CN109122321A - A kind of breeding method of the tissue culture method for obtaining high-quality bletilla pseudobulb and bletilla seedling - Google Patents
A kind of breeding method of the tissue culture method for obtaining high-quality bletilla pseudobulb and bletilla seedling Download PDFInfo
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- CN109122321A CN109122321A CN201811068555.4A CN201811068555A CN109122321A CN 109122321 A CN109122321 A CN 109122321A CN 201811068555 A CN201811068555 A CN 201811068555A CN 109122321 A CN109122321 A CN 109122321A
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- 241001313855 Bletilla Species 0.000 title claims abstract description 103
- 238000012136 culture method Methods 0.000 title claims abstract description 25
- 238000009395 breeding Methods 0.000 title claims abstract description 11
- 239000001963 growth medium Substances 0.000 claims abstract description 36
- 238000005728 strengthening Methods 0.000 claims abstract description 30
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 20
- 229920001817 Agar Polymers 0.000 claims abstract description 19
- 239000008272 agar Substances 0.000 claims abstract description 19
- 235000021552 granulated sugar Nutrition 0.000 claims abstract description 17
- 235000013575 mashed potatoes Nutrition 0.000 claims abstract description 7
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910001414 potassium ion Inorganic materials 0.000 claims abstract description 5
- 235000013399 edible fruits Nutrition 0.000 claims description 20
- 241000196324 Embryophyta Species 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 238000004659 sterilization and disinfection Methods 0.000 claims description 14
- 239000000306 component Substances 0.000 claims description 13
- 239000000843 powder Substances 0.000 claims description 13
- 235000019441 ethanol Nutrition 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 239000011159 matrix material Substances 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- 235000021186 dishes Nutrition 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 4
- 239000008399 tap water Substances 0.000 claims description 4
- 235000020679 tap water Nutrition 0.000 claims description 4
- 238000002054 transplantation Methods 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 15
- 238000011109 contamination Methods 0.000 abstract description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 12
- 229910052700 potassium Inorganic materials 0.000 description 12
- 239000011591 potassium Substances 0.000 description 12
- 230000004083 survival effect Effects 0.000 description 11
- 238000012258 culturing Methods 0.000 description 8
- 230000001954 sterilising effect Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 239000012869 germination medium Substances 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- 241001313857 Bletilla striata Species 0.000 description 4
- 239000012533 medium component Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000012549 training Methods 0.000 description 4
- 238000005286 illumination Methods 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 241000233855 Orchidaceae Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 230000000249 desinfective effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000003337 fertilizer Substances 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000008832 photodamage Effects 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 238000009331 sowing Methods 0.000 description 2
- 241001523383 Achnatherum Species 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 241001167064 Bletilla formosana Species 0.000 description 1
- 241001656044 Bletilla ochracea Species 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 240000008790 Musa x paradisiaca Species 0.000 description 1
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 230000002609 anti-worm Effects 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000032297 kinesis Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
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Classifications
-
- 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
-
- 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
- A01G22/25—Root crops, e.g. potatoes, yams, beet or wasabi
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to bletilla seedlings to cultivate field, in particular to a kind of tissue culture method for obtaining high-quality bletilla pseudobulb and the breeding method of bletilla seedling.A kind of strengthening seedling and rooting culture medium, its component includes: containing potassium ion 18-20mM, 60 ± 2g/L of white granulated sugar, 6-BA 1.4-1.5mg/L, 0.3 NAA 0.2 ± 0.02mg/L of ± 0.02mg/L, IBA and 100 ± 5g/L of mashed potato in 1/2MS culture medium, and the concentration of agar is 4.5 ± 0.1g/L.The sprouting seedling of bletilla is transferred to the strengthening seedling and rooting culture medium culture, culture 4 months, the high-quality bletilla pseudobulb obtained accounts for 85% or more of its sum, and, the present invention for the first time by bletilla tissue culture method strong seedling culture and the culture of rootage stage merge into strengthening seedling and rooting culture, the step in production process is reduced, saves artificial and production cost, while avoiding the germ contamination in production process.
