CN105325302A - Method for bletilla striata seedling production based on liquid medium - Google Patents
Method for bletilla striata seedling production based on liquid medium Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention belongs to the technical field of traditional Chinese medicine materials and particularly relates to a method for bletilla striata seedling production based on a liquid medium. The to-be-solved technical problem is how to provide a novel method for quickly acquiring bletilla striata seedlings. The method comprises a step a of sterilization, a step b of germination, a step c of growth into buds, a step d of rooting culture and a step e of seedling hardening of adult seedlings and transplantation. The method is simple and convenient to operate and low in cost, and each bottle of liquid medium can be used for germinating several tens of thousands of seed. The seeds are germinated tidily, the seedlings emerge consistently, and the pollution rate is low. According to the method, the efficient quick production of the bletilla striata tissue culture seedlings is achieved finally, operating procedures are effectively simplified, the production cost is reduced, and large-scale production is facilitated.
Description
Technical field
The invention belongs to traditional Chinese medicine planting technology field, particularly a kind of bletilla striata seedling production method based on liquid nutrient medium.
Background technology
The bletilla striata is the perennial root herbaceous plant that the orchid family bletilla striata belongs to, and is national key protected wild plants.Bletilla striata large flower and brilliant color, has higher ornamental value; Simultaneously the bletilla striata has the block pseudobulb of plump meat, is one of important traditional Chinese medicine.
Under nature wild state, ripe bletilla striata seeds is Powdered, very tiny, many in spindle under the microscope, is about 1.2mm, wide about 0.2mm.The structure of its seed is very simple, is made up of the seed coat of layer of transparent and an embryo.Seed coat is cell monolayer composition, and organelle and protoplasm disappear all, are the translucent dead cells of one deck; Embryo is still in undifferentiated state in shape, is proembryo stage, and suspensor is degenerated, without endosperm.The seed of pollination after 15 weeks of blooming has embryo rate to be only about 59%, few Germination And Seedling.For above-mentioned reasons, bletilla striata seeds nature germination rate is extremely low, breeding difficulty, and people rob excavating of formula for a long time and cause its wild resource frequently to face exhaustion in addition, and therefore existing bletilla striata resource cannot meet the very big demand in market.
Artificial breeding is the effective measure improving bletilla striata output at present, and existing mating system is main mainly with plant division greatly, and to be cut into small pieces plantation by stem tuber, but reproduction coefficient is low, consumption is large, and large area, for kind still comparatively difficulty, is difficult to the needs meeting large-scale production.
Comprise the orchid of the bletilla striata, by the mode of aseptic seeding and tissue cultures, a large amount of seedlings can be obtained at short notice, one of can yet be regarded as propagation method fast and effectively.Prior art mainly through by seed broadcasting add hormone solid culture medium on sprout, regeneration aseptic seedling, or induction protocorm, through propagation take root after carry out numerous soon.These methods relate to the medium of more than 4-5 kind mostly, operate comparatively loaded down with trivial details, and the cycle are longer, the shortlyest can become transplantation of seedlings in 110 days, and therefore production cost is higher relatively.
In the tissue rapid propagation technology of the bletilla striata, the kind of hormone and proportioning are very crucial, directly affect the germination rate of seed, the inductivity of protocorm and growth rate.Be that interpolation comprises cytokinin 6-BA, KT and auxins NAA, 2 for the medium of seed germination in existing method, the solid culture medium of 4-D etc., germination rate is not at 50%-95% etc., the seed germination cycle is longer general at about 30-45 days, and seed germination rate affects greatly by seed age, maturescent seed (after general 20 week age) germination rate is high, and the seed germination rate before 15-20 age in week is extremely low or do not sprout.TDZ (Thidiazuron) is a kind of new plant growth regulator, has very strong cytokine activity, its activity higher than general cytokinin tens times to hundred times.Based on TDZ short cell division activity efficiently, in the tissue-culturing rapid propagation of the bletilla striata, there is researcher TDZ is used for the propagation of protocorm and obtains good result, but have no the report using it for seed germination.TDZ is slightly expensive relative to common hormones such as BA, and large for breeding usage amount, the time is long, increases cost.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of new method for obtaining bletilla striata seedling fast.
