CN105145352A - Efficient tissue culture and rapid propagation technology for seedlings of bletilla striata - Google Patents
Efficient tissue culture and rapid propagation technology for seedlings of bletilla striata Download PDFInfo
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Abstract
The invention discloses an efficient tissue culture and rapid propagation technology for seedlings of bletilla striata. According to the technology, bletilla striata seeds are sown on a seed germination culture medium for sterile germination, the obtained seedlings are inoculated to a multiplication and subculture medium one by one for multiplication and subculture, clustering seedlings are obtained and inoculated to a rooting culture medium for rooting culture, the obtained tissue culture seedlings are planted on a prepared seedling hardening substrate, and the seedlings are transplanted to a large filed for plantation after seedling hardening, wherein the seed germination culture medium comprises MS (magnesium sulfate), 30g/L of sugar, 8g/L of agar and 10g/L-12g/L of potatoes; the multiplication and subculture medium comprises MS, 1.0 mg/L of KT (kinetin), 0.2 mg/L of NAA (naphthalene acetic acid), 30g/L of sugar and 9g/L of agar; the rooting culture medium comprises MS, 0.2 mg/L of NAA, 1.0mg/L of IAA (indole acetic acid), 30g/L of sugar, 9g/L of agar and 25-30 g/L of potatoes; the seedling hardening substrate comprises medium moor peat soil, perlite and wood dust in the volume ratio being 5:2:3. The technology is mature, a system is perfect, the problems of low natural germination rate of bletilla striata and difficulty in obtaining of seedlings can be effectively solved, and the technology has better popularization and application value and broad market prospect.
Description
Technical field
The invention belongs to the technical field utilizing tissue cultures to carry out medicinal plant nursery, particularly relate to the efficient group culturation rapid propagating technology of a kind of bletilla seedling.
Background technology
Bletilla
bletillastriatafor the orchid family bletilla belongs to herbaceos perennial, be a kind of medicinal herbs most in use, be used as medicine with stem tuber, there is effect of astringing to arrest bleeding, detumescence and promoting granulation, be used for the treatment of spitting of blood, spit blood, traumatism and bleeding, sore swollen toxin, the diseases such as chapped skin.
Because bletilla market mainly relies on wild resource, private is blindly adopted and is dug excessively, causes bletilla wild resource day by day exhausted.Bletilla seed is minimum, and does not have endosperm, and natural germination rate is extremely low, and division propagation conventional at present, reproduction coefficient is low, cannot meet the demand of large-scale production.Therefore, the restraining factors that bletilla seedling becomes bletilla large-scale planting can be obtained fast.For addressing this problem, tissue culture technique acquisition bletilla plantlet in vitro is taked progressively to become the important measures of bletilla seedling acquisition, and achieve greater advance, there is the breeding technique of many bletilla seedling aspects, distinct methods is adopted to obtain bletilla seedling, but for later stage plantlet in vitro hardening, domestication link technology indefinite, effect is also good not.
Summary of the invention
The object of the invention is for seed germination rate in current bletilla artificial cultivation low; the problem of natural propagation difference; a kind of bletilla seedling group culturation rapid propagating technology is provided; not only can obtain bletilla plantlet in vitro efficiently; also the hardening of bletilla plantlet in vitro, domestication mechanism are studied; utilize tissue culture technique, a large amount of bletilla seedlings can be obtained, for bletilla large-scale planting is laid a good foundation.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: the efficient group culturation rapid propagating technology of a kind of bletilla seedling, by the bletilla seed broadcasting in aseptic bletilla capsule on seed germination medium, carry out seed asepsis sprouting, then by after sprouting obtain seedling plant division and be seeded in proliferation and subculture medium, carry out proliferation and subculture cultivation, be seeded in after obtaining bletilla tufted seedling in root media, carry out culture of rootage, again the bletilla plantlet in vitro obtained is planted and carry out hardening in prepared hardening matrix, field planting is transplanted after hardening, wherein, the method obtaining aseptic bletilla capsule is: choose maturation but still uncracked bletilla capsule, first drain with running water surface impurity, then use the cotton ball soaked in alcohol of 75% clean for capsule surface wipes, transfer them to again in superclean bench, after alcoholic solution with 75% soaks 30 seconds, successively with the mercuric chloride solution of 0.1-0.5% sterilization 6min and aseptic water washing 2-3 time, blot the moisture on surface again with aseptic filter paper after, obtain aseptic bletilla capsule, described seed asepsis sprouting and the method for hardening as follows:
(1) method of seed asepsis sprouting is: be placed in culturing room by the seed germination medium broadcasting sowing bletilla seed and carry out light culture, cultivation temperature is 25 DEG C, when the seed on medium starts to sprout green, transferred to light to cultivate, intensity of illumination is 2000-2500lx, illumination is 12 hours every days, and Continuous irradiation, light is cultivated after 15 ~ 20 days and is divided into Multiple Buds
