CN105028215B - It is a kind of for the root media of bletilla striata tissue culture and the tissue culture method of the bletilla striata - Google Patents
It is a kind of for the root media of bletilla striata tissue culture and the tissue culture method of the bletilla striata Download PDFInfo
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- CN105028215B CN105028215B CN201510593409.3A CN201510593409A CN105028215B CN 105028215 B CN105028215 B CN 105028215B CN 201510593409 A CN201510593409 A CN 201510593409A CN 105028215 B CN105028215 B CN 105028215B
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Abstract
The invention provides a kind of root media for bletilla striata tissue culture and the tissue culture method of the bletilla striata.A kind of root media for bletilla striata tissue culture, including nutriment and sawdust, and every liter of root media contains 160 180g sawdusts.The tissue culture method of the bletilla striata comprises the following steps：Bletilla striata seeds are made to transfer to by sprouting and breaking up and carry out culture of rootage in above-mentioned root media.The bletilla striata seedling that the root media of the present invention is cultivated easily is transplanted, and has the advantages that transplanting survival rate is high, cost is low, need not clean nursery.
Description
Technical field
The present invention relates to tissue culture field, in particular to a kind of root media for bletilla striata tissue culture and the bletilla striata
Tissue culture method.
Background technology
The bletilla striata is perennial herb bulbous plant, and plant is high 18-60 centimetres, is mainly distributed on China, Japan and Burma
The north, the main florescence is but different because of various regions weather in spring, and late Winter Solstice early summer may all bloom.The bletilla striata has extensive medicinal
Value and Ornamental value.The bletilla striata is mainly used in astringing to arrest bleeding, detumescence and promoting granulation, can potted plant indoor appreciation, can also intersperse in more shady
One jiao of flower stand, flower border or the garden covered.
Contain ten hundreds of trickle seeds in the capsule of the bletilla striata, after sterilization, select suitable culture medium, carry out white
The axenic germination of splendid achnatherum seed, in production to use plate method nursery to plant, i.e. " seed sprouting-differentiation-induction more
Take root seedling " mode mass produced, after last process of rooting culture, bletilla tissue culture seedlings can be moved with bottle outlet
Plant, but traditional root media is agar medium, it is easily attached to bletilla striata seedling root, is not easy to take out from vial,
Also be inconvenient to clean, easily hurt seedling root, have a strong impact on transplanting survival rate.
In view of this, it is special to propose the present invention.
The content of the invention
The first object of the present invention is to provide a kind of root media for bletilla striata tissue culture, described root media
Easily transplanting bletilla striata seedling, have the advantages that transplanting survival rate is high, cost is low, need not clean nursery.
The second object of the present invention is to provide a kind of tissue culture method of the bletilla striata, the bletilla striata that described tissue culture method is turned out
Seedling has transplanting survival rate height, low cost and other advantages.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of root media for bletilla striata tissue culture, including nutriment and sawdust, and every liter of culture of rootage
Base sawdust containing 160-180g.
Above-mentioned root media instead of traditional coagulator-agar powder using sawdust, thus bletilla striata when avoiding transplanting
The problem of seedling root needs cleaning by agar adhesion, will not injure seedling root, so as to improve the transplanting survival rate of bletilla striata seedling because of cleaning.
Simultaneously compared with agar powder, sawdust can recycle, and cost is relatively low.In addition, the sawdust of 160-180g/L concentration is suitable
Bletilla striata seedling is taken root.
In above-mentioned root media, sawdust can use the sawdust of any plant, such as：Wax gourd tree sawdust, pine tree saw
End, Chinese fir sawdust and eucalyptus sawdust etc., all sawdusts all have the effect of ventilative water conservation, and wherein wax gourd tree and Chinese fir sawdust are containing fibre
Peacekeeping carboritride is higher, is adapted to needed for plant growth, and pine tree and eucalyptus are higher containing terpene substances, have and suppress plant growth
Effect after pile fermentation, it is necessary to can just use.
Preferably, the nutriment includes：The MS culture mediums of original concentration, the 0.1-2.0mg/L basic elements of cell division and
0.1-1.0mg/L methyl α-naphthyl acetates.
