CN105103714B - Manual germination accelerating method and seedling raising method of Bletilla sfriata (Thunb.)Reiehb.f. - Google Patents
Manual germination accelerating method and seedling raising method of Bletilla sfriata (Thunb.)Reiehb.f. Download PDFInfo
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- CN105103714B CN105103714B CN201510528672.4A CN201510528672A CN105103714B CN 105103714 B CN105103714 B CN 105103714B CN 201510528672 A CN201510528672 A CN 201510528672A CN 105103714 B CN105103714 B CN 105103714B
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Abstract
The invention provides a manual germination accelerating method. The method comprises the following steps: (1) inoculating seeds into a culture medium for cultivation, wherein the culture medium contains locust bean gum with mass percent being 0.1%-2.0%; and (2) inoculatingpaenibacillus xylanilyticusYUPP-1 onto the culture medium, and degrading the culture medium. The invention also provides application of the method in seedling raising of Bletilla sfriata (Thunb.)Reiehb.f. The application comprises the following steps: laying a substrate on the ground after performing germination accelerating by the method, spreading symbiotic strains of Bletilla sfriata (Thunb.)Reiehb.f. on a compartment surface to obtain a seedbed, and sowing buds of Bletilla sfriata (Thunb.)Reiehb.f. on the seedbed for cultivating and seedling raising. The manual germination accelerating method can be used for solving the problem that solid culture media carried on the roots of seedlings cannot be cleaned thoroughly, thereby enhancing the survival rate of seedlings remarkably; when the manual germination accelerating method is used by Bletilla sfriata (Thunb.)Reiehb.f. and is further applied to large-scale rapid seedling raising, the survival rate is improved to 95% or above, no dead seedling or rejuvenation period exists during transplanting to a field, and adult seedlings can be obtained by about 4-6 months in advance; the manual germination accelerating method is simple to operate and suitable for large-scale production, and has important guiding significance on manual large-scale plantation of orchidacea medicinal plants.
Description
Technical field
The invention belongs to agricultural science and technology technical field, more particularly to a kind of artificial germination method and Pseudobulbus Bletillae (Rhizoma Bletillae) method for culturing seedlings.
Background technology
The Pseudobulbus Bletillae (Rhizoma Bletillae), i.e. Bletilla sfriata (Thunb.) Reiehb.f., are land form orchid, and it is main medicinal
Part is dry Pseudobulbus Bletillae (Rhizoma Bletillae) tuber, main to contain Bletilla glucomannan, and its content is up to 50%.First, Bletilla glucomannan non-stimulated work nontoxic to skin
With being green safety product；Secondly, the effects such as Bletilla glucomannan has hemostasis tonifying the lung, myogenic pain relieving, for treating interior traumatic hemorrhage,
Sore swollen toxin, promotes skin repair to have special effect, is the primary raw material of the medicines such as YUNNAN BAIYAO, sunflower WEIKANGLING, bandage；Again
Person, Bletilla glucomannan also has functions that to remove interior free yl, delay skin aging, has extensive use in cosmetic industry；Most
Afterwards, Bletilla glucomannan has very high sticky nature, can act on as thickening agent, suspending agent, co-emulsifier etc., has in food service industry
Extensive purposes.National annual demand Pseudobulbus Bletillae (Rhizoma Bletillae) dry product about 2000-3000 tons, and annual reality supply only has 300 tons or so, lacks
It is mouthful huge, cause Pseudobulbus Bletillae (Rhizoma Bletillae) price to rise every year, the close 800 yuan/kg of present Pseudobulbus Bletillae (Rhizoma Bletillae) dry product.
The Pseudobulbus Bletillae (Rhizoma Bletillae) and the seed simple structure of other orchids, are proembryo stage when ripe, very tiny and without endosperm, many
Several sons are difficult rudiment seedling without the nutrition storage tissues needed for sprouting, under naturalness, only when bletilla striata seeds fall it is dark,
It is moist and could smooth rudiment seedling containing fungal component environment.Look forward to as Pseudobulbus Bletillae (Rhizoma Bletillae) price is high, wild resource is gradually excavated and connect
Near exhausted, this has encouraged further riseing for Pseudobulbus Bletillae (Rhizoma Bletillae) price again in turn.The maximum difficulty that at present Pseudobulbus Bletillae (Rhizoma Bletillae) planting industry faces is
Seedling critical shortage.
The natural propagation of the Pseudobulbus Bletillae (Rhizoma Bletillae) is that based on division propagation, planting biennial pseudobulb can only grow 1 new pseudobulb,
Increase by 2 within 3rd year, 4 are increased again within the 4th year, its breeding cycle is long, and reproductive efficiency is low, it is difficult to meet the needs of a large amount of cultivations.