seedling, complete axenic germination;
Wherein, consisting of of described seed germination medium: MS, sucrose, agar and potato, wherein the addition of sucrose is 30g/L, the addition of agar is 8g/L, the interpolation form of potato is pasty state, and its addition is 10-12g/L, and the pH of seed germination medium is 5.5 ~ 6.0;
(2) method of hardening is: the tissue culture flasks of getting the bletilla plantlet in vitro filled through culture of rootage gained, open bottle cap, after spraying the sterile water of 5 ~ 8ml wherein, place 2 ~ 3 days in culturing room, then tissue culture flasks is shifted out culturing room and be placed on outdoor 2 ~ 3 days, during outside scenery, temperature remains on 20 ~ 25 DEG C, humidity remains on 30 ~ 40%, then the bletilla plantlet in vitro in tissue culture flasks is taken out, wash the medium of plantlet in vitro root with distilled water after, soak 30 seconds in the NAA of 0.2mg/L, plant in hardening matrix, hardening can transplant field planting after 90 days,
Wherein, the composition of described hardening matrix comprises medium peat soil, perlite and wood fragments bits, wood fragments bits are sawdust, its preparation method is: first by medium peat soil through high-temperature heat sterilization, sterilising temp 121 DEG C, sterilization time is 20min, then by medium peat soil, perlite and wood fragments bits are according to after the ratio mixing of volume ratio 5:2:3, the distilled water adding sterilizing is mixed thoroughly, its water content is made to be 50 ~ 55%, then made hardening matrix is positioned in seedling-cultivating tray, and to be placed on sunshade rate be for subsequent use in the shed of 50%, humidity in shed is 50 ~ 55%, temperature is 15 ~ 25 DEG C.
Further, the preparation method of described seed germination medium is: first potato cleaning is fallen surface impurity, then peeling is put into distilled water and is heated, continue after boiling to boil 30min, then potato is taken out and smash into mashed potato to pieces, and the distilled water after boiling is cooled to room temperature and adds in mashed potato and be mixed into pasty state, finally the potato of pasty state and sucrose, agar are added in MS successively according to described addition, and stir.
Further, in proliferation and subculture is cultivated, its cultural method is: in superclean bench, be seeded in after the Multiple Buds seedling plant division that seed asepsis sprouting is obtained in the tissue culture flasks filling proliferation and subculture medium, the strain of every bottle graft kind 20 ~ 25, and by postvaccinal tissue culture flasks as illumination cultivation in culturing room, cultivation temperature is 25 DEG C, intensity of illumination is 2000-2500lx, and between culture period, the light application time of every day is 12h, within 50 days, obtains the bletilla tufted seedling of 3 ~ 5cm afterwards;
The formulation ratio of described proliferation and subculture medium is: MS+KT1.0mg/L+NAA0.2mg/L+ sucrose 30g/L+ agar 9g/L.
Further, in the process of culture of rootage, its cultural method is as follows: in superclean bench, proliferation and subculture is cultivated the tufted seedling obtained and be divided into individual plant, be seeded in and fill in the tissue culture flasks of root media, the strain of every bottle graft kind 20 ~ 25, is placed in culturing room and cultivates, cultivation temperature is 25 DEG C, intensity of illumination is 2000-2500lx, and illumination is 12 hours every days, can obtain plant height 5-8cm after 40 days, the bletilla plantlet in vitro of the long 2-3cm of root, rooting rate reaches more than 95%;
The formulation ratio of described root media is: MS+NAA0.2mg/L+IAA1.0mg/L+ sucrose 30g/L+ agar 9g/L+25-30g/L potato.
Further, in described hardening matrix, add the carbendazim solution of 0.1g/L.
Compared with prior art, the present invention has following beneficial effect:
The first, the technology of the present invention is ripe, and system is perfect, and effectively can solve bletilla nature germination rate low, seedling obtains the present situation of difficulty, has good popularization, using value, wide market;
Second, in method provided by the present invention, any hormone is not added in the seed germination medium that the seed asepsis sprouting stage is used, but utilize the natural materials in treated potato to provide nutrient for bletilla seed germination, light culture and light is adopted to cultivate the cultural method combined in addition, bletilla seed germination can be made in 30 days to obtain Multiple Buds, hormone is added relative to external source, both toxigenic capacity was reduced, additionally reduce the utilization of incubation to hormone, more meet the theory of green agriculture, and, as calculated, the germination rate of bletilla seed reaches more than 98%,
3rd, the hardening medium constituent that the present invention provided in the hardening stage easily obtains, reasonable mixture ratio, ensures the supply of the water conservation in bletilla training tissue culture seedling, domestication process, ventilative and nutriment, significantly improves the survival rate of bletilla training tissue culture seedling, domestication.
Accompanying drawing explanation
Fig. 1 adopts different seed germination medium on the impact of seed germination rate in the seed asepsis sprouting stage.
Fig. 2 adopts different root media on the impact of rooting rate in the culture of rootage stage.