The bletilla striata seedling stalk cultivated using this nutriment is sturdy.Further to improve the matter for the bletilla striata seedling cultivated
Amount, can also use following nutriment
The modified form MS culture mediums of original concentration, the 0.1-2.0mg/L basic elements of cell division and 0.1-1.0mg/L methyl α-naphthyl acetates；Institute
The formula for stating modified form MS culture mediums is：Potassium nitrate 1890-1900mg/L, ammonium nitrate 1650-1660mg/L, calcium chloride dihydrate
440-445mg/L, epsom salt 370-375mg/L, potassium dihydrogen phosphate 165-170mg/L, KI 0.75-0.83mg/L,
Boric acid 5-6.2mg/L, four water manganese sulfate 22.3-24mg/L, white vitriol 8.6-10mg/L, sodium molybdate 0.2-0.25mg/L,
Cupric sulfate pentahydrate 0.025-0.03mg/L, cobalt chloride 0.025-0.03mg/L, ferrous sulfate 27.8-30mg/L, ethylenediamine tetrem
Sour 24.3-26mg/L, inositol 95-100mg/L, nicotinic acid 0.8-1mg/L, puridoxine hydrochloride 0.8-1mg/L, nicotinic acid thiamine 0.8-
1mg/L, glycine 3-4mg/L, pH 5.8-6.0.
On the basis of above-mentioned nutriment, following material can be added：60-70g/L bananas juices, 30-40g/L potatoes
Juice, 30-40g/L sucrose, to shorten cultivation cycle, improve the robustness of bletilla striata seedling.Specifically, murphy juice plays offer nutrients
Matter and somatotrophic effect.Bananas juice, beneficial to bletilla striata seedling rooting strengthening root, improve survival rate of the bletilla striata in outdoor hardening.Sucrose, carry
For carbon source, make bletilla striata seedling robust growth, and reduce the pollution of microorganism to a certain extent.
It is highly preferred that the concentration of the basic element of cell division is 0.1-0.2mg/L, the concentration of methyl α-naphthyl acetate is preferably 0.8-1.0mg/L,
The basic element of cell division preferably selects 6-BA.
Preferably, the formula of the modified form MS culture mediums is：Potassium nitrate 1895-1900mg/L, ammonium nitrate 1650-
1655mg/L, calcium chloride dihydrate 440-442mg/L, epsom salt 370-373mg/L, potassium dihydrogen phosphate 165-170mg/L, iodine
Change potassium 0.8-0.83mg/L, boric acid 5.5-6.2mg/L, four water manganese sulfate 22.3-23.6mg/L, white vitriol 8.6-10mg/
L, sodium molybdate 0.2-0.25mg/L, cupric sulfate pentahydrate 0.025-0.03mg/L, cobalt chloride 0.028-0.03mg/L, ferrous sulfate
27.8-29mg/L, ethylenediamine tetra-acetic acid 24.3-25mg/L, inositol 98-100mg/L, nicotinic acid 0.8-0.9mg/L, puridoxine hydrochloride
0.8-0.9mg/L, nicotinic acid thiamine 0.8-0.9mg/L, glycine 3-3.5mg/L, pH 5.8-6.0.
The tissue culture method of the bletilla striata, comprises the following steps：
Bletilla striata seeds are made to transfer to by sprouting and breaking up and carry out culture of rootage in root media described above.
Root media used instead of traditional coagulator-agar powder using sawdust in above-mentioned tissue culture, thus avoid
The problem of bletilla striata seedling root needs cleaning by agar adhesion during transplanting, therefore the bletilla striata seedling that the tissue culture method obtains will not be because of cleaning
Seedling root is injured, so as to improve transplanting survival rate.
Preferably, the culture medium used in the germination process is：The modified form MS culture mediums of original concentration, 0.5-
0.6mg/L methyl α-naphthyl acetates and 4.3-4.5g/L agar powders；Preferably, in addition to 30-40g/L murphy juices, 30-40g/L sucrose.
The effect of sprouting has vital influence to the quality of bletilla striata seedling, can promote protocorm using above-mentioned culture medium
Adventitious buds proliferation is induced, makes that the root of growth is more sturdy, and blade is more roomy.In addition, contain starch and other unknown lifes in murphy juice
The long factor, contribute to the sprouting of bletilla striata seeds and the propagation of protocorm.The bletilla striata seeds sprouting stage can not carry out photosynthesis, lack
For weary autophyting ability, it is necessary to which external source carbon source provides energy, sucrose molecule can pass in and out plant cell, it is possible to provide carbon source, sprout seed
Hair and protocorm increment.Agar powder, high-temperature digestion, air-setting are solid gum, are easy to other nutriment mixing solidifications white
Splendid achnatherum seed or protocorm preferably absorb nutrient.