Simultaneously because after the continuous generation of proliferation 3, the mother bulb stem of 1st generation is difficult to rudiment again, and this gives the seedling raising mannerses of traditional plant division
Larger difficulty is brought, some mother bulb stems are unable to sprout growth in actual production.
The aseptic seedling brought out based on bletilla striata seeds by tissue culture mode is at present can in a large number for the main side of Pseudobulbus Bletillae (Rhizoma Bletillae) Seedling
Formula.But tissue culture aseptic seedling maximum difficulty be the Solid agar culture that carries of seedling root cannot wash clean, cause artificial taming and dociling
Mortality rate during change by courses of infection is very high, and the survival rate of Pseudobulbus Bletillae (Rhizoma Bletillae) Seedling is low, it is difficult to meet the needs of Pseudobulbus Bletillae (Rhizoma Bletillae) industry development.Cause
This, the cultivation for strengthening Pseudobulbus Bletillae (Rhizoma Bletillae) high-quality seedling is most important.
Also experiment does direct sowing and seedling, but the condition of live Seedling requires quite harshness, needs fungal component to be inoculated with, Yi Jihe
Suitable temperature, humidity and pH value.Live Seedling defect is that germination rate is less than 10% and long the time required to rudiment, about needs 3-4 month
Ability rudiment differentiation is sprouted, and grows up to 1 year half time of Seedling needs of 5-10cm.
The content of the invention
In view of this, the invention provides a kind of artificial germination method and Pseudobulbus Bletillae (Rhizoma Bletillae) method for culturing seedlings, the artificial germination method solution
The problem that the solid medium that seedling root of having determined is carried cannot be cleaned, and the survival rate of method for culturing seedlings is high, seedling raise period is short.
First aspect present invention provides a kind of artificial germination method, comprises the following steps：
(1) seed is inoculated in culture medium and is cultivated, the culture medium includes the Robinia pseudoacacia L. that mass percent is 0.1-2.0%
Locust beam gum；
(2) solution xylan series bacillus are inoculated in culture medium, culture medium of degrading, the solution xylan class spore bar
Bacterium is preserved in Wuhan University's China typical culture collection center on January 8th, 2009, and preserving number is CCTCC NO:M
209014, it is named as solution xylan series bacillus Paenibacillus xylanilyticus YUPP-1.
The bacteria characteristic of solution xylan series bacillus YUPP-1：
Gram-positive or feminine gender, shaft-like, size is 0.3-0.8 × 3-5 μm, and suitable growth temperature is 20-30 DEG C,
Amphimicrobian, produces oval spore, the life of spore end.Salt ionic concentration is unfavorable for its growth and breeding higher than 1%.It is in meat soup solid
The dirty white of media surface bacterium colony, surface wettability is smooth.
Second aspect present invention provides the Pseudobulbus Bletillae (Rhizoma Bletillae) method for culturing seedlings for applying above-mentioned artificial germination method, including following step
Suddenly：
(1) accelerating germination is carried out to the Pseudobulbus Bletillae (Rhizoma Bletillae) using above-mentioned artificial germination method；
(2) substrate being spread on the ground, then Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component being spread on the face of railway carriage or compartment and obtain final product seedbed, Pseudobulbus Bletillae (Rhizoma Bletillae) bud is sowed to seedbed
Culture nursery；The preparation method of the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component comprises the steps：By sterilization Pseudobulbus Bletillae (Rhizoma Bletillae) tuber in culture medium in 23-
Cultivate 3-5 days at 27 DEG C, by hyphal colonization in Pseudobulbus Bletillae (Rhizoma Bletillae) tuber, selection is not result in the dead mycelia of Pseudobulbus Bletillae (Rhizoma Bletillae) aseptic seedling, as
Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component.
The invention has the beneficial effects as follows：The artificial germination method that the present invention is provided is by the addition thorn in the culture medium of accelerating germination
Locust bean gum, and addition solution xylan series bacillus YUPP-1 allows the special culture medium to liquefy completely after the completion of accelerating germination,
Solve the problems, such as that the solid medium that seedling root is carried cannot be cleaned, significantly improve the survival rate of Seedling；By the artificial germination
Method is gone forward side by side a stepping professional etiquette modelling fast seedling growing for the Pseudobulbus Bletillae (Rhizoma Bletillae), and the transplanting survival rate for overcoming tissue culture mode nursery not high lacks
Fall into, survival rate is improved to more than 95% by less than 50%, effect substantially, transplants land for growing field crops without seedling death phenomenon and seedling-slowing stage, while
Substantially shorten the seedling raise period of live Seedling, 4-6 month or so seedling, and simple to operate, suitable large-scale production, reduction can be shifted to an earlier date
Seedling cost, nursery speed and efficiency are improve, to the artificial implant mass of orchidaceae medicinal plant there is important guidance
Meaning.