Technical scheme of the present invention is a kind of bletilla striata seedling production method based on liquid nutrient medium, comprises the steps:
A, sterilizing: by bletilla striata capsule running water more than the running water 30min in 15 ~ 20 week age of pollination of blooming, carry out surface sterilizing, then strip off, seed is carried out disinfection;
B, sprouting: the seed after sterilization is placed in the liquid nutrient medium of MS+0.5 ~ 2.0mg/LTDZ+30g/L white granulated sugar, pH5.8,23 ~ 27 DEG C, 90 ~ 120rpm dark culturing, after 5 days, shakes cultivation 15 ~ 20 days, seed germination under proceeding to the photoperiod of illumination 16h/ light culture 8h;
C, grow into bud: be inoculated in by the seed sprouted on the solid culture medium of MS+0.1 ~ 0.5mg/L6-BA (6-benzyl aminoadenine)+30g/L white granulated sugar+6g/L agar, pH5.8,25 DEG C, the 16/8h photoperiod cultivates; Cultivate 20 ~ 30 days, namely obtain seedling;
D, culture of rootage: seedling inoculation is on the medium of 1/2MS+0.1 ~ 1.0mg/LNAA (methyl α-naphthyl acetate)+30g/L white granulated sugar+0.5 ~ 2g/L active carbon (active carbon is conducive to avoiding brownization)+6g/L agar, pH5.8,25 DEG C, the 16/8h photoperiod cultivates; 30 ~ 40 obtain seedling everyday afterwards,
E, seedling is carried out hardening, transplant.
Concrete, in step a, bletilla striata capsule surface sterilizing adopts 75% ethanol to carry out surface sterilization 30s, aseptic water washing 1 ~ 2 time.
Concrete, the operation carried out disinfection to seed in step a is as follows: shake off seed in sterile chamber, adds that 5% liquor natrii hypochloritis is suitable soaks seed 20 minutes completely; With 50 order cell sieve filter seed of sterilizing; Use aseptic water washing, filtration again, 4 ~ 6 times repeatedly, wash away the thimerosal of remained on surface; The sieved filter of cell, the seed after being sterilized.
Preferably, 1 ~ 2 polysorbas20 is dripped in described liquor natrii hypochloritis.
Concrete, add 1.0mg/LTDZ in step b medium.
Concrete, 25 DEG C, 100rpm light culture in step b.
Concrete, add 0.2mg/L6-BA in step c medium.
Concrete, add 0.1 ~ 0.5mg/LNAA and 2g/L active carbon in medium in steps d.
Concrete, described in step e, the operation of hardening is as follows: when the root growth of seedling is long to 3 ~ 5cm, opens bottle cap gradually and adapts to 3 ~ 5 days.
Concrete, the operation of transplanting described in step e is as follows: by the seedling after hardening, cleans root agar with clear water, and be transplanted in the cave dish adding matrix, matrix is watered permeable, and the low light level is cultivated, and keeps matrix moistening.
TDZ is applied in the sprouting of the bletilla striata by the present invention, also selects liquid suspension culture germination seed simultaneously, instead of conventional solid culture primary surface is sprouted.Bletilla striata seeds germination rate is extremely low, relies on by force to age in kernel maturing week, and before general 22 week age, seed is there are no sprouting report, sprout time long general at least one moon of needs.Growth cycle is long, generally needs half a year more than to transplanting.The present invention adopts different hormone combinations, particularly TDZ to be used for germination seed (being generally used for callus induction and induced bud), sends out seed, effectively raises germination rate, shortens the seedling time with liquid nutrient medium.
Beneficial effect of the present invention: adopt Post flowering 15 thoughtful 20 weeks maturescent bletilla striata seeds to carry out liquid suspension culture, can significantly improve seed germination rate and reach 99%, accelerates (remarkable result, key technology) about the formation speed to 15 day of protocorm.The method is easy and simple to handle, with low cost, and every bottle of liquid nutrient medium can sprout tens thousand of seeds, and seed germination is neat, and emerge consistent, pollution rate is low; Cultivate in conjunction with the solid culture medium simplified, greatly shorten the bletilla striata group training cycle, a large amount of healthy and strong aseptic seedling can be obtained the soonest be used for transplanting in 75-90 days; Sprout and Multiplying culture through induced bundle, can at 30-45 days internal breeding 8-10 doubly; The plantlet in vitro protocorm obtained through this method is thick, rooting rate 100%, transplants survival rate up to 99%.The present invention finally achieves the efficient of bletilla striata plantlet in vitro and produces fast, and effectively simplifies procedures, and reduces production cost, is convenient to carry out large-scale production.
Accompanying drawing explanation
Fig. 1 is the bletilla striata seeds (A: latter 5 days of seed inoculation sprouted in liquid nutrient medium; B: latter 10 days of seed inoculation; C: latter 15 days of seed inoculation.)