Fig. 3 adopts different hardening matrix on the impact of survival rate.
Embodiment
Below by embodiment and test example, the present invention is further illustrated.
Embodiment 1: the efficient group culturation rapid propagating technology of a kind of bletilla seedling, its step comprises material preparation, seed asepsis sprouting, proliferation and subculture cultivation, culture of rootage and hardening and domestication, and concrete grammar is as follows:
1, material prepares
Choose maturation but still uncracked bletilla capsule, first drain with running water surface impurity, then use the cotton ball soaked in alcohol of 75% clean for capsule surface wipes, transfer them to again in superclean bench, after alcoholic solution with 75% soaks 30 seconds, successively with 0.1% mercuric chloride solution sterilization 6min and aseptic water washing 2-3 time, then after blotting surperficial moisture with aseptic filter paper, obtain aseptic bletilla capsule.
2, seed asepsis sprouting
2.1, the preparation of seed germination medium
The mashed sweet potato of the sucrose of 30g/L, the agar of 8g/L and 10g/L is added successively in MS medium, stirring and evenly mixing, and adjust pH to 5.5, and it is for subsequent use to be placed in blake bottle, wherein, the preparation method of mashed sweet potato is: potato cleaning is fallen surface impurity, then peeling is put into distilled water and is heated, continue after boiling to boil 30min, then potato is taken out and smash into mashed potato to pieces, and the distilled water after boiling is cooled to room temperature and adds in mashed potato and be mixed into pasty state.
2.2, inoculation is sprouted
With blade, aseptic bletilla capsule is cut in superclean bench, capsule is clamped with tweezers, go deep in blake bottle, by vibrations tweezers, bletilla seed is sown on seed germination medium equably, then this blake bottle is placed in culturing room and carries out light culture, cultivation temperature is 25 DEG C, light culture about 10 days, when seed starts to sprout green, transfer light to and cultivate, intensity of illumination is 2000lx, light application time is Continuous irradiation 12 hours every day, can be divided into a large amount of Multiple Buds after illumination cultivation 15-20 days.Because bletilla seed is minimum, cannot calculate seed number, calculate with the floor space of medium, the germination rate of seed reaches more than 98%.
3, proliferation and subculture is cultivated
3.1, the preparation of proliferation and subculture medium
The formulation ratio of proliferation and subculture medium is: MS+KT1.0mg/L+NAA0.2mg/L+ sucrose 30g/L+ agar 9g/L, its preparation method is as follows: after first claiming 30g sucrose dissolved in water, add 9g agar powder wherein, and heat, after agar powder dissolves completely, add KT and NAA solution respectively wherein again, the concentration of KT and NAA is made to be respectively 1.0mg/L and 0.2mg/L, then 1000mL is settled to, pH is now 5.5-6.0, being divided in base diameter after constant volume is in the tissue culture flasks of 70cm, the dispensed loading amount of each tissue culture flasks is 30mL, then sterilizing is carried out, to be cooled after sterilizing, proliferation and subculture medium can be obtained after solidifying.
3.2, inoculated and cultured
In superclean bench, be seeded in after the Multiple Buds seedling plant division that seed asepsis sprouting is obtained in the tissue culture flasks filling proliferation and subculture medium, the strain of every bottle graft kind 20, if be not easy separately, also can 3 ~ 5 strains to inoculate, and by postvaccinal tissue culture flasks as illumination cultivation in culturing room together, cultivation temperature is 25 DEG C, intensity of illumination is 2000lx, and between culture period, the light application time of every day is 12h, within 50 days, obtains the bletilla tufted seedling of 3 ~ 5cm afterwards.
4, culture of rootage
4.1, the preparation of root media
The formulation ratio of root media is: MS+NAA0.2mg/L+IAA1.0mg/L+ sucrose 30g/L+ agar 9g/L+25g/L potato, the interpolation form of potato is mashed sweet potato, the preparation method of mashed sweet potato is identical with the method described in above-mentioned steps 2.1, the preparation method of root media is as follows: after first claiming 30g sucrose dissolved in water, add 9g agar powder wherein, and heat, after agar powder dissolves completely, add IAA and NAA solution respectively wherein again, the concentration of IAA and NAA is made to be respectively 1.0mg/L and 0.2mg/L, then 1000mL is settled to, pH is now 5.5-6.0, being divided in base diameter after constant volume is in the tissue culture flasks of 70cm, the dispensed loading amount of each tissue culture flasks is 30mL, then sterilizing is carried out, to be cooled after sterilizing, root media can be obtained after solidifying.
4.2, tufted seedling is inoculated
In superclean bench proliferation and subculture cultivate obtain tufted seedling and be divided into individual plant, be seeded in the root media obtained by 4.1, each tissue culture flasks inoculation 20 ~ 25 strains, then be placed in culturing room and cultivate, cultivation temperature is 25 DEG C, and intensity of illumination is 2000lx, illumination is 12 hours every days, can obtain plant height 5-8cm after about 40 days, the bletilla plantlet in vitro of the long 2-3cm of root, rooting rate reaches more than 95%.