Specific embodiment
First aspect present invention provides a kind of artificial germination method, comprises the following steps：
(1) seed is inoculated in culture medium and is cultivated, the culture medium includes the Robinia pseudoacacia L. that mass percent is 0.1-2.0%
Locust beam gum；
(2) solution xylan series bacillus are inoculated in culture medium, culture medium of degrading, the solution xylan class spore bar
Bacterium is preserved in Wuhan University's China typical culture collection center on January 8th, 2009, and preserving number is CCTCC NO:M
209014, it is named as solution xylan series bacillus Paenibacillus xylanilyticus YUPP-1.
By adding appropriate locust bean gum, and the addition solution xylan class bud after the completion of accelerating germination in the culture medium of accelerating germination
Spore bacillus YUPP-1 allows the special culture medium to liquefy completely, solves the solid medium of seedling root carrying and cannot clean
Problem, significantly improve the survival rate of Seedling.
Preferably, step (1) culture medium is to add mashed potatoes, activated carbon, agar, NAA, 6- in 1/2MS culture medium
BA and locust bean gum, the concentration for making mashed potatoes is 10-100g/L, and the concentration of activated carbon is 0.1-2g/L, the quality percentage of agar
Than for 0.1-1%, the concentration of NAA is 1.0-1.2mg/L, and the concentration of 6-BA is 0.5-1.0mg/L, the quality hundred of locust bean gum
Divide than being 0.1-2.0%.The culture medium prescription provides the nutrient substance needed for seed is sprouted, and carries out at accelerating germination under the culture medium
The seed of reason is put up the best performance on germination rate and growth of seedling state.
It is further preferred that the pH value of step (1) culture medium is 5.5-6.0.Seed is conducive to sprout under the pH value.
Preferably, step (1) seed is Orchid Seeds.
Second aspect present invention provides the Pseudobulbus Bletillae (Rhizoma Bletillae) method for culturing seedlings for applying above-mentioned artificial germination method, including following step
Suddenly：
(1) accelerating germination is carried out to the Pseudobulbus Bletillae (Rhizoma Bletillae) using above-mentioned artificial germination method；
(2) substrate being spread on the ground, then Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component being spread on the face of railway carriage or compartment and obtain final product seedbed, Pseudobulbus Bletillae (Rhizoma Bletillae) bud is sowed to seedbed
Culture nursery；The preparation method of the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component comprises the steps：By sterilization Pseudobulbus Bletillae (Rhizoma Bletillae) tuber in culture medium in 23-
Cultivate 3-5 days at 27 DEG C, by hyphal colonization in Pseudobulbus Bletillae (Rhizoma Bletillae) tuber, selection is not result in the dead mycelia of Pseudobulbus Bletillae (Rhizoma Bletillae) aseptic seedling, as
Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component.
Preferably, the compound method of step (2) culture medium includes：By Pseudobulbus Bletillae (Rhizoma Bletillae) tuber in water it is well-done after be filtrated to get
Filtrate, to by addition gained filtrate, agar in PDA culture medium, makes filtrate quality percentage ratio for 0.1-0.3%, agar quality hundred
Divide than being 1.0-1.4%, when being cooled to 40-50 DEG C after sterilizing, kalamycin and ampicillin added in the medium so as to
Final concentration is respectively 20-30 μ g/mL and 95-105 μ g/mL, mixes, solidification.The culture of the formula is adopted for selective
Culture medium, can avoid the growth of miscellaneous bacteria, beneficial to obtaining purer mycelia.
It is further preferred that spread Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component described in step (2) on the face of railway carriage or compartment reaching for bacterial content in making every gram of substrate
108-109Individual spore.
It is further preferred that the method for step (2) sterilization comprises the steps：It is soaked in after Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is cleaned
Rinse in ethanol, during 4% liquor natrii hypochloritises will be proceeded to after Pseudobulbus Bletillae (Rhizoma Bletillae) stripping and slicing, after sterilization 8-15min rinsed with sterile water is used.
Preferably, nitrogen content is 1.0-2.0g/kg in step (2) substrate, and phosphorus content is 0.5-2.0g/kg, and potassium contains
Measure as 0.5-2.0g/kg, quality of organic matter percentage ratio is 40-70%.
A kind of artificial germination method and Pseudobulbus Bletillae (Rhizoma Bletillae) method for culturing seedlings provided the present invention below in conjunction with embodiment is entered one
Step explanation.
Embodiment 1
Place is selected in the farm of government's bank of Zhongxiang District Hubei Province city lake and completes the present embodiment, and the farm is located at 30 ° or so of north latitude, sea
About 200m is pulled out, physical features relatively flat has standardized controllable greenhouse 3000m2, the interior row of laying seedbed 16, every row seedbed width
2m, long 70m, greenhouse East and West direction and are both needed to above with the sunshade net sunshade of 6 pin densities, and shading rate reaches 90-95%.