Fig. 2 is the bletilla striata seedling (A: seed latter 20 days of inoculation (be transferred to solid culture medium upper 5 day) grown in solid culture medium; B: seed latter 30 days of inoculation (be transferred to solid culture medium upper 15 day); C: seed latter 45 days of inoculation (be transferred to solid culture medium upper 30 day); D: be transferred to the solid culture medium bletilla striata population growth situation of 30 days.)
Fig. 3 is the bletilla striata seedling (A: after seed inoculation the 55th day (switching root media 10 days) grown in root media; B: after seed inoculation the 75th day (switching root media 30 days); C: the switching root media bletilla striata population growth situation of 30 days.)
Fig. 4 is the bletilla striata plantlet in vitro (A: after seed inoculation the 90th day (transplanting the same day) of transplanting and field planting; B: after seed inoculation the 100th day (transplanting latter 10 days); C: seed inoculation latter 105 days (transplanting latter 15 days); D: seed inoculates latter 120 days (the field planting same day).)
Fig. 5 is the bletilla striata seedling (A: seedling inoculation was to proliferated culture medium same day of Multiplying culture; B: after seedling cultivates 30 days on proliferated culture medium.)
Embodiment
The embodiment 1 prematurity bletilla striata is cultivated
1, the surface of the seed sterilizing and sprouting
Choose and bloom pollination after 15 weeks, the bletilla striata capsule (containing seed) that do not ftracture carries out surface sterilization 30s, aseptic water washing 1-2 time with 75% ethanol on superclean bench.With scalpel and the careful strip off fruit of tweezers, careful for seed is shaken off in aseptic tissue culture bottle, add appropriate 5% liquor natrii hypochloritis (drip 1-2 and drip polysorbas20) and sterilize 20 minutes.With 50 order cell sieve filter seed of sterilizing; Use aseptic water washing, filtration again, 4-6 time repeatedly, wash away the thimerosal of remained on surface.By upper for the cell sieve seed filtered, with MS+1.0mg/LTDZ+30g/L white granulated sugar, the liquid seeds germination medium of pH5.8 is flushed in triangular flask.25 DEG C, 100rpm, dark culturing 15-20 days.
After about 3 days, namely seed expands and shows money or valuables one carries unintentionally, and cultivate after 5 days, embryo cellular colours changes light green into from faint yellow, shows the generation of existing chloroplast, and seed is sprouted (Fig. 1-A).Under the photoperiod proceeding to 16h/8h after illumination cultivation 5 days (inoculating 10 days), the vegetative cone can observing most of seed breaks through seed coat from the micropylar end side of seed, presents spheroidal, forms protocorm (Fig. 1-B).Inoculate protocorm top after 15 days and differentiate leaf primordium, elongated, develop into blade (Fig. 1-C) gradually.
2, seed growth becomes bud
By inoculation 15 days, the seed sprouted removed liquid through 50 order cell sievings, seed protocorm was inoculated in MS+0.2mg/L6-BA+30g/L white granulated sugar+6g/L agar, on the solid induction buds sprouting medium of pH5.8, disperseed seedling as far as possible, about 100, every ware.25 DEG C, the 16/8h photoperiod cultivates.
After seed inoculates 20 days (be transferred to solid culture medium upper 5 day), seed base portion protocorm starts to expand, and top blade starts to extend (Fig. 2-A).Be transferred to solid culture medium after 15 days, protocorm expands further, and diameter is about 3mm, and young shoot is formed gradually, is about 0.5cm (Fig. 2-B).Cultivate after 30 days, young shoot is all grown to seedling, and have significant blade and root morphology difference, young shoot is about about 2cm, and root is about about 0.5cm, and young shoot planting percent is up to 100% (Fig. 2-C, D)
3, culture of rootage
By the seedling inoculation in plate to 1/2MS+0.1mg/LNAA+30g/L white granulated sugar+2g/L active carbon+6g/L agar, on the solid root media of pH5.8, every bottle of 25-30 strain, 25 DEG C, the 16/8h photoperiod cultivates, and promotes the growth of seedling and root, improves strengthening seedling and rooting rate.
Switching root media is after 10 days, and the height of seedling obviously increases, and reaches 3-5cm, and the protocorm of seedling base portion increases obviously, and diameter reaches more than 5mm, and (Fig. 3 A) is taken root obviously in bottom.Culture of rootage is after 30 days, and plantlet in vitro is grown to healthy and strong seedling, highly reaches more than 8-10cm, the healthy and strong color of blade is dark green, and base portion protocorm increases to diameter and is about 1cm, and rooting rate reaches 100%, mean elements 5-6 root, root length can reach more than 3-5cm (Fig. 3 B, 3C).
