colonized onto seedling disk in the production process of whole bletilla seedling, educated with other
Seedling-growing method compares, and has saved substantial amounts of labor cost.Plus bletilla tissue culture process of the invention in its culturing room only with 2
The time (other bletilla tissue-cultured seedling are at least cultivated more than 6 months in culturing room, are consumed energy very big) of or so the moon, energy resource consumption is also non-
It is often few.Additionally, the present invention is also combined using seedlings nursing plate and seedling culture frame, the big deficiency of bletilla nursery floor space has been saved.
Therefore, the present invention successfully solves the production technology problem of current bletilla seedling, and " two low two is high " for realizing the production of bletilla seedling is (low
Cost and low power consuming, high-quality and high efficiency), be conducive to large-scale industrialized production nursery, further reduce peasant household's bletilla seedling
Take purchase cost.
Although illustrate and describing the present invention with specific embodiment, but will be appreciated that without departing substantially from of the invention
Many other changes and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of method that fast energy-saving cultivates bletilla seedling, it is characterised in that comprise the following steps：
(a), by the sowing of aseptic bletilla seed, sprouting culture is carried out, be 1-2cm to the bletilla height of seedling degree for sprouting culture, moved
Plant in seedlings nursing plate, cover preservative film, be positioned over the transition culture of slow seedling room；
It is carried in greenhouse after the completion of the culture of (b), slow seedling room, it is long to 16-20cm, a diameter of 1- of root pseudobulb to bletilla seedling
2cm, carries out slow seedling 10-15 days before Field planting, then directly carries out Field planting；
In step (a), the medium component used by the sprouting culture is：In 1/2MS culture mediums containing potassium ion 15-20mM,
Sucrose 58-62g/L, 6-BA 0.8-1.2mg/L, NAA1.5-2.5mg/L and agar powder 4.3-4.7g/L.
2. the method that fast energy-saving according to claim 1 cultivates bletilla seedling, it is characterised in that described in step (a)
Aseptic bletilla seed is obtained by following steps：
The bletilla Fruit pod of full maturity and cracking is cut off, the bletilla seed for obtaining soaks 4-6min with 75% ethanol, then
Remove 75% ethanol.
3. the method that fast energy-saving according to claim 1 cultivates bletilla seedling, it is characterised in that the aseptic bletilla seed
Obtained by following steps：
The crust of the full maturity of collection and uncracked bletilla Fruit pod is cleaned up, then aseptically with 75%
Ethanol soaks 4-6min, with aseptic water washing 3-4 times, is burnt 3-5 times with alcolhol burner flame envelope afterwards, is placed in and water is blotted on aseptic paper
Point, cut off and obtain aseptic bletilla seed.
4. the method that fast energy-saving according to claim 1 cultivates bletilla seedling, it is characterised in that described in step (a)
Sprouting the condition cultivated is：Temperature is 22-24 DEG C, and light intensity is 1500-2500LX, and light application time is 11-13h/d；The sprouting
The time of culture is preferably 55-65 days.
5. the method that fast energy-saving according to claim 1 cultivates bletilla seedling, it is characterised in that in the seedlings nursing plate into
It is divided into：It is the 1/2MS nutrient solutions of 15-20mM that 6-BA and potassium concentration are filled in fertile soil, and 6-BA is contained in the fertile soil
0.8-1.2mg/L, contains the 1/2MS nutrient solutions 0.8-1.2mg/L that potassium concentration is 15-20mM in the fertile soil.
6. the method that fast energy-saving according to claim 5 cultivates bletilla seedling, it is characterised in that in the seedlings nursing plate, in vain
And the spacing in the rows of seedling is 28-33mm, line-spacing is 33-38mm.
7. the method that fast energy-saving according to claim 1 cultivates bletilla seedling, it is characterised in that the preservative film distance is white
And seedling top 450-550mm；
The time of the transition culture is 14-16 days.
8. the method that fast energy-saving according to claim 7 cultivates bletilla seedling, it is characterised in that the transition culture is：
The condition of culture of preceding 9-10d is：22~24 DEG C of temperature, 1000~1500LX of light intensity, light application time is 11-13h/d；；
The condition of culture of 5-6d is afterwards：20~26 DEG C of temperature, light intensity are natural light, daily light irradiation time is natural lighting duration.
9. the method that fast energy-saving according to claim 1 cultivates bletilla seedling, it is characterised in that the greenhouse is duplicature
Greenhouse；
In greenhouse culture, seedlings nursing plate is placed on culturing rack, throws off the preservative film covered on seedlings nursing plate, is cultivated 6~8 months；
Further, insect prevention and diseases prevention are noted in greenhouse incubation, wherein to carry out being sterilized once in canopy every other month；Root
Foliar fertilizer is provided according to the growing state of seedling.
10. the method that the fast energy-saving according to claim any one of 1-9 cultivates bletilla seedling, it is characterised in that crop field is fixed
During plant, every mu of 6000~8000 plants of seedling in ground.
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| CN109122321A (en) * | 2018-09-13 | 2019-01-04 | 南京大学 | A kind of breeding method of the tissue culture method for obtaining high-quality bletilla pseudobulb and bletilla seedling |
| CN110679421A (en) * | 2019-11-06 | 2020-01-14 | 安徽东方金桥农林科技股份有限公司 | Bletilla striata seedling cultivation method |
| CN114051930A (en) * | 2021-11-23 | 2022-02-18 | 河北农业大学 | Inoculation and seedling raising method for tissue culture and rapid propagation of raspberry seeds |
| CN115589948A (en) * | 2022-10-25 | 2023-01-13 | 云南省林业和草原科学院（Cn） | Bletilla striata non-symbiotic germination culture medium and propagation method |
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