Bletilla striata seeds are carried out into artificial germination in experiment interior, concrete steps include：
(1) storage of bletilla striata seeds and sterilization：Pseudobulbus Bletillae (Rhizoma Bletillae) Fruit pod is closed with plastic film bag encapsulation after the 10-11 months harvest,
4 DEG C of refrigerators are stored in, this kind of method can enable bletilla striata seeds survive 6-9 month.Pseudobulbus Bletillae (Rhizoma Bletillae) Fruit pod disinfecting process should be in ultra-clean work
Complete on platform, first rinsed well with tap water, then blotted in the water of Fruit pod remained on surface with aseptic filter paper, be then soaked in
2-3min is rinsed in 75% ethanol carries out surface sterilization, then Fruit pod is proceeded to incision in 4% liquor natrii hypochloritises, obtains the Pseudobulbus Bletillae (Rhizoma Bletillae)
Seed, removes Fruit pod shell, after sterilization 10min, bletilla striata seeds is transferred in the 50mL centrifuge tubes of sterilizing on superclean bench,
Close the lid, ready to balance is centrifuged 5min, the liquor natrii hypochloritises of tipping 8% on superclean bench after 8000rpm；In centrifuge tube
In add 75% ethanol 30mL, blow and beat even bletilla striata seeds with sterilizing pipette tips, sterilize 2min, 8000rpm centrifugation 5min, ultra-clean
Tipping supernatant on workbench, then rinsed 3 times according to said method with sterile deionized water, tipping supernatant finally leaves bletilla striata seeds,
The bletilla striata seeds for as disinfecting.
(2) culture medium for being adapted to accelerating germination is prepared：Add mashed potatoes, activated carbon, agar, NAA, 6-BA in 1/2MS culture medium
And locust bean gum, the concentration for making mashed potatoes is 100g/L, and the concentration of activated carbon is 0.5g/L, and the mass percent of agar is
The concentration of 0.1%, NAA is 1.0mg/L, and the concentration of 6-BA is 0.5mg/L, and the mass percent of locust bean gum is 0.5%.
The 1/2MS culture medium refers to the MS culture medium that a great number of elements and calcium salt content halve.The 1/2MS that the present embodiment is adopted
Culture medium compound method is as follows：A great number of elements mother solution 25ml, Calcisolution 25ml, trace element mother solution 10ml are taken, vitamin is female
Liquid 10ml, mother liquid of iron salt 5ml, plus inositol 0.1g, 20-30g of sucrose or white sugar, distilled water is settled to 1L.
Wherein, a great number of elements mother liquor method is：NH4NO316.5g；MgSO4·7H2O 3.7g；KH2PO41.7g；
KNO319g, is dissolved in water, and is settled to 500ml；
Calcisolution compound method is：CaCl23.32g, is dissolved in water, and is settled to 500ml；
Trace element mother liquor method is：KI 0.0415g；Na2MoO4·2H2O 0.0125g；H3BO30.31g；
CuSO4·5H2O 0.00125g；MnSO4·H2O 0.845g；CoCl2·6H2O 0.00125g；ZnSO4·7H2O 0.43g, plus
Water dissolution, is settled to 500ml；
Mother liquid of iron salt compound method is：EDETATE SODIUM 3.725g, plus 100ml water heating for dissolving, FeSO4·7H2O
Both mix after 2.785g, plus 100ml water dissolutioies, add water and are made into 500ml；
Vitamin stock solution compound method is：Pyridoxine hydrochloride (VB6) 0.025g；Thiamine hydrochloride (VB1) 0.025g；Nicotinic acid
0.025g, glycine 0.1g, is dissolved in water, and is settled to 500ml.
(3) accelerating germination of bletilla striata seeds：A certain amount of sterilized water is added to the bletilla striata seeds after sterilization, by 1 Fruit pod 20mL without
The ratio of bacterium water is watered, and with the steel spoon of sterilizing seed is stirred evenly, and then per bottle is put into 2 spoonfuls of seed liquor in accelerating germination solid culture base table
Face, about 500-1000 grain seeds, jog culture medium is uniformly distributed in media surface to seed；Covered with black plastic bag,
Light culture 10 days at 25-28 DEG C, make to absorb nutrition expansion rudiment, seed greening containing kind of blastocyte.Then simulation nature is used instead
Alternate illumination condition, i.e. make intensity of illumination reach 1500lx additional natural scattered lights using fluorescent tube daytime, night keeps
Dark, through 30 days or so, Pseudobulbus Bletillae (Rhizoma Bletillae) seedling differentiation went out 1-2 leaves.