5, hardening and domestication
5.1, the preparation of hardening matrix
The composition of hardening matrix comprises medium peat soil, perlite and wood fragments bits, wood fragments bits are sawdust, its preparation method is: first by medium peat soil through high-temperature heat sterilization, sterilising temp 121 DEG C, sterilization time is 20min, then by medium peat soil, perlite and wood fragments bits are according to after the ratio mixing of volume ratio 5:2:3, the distilled water adding sterilizing is mixed thoroughly, its water content is made to be 50 ~ 55%, then made hardening matrix is positioned in seedling-cultivating tray, and to be placed on sunshade rate be for subsequent use in the shed of 50%, the humidity controlled in shed is 50%, temperature is 15 DEG C.
5.2, hardening, domestication
Growth selection is healthy and strong, the bletilla plantlet in vitro of well developed root system, open the tissue cultures bottle cap at its place, sterile water is sprayed in bottle, every bottle is sprayed 5ml, then to be placed in culturing room after 2 ~ 3 days, tissue culture flasks is shifted out culturing room and place 2 ~ 3 days again, during outdoor, temperature is 20 DEG C, humidity is 30%, then from blake bottle, bletilla plantlet in vitro is taken out, and clean up the residual medium of root with distilled water, the NAA being placed in 0.2mg/L soaks 30 seconds, plant in the hardening matrix made by 5.1, hardening can transplant field planting after 90 days, hardening survival rate is about 95%.
Embodiment 2: the efficient group culturation rapid propagating technology of a kind of bletilla seedling, its step comprises material preparation, seed asepsis sprouting, proliferation and subculture cultivation, culture of rootage and hardening and domestication, and concrete grammar is as follows:
1, material prepares
Choose maturation but still uncracked bletilla capsule, first drain with running water surface impurity, then use the cotton ball soaked in alcohol of 75% clean for capsule surface wipes, transfer them to again in superclean bench, after alcoholic solution with 75% soaks 30 seconds, successively with 0.5% mercuric chloride solution sterilization 6min and aseptic water washing 2-3 time, then after blotting surperficial moisture with aseptic filter paper, obtain aseptic bletilla capsule.
2, seed asepsis sprouting
2.1, the preparation of seed germination medium
The mashed sweet potato of the sucrose of 30g/L, the agar of 8g/L and 12g/L is added successively in MS medium, stirring and evenly mixing, and adjust pH to 6.0, and it is for subsequent use to be placed in blake bottle, wherein, the preparation method of mashed sweet potato is: potato cleaning is fallen surface impurity, then peeling is put into distilled water and is heated, continue after boiling to boil 30min, then potato is taken out and smash into mashed potato to pieces, and the distilled water after boiling is cooled to room temperature and adds in mashed potato and be mixed into pasty state.
2.2, inoculation is sprouted
With blade, aseptic bletilla capsule is cut in superclean bench, capsule is clamped with tweezers, go deep in blake bottle, by vibrations tweezers, bletilla seed is sown on seed germination medium equably, then this blake bottle is placed in culturing room and carries out light culture, cultivation temperature is 25 DEG C, light culture about 10 days, when seed starts to sprout green, transfer light to and cultivate, intensity of illumination is 2500lx, light application time is Continuous irradiation 12 hours every day, can be divided into a large amount of Multiple Buds after illumination cultivation 15-20 days.Because bletilla seed is minimum, cannot calculate seed number, calculate with the floor space of medium, the germination rate of seed reaches more than 98%.
3, proliferation and subculture is cultivated
3.1, the preparation of proliferation and subculture medium
The formulation ratio of proliferation and subculture medium is: MS+KT1.0mg/L+NAA0.2mg/L+ sucrose 30g/L+ agar 9g/L, its preparation method is as follows: after first claiming 30g sucrose dissolved in water, add 9g agar powder wherein, and heat, after agar powder dissolves completely, add KT and NAA solution respectively wherein again, the concentration of KT and NAA is made to be respectively 1.0mg/L and 0.2mg/L, then 1000mL is settled to, pH is now 5.5-6.0, being divided in base diameter after constant volume is in the tissue culture flasks of 70cm, the dispensed loading amount of each tissue culture flasks is 30mL, then sterilizing is carried out, to be cooled after sterilizing, proliferation and subculture medium can be obtained after solidifying.
3.2, inoculated and cultured
In superclean bench, be seeded in after the Multiple Buds seedling plant division that seed asepsis sprouting is obtained in the tissue culture flasks filling proliferation and subculture medium, the strain of every bottle graft kind 20, if be not easy separately, also can 3 ~ 5 strains to inoculate, and by postvaccinal tissue culture flasks as illumination cultivation in culturing room together, cultivation temperature is 25 DEG C, intensity of illumination is 2500lx, and between culture period, the light application time of every day is 12h, within 50 days, obtains the bletilla tufted seedling of 3 ~ 5cm afterwards.