Description
Technical field
The present invention relates to bletilla seedlings to cultivate field, in particular to a kind of tissue culture side for obtaining high-quality bletilla pseudobulb
The breeding method of method and bletilla seedling.
Background technique
It is the raw herbaceous plant in orchid family (Orchidaceae) ground that bletilla (splendid achnatherum), which belongs to (Bleatta Reichb.f.), with higher
Medical value and ornamental value.Its pseudobulb is a kind of traditional Chinese medicine, has and holds back gas, seep phlegm, stop blooding, the function such as the carbuncle that disappears
Effect, bletilla category are distributed widely in Asian countries, are bletilla (Bletilla striata respectively in China altogether there are four kind
(Thunb.) H.G.Reichenbach), magnificent bletilla (Bletilla kinesis (Rolfe) Schltr.), chrysanthemum bletilla
(Bletilla ochracea Schltr.) and small bletilla (Bletilla formosana (Hayata) Schltr.).Bletilla kind
The natural germination rate of son is low, and the mankind seriously break the destruction in habitat and excessive excavation, bletilla plant wild resource in addition
It is bad, it is extremely urgent to the protection of its wild resource.
However, obtaining bletilla field planting seedling using the predominance of bletilla fruit pod explant is most common method.
But because bletilla fruit pod explant sprout and take root under field conditions (factors) it is extremely difficult, therefore, manually usually by organizing to train to it
Feeding method is realized, tissue-cultured seedling domesticated seedlings are generally become.As the bletilla tissue culture patent of invention having had has: a kind of bletilla seedling
Efficient group culturation rapid propagating technology (patent No.: CN105145352A)；Bletilla factorial seedling-culturing method (patent No.:
CN104719152A).But these tissue culture methods are primarily present that the tissue culture time is long, tissue culture process operating procedure is many and diverse, directly fixed
Plant that survival rate is low and the too small survival rate for directly affecting greenhouse domestication of bletilla pseudobulb and the yield etc. of later field plantation
Deficiency.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of strengthening seedling and rooting culture medium, for the first time by the strong sprout in bletilla tissue culture method
Culture and culture of rootage stage merge as strengthening seedling and rooting culture, reduce the step in production process, save artificial and production
Cost, while avoiding the germ contamination in production process.
The second object of the present invention is to provide a kind of tissue culture method for obtaining high-quality bletilla pseudobulb, makes industrial fast breeding
The time cycle high-quality bletilla pseudobulb that shorten to 5 months, and produce acquisition of production bletilla seedling account for the 85% of its sum with
On.
The third object of the present invention is a kind of breeding method of bletilla seedling, and the survival rate of greenhouse domestication is 100%, improves
The yield of crop field final products produces the finished product period at 3~4 years or so.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of strengthening seedling and rooting culture medium, component include: to contain potassium ion 18-20mM, white granulated sugar 60 in 1/2MS culture medium
± 2g/L, 6-BA 1.4-1.5mg/L, 100 ± 5g/ of 0.3 ± 0.02mg/L of NAA, 0.2 ± 0.02mg/L of IBA and mashed potato
L；
Further, the culture medium is solid medium, and the components also include agar, the concentration of the agar is
4.5±0.1g/L。
Strengthening seedling and rooting culture medium provided by the invention, for the strengthening seedling and rooting culture of bletilla, for the first time by bletilla tissue culture method
In strong seedling culture and culture of rootage stage merge into strengthening seedling and rooting culture, reduce the step in production process, save manually and
Production cost, while avoiding the germ contamination in production process.
Strengthening seedling and rooting culture medium provided by the invention, by screening, the obtained culture medium mentions to obtain high-quality bletilla seedling
For good basis.
Strengthening seedling and rooting culture medium can may be solid medium for fluid nutrient medium.