After completing accelerating germination, Pseudobulbus Bletillae (Rhizoma Bletillae) nursery is further carried out, concrete steps include：
(1) culture medium liquefaction and seedling taking：First, the bacterial strain of activated overnight degraded locust bean gum --- solution xylan class spore
Bacillus YUPP-1, by 1% inoculum concentration inoculated and cultured 20h after, the culture medium of bletilla striata seeds accelerating germination is then inoculated in by 5% inoculum concentration
On, concentration is about 1 × 108Cfu/mL, quiescent culture 24h, solid medium liquefies after locust bean gum is decomposed, and will be broken up with water
The Pseudobulbus Bletillae (Rhizoma Bletillae) bud for going out 1-2 piece leaves is washed upside down on gauze, washes away unnecessary nutrient, the Pseudobulbus Bletillae (Rhizoma Bletillae) bud that as can be sowed.
(2) prepared by seedbed：Ground is flattened, Culture media, the N of the substrate, P, k, organic matter are then spread above
Content is respectively：1.5g/kg, 1.5g/kg, 1.0g/kg, 50%, substrate railway carriage or compartment face width 1.5-2m, high 10-15cm, length can basis
Plot determines.Before the paving Pseudobulbus Bletillae (Rhizoma Bletillae) bud of seedbed, need to spread last layer Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component again on the face of railway carriage or compartment, mix homogeneously with dry matrices, make
Bacterial content reaches 1 × 108Spore/g, is then spread on the face of railway carriage or compartment, and then with mist-like shower nozzle, by railway carriage or compartment face, entirely spray is saturating.
The Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component obtains according to the following steps：
First, Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is sterilized, concrete grammar includes：Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is cleaned, then on superclean bench, will
It is soaked in 75% ethanol and rinses 10min, and 0.5cm (length) × 0.5cm is cut in the liquor natrii hypochloritises that the Pseudobulbus Bletillae (Rhizoma Bletillae) is proceeded to 4%
The fritter of (width) × 0.3cm (height), it is with rinsed with sterile water 3 times then with aseptic filter paper that Pseudobulbus Bletillae (Rhizoma Bletillae) block surface is residual after sterilization 10min
The water for staying is blotted, the Pseudobulbus Bletillae (Rhizoma Bletillae) endogenetic fungus separation material for as disinfecting.
Then, plating medium is prepared, concrete grammar includes：Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is well-done in the water for waiting quality, filter
To filtrate, to by addition gained filtrate, agar in PDA culture medium, filtrate quality percentage ratio is set to be 0.2%, agar quality percentage
Than for 1.2%, when 45 DEG C are cooled to after sterilizing, kalamycin and ampicillin being added in the medium so as to final concentration point
Not Wei 25 μ g/mL and 100 μ g/mL, mix, be down flat plate after flat board solidification after it is standby.
The PDA culture medium formula is：Rhizoma Solani tuber osi 200g, glucose 20g, agar 20g, water 1000Ml.
Finally, it is inoculated with, concrete grammar includes：The Pseudobulbus Bletillae (Rhizoma Bletillae) block of sterilization is attached to into above-mentioned plating medium surface, 25 DEG C
Lower culture 4 days, the mycelia that purification grows selects germination and growth at first, and front end growth is neat, without the substantially miscellaneous bacteria such as red, yellowish green
Mycelia, then on mycelium inoculation to Pseudobulbus Bletillae (Rhizoma Bletillae) aseptic seedling, can will smoothly colonize in Pseudobulbus Bletillae (Rhizoma Bletillae) tuber and be not result in that Pseudobulbus Bletillae (Rhizoma Bletillae) Seedling is dead
The mycelia for dying is required Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component.
The Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component obtained using above method screening is respectively provided with facilitation to Pseudobulbus Bletillae (Rhizoma Bletillae) growth, identified, this
The Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component obtained in enforcement is Rhizoctonia and non-pathogenic Fusarium spp. each 50%.
(3) seedbed paving Seedling：In paving Seedling the previous day, seedbed is sprayed into saturating with water, it is with water that clean Pseudobulbus Bletillae (Rhizoma Bletillae) Seedling is dilute during paving Seedling
Release, be then poured onto on seedbed with downpour, substantially seed bud thickness of sowing is 500-1000/square meter.Daily with spray within 1 month
Head spraying makes railway carriage or compartment face keep moistening for 1 hour, and humidity is maintained at more than 80% in air, and every time ground can not have obvious product after spraying
Water, warm temperature of shed is controlled between 18 DEG C -28 DEG C.
(4) seedbed water management：After seedling is spread in seedbed, to be sprayed water daily, seedbed is drenched, but there can not be obvious hydrops；
Temperature of shed is controlled at 18 DEG C -28 DEG C；Kept seedbed humidity to reach 100% in 15 days, after Seedling field planting is stood, can suitably subtract
The number of times of few moisture spray, at this moment, can keep railway carriage or compartment surface humidity in 80-90%.