4, culture of rootage
4.1, the preparation of root media
The formulation ratio of root media is: MS+NAA0.2mg/L+IAA1.0mg/L+ sucrose 30g/L+ agar 9g/L+30g/L potato, the interpolation form of potato is mashed sweet potato, the preparation method of mashed sweet potato is identical with the method described in above-mentioned steps 2.1, the preparation method of root media is as follows: after first claiming 30g sucrose dissolved in water, add 9g agar powder wherein, and heat, after agar powder dissolves completely, add IAA and NAA solution respectively wherein again, the concentration of IAA and NAA is made to be respectively 1.0mg/L and 0.2mg/L, then 1000mL is settled to, pH is now 5.5-6.0, being divided in base diameter after constant volume is in the tissue culture flasks of 70cm, the dispensed loading amount of each tissue culture flasks is 30mL, then sterilizing is carried out, to be cooled after sterilizing, root media can be obtained after solidifying.
4.2, tufted seedling is inoculated
In superclean bench proliferation and subculture cultivate obtain tufted seedling and be divided into individual plant, be seeded in the root media obtained by 4.1, each tissue culture flasks inoculation 20 ~ 25 strains, then be placed in culturing room and cultivate, cultivation temperature is 25 DEG C, and intensity of illumination is 2500lx, illumination is 12 hours every days, can obtain plant height 5-8cm after about 40 days, the bletilla plantlet in vitro of the long 2-3cm of root, rooting rate reaches more than 95%.
5, hardening and domestication
5.1, the preparation of hardening matrix
The composition of hardening matrix comprises medium peat soil, perlite and wood fragments bits, wood fragments bits are sawdust, its preparation method is: first by medium peat soil through high-temperature heat sterilization, sterilising temp 121 DEG C, sterilization time is 20min, then by medium peat soil, perlite and wood fragments bits are according to after the ratio mixing of volume ratio 5:2:3, the distilled water adding sterilizing is mixed thoroughly, its water content is made to be 50 ~ 55%, then made hardening matrix is positioned in seedling-cultivating tray, and to be placed on sunshade rate be for subsequent use in the shed of 50%, the humidity controlled in shed is 55%, temperature is 25 DEG C.
5.2, hardening, domestication
Growth selection is healthy and strong, the bletilla plantlet in vitro of well developed root system, open the tissue cultures bottle cap at its place, sterile water is sprayed in bottle, every bottle is sprayed 8ml, then to be placed in culturing room after 2 ~ 3 days, tissue culture flasks is shifted out culturing room and place 2 ~ 3 days again, during outdoor, temperature is 25 DEG C, humidity is 40%, then from blake bottle, bletilla plantlet in vitro is taken out, and clean up the residual medium of root with distilled water, the NAA being placed in 0.2mg/L soaks 30 seconds, plant in the hardening matrix made by 5.1, hardening can transplant field planting after 90 days, hardening survival rate is about 95%.
Embodiment 3: the efficient group culturation rapid propagating technology of a kind of bletilla seedling, its step comprises material preparation, seed asepsis sprouting, proliferation and subculture cultivation, culture of rootage and hardening and domestication, and concrete grammar is as follows:
1, material prepares
Choose maturation but still uncracked bletilla capsule, first drain with running water surface impurity, then use the cotton ball soaked in alcohol of 75% clean for capsule surface wipes, transfer them to again in superclean bench, after alcoholic solution with 75% soaks 30 seconds, successively with 0.3% mercuric chloride solution sterilization 6min and aseptic water washing 2-3 time, then after blotting surperficial moisture with aseptic filter paper, obtain aseptic bletilla capsule.
2, seed asepsis sprouting
2.1, the preparation of seed germination medium
The mashed sweet potato of the sucrose of 30g/L, the agar of 8g/L and 10g/L is added successively in MS medium, stirring and evenly mixing, and adjust pH to 5.8, and it is for subsequent use to be placed in blake bottle, wherein, the preparation method of mashed sweet potato is: potato cleaning is fallen surface impurity, then peeling is put into distilled water and is heated, continue after boiling to boil 30min, then potato is taken out and smash into mashed potato to pieces, and the distilled water after boiling is cooled to room temperature and adds in mashed potato and be mixed into pasty state.
2.2, inoculation is sprouted
With blade, aseptic bletilla capsule is cut in superclean bench, capsule is clamped with tweezers, go deep in blake bottle, by vibrations tweezers, bletilla seed is sown on seed germination medium equably, then this blake bottle is placed in culturing room and carries out light culture, cultivation temperature is 25 DEG C, light culture about 10 days, when seed starts to sprout green, transfer light to and cultivate, intensity of illumination is 2300lx, light application time is Continuous irradiation 12 hours every day, can be divided into a large amount of Multiple Buds after illumination cultivation 15-20 days.Because bletilla seed is minimum, cannot calculate seed number, calculate with the floor space of medium, the germination rate of seed reaches more than 98%.