Preferably, the culture medium used in the atomization is：The modified form MS culture mediums of original concentration, 2.0-
The 3.0mg/L basic elements of cell division, 0.2-0.3mg/L methyl α-naphthyl acetates and 4.3-4.5g/L agar powders.
The differentiation speed and differentiation effect of bud can be improved using this culture medium.
Preferably, 30-40g/L murphy juices, 30-40g/L sucrose are also included in above-mentioned sprouting and differential period.For sprout and
Differential period provides sufficient nutrition, improves tissue culture speed, improves the robustness of bletilla striata seedling.
Preferably, the sawdust is the sawdust cleaned by liquor natrii hypochloritis.
Sodium chlorate solution can both sterilize, and can remove the pollutant in sawdust again, gnotobasis is provided for tissue culture.Specifically
The method of cleaning may be referred to：Sawdust is put into after being soaked 1 hour in 10% liquor natrii hypochloritis of 5 times of dilution, extruding filters off
Liquor natrii hypochloritis, place into clear water and embathe 1 time, extruding filters off the aqueous solution, standby.
Compared with prior art, beneficial effects of the present invention are：
(1) root media used in bletilla striata tissue culture is improved, to improve transplanting survival rate, and lowers culture medium cost.
(2) improved MS medium, to shorten cultivation cycle, reduce pollution rate, reduce cost, improve survival rate.
(3) germination medium and differential medium used in bletilla striata tissue culture are improved, to improve the healthy and strong situation of bletilla striata seedling.
(4) nutriment such as murphy juice and bananas juice is added, to improve the root development situation of bletilla striata seedling.
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
There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 is bletilla striata seedling of the embodiment 10 after culture of rootage one month；
Fig. 2 is bletilla striata seedling of the control group after culture of rootage one month；
Fig. 3 is bletilla striata seedling of the embodiment 10 in culture of rootage after two months；
Fig. 4 is bletilla striata seedling of the control group in culture of rootage after two months.
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 suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products that can be obtained by commercially available purchase.
Hereafter the sawdust used in all embodiments all cleans by following methods：
Sawdust is put into after being soaked 1 hour in 10% liquor natrii hypochloritis of 5 times of dilution, it is molten that extruding filters off sodium hypochlorite
Liquid, place into clear water and embathe 1 time, extruding filters off the aqueous solution, standby.
Embodiment 1
A kind of root media for bletilla striata tissue culture：
MS culture mediums, 0.1mg/L6-BA, 1.0mg/L methyl α-naphthyl acetates and 160g/L wax gourd sawdusts.
Embodiment 2
A kind of root media for bletilla striata tissue culture：
MS culture mediums, 2.0mg/L6-BA, 0.1mg/L methyl α-naphthyl acetates and 180g/L wax gourd sawdusts.
Embodiment 3
A kind of root media for bletilla striata tissue culture：
MS culture mediums, 0.2mg/L6-BA, 0.8mg/L methyl α-naphthyl acetates and 180g/L wax gourd sawdusts.
Embodiment 4
A kind of root media for bletilla striata tissue culture：
MS culture mediums, 0.1mg/L6-BA, 1.0mg/L methyl α-naphthyl acetates, 60g/L bananas juices, 30g/L murphy juices, 30g/L sucrose
With 160g/L wax gourd sawdusts.
Embodiment 5
A kind of root media for bletilla striata tissue culture：
MS culture mediums, 0.1mg/L6-BA, 1.0mg/L methyl α-naphthyl acetates, 70g/L bananas juices, 40g/L murphy juices, 40g/L sucrose
With 160g/L Chinese fir sawdusts.
Embodiment 6
A kind of root media for bletilla striata tissue culture：
Modified form MS culture mediums, 0.1mg/L6-BA, 1.0mg/L methyl α-naphthyl acetates, 70g/L bananas juices, 40g/L murphy juices, 40g/
L sucrose and 160g/L wax gourd sawdusts；
Modified form MS culture mediums：Formula is as shown in table 1.
The modified form MS culture mediums of the embodiment 1 of table 1
Embodiment 7
A kind of root media for bletilla striata tissue culture：
Modified form MS culture mediums, 0.1mg/L6-BA, 1.0mg/L methyl α-naphthyl acetates, 70g/L bananas juices, 40g/L murphy juices, 40g/
L sucrose and 160g/L pine tree sawdusts；Modified form MS culture mediums：Formula is as shown in table 1.
The modified form MS culture mediums of the embodiment 2 of table 2
Embodiment 8
A kind of root media for bletilla striata tissue culture：
Modified form MS culture mediums, 0.1mg/L6-BA, 1.0mg/L methyl α-naphthyl acetates, 70g/L bananas juices, 40g/L murphy juices, 40g/
L sucrose and 160g/L eucalyptus sawdusts；Modified form MS culture mediums：Formula is as shown in table 1.