Seedbed nutritional care：After the field planting of Pseudobulbus Bletillae (Rhizoma Bletillae) bud, at interval of 10 days, with urea quality percentage ratio 1%, potassium dihydrogen phosphate
Mass percent 0.5%, the aqueous solution of white sugar quality percentage ratio 0.5% are sprayed, and every time can not there be substantially ground after spraying
Hydrops so as to Rapid development seedling.
Weed in seedbed is prevented and kill off：Due to watering everyday, humidity is larger on seedbed, it should be noted that the growth of aquatic weed, pulls out in time
Except these weeds, in order to avoid contention nutrient.
The artificial germination method provided using the present embodiment, through 30 days, Pseudobulbus Bletillae (Rhizoma Bletillae) Seedling can differentiate 1-2 leaves, by connecing
Enter to solve xylan series bacillus YUPP-1 as culture medium decomposer, make accelerating germination culture medium be liquefied, directly rinse children with water
Seedling can remove seedling root culture medium, can obtain for sowing seedling, it is simple and convenient, overcome in routine techniquess due to
Seedling root carry solid medium cannot wash clean, cause during domestication by courses of infection make mortality rate it is high lack
Fall into.Using the method accelerating germination, then in conjunction with fungal component seedbed, survival rate to more than 95%, compared to conventional seedbed system method
It is significantly improved.1 year half time of Seedling needs of 5-10cm is grown up to using conventional seedbed system method, and in the present embodiment, transplants land for growing field crops
Without seedling death phenomenon and seedling-slowing stage, the Pseudobulbus Bletillae (Rhizoma Bletillae) Seedling for obtaining plant height 10cm or so for 6-9 month is cultivated in seedbed, seedling raise period substantially contracts
It is short, it is simple to operate, be adapted to large-scale production, reduce seedling cost, improve nursery speed and efficiency.Embodiment 2
Embodiment 2 is substantially the same manner as Example 1, and difference is：
In the present embodiment, bletilla striata seeds are carried out into artificial germination in experiment interior, used culture medium is：1/2MS is trained
Add mashed potatoes, activated carbon, agar, NAA, 6-BA and locust bean gum in foster base, the concentration for making mashed potatoes is 10g/L, activated carbon
Concentration be 2.0g/L, the mass percent of agar is 1.2mg/L for the concentration of 1.0%, NAA, and the concentration of 6-BA is 1.0mg/
L, the mass percent of locust bean gum is 2.0%, pH value 6.0.
In the nursery stage, seedbed is prepared as follows：Ground is flattened, matrix nutrition is then spread above
Soil, the N of the substrate, P, k, the content of organic matter are respectively：1.0g/kg, 0.5g/kg, 0.5g/kg, 70%, substrate railway carriage or compartment face width
2m, high 15cm, length can be determined according to plot.Before the paving Pseudobulbus Bletillae (Rhizoma Bletillae) bud of seedbed, need to spread last layer Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component again on the face of railway carriage or compartment, with
Dry matrices mix homogeneously, makes bacterial content reach 1 × 109Spore/g, is then spread on the face of railway carriage or compartment, then with mist-like shower nozzle by railway carriage or compartment
Entirely spray is saturating in face.
The Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component obtains according to the following steps：
First, Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is sterilized, concrete grammar includes：Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is cleaned, then on superclean bench, will
It is soaked in 75% ethanol and rinses 10min, and 0.5cm (length) × 0.5cm is cut in the liquor natrii hypochloritises that the Pseudobulbus Bletillae (Rhizoma Bletillae) is proceeded to 4%
The fritter of (width) × 0.3cm (height), it is with rinsed with sterile water 3 times then with aseptic filter paper that Pseudobulbus Bletillae (Rhizoma Bletillae) block surface is residual after sterilization 15min
The water for staying is blotted, the Pseudobulbus Bletillae (Rhizoma Bletillae) endogenetic fungus separation material for as disinfecting.
Then, plating medium is prepared, concrete grammar includes：Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is well-done in the water for waiting quality, filter
To filtrate, to by addition gained filtrate, agar in PDA culture medium, filtrate quality percentage ratio is set to be 0.3%, agar quality percentage
Than for 1.4%, when 50 DEG C are cooled to after sterilizing, kalamycin and ampicillin being added in the medium so as to final concentration point
Not Wei 30 μ g/mL and 105 μ g/mL, mix, be down flat plate after flat board solidification after it is standby.