Further, the culture medium is solid medium, and the components also include agar, the concentration of the agar is
4.5±0.1g/L。
Strengthening seedling and rooting culture medium used in the present invention is solid medium.
In the present invention, the content of the component of strengthening seedling and rooting culture medium is in terms of water such as distilled water or distilled water.
The present invention also provides a kind of tissue culture method for obtaining high-quality bletilla pseudobulb, the sprouting seedling of bletilla is transferred to above-mentioned
Strengthening seedling and rooting culture medium culture.
Bletilla sprouts seedling through strengthening seedling and rooting culture medium provided by the invention culture 4 months, the high-quality bletilla pseudobulb of acquisition
Account for 85% or more of its sum.Wherein, the standard of high-quality bletilla pseudobulb is that bletilla pseudobulb is not less than 0.5g.
Further, the condition of culture of the strengthening seedling and rooting culture medium are as follows: 22~24 DEG C of temperature, light intensity 1500~
2500LX, light application time 11-13h/d.
Further, culture bottle used in the strengthening seedling and rooting culture medium is the tissue culture vial that volume is 650ml, wide opening
Ventilative with bottle cap, each bottle fills strengthening seedling and rooting culture medium described in 150 ± 5ml.
Further, the 50-60 plants of sprouting seedlings are planted in each tissue culture vial.
It is cultivated by the culture bottle, not only ensure that reasonable planting density, but also the nutrition of culture medium can be made full use of
Component.
Further, the time of the strengthening seedling and rooting culture medium culture is 120 ± 5d.
Further, the plant height of the bletilla seedling obtained after cultivating 4 months is 5-6cm, and microballoon stem weight is not less than 0.5g's
85% or more.
The high-quality seedling obtained relative to existing tissue culture method only accounts for 20%-40%, and of the invention up to 85% or more, has
There is apparent advantage.High-quality seedling is the seedling with high-quality bletilla pseudobulb.
Further, the sprouting seedling is prepared by the following method:
Sterile bletilla seed is sowed, sprouting culture is carried out；
It is described to sprout culture medium component used are as follows: to contain potassium ion 18-20mM, white granulated sugar in 1/2MS culture medium
60 ± 2g/L, 4.5 ± 0.2g/L of 1.0 ± 0.2mg/L of 6-BA, 0.2 ± 0.05mg/L of NAA and agar powder.
Sucrose in germination medium used in the present invention replaces with white granulated sugar, while save the cost, imitates to sprouting
Fruit, which has no, to be significantly affected.
Further, the sprouting condition of culture are as follows: 22~24 DEG C of temperature, 1500~2500LX of light intensity, light application time are
11-13h/d。
Further, the time for sprouting culture is 30 ± 2d.
By sprouting in one month or so, obtain sprouting seedling.
Further, the sterile bletilla seed is obtained by following steps:
Mature uncracked bletilla fruit pod is cleaned, disinfection is removed moisture removal, cut off, 75% second of obtained bletilla seed
Alcohol impregnates 4-6min, then removes 75% ethyl alcohol.
Further, the sprouting culture is carried out using 900mm diameter Petri dishes.
Further, the seed number of the culture medium of each culture dish is 200~300.
By the culture of debita spissitudo, obtained suitable sprouting seedling.
The breeding method of bletilla seedling provided by the invention, the time cycle of production bletilla seedling are 5 months or so.
The present invention also provides a kind of breeding methods of bletilla seedling, comprising the following steps:
In the bletilla transplantation of seedlings that above-mentioned tissue culture method obtains to greenhouse domestication matrix, until seedling is long to 16-20cm, root vacation squama
Stem diameter is 3-5cm, carries out before Field planting slow seedling 10-15 days, then directly carries out Field planting.
High-quality bletilla pseudobulb provided by the invention carries out greenhouse domestication as bletilla seedling, and survival rate 100% improves
The yield of crop field final products produced the finished product period in 3~4 years.