4, hardening is transplanted
When the root growth of seedling is long to 3-5cm (after general seed germination 75-90 days), first opens bottle cap gradually and adapt to 3-5 days, period is noted keeping bottle humidity.Clean root agar with clear water, avoid damage root and protocorm (Fig. 4 A); The bletilla striata group of stalwartness training seedling be transplanted in the cave dish adding matrix, matrix is watered permeable, and low light level moisturizing is noted in daily management.Transplant after 7-10 days, bletilla striata plantlet in vitro survival rate is up to 99% (Fig. 4 B).Transplant bletilla striata seedling after 15 days to survive, growth is normal, and plant height obviously increases, and can reach more than 12cm (Fig. 4 C).The bletilla striata transplants size and the vigor that the key of surviving is protocorm, and generally have the seedling of protocorm all can transplant survival, survival rate can reach more than 95%.Transplant the cave seedling buried about 30 days and get final product field planting, conventional field management (Fig. 4 D).
Embodiment 2 is cultivated by the nearly ripe bletilla striata
1, the surface of the seed sterilizing and sprouting:
Post flowering more than 20 weeks, maturescent bletilla striata capsule (not ftractureing, containing seed) binds up with gauze running water running water 30min; Superclean bench carries out surface sterilization 30s, aseptic water washing 1-2 time with 75% ethanol.With scalpel and the careful strip off fruit of tweezers, careful for seed is dialled in aseptic tissue culture bottle, add appropriate 5% liquor natrii hypochloritis (dripping tween 2-3 to drip) sterilization 20min.(50 order) filter seed is sieved with the cell of sterilizing; Use aseptic water washing, filtration again, 4-6 time repeatedly, wash away the thimerosal of remained on surface.By upper for the cell sieve seed filtered, with MS+2.0mg/LTDZ+30g/L white granulated sugar, the liquid seeds of pH5.8 is sprouted liquid nutrient medium and is flushed in triangular flask.25 DEG C, 100rpm, dark culturing 15 days.
2, seed growth becomes bud:
Seed protocorm, through cell sieve (50 order) elimination liquid, is inoculated in MS+0.1mg/L6-BA+30g/L white granulated sugar+6g/L agar by the seed sprouted, seed-dispersed that the induction buds sprouting solid culture medium of pH5.8 is tried one's best, 100, every ware.25 DEG C, the 16/8h photoperiod cultivates 20-30 days.
3, culture of rootage:
By the seedling that generates and the seedling inoculation of propagation to 1/2MS+0.5mg/LNAA+30g/L white granulated sugar+0.5g/L active carbon+6g/L agar, on the root media of pH5.8, strong sprout, every bottle of 25-30 strain, 25 DEG C, the 16/8h photoperiod cultivated 30-40 days to improve rooting rate.Within 30 days, seedling rooting rate reaches 100%, mean elements 5-6, the long 3-5cm of root, can carry out acclimatization and transplants.Even if the seedling do not taken root due to protocorm obviously thick, also can directly transplant.
4, acclimatization and transplants:
When the root growth of seedling is long to 3-5cm, first opens bottle cap gradually and adapt to 3-5 days, note keeping humidity, then clean root agar, be transplanted in the cave dish adding matrix, the moisturizing of first week low light level cover film, in hardening canopy growth 15-30d.Grow normal cave seedling and get final product field planting.
Embodiment 3 is cultivated by the ripe bletilla striata
1, the surface of the seed sterilizing and sprouting:
The bletilla striata capsule (not ftractureing, containing seed) of maturation is bound up with gauze running water running water 30min; Superclean bench carries out surface sterilization 30s, aseptic water washing 1-2 time with 75% ethanol.With scalpel and the careful strip off fruit of tweezers, careful for seed is dialled in aseptic tissue culture bottle, add appropriate 5% liquor natrii hypochloritis (dripping tween 2-3 to drip) sterilization 20min.(50 order) filter seed is sieved with the cell of sterilizing; Use aseptic water washing, filtration again, 4-6 time repeatedly, wash away the thimerosal of remained on surface.By upper for the cell sieve seed filtered, with MS+0.5mg/LTDZ+30g/L white granulated sugar, the liquid seeds of pH5.8 is sprouted liquid nutrient medium and is flushed in triangular flask.25 DEG C, 100rpm, dark culturing 15 days.
2, seed growth becomes bud:
Seed protocorm, through cell sieve (50 order) elimination liquid, is inoculated in MS+0.5mg/L6-BA+30g/L white granulated sugar+6g/L agar by the seed sprouted, seed-dispersed that the induction buds sprouting solid culture medium of pH5.8 is tried one's best, 100, every ware.25 DEG C, the 16/8h photoperiod cultivates 20-30 days.