Finally, it is inoculated with, concrete grammar includes：The Pseudobulbus Bletillae (Rhizoma Bletillae) block of sterilization is attached to into above-mentioned plating medium surface, 23 DEG C
Lower culture 5 days, the mycelia that purification grows selects germination and growth leading portion growth at first neat, without the substantially miscellaneous bacteria such as red, yellowish green
Mycelia, then on mycelium inoculation to Pseudobulbus Bletillae (Rhizoma Bletillae) aseptic seedling, will be not result in that Pseudobulbus Bletillae (Rhizoma Bletillae) Seedling is dead, and mycelia can be smoothly colonized in Pseudobulbus Bletillae (Rhizoma Bletillae) block
In stem, as required Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component.
Embodiment 3
Embodiment 3 is substantially the same manner as Example 1, and difference is：
In the present embodiment, bletilla striata seeds are carried out into artificial germination in experiment interior, used culture medium is：1/2MS is trained
Add mashed potatoes, activated carbon, agar, NAA, 6-BA and locust bean gum in foster base, the concentration for making mashed potatoes is 60g/L, activated carbon
Concentration be 1.2g/L, the mass percent of agar is 1.1mg/L for the concentration of 0.5%, NAA, and the concentration of 6-BA is 0.8mg/
L, the mass percent of locust bean gum is 1.5%, pH value 5.8.
In the nursery stage, seedbed is prepared as follows：Ground is flattened, matrix nutrition is then spread above
Soil, the N of the substrate, P, k, the content of organic matter are respectively：2.0g/kg, 2.0g/kg, 2.0g/kg, 40%, substrate railway carriage or compartment face width
2m, high 15cm, length can be determined according to plot.Before the paving Pseudobulbus Bletillae (Rhizoma Bletillae) bud of seedbed, need to spread last layer Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component again on the face of railway carriage or compartment, with
Dry matrices mix homogeneously, makes bacterial content reach 1 × 108Spore/g, is then spread on the face of railway carriage or compartment, then with mist-like shower nozzle by railway carriage or compartment
Entirely spray is saturating in face.
The Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component obtains according to the following steps：
First, Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is sterilized, concrete grammar includes：Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is cleaned, then on superclean bench, will
It is soaked in 75% ethanol and rinses 10min, and 0.5cm (length) × 0.5cm is cut in the liquor natrii hypochloritises that the Pseudobulbus Bletillae (Rhizoma Bletillae) is proceeded to 4%
The fritter of (width) × 0.3cm (height), it is with rinsed with sterile water 3 times then with aseptic filter paper that Pseudobulbus Bletillae (Rhizoma Bletillae) block surface is residual after sterilization 15min
The water for staying is blotted, the Pseudobulbus Bletillae (Rhizoma Bletillae) endogenetic fungus separation material for as disinfecting.
Then, plating medium is prepared, concrete grammar includes：Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is well-done in the water for waiting quality, filter
To filtrate, to by addition gained filtrate, agar in PDA culture medium, filtrate quality percentage ratio is set to be 0.1%, agar quality percentage
Than for 1.2%, when 48 DEG C are cooled to after sterilizing, kalamycin and ampicillin being added in the medium so as to final concentration point
Not Wei 28 μ g/mL and 98 μ g/mL, mix, be down flat plate after flat board solidification after it is standby.
Finally, it is inoculated with, concrete grammar includes：The Pseudobulbus Bletillae (Rhizoma Bletillae) block of sterilization is attached to into above-mentioned plating medium surface, 24 DEG C
Lower culture 4 days, the mycelia that purification grows selects germination and growth leading portion growth at first neat, without the substantially miscellaneous bacteria such as red, yellowish green
Mycelia, then on mycelium inoculation to Pseudobulbus Bletillae (Rhizoma Bletillae) aseptic seedling, will be not result in that Pseudobulbus Bletillae (Rhizoma Bletillae) Seedling is dead, and mycelia can be smoothly colonized in Pseudobulbus Bletillae (Rhizoma Bletillae) block
In stem, as required Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component.
The foregoing is only presently preferred embodiments of the present invention, not to limit the present invention, all spirit in the present invention and
Within principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
Claims (8)
1. a kind of artificial germination method, it is characterised in that：The method comprising the steps of：
(1) bletilla striata seeds are inoculated in culture medium and are cultivated, the culture medium includes the Robinia pseudoacacia L. that mass percent is 0.1-2.0%
Locust beam gum；
(2) solution xylan series bacillus are inoculated in culture medium, culture medium of degrading, the solution xylan series bacillus
Preserving number is CCTCC NO:M 209014, is named as solution xylan series bacillus (Paenibacillus
xylanilyticus)YUPP-1。
2. artificial germination method as claimed in claim 1, it is characterised in that：Step (1) culture medium is 1/2MS culture medium
Middle addition mashed potatoes, activated carbon, agar, NAA, 6-BA and locust bean gum, the concentration for making mashed potatoes is 10-100g/L, activated carbon
Concentration be 0.1-2g/L, the mass percent of agar is 0.1-1%, and the concentration of NAA is 1.0-1.2mg/L, the concentration of 6-BA
For 0.5-1.0mg/L, the mass percent of locust bean gum is 0.1-2.0%.