Further, the bletilla transplantation of seedlings goes forward also to carry out the following processing to greenhouse domestication matrix: the culture of bletilla seedling
Container opens bottleneck, places 2-3 days or so, the then taking-up with tweezers by seedling gently, then with tap water by the culture of its root
Base is rinsed well and dries moisture.
Further, in the greenhouse domestication matrix, length and width 2 ± 0.2cm × 2 ± 0.2cm, incubation time are divided between spacing in the rows
It is 5~6 months.
Further, the greenhouse is duplicature greenhouse, and it is primary to carry out disinfection in canopy every other month.
Further, when the Field planting, per acre with seedling at 8000~10000 plants.
In addition, the formula of the 1/2MS culture medium in the present invention is as shown in table 1.
The 1 specific ingredient of MS culture medium of table
Compared with prior art, the invention has the benefit that
(1) during tissue culture, for the first time by bletilla tissue culture method strong seedling culture and the culture of rootage stage merge
For strengthening seedling and rooting culture, the step in production process is reduced, saves artificial and production cost, while avoiding in production process
Germ contamination.
(2) 5% cost has been saved compared with sucrose for sucrose using white granulated sugar the Tang Dynasty for the first time.
(3) strengthening seedling and rooting culture medium provided by the invention, finally screens by a large amount of optimum organizations and obtains, relative to it
His culture medium, under same culture conditions, the high-quality seedling rate which obtains is substantially improved, and improves to 40% or more.
(4) tissue culture method provided by the invention for obtaining high-quality bletilla pseudobulb makes industrial fast breeding production bletilla seedling
Time cycle shorten to 5 months.
(5) high-quality bletilla pseudobulb provided by the invention carries out greenhouse domestication as bletilla seedling, and survival rate 100% mentions
The high yield of crop field final products, produced the finished product period in 3~4 years.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is that the embodiment of the present invention 1 sprouts 1 month culture medium growing state figure of stage sprouting；
Fig. 2 is culture 4 months growing state figures of 1 strengthening seedling and rooting cultivation stage of the embodiment of the present invention；
Fig. 3 is the growing state figure that 1 greenhouse of the embodiment of the present invention is tamed 6 months；
Fig. 4 is the result figure that 1 different embodiments of the embodiment of the present invention obtain tissue-cultured seedling.
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 invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
A kind of tissue culture method obtaining high-quality bletilla pseudobulb, including following key step:
1) fruit pod is collected and is saved.In the bletilla that crop field is planted under natural conditions, generally opened in annual or so mid-April
Flower, fruit pod is in September part of current year or so maturation.Therefore, we select to collect full maturity in or so October but not crack
Bletilla fruit pod, dried up after 75% alcohol disinfecting in gnotobasis after drying in the cool, last Sterile vacuum environment storage
It is spare.
2) fruit pod processing before cultivating.Uncracked bletilla capsule is used into 75% ethyl alcohol soaking disinfection on superclean bench
5min or so, then with aseptic water washing 3 times, it is placed in suck dry moisture on the filter paper of sterilizing.
3) culture is sprouted in culture dish sowing.Bletilla fruit pod will be disinfected with the scissors of sterilization to cut off from middle part,
Seed is taken out, is inoculated into rapidly on the culture medium of configured 9cm diameter Petri dishes.Medium component are as follows: 1/2MS (1/2MS
(potassium concentration 20/mM)+6-BA+NAA+ white granulated sugar.Each fruit pod sows culture dish and handles 10~12, every culture dish training
About 200~300, seed for supporting base, are then sealed the mouth of culture medium with sealed membrane, and condition of culture is 19~23 DEG C of temperature, light
Culture 1 month of strong 1500~2500LX, daily illumination 12h and dark 12h.