The modified form MS culture mediums of the embodiment 3 of table 3
Embodiment 9
The tissue culture method of the bletilla striata：
Prepare seed germination medium：MS culture medium+NAA0.5mg/L+40g/L murphy juice+40g/L sucrose+4.3g/L fine jades
Cosmetics.
Prepare differential medium：MS culture medium+6-BA2.0mg/L+NAA0.2mg/L+40g/L murphy juice+40g/L sucrose+
4.3g/L agar powder.
Root media such as embodiment 1.
First seed is sprouted 30 days, then transfers to differential medium, is cultivated 30 days, finally moves to root media training
Support 60 days, three above process condition of culture is identical：25 DEG C, intensity of illumination 1500lx, light application time 10h/d of temperature.
Embodiment 10
The tissue culture method of the bletilla striata：
Prepare seed germination medium：MS culture medium+NAA0.5mg/L+40g/L murphy juice+40g/L sucrose+4.3g/L fine jades
Cosmetics.
Prepare differential medium：MS culture medium+6-BA2.0mg/L+NAA0.2mg/L+40g/L murphy juice+40g/L sucrose+
4.3g/L agar powder.
Root media such as embodiment 6.
First seed is sprouted 30 days, then transfers to differential medium, is cultivated 30 days, finally moves to root media training
Support 60 days, three above process condition of culture is identical：25 DEG C, intensity of illumination 1500lx, light application time 10h/d of temperature.
Embodiment 11
The tissue culture method of the bletilla striata：
Prepare seed germination medium：Modified form MS culture medium+NAA0.5mg/L+40g/L murphy juice+40g/L sucrose+
4.3g/L agar powder.
Prepare differential medium：Modified form MS culture medium+6-BA2.0mg/L+NAA0.2mg/L+40g/L murphy juices+40g/
L sucrose+4.3g/L agar powders.
Modified form MS culture mediums such as embodiment 6.
Root media such as embodiment 6.
First seed is sprouted 30 days, then transfers to differential medium, is cultivated 30 days, finally moves to root media training
Support 60 days, three above process condition of culture is identical：25 DEG C, intensity of illumination 1500lx, light application time 10h/d of temperature.
Embodiment 12
The tissue culture method of the bletilla striata：
Prepare seed germination medium：Modified form MS culture medium+NAA0.6mg/L+30g/L murphy juice+30g/L sucrose+
4.5g/L agar powder.
Prepare differential medium：Modified form MS culture medium+6-BA3.0mg/L+NAA0.3mg/L+30g/L murphy juices+30g/
L sucrose+4.5g/L agar powders.
Modified form MS culture mediums such as embodiment 6.
Root media such as embodiment 6.
First seed is sprouted 30 days, then transfers to differential medium, is cultivated 30 days, finally moves to root media training
Support 60 days, three above process condition of culture is identical：25 DEG C, intensity of illumination 1500lx, light application time 10h/d of temperature.
Embodiment 13
The tissue culture method of the bletilla striata：
Prepare seed germination medium：MS culture medium+NAA0.5mg/L+40g/L murphy juice+40g/L sucrose+4.3g/L fine jades
Cosmetics.
Prepare differential medium：MS culture medium+6-BA2.0mg/L+NAA0.2mg/L+40g/L murphy juice+40g/L sucrose+
4.3g/L agar powder.
Root media such as embodiment 4.
First seed is sprouted 30 days, then transfers to differential medium, is cultivated 30 days, finally moves to root media training
Support 60 days, three above process condition of culture is identical：25 DEG C, intensity of illumination 1500lx, light application time 10h/d of temperature.
Experiment：
Embodiment 9-13 tissue culture result is evaluated, and by prior art as a control group.
The tissue culture method of control group：Seed germination medium：MS+NAA0.5mg/L+ murphy juice 40g/L+ sucrose 40g/L+
Agar powder, cultivate 30 days, be transferred in differential medium and cultivate.Differential medium：MS+6-BA2.0mg/L+NAA0.2mg/L+
Murphy juice 40g/L+ sucrose 40g/L+ agar powders, cultivate 30 days, be transferred in root media and cultivate.Root media：MS+6-
BA0.1mg/L+NAA1.0mg/L+ murphy juice 40g/L+ bananas juice 70g/L+ sucrose 40g/L+ agar.Agar in all culture mediums
Powder dosage is 4.4g/L.Culture bottle outlet transplanting in 80-90 days.