3, proliferation and subculture is cultivated
3.1, the preparation of proliferation and subculture medium
The formulation ratio of proliferation and subculture medium is: MS+KT1.0mg/L+NAA0.2mg/L+ sucrose 30g/L+ agar 9g/L, its preparation method is as follows: after first claiming 30g sucrose dissolved in water, add 9g agar powder wherein, and heat, after agar powder dissolves completely, add KT and NAA solution respectively wherein again, the concentration of KT and NAA is made to be respectively 1.0mg/L and 0.2mg/L, then 1000mL is settled to, pH is now 5.5-6.0, being divided in base diameter after constant volume is in the tissue culture flasks of 70cm, the dispensed loading amount of each tissue culture flasks is 30mL, then sterilizing is carried out, to be cooled after sterilizing, proliferation and subculture medium can be obtained after solidifying.
3.2, inoculated and cultured
In superclean bench, be seeded in after the Multiple Buds seedling plant division that seed asepsis sprouting is obtained in the tissue culture flasks filling proliferation and subculture medium, the strain of every bottle graft kind 20, if be not easy separately, also can 3 ~ 5 strains to inoculate, and by postvaccinal tissue culture flasks as illumination cultivation in culturing room together, cultivation temperature is 25 DEG C, intensity of illumination is 2300lx, and between culture period, the light application time of every day is 12h, within 50 days, obtains the bletilla tufted seedling of 3 ~ 5cm afterwards.
4, culture of rootage
4.1, the preparation of root media
The formulation ratio of root media is: MS+NAA0.2mg/L+IAA1.0mg/L+ sucrose 30g/L+ agar 9g/L+25g/L potato, the interpolation form of potato is mashed sweet potato, the preparation method of mashed sweet potato is identical with the method described in above-mentioned steps 2.1, the preparation method of root media is as follows: after first claiming 30g sucrose dissolved in water, add 9g agar powder wherein, and heat, after agar powder dissolves completely, add IAA and NAA solution respectively wherein again, the concentration of IAA and NAA is made to be respectively 1.0mg/L and 0.2mg/L, then 1000mL is settled to, pH is now 5.5-6.0, being divided in base diameter after constant volume is in the tissue culture flasks of 70cm, the dispensed loading amount of each tissue culture flasks is 30mL, then sterilizing is carried out, to be cooled after sterilizing, root media can be obtained after solidifying.
4.2, tufted seedling is inoculated
In superclean bench proliferation and subculture cultivate obtain tufted seedling and be divided into individual plant, be seeded in the root media obtained by 4.1, each tissue culture flasks inoculation 20 ~ 25 strains, then be placed in culturing room and cultivate, cultivation temperature is 25 DEG C, and intensity of illumination is 2300lx, illumination is 12 hours every days, can obtain plant height 5-8cm after about 40 days, the bletilla plantlet in vitro of the long 2-3cm of root, rooting rate reaches more than 95%.
5, hardening and domestication
5.1, the preparation of hardening matrix
The composition of hardening matrix comprises medium peat soil, perlite and wood fragments bits, wood fragments bits are sawdust, its preparation method is: first by medium peat soil through high-temperature heat sterilization, sterilising temp 121 DEG C, sterilization time is 20min, then by medium peat soil, perlite and wood fragments bits are according to after the ratio mixing of volume ratio 5:2:3, the distilled water adding sterilizing is mixed thoroughly, its water content is made to be 50 ~ 55%, then made hardening matrix is positioned in seedling-cultivating tray, and to be placed on sunshade rate be for subsequent use in the shed of 50%, the humidity controlled in shed is 50%, temperature is 20 DEG C.
5.2, hardening, domestication
Growth selection is healthy and strong, the bletilla plantlet in vitro of well developed root system, open the tissue cultures bottle cap at its place, sterile water is sprayed in bottle, every bottle is sprayed 6ml, then in culturing room after 2 ~ 3 days, tissue culture flasks is shifted out culturing room and place 2 ~ 3 days again, during outdoor, temperature is 22 DEG C, humidity is 35%, then from blake bottle, bletilla plantlet in vitro is taken out, and clean up the residual medium of root with distilled water, the NAA being placed in 0.2mg/L soaks 30 seconds, plant in the hardening matrix made by 5.1, hardening can transplant field planting after 90 days, hardening survival rate is about 95%.
Below in conjunction with concrete test example, by the control experiment of design, beneficial effect of the present invention is described.
Test example 1: this test example adopts different seed germination medium on the impact of seed germination rate for studying the seed asepsis sprouting stage, and the seed germination medium formula of each experimental group is as follows:
1 group: MS+ sucrose 30g/L+ agar 8g/L+10g/L potato
2 groups: MS+ sucrose 30g/L+ agar 8g/L+12g/L potato
3 groups: MS+ sucrose 30g/L+ agar 8g/L
4 groups: MS+ sucrose 30g/L+ agar 8g/L+NAA1.0mg/L
5 groups: MS+ sucrose 30g/L+ agar 8g/L+NAA1.0mg/L+10g/L potato
6 groups: MS+ sucrose 30g/L+ agar 8g/L+NAA1.0mg/L+12g/L potato
Seed germination medium in above-mentioned 6 groups of test examples is carried out bletilla seed asepsis sprouting respectively according to the method described in the present invention, and calculates seed germination rate.