4) strengthening seedling and rooting culture.Bletilla explant (seed) germination and growth on equal culture dishes culture medium was by 1 month or so
Time, be transplanted in the tissue culture bottle equipped with strengthening seedling and rooting culture medium with tweezers, each bottle is 50-60 root or so.Culture
Based component are as follows: 1/2MS (potassium concentration 20/mM)+6-BA+IBA+NAA+ white granulated sugar+mashed potato).It is then placed into group
It is cultivated on training room culturing rack, condition of culture are as follows: 22~24 DEG C of temperature, 1000~1500LX of light intensity, daily light irradiation time are 12h light
According to dark with 12h.Incubation time is 4 months.
5) greenhouse domestication and management.4 months being cultivated in culturing room, (seedling plant height about 5-6cm, microballoon stem weight are 0.5g left
It is right.) tissue culture bottle move on in greenhouse, open bottleneck, place 2-3 days or so, the then taking-up with tweezers by seedling gently, then with oneself
The culture medium of its root is rinsed well and dries moisture by water.It is finally colonized in the processed matrix of domestication well of greenhouse,
Length and width 2cmm is divided between spacing in the rows, cultivating is 6~8 months.Condition of culture is duplicature greenhouse, and the period in summer (prevents high temperature and strong
Light damages bletilla striata seedling) plastic house sunshade net is given, other conditions are greenhouse natural conditions.Pay attention in incubation anti-
Worm and diseases prevention, wherein it is primary to carry out disinfection in canopy every other month.Also certain foliar fertilizer is provided according to the growing state of seedling.
6) Field planting.The domesticated seedlings for having carried out greenhouse domestication 6 months or more are transported to the crop field put in order to carry out
Field planting.Per acre with seedling at 8000~10000 plants or so, survival rate is 100% or more after field planting.
Embodiment 1
Embodiment 1
Test site: Nanjing University
Test period: 2015.8-2017.12
Test material: it is acquired respectively for test variety bletilla from Guizhou Province Nayong County and Sansui County
A kind of tissue culture method obtaining high-quality bletilla pseudobulb, including following key step:
1. fruit pod is collected and is saved.In the bletilla that crop field is planted under natural conditions, generally opened in annual or so mid-April
Flower, fruit pod is in September part of current year or so maturation.Therefore, selection collects full maturity but uncracked white in or so October
And fruit pod, it is dried up after 75% alcohol disinfecting in gnotobasis after drying in the cool, last Sterile vacuum environment is in store for.
2. bletilla striata fruit pod and seed disinfection.Uncracked bletilla capsule is cleaned with washing powder solution, in tap water undershoot
5~10min is washed, then using 75% ethyl alcohol soaking disinfection 5min or so on superclean bench, then with aseptic water washing 3~4
It is secondary, then in the case where alcolhol burner flame envelope goes out slight bright burning 3-5, it is careful not to hardening crack bletilla fruit pod, is finally placed in the filter paper of sterilizing
Upper suck dry moisture.It if there is the bletilla fruit pod of cracking, can be cut off with diameter, fall its seed clean, utilize blank sheet of paper will
Its in the EP pipe, then toward fill in seed-bearing EP pipe plus 75% ethyl alcohol sterilization 5min, with micro-shifting device by seed with
Alcohol be sucked out, be placed on filter paper dried up in super-clean bench it is spare.
3. the preparation of germination medium.Disposable sterilized pure polystyrene 900mm culture dish is selected, it will be formulated
Good and 121 DEG C of autoclave sterilizations solid improves 1/2MS culture medium and pours into culture dish, and each culture dish pours into
The culture medium of 25mL.The main component of 1/2MS solid medium after wherein improving are as follows: 1/2MS (potassium concentration 20/mM)+
White granulated sugar (60g/L)+6-BA (1mg/L)+NAA (0.2mg/L)+agar powder (4.5g/L).