3, culture of rootage:
By the seedling that generates and the seedling inoculation of propagation to 1/2MS+1.0mg/LNAA+30g/L white granulated sugar+1.0g/L active carbon+6g/L agar, on the root media of pH5.8, strong sprout, every bottle of 25-30 strain, 25 DEG C, the 16/8h photoperiod cultivated 30-40 days to improve rooting rate.Within 30 days, seedling rooting rate reaches 100%, mean elements 5-6, the long 3-5cm of root, can carry out acclimatization and transplants.Even if the seedling do not taken root due to protocorm obviously thick, also can directly transplant.
4, acclimatization and transplants:
When the root growth of seedling is long to 3-5cm, first opens bottle cap gradually and adapt to 3-5 days, note keeping humidity, then clean root agar, be transplanted in the cave dish adding matrix, the moisturizing of first week low light level cover film, in hardening canopy growth 15-30d.Grow normal cave seedling and get final product field planting.
Claims (10)
1., based on a bletilla striata seedling production method for liquid nutrient medium, it is characterized in that: comprise the steps:
A, sterilizing: by bletilla striata capsule running water more than the running water 30min in 15 ~ 20 week age of pollination of blooming, carry out surface sterilizing, then strip off, seed is carried out disinfection;
B, sprouting: the seed after sterilization is placed in the liquid nutrient medium of MS+0.5 ~ 2.0mg/LTDZ+30g/L white granulated sugar, pH5.8,23 ~ 27 DEG C, 90 ~ 120rpm dark culturing, after 15 ~ 20 days, shakes cultivation 15 ~ 20 days, seed germination under proceeding to the photoperiod of illumination 16h/ light culture 8h;
C, grow into bud: be inoculated in by the seed sprouted on the solid culture medium of MS+0.1 ~ 0.5mg/L6-BA (6-benzyl aminoadenine)+30g/L white granulated sugar+6g/L agar, pH5.8,25 DEG C, the 16/8h photoperiod cultivates; Cultivate 20 ~ 30 days, namely obtain seedling;
D, culture of rootage: seedling inoculation is on the medium of 1/2MS+0.1 ~ 1.0mg/LNAA (methyl α-naphthyl acetate)+30g/L white granulated sugar+0.5 ~ 2g/L active carbon+6g/L agar, pH5.8, and 25 DEG C, the 16/8h photoperiod cultivates; Seedling is obtained after 30 ~ 40 days,
E, seedling is carried out hardening, transplant.
2. the method for claim 1, is characterized in that: in step a, bletilla striata capsule surface sterilizing adopts 75% ethanol to carry out surface sterilization 30s, aseptic water washing 1 ~ 2 time.
3. method as claimed in claim 1 or 2, is characterized in that: the operation carried out disinfection to seed in step a is as follows: shake off seed in sterile chamber, adds that 5% liquor natrii hypochloritis is suitable soaks seed 20 minutes completely; With 50 order cell sieve filter seed of sterilizing; Use aseptic water washing, filtration again, 4 ~ 6 times repeatedly, wash away the thimerosal of remained on surface; The sieved filter of cell, the seed after being sterilized.
4. method as claimed in claim 3, is characterized in that: drip 1 ~ 2 polysorbas20 in described liquor natrii hypochloritis.
5. the method as described in any one of Claims 1 to 4, is characterized in that: add 1.0mg/LTDZ in step b medium.
6. the method as described in any one of Claims 1 to 5, is characterized in that: 25 DEG C, 100rpm light culture in step b.
7. the method as described in any one of claim 1 ~ 6, is characterized in that: add 0.2mg/L6-BA in step c medium.
8. the method as described in any one of claim 1 ~ 7, is characterized in that: add 0.1 ~ 0.5mg/LNAA and 2g/L active carbon in medium in steps d.
9. the method as described in any one of claim 1 ~ 8, is characterized in that: described in step e, the operation of hardening is as follows: when the root growth of seedling is long to 3 ~ 5cm, opens bottle cap gradually and adapts to 3 ~ 5 days.
10. the method as described in any one of claim 1 ~ 9, is characterized in that: the operation of transplanting described in step e is as follows: by the seedling after hardening, cleans root agar with clear water, be transplanted in the cave dish adding matrix, matrix is watered permeable, and the low light level is cultivated, and keeps matrix moistening.
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| CN107155882A (en) * | 2017-05-05 | 2017-09-15 | 江苏东郁园林科技有限公司 | A kind of medicinal bletilla striata aseptic seeding fast seedling-cultivating method |
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