As a result as shown in table 4 and Fig. 1-4.Wherein, Fig. 1 is bletilla striata seedling of the embodiment 10 after culture of rootage one month, Fig. 2
For bletilla striata seedling of the control group after culture of rootage one month, Fig. 3 is bletilla striata seedling of the embodiment 10 in culture of rootage after two months, figure
4 be bletilla striata seedling of the control group in culture of rootage after two months.
The growing state of the bletilla striata seedling of table 4
Although illustrate and describing the present invention with specific embodiment, but will be appreciated that without departing substantially from the present invention's
Many other change 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 (1)
1. the tissue culture method of the bletilla striata, it is characterised in that comprise the following steps：
Prepare seed germination medium：Modified form MS culture mediums, NAA0.5mg/L, 40g/L murphy juice, 40g/L sucrose and 4.3g/
L agar powders；
Prepare differential medium：Modified form MS culture mediums, 6-BA2.0mg/L, NAA0.2mg/L, 40g/L murphy juice, 40g/L sugarcanes
Sugar and 4.3g/L agar powders；
Prepare root media：Modified form MS culture mediums, 0.1mg/L6-BA, 1.0mg/L methyl α-naphthyl acetate, 70g/L bananas juices, 40g/L
Murphy juice, 40g/L sucrose and 160g/L wax gourd tree sawdusts；
First seed is sprouted 30 days in the seed germination medium, then transfers to the differential medium, culture 30
My god, the root media culture 60 days is finally moved to, three above process condition of culture is identical：25 DEG C of temperature, intensity of illumination
1500lx, light application time 10h/d；
The formula of the modified form MS culture mediums is：Potassium nitrate 1900mg/L, ammonium nitrate 1650mg/L, calcium chloride dihydrate 440mg/
L, epsom salt 370mg/L, potassium dihydrogen phosphate 170mg/L, KI 0.83mg/L, boric acid 6.2mg/L, four water manganese sulfates
22.3mg/L, white vitriol 8.6mg/L, sodium molybdate 0.25mg/L, cupric sulfate pentahydrate 0.025mg/L, cobalt chloride 0.025mg/
L, ferrous sulfate 27.8mg/L, ethylenediamine tetra-acetic acid 24.3mg/L, inositol 100mg/L, nicotinic acid 0.8mg/L, puridoxine hydrochloride
0.8mg/L, thiamine hydrochloride 0.8mg/L, and glycine 3mg/L；PH is 5.8；
The sawdust cleans by following methods：
Sawdust being put into after being soaked 1 hour in 10% liquor natrii hypochloritis of 5 times of dilution, extruding filters off liquor natrii hypochloritis, then
It is put into clear water and embathes 1 time, extruding filters off the aqueous solution, standby.
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| CN106613264B (en) * | 2016-12-29 | 2020-04-17 | 铜仁昱丰农业发展有限公司 | Bletilla striata tissue culture seedling transplanting domestication technology |
| CN106973789A (en) * | 2017-03-29 | 2017-07-25 | 师宗县福禄农业科技开发有限公司 | A kind of root media and its method for culturing seedlings of bletilla striata tissue culture |
| CN108124704A (en) * | 2017-12-27 | 2018-06-08 | 康美药业（文山）药材种植管理有限公司 | A kind of oldenlandia diffusa subculture medium and preparation method thereof |
| CN108094209A (en) * | 2017-12-28 | 2018-06-01 | 临沧市云瑞堂生物科技有限公司 | A kind of bletilla striata quickly breeds the tissue culture method of seedling |
| CN109089851A (en) * | 2018-06-21 | 2018-12-28 | 四川千草生物技术股份有限公司 | A kind of bletilla striata live streaming fast breeding method |
| CN110283775B (en) * | 2019-07-12 | 2021-03-19 | 遵义医科大学 | Method for promoting synthesis of secondary metabolites in bletilla striata suspension culture cells |
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Effective date of registration: 20190305 Address after: 650000 6th Floor, Block A, Sunshine Building, 999 Kegao Road, Kunming City, Yunnan Province Patentee after: Kangmei Pharmaceutical (Kunming) Germplasm Resources Co., Ltd. Address before: 663000 Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province, No. 4-21, Wenshan Biovalley of Chinese Traditional Medicine, South Kaihua Road, Wenshan City Patentee before: Kangmei Pharmaceutical (Wenshan) medicinal material planting Management Co., Ltd. |
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