4. sowing bletilla seed.Uncracked bletilla fruit pod will be disinfected with the scissors of sterilization to cut from middle part
It opens, takes out seed, be inoculated into rapidly on the culture medium of configured 900mm diameter Petri dishes.Each fruit pod sows culture dish
Processing 10~12, then about 200~300, the seed of every culture dish culture medium is sealed the mouth of culture medium with sealed membrane.And
The bletilla fruit pod seed of cracking after the poison that disappeared, with toothpick by seed point on culture medium, after dibbling, by culture medium seal
Membrana oralis sealing.
5. sprouting culturing room's culture.The culture dish for having sowed seed is laid flat to be placed on culturing room's culturing rack and is cultivated.
Its condition of culture are as follows: 22~24 DEG C of temperature, 1500~2500LX of light intensity, daily light irradiation time: 12h illumination and 12h are dark, culture
Time is 1 month, and growing state is shown in Fig. 1.
6. the preparation of Rooting and hardening-off culture base.Selecting volume is the tissue culture vial of 650ml, and wide opening and bottle cap are ventilative, often
A bottle is 150ml culture medium, through 121 DEG C of autoclave sterilizations after preparing.Medium component are as follows: (potassium concentration is 1/2MS
20/mM)+white granulated sugar (60g/L)+6-BA (1.5mg/L)+NAA (0.3mg/L)+IBA (0.2mg/L) agar powder (4.5g/L)+horse
Bell mashed potatoes (100g/L).
7. transplanting seedlings to taking root and strong seedling culture base.It will be cultivated 1 month or so on sprouting culture and sprout seedling, sterile
It is transferred in tissue culture bottle in clean bench with the tweezers after having sterilized, each bottle connects 50-60 plants.
8. training orientation.The tissue culture bottle connected is placed on the tissue culture frame of tissue culture room and is cultivated.Condition of culture are as follows: temperature
22~24 DEG C, 1500~2500LX of light intensity, daily light irradiation time: 12h illumination and 12h are dark, and incubation time is 4 months, life
Long situation is shown in Fig. 2.
9. greenhouse domestication and training orientation.It will cultivate that (seedling plant height about 5-6cm, microballoon stem weight were in 4 months in culturing room
0.5g or so) tissue culture bottle move on in greenhouse, open bottleneck, place 2-3 days or so, the then taking-up with tweezers by seedling gently,
The culture medium of its root is rinsed well with tap water again and dries moisture.It is finally colonized and tames base well greenhouse is processed
Length and width 2cmm is divided into matter, between spacing in the rows, cultivating is 5~6 months (Fig. 3).Condition of culture is duplicature greenhouse, and the period in summer is (anti-
Only high temperature and strong light damage bletilla striata seedling) plastic house sunshade net is given, other conditions are greenhouse natural conditions.It cultivated
Insect prevention and diseases prevention are paid attention in journey, wherein it is primary to carry out disinfection in canopy every other month.Also to be provided according to the growing state of seedling
Certain foliar fertilizer.
10. completely natural slow seedling before field planting.Until the seedling of greenhouse culture grows to 16~20cm, root pseudobulb diameter
When for 3~5cm.The ventilation opening for opening culturing room's greenhouse, 10~15d of slow seedling is left before carrying out Field planting to the cultivating seedling in greenhouse
It is right.
11. Field planting.The domesticated seedlings for having carried out greenhouse domestication 6 months or more are transported to the crop field put in order to carry out
Field planting.Per acre with seedling at 8000~10000 plants or so, survival rate is 100% after field planting.
Embodiment 2
Unlike embodiment 1, germination medium are as follows: 1/2MS (potassium concentration 20/mM)+sucrose (60g/L)
+ 6-BA (1mg/L)+NAA (0.2mg/L)+agar powder (4.5g/L).
Rooting and hardening-off culture base are as follows: 1/2MS (potassium concentration 20/mM)+sucrose (60g/L)+6-BA (1mg/L)+NAA
(0.2mg/L)+IBA (0.2mg/L) agar powder (4.5g/L)+potato (100g/L).Other are all the same.