Result of the test is shown in accompanying drawing 1, from accompanying drawing 1: when not adding potato and exogenous hormone NAA in medium, the germination rate of its inoculation after 30 days only has 60%, not as good as the germination rate added after potato or NAA, and adopt no matter the seed germination rate in the medium adding potato is inoculation 7 days, all far above the germination rate of the medium of interpolation NAA when 30 days, especially in inoculation after 7 days, effect is more obvious, and, by 1 group and 5 groups, 2 combinations 6 groups can be found out, if again add the sprouting that instead NAA is unfavorable for bletilla seed after adding potato, therefore, the present invention's seed germination medium used is without the need to adding exogenous hormone, effectively can promote the sprouting of bletilla seed, and compared with adding the medium of hormone, higher germination rate can be obtained.
Test example 2: this test example adopts different root media on the impact of rooting rate for studying cultivation stage of taking root, and the prescription of rooting medium of each experimental group is as follows:
1 group:
MS+NAA0.2mg/L+IAA1.0mg/L+ sucrose 30g/L+ agar 9g/L+25g/L potato
2 groups:
MS+NAA0.2mg/L+IAA1.0mg/L+ sucrose 30g/L+ agar 9g/L+28g/L potato
3 groups:
MS+NAA0.2mg/L+IAA1.0mg/L+ sucrose 30g/L+ agar 9g/L+30g/L potato
4 groups:
MS+NAA0.2mg/L+IAA1.0mg/L+ sucrose 30g/L+ agar 9g/L
5 groups:
MS+NAA0.2mg/L+ sucrose 30g/L+ agar 9g/L
6 groups:
MS+IAA1.0mg/L+ sucrose 30g/L+ agar 9g/L
Wherein, 1,2,3 groups is experimental group, 4,5,6 groups is control group, carry out culture of rootage according to the method described in the present invention, and calculate rooting rate, its result of the test is shown in accompanying drawing 2 as follows, and from accompanying drawing 2: add potato in root media after, its rooting rate is significantly higher than the root media not adding potato.
Test example 3: this test example for studying hardening, the domestication stage adopts different hardening matrix on the impact of survival rate, the hardening matrix formulations of each experimental group is as follows:
Group 1: according to volume ratio, medium peat soil: perlite: wood fragments bits=5:2:3
Group 2: the peat soil of employing group 1 equivalent
Group 3: the perlite of employing group 1 equivalent
Group 4: the wood fragments bits of employing group 1 equivalent
Wherein, group 1 is experimental group, group 2,3,4 is control group, then, carries out hardening, the domestication of bletilla plantlet in vitro according to the method for hardening of the present invention, domestication, and calculate its survival rate, result of the test is shown in accompanying drawing 3, from accompanying drawing 3, after employing group 1 i.e. hardening matrix of the present invention, its survival rate can reach 98%, far above the survival rate of other three control groups.
Claims (5)
1. the efficient group culturation rapid propagating technology of bletilla seedling, by the bletilla seed broadcasting in aseptic bletilla capsule on seed germination medium, carry out seed asepsis sprouting, then by after sprouting obtain the plant division of Multiple Buds seedling and be seeded in proliferation and subculture medium, carry out proliferation and subculture cultivation, be seeded in after obtaining bletilla tufted seedling in root media, carry out culture of rootage, obtain bletilla plantlet in vitro, again the bletilla plantlet in vitro obtained is planted and carry out hardening in prepared hardening matrix, field planting is transplanted after hardening, wherein, the method obtaining aseptic bletilla capsule is: choose maturation but still uncracked bletilla capsule, first drain with running water surface impurity, then use the cotton ball soaked in alcohol of 75% clean for capsule surface wipes, transfer them to again in superclean bench, after alcoholic solution with 75% soaks 30 seconds, successively with the mercuric chloride solution of 0.1-0.5% sterilization 6min and aseptic water washing 2-3 time, blot the moisture on surface again with aseptic filter paper after, obtain aseptic bletilla capsule, it is characterized in that: described seed asepsis sprouting and the method for hardening as follows:
(1) method of seed asepsis sprouting is: be placed in culturing room by the seed germination medium broadcasting sowing bletilla seed and carry out light culture, cultivation temperature is 25 DEG C, when the seed on medium starts to sprout green, transferred to light to cultivate, continuous illumination 12 hours every days, intensity of illumination is 2000-2500lx, and light cultivates the seedling being divided into Multiple Buds after 15 ~ 20 days, completes axenic germination;
Wherein, consisting of of described seed germination medium: MS, sucrose, agar and potato, wherein the addition of sucrose is 30g/L, the addition of agar is 8g/L, the interpolation form of potato is pasty state, and its addition is 10-12g/L, and the pH of seed germination medium is 5.5 ~ 6.0;