3. artificial germination method as claimed in claim 2, it is characterised in that：The pH value of step (1) culture medium is 5.5-
6.0。
4. a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) method for culturing seedlings of the artificial germination method applied described in claim 1, it is characterised in that：Including following
Step：
A carries out accelerating germination using the artificial germination method described in claim 1 to the Pseudobulbus Bletillae (Rhizoma Bletillae)；
B spreads on the ground substrate, then Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component is spread on the face of railway carriage or compartment obtains final product seedbed, Pseudobulbus Bletillae (Rhizoma Bletillae) bud is sowed to seedbed into culture and is educated
Seedling；The preparation method of the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component comprises the steps：By sterilization Pseudobulbus Bletillae (Rhizoma Bletillae) tuber in culture medium at 23-27 DEG C
Culture 3-5 days, by hyphal colonization in Pseudobulbus Bletillae (Rhizoma Bletillae) tuber, selection is not result in the dead mycelia of Pseudobulbus Bletillae (Rhizoma Bletillae) aseptic seedling, and the as Pseudobulbus Bletillae (Rhizoma Bletillae) is total to
Raw bacterium.
5. the Pseudobulbus Bletillae (Rhizoma Bletillae) method for culturing seedlings of artificial germination method is applied as claimed in claim 4, it is characterised in that：Described in step b
The compound method of culture medium includes：Filtrate is filtrated to get after Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is boiled into 20-30min in water, to by PDA culture medium
Addition gained filtrate, agar, make filtrate quality percentage ratio be 0.1-0.3%, and agar mass percent is 1.0-1.4%, is sterilized
After be cooled to 40-50 DEG C, kalamycin and ampicillin are added in the medium so as to which final concentration is respectively 20-30 μ g/mL
With 95-105 μ g/mL, mix, solidification.
6. the Pseudobulbus Bletillae (Rhizoma Bletillae) method for culturing seedlings of artificial germination method is applied as claimed in claim 5, it is characterised in that：Described in step b
Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component is spread on the face of railway carriage or compartment to make in every gram of substrate bacterial content up to 108-109Individual spore.
7. the Pseudobulbus Bletillae (Rhizoma Bletillae) method for culturing seedlings of artificial germination method is applied as claimed in claim 5, it is characterised in that：Described in step b
The method of sterilization comprises the steps：It is soaked in after Pseudobulbus Bletillae (Rhizoma Bletillae) tuber is cleaned in ethanol and rinses, 4% will be proceeded to after Pseudobulbus Bletillae (Rhizoma Bletillae) stripping and slicing
In liquor natrii hypochloritises, after sterilization 8-15min rinsed with sterile water is used.
8. the Pseudobulbus Bletillae (Rhizoma Bletillae) method for culturing seedlings of artificial germination method is applied as claimed in claim 4, it is characterised in that：Described in step b
Nitrogen content is 1.0-2.0g/kg in substrate, and phosphorus content is 0.5-2.0g/kg, and potassium content is 0.5-2.0g/kg, quality of organic matter
Percentage ratio is 40-70%.
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| CN105519350B (en) * | 2015-12-14 | 2018-07-03 | 广西壮族自治区药用植物园 | The method of quick production bletilla seedling |
| CN108094105A (en) * | 2018-01-21 | 2018-06-01 | 大姚县春苗中药材种植有限公司 | The cultivation implantation methods of the bletilla striata |
| CN108719070A (en) * | 2018-06-12 | 2018-11-02 | 中国科学院武汉植物园 | A kind of tissue culture seed disinfection of bletilla and inoculation method |
| CN110338047B (en) * | 2019-06-28 | 2021-09-17 | 长江大学 | Wild-imitating efficient direct seeding seedling method for bletilla striata seeds |
| CN111903518A (en) * | 2020-07-28 | 2020-11-10 | 安徽东方金桥农林科技股份有限公司 | Rapid propagation method of bletilla striata plants by taking bletilla striata rhizome as explant |
| CN113080001B (en) * | 2021-04-19 | 2023-04-07 | 福建坤加建设有限公司 | Garden orchid seedling growing method |
| CN113207590A (en) * | 2021-04-29 | 2021-08-06 | 南京工业大学大丰海洋产业研究院 | Bletilla direct seeding and seedling raising method utilizing symbiotic bacteria |
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| CN104041276A (en) * | 2014-06-13 | 2014-09-17 | 江苏茅山地道中药材种植有限公司 | Pollution-free bletilla striata cultivation method |
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