The domesticated seedlings for having carried out greenhouse domestication 6 months or more are transported to the crop field put in order to be colonized.Per acre
With seedling at 8000~10000 plants or so, survival rate is 90% after field planting.
Embodiment 3
Unlike embodiment 1, germination medium are as follows: 1/2MS (potassium concentration 20/mM)+white granulated sugar (60g/
L)+6-BA (1mg/L)+NAA (0.2mg/L)+agar powder (4.5g/L).
Rooting and hardening-off culture base are as follows: 1/2MS (potassium concentration 20/mM)+white granulated sugar (60g/L)+6-BA (1mg/L)+
NAA (0.2mg/L)+IBA (0.2mg/L) agar powder (4.5g/L)+banana puree (100g/L).Other are all the same.
The domesticated seedlings for having carried out greenhouse domestication 6 months or more are transported to the crop field put in order to be colonized.Per acre
With seedling at 8000~10000 plants or so, survival rate is 80% after field planting.
The strain number of high-quality seedling, medium seedling and inferior seedling that statistics different embodiments obtain, and calculate the percentage shared by it
Than as a result as shown in Figure 4.Wherein, high-quality seedling is the bletilla seedling that microballoon stem weight is not less than 0.5g, and medium seedling is microballoon stem weight
Less than 0.5g and more than or equal to the bletilla seedling of 0.14g, inferior seedling is the bletilla seedling less than 0.14g.
From fig. 4, it can be seen that the cultivation results of germination medium provided by the invention and Rooting and hardening-off culture base are significantly excellent
In the culture medium and combinations thereof of other groups.
Embodiment 2
A kind of tissue culture method obtaining high-quality bletilla pseudobulb is sprouted unlike the embodiment 1 in embodiment 1
Medium component are as follows: 1/2MS (potassium concentration 20/mM)+white granulated sugar (58g/L)+6-BA (1.2mg/L)+NAA (0.25mg/
L)+agar powder (4.7g/L).
Rooting and hardening-off culture based component are as follows: 1/2MS (potassium concentration 18/mM)+white granulated sugar (58g/L)+6-BA
(1.4mg/L)+NAA (0.32mg/L)+IBA (0.22mg/L)+agar powder (4.6g/L)+mashed potato (95g/L).
Obtained high-quality bletilla seedling accounting is almost the same with embodiment 1.
The domesticated seedlings for having carried out greenhouse domestication 6 months or more are transported to the crop field put in order to be colonized.Per acre
With seedling at 8000~10000 plants or so, survival rate is 100% after field planting.
Embodiment 3
A kind of tissue culture method obtaining high-quality bletilla pseudobulb is sprouted unlike the embodiment 1 in embodiment 1
Medium component are as follows: 1/2MS (potassium concentration 20mM)+white granulated sugar (62g/L)+6-BA (0.8mg/L)+NAA (0.15mg/
L)+agar powder (4.3g/L).
Rooting and hardening-off culture based component are as follows: 1/2MS (potassium concentration 18/mM)+white granulated sugar (62g/L)+6-BA
(1.5mg/L)+NAA (0.28mg/L)+IBA (0.18mg/L)+agar powder (4.4g/L)+mashed potato (105g/L).
Obtained high-quality bletilla seedling accounting is almost the same with embodiment 1.
The domesticated seedlings for having carried out greenhouse domestication 6 months or more are transported to the crop field put in order to be colonized.Per acre
With seedling at 8000~10000 plants or so, survival rate is 100% after field planting.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where 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 strengthening seedling and rooting culture medium, which is characterized in that its component includes: to contain potassium ion 18- in 1/2MS culture medium
20mM, 60 ± 2g/L of white granulated sugar, 6-BA 1.4-1.5mg/L, 0.3 NAA, 0.2 ± 0.02mg/L of ± 0.02mg/L, IBA and horse
100 ± 5g/L of bell mashed potatoes；
Further, the culture medium is solid medium, the components also include agar, the concentration of the agar is 4.5 ±
0.1g/L。
2. a kind of tissue culture method for obtaining high-quality bletilla pseudobulb, which is characterized in that the sprouting seedling of bletilla is transferred to claim 1
The strengthening seedling and rooting culture medium culture.