(2) method of hardening is: the tissue culture flasks of getting the bletilla plantlet in vitro filled through culture of rootage gained, open bottle cap, after spraying the sterile water of 5 ~ 8ml wherein, place 2 ~ 3 days in culturing room, then tissue culture flasks is shifted out culturing room and be placed on outdoor 2 ~ 3 days, during outside scenery, temperature remains on 20 ~ 25 DEG C, humidity remains on 30 ~ 40%, then the bletilla plantlet in vitro in tissue culture flasks is taken out, wash the medium of plantlet in vitro root with distilled water after, soak 30 seconds in the NAA of 0.2mg/L, plant in hardening matrix, hardening can transplant field planting after 90 days,
Wherein, the composition of described hardening matrix comprises medium peat soil, perlite and wood fragments bits, wood fragments bits are sawdust, its preparation method is: first by medium peat soil through high-temperature heat sterilization, sterilising temp 121 DEG C, sterilization time is 20min, then by medium peat soil, perlite and wood fragments bits are according to after the ratio mixing of volume ratio 5:2:3, the distilled water adding sterilizing is mixed thoroughly, its water content is made to be 50 ~ 55% namely make hardening matrix, then made hardening matrix is positioned in seedling-cultivating tray, and to be placed on sunshade rate be for subsequent use in the shed of 50%, humidity in shed is 50 ~ 55%, temperature is 15 ~ 25 DEG C.
2. the efficient group culturation rapid propagating technology of a kind of bletilla seedling according to claim 1, it is characterized in that: the preparation method of described seed germination medium is: first potato cleaning is fallen surface impurity, then peeling is put into distilled water and is heated, continue after boiling to boil 30min, then potato is taken out and smash into mashed potato to pieces, and the distilled water after boiling is cooled to room temperature and adds in mashed potato and be mixed into pasty state, finally the potato of pasty state and sucrose, agar are added in MS successively according to described addition, and stir.
3. the efficient group culturation rapid propagating technology of a kind of bletilla seedling according to claim 1, it is characterized in that: described proliferation and subculture cultural method is: in superclean bench, be seeded in after the Multiple Buds seedling plant division that seed asepsis sprouting is obtained in the tissue culture flasks filling proliferation and subculture medium, the strain of every bottle graft kind 20 ~ 25, and by postvaccinal tissue culture flasks as illumination cultivation in culturing room, cultivation temperature is 25 DEG C, intensity of illumination is 2000-2500lx, between culture period, the light application time of every day is 12h, within 50 days, obtains the bletilla tufted seedling of 3 ~ 5cm afterwards;
The formulation ratio of described proliferation and subculture medium is: MS+KT1.0mg/L+NAA0.2mg/L+ sucrose 30g/L+ agar 9g/L.
4. the efficient group culturation rapid propagating technology of a kind of bletilla seedling according to claim 1, it is characterized in that: the method for described culture of rootage is as follows: in superclean bench, proliferation and subculture is cultivated the tufted seedling obtained and be divided into individual plant, be seeded in and fill in the tissue culture flasks of root media, the strain of every bottle graft kind 20 ~ 25, be placed in culturing room and cultivate, cultivation temperature is 25 DEG C, intensity of illumination is 2000-2500lx, illumination is 12 hours every days, plant height 5-8cm can be obtained, the bletilla plantlet in vitro of the long 2-3cm of root after 40 days;
The formulation ratio of described root media is: MS+NAA0.2mg/L+IAA1.0mg/L+ sucrose 30g/L+ agar 9g/L+25-30g/L potato.
5. the efficient group culturation rapid propagating technology of a kind of bletilla seedling according to claim 1, is characterized in that: the carbendazim solution adding 0.1g/L in described hardening matrix.
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| CN110447540A (en) * | 2019-09-11 | 2019-11-15 | 遵义医科大学 | A kind of tissue cultivating method of bletilla seedling |
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| CN114902920A (en) * | 2022-04-27 | 2022-08-16 | 贵州省农作物品种资源研究所 | Bletilla striata cultivation method, bletilla striata extract, preparation method of bletilla striata extract and oxidation resistance detection method |
| CN114902920B (en) * | 2022-04-27 | 2024-04-09 | 贵州省农作物品种资源研究所 | Bletilla striata cultivation method, bletilla striata extract, preparation method thereof and oxidation resistance detection method |
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Application publication date: 20151216 Assignee: Linyun family farm, Panhe Township, Lushi County Assignor: Henan University of Science and Technology Contract record no.: X2019980000499 Denomination of invention: Efficient tissue culture and rapid propagation technology for seedlings of bletilla striata Granted publication date: 20170606 License type: Common License Record date: 20191105 |