3. the tissue culture method according to claim 2 for obtaining high-quality bletilla pseudobulb, which is characterized in that the strengthening seedling and rooting
The condition of culture of culture medium are as follows: 22~24 DEG C of temperature, 1500~2500LX of light intensity, light application time 11-13h/d.
4. the tissue culture method according to claim 2 for obtaining high-quality bletilla pseudobulb, which is characterized in that the strengthening seedling and rooting
Culture bottle used in culture medium is the tissue culture vial that volume is 650ml, and wide opening and bottle cap are ventilative, and each bottle fills 150 ± 5ml
The strengthening seedling and rooting culture medium；
Further, the 50-60 plants of sprouting seedlings are planted in each tissue culture vial.
5. the tissue culture method according to claim 2 for obtaining high-quality bletilla pseudobulb, which is characterized in that the strengthening seedling and rooting
The time of culture medium culture is 120 ± 5d；
Further, the plant height of bletilla seedling obtained after cultivating 4 months is 5-6cm, and microballoon stem weight exists not less than 0.5g
85% or more.
6. according to the described in any item tissue culture methods for obtaining high-quality bletilla pseudobulb of claim 2-5, which is characterized in that described
Seedling is sprouted to be prepared by the following method:
Sterile bletilla seed is sowed, sprouting culture is carried out；
It is described to sprout culture medium component used are as follows: in 1/2MS culture medium containing potassium ion 18-20mM, white granulated sugar 60 ±
4.5 ± 0.2g/L of 1.0 ± 0.2mg/L of 2g/L, 6-BA, 0.2 ± 0.05mg/L of NAA and agar powder；
Further, the sprouting condition of culture are as follows: 22~24 DEG C of temperature, 1500~2500LX of light intensity, light application time 11-
13h/d；
Further, the time for sprouting culture is 30 ± 2d.
7. the tissue culture method according to claim 6 for obtaining high-quality bletilla pseudobulb, which is characterized in that the sterile bletilla
Seed is obtained by following steps:
Mature uncracked bletilla fruit pod is cleaned, disinfection is removed moisture removal, cut off, obtained bletilla seed is soaked with 75% ethyl alcohol
4-6min is steeped, 75% ethyl alcohol is then removed.
8. the tissue culture method according to claim 6 for obtaining high-quality bletilla pseudobulb, which is characterized in that the sprouting culture
It is carried out using 900mm diameter Petri dishes；
Further, the seed number of the culture medium of each culture dish is 200~300.
9. a kind of breeding method of bletilla seedling, which comprises the following steps:
The bletilla transplantation of seedlings that the described in any item tissue culture methods for obtaining high-quality bletilla pseudobulbs of claim 2-8 obtain is to greenhouse
It tames in matrix, until seedling is long to 16-20cm, root pseudobulb diameter is 3-5cm, carries out before Field planting slow seedling 10-15 days, so
Field planting is directly carried out afterwards.
10. the breeding method of bletilla seedling according to claim 9, which is characterized in that the bletilla transplantation of seedlings to greenhouse is tamed and dociled
Change matrix to go forward also to carry out the following processing: the culture vessel of bletilla seedling opens bottleneck, places 2-3 days or so, then will with tweezers
Seedling takes out, then rinses the culture medium of its root well with tap water and dry moisture；
Further, in the greenhouse domestication matrix, length and width 2 ± 0.2cm × 2 ± 0.2cm, incubation time 5 are divided between spacing in the rows
~6 months；
Further, the greenhouse is duplicature greenhouse, and it is primary to carry out disinfection in canopy every other month；
Further, when the Field planting, per acre with seedling at 8000~10000 plants.
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