CN104186350A - Method for reproducing Biyun tea tree test-tube plantlets - Google Patents
Method for reproducing Biyun tea tree test-tube plantlets Download PDFInfo
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- CN104186350A CN104186350A CN201410476545.XA CN201410476545A CN104186350A CN 104186350 A CN104186350 A CN 104186350A CN 201410476545 A CN201410476545 A CN 201410476545A CN 104186350 A CN104186350 A CN 104186350A
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Abstract
The invention discloses a method for efficiently and rapidly reproducing Biyun tea tree test-tube plantlets. The method comprises the following steps: disinfecting and bleaching Biyun tea tree annual semi-ligneous branches; shearing the branches into small sections with one to two axillary buds and inoculating to a solid culture medium for culturing; inoculating to a multiplication medium for culturing after the axillary buds sprout, inoculating every 45 days until required seedling number is obtained, and then, inoculating to a rooting medium for rooting culture; 50 days later, when Biyun tea tree seedlings grow two to three roots, carrying out domestication and transplanting. The method can be directly used for factory large-scale seedling production and has the advantages of little pollution, high reproducing speed, low mutation rate, tidy and health seedlings, low production cost, good convenience in transportation, high transplanting survival rate and the like.
Description
Technical field
We's invention belongs to field of plant growing technology, is specifically related to a kind of method of breeding green cloud tea tree test-tube plantlet.
Background technology
Green cloud tea tree, clonal cultivar kind, has output high, the advantages such as strong stress resistance.The tealeaves profile of its making is carefully tight, and color and luster is emerald green, and fragrance is invigorating pure, the fresh alcohol of flavour, and be rich in the materials such as amino acid, Tea Polyphenols, catechin, caffeine, be a kind of local tea variety that Important Economic is worth that has.Along with the development of green tea industry, green cloud tea tree planting scale constantly expands, also constantly soaring to the demand of good tea tree seedling, traditional cottage propagation method can not meet market demand.
Summary of the invention
Technical problem to be solved by this invention, is to provide a kind of method of breeding green cloud tea tree test-tube plantlet.
A method of breeding green cloud tea tree test-tube plantlet, is characterized in that, the method comprises the following steps:
(1) branch of the green cloud tea tree of clip, is cut into the long segment of 10cm after removing blade, cleans 10min, then rinse 10min with flowing water with washing powder, soaks, then be positioned over and in mercuric chloride solution, soak 7min with ethanol water;
(2) the aseptic skill bar of green cloud tea tree step (1) being obtained is cut into the segment with 1-2 axillalry bud, is inoculated in solid culture medium and cultivates, and after axillary bud sprouting, gets axillalry bud standby; Wherein, described solid culture medium consists of: methyl α-naphthyl acetate 0.5～1mg/L, sucrose 25～30g/L, MS medium, agar 4.5～6g/L, pH 5.6～5.8;
(3) axillalry bud step (2) being obtained is placed in proliferation and subculture medium and cultivates the green cloud tea tree seedling that obtains growing thickly; Wherein, shoot proliferation medium consists of: methyl α-naphthyl acetate 0.4～1.2mg/L, 6-benzyl aminopurine 2-3mg/L, sucrose 25～30g/L, MS medium, agar 4.5～6g/L, pH 5.6～5.8;
(4) the green cloud tea tree seedling cutting and separating on superclean bench of growing thickly obtaining in step (3) is become to individual plant, transfer in strengthening seedling and rooting medium and cultivate, green cloud tea seedling grows up to the seedling that 4-5cm is high, have 2-3 bar root; Wherein, strengthening seedling and rooting medium consists of: methyl α-naphthyl acetate 0.2～0.5mg/L, sucrose 25～30g/L, 1/3MS medium, agar 4.5～6g/L, pH 5.6～5.8;
(5) seedling step (4) being obtained is taken out with clear water and rinse to be removed medium in bottle, and dislocation is covered with on the seedbed of Nutrition Soil, and the moisturizing of sheltering from heat or light, and until seedling, grows to 10cm when high, carries out field-transplanting.
Wherein, the branch described in step (1) is for giving birth to then semi-lignified branch.
Wherein, the described ethanol of step (1) is water-soluble, and the volumn concentration of ethanol is 75%, and ethanol water soak time is 45～60s.
Wherein, in the described mercuric chloride solution of step (1), the quality percentage composition of mercury chloride is 0.1%～0.15%, and mercuric chloride solution soak time is 7min.
Wherein, the condition of culture of step (2) is: 22～25 ℃ of temperature, illuminance 300～1000 luxs, under light application time 11～13h/d condition, cultivate 40～50d.
Wherein, the described solid culture medium of step (2) consists of: methyl α-naphthyl acetate 0.5mg/L, sucrose 30g/L, MS medium, agar 6g/L, pH 5.8.
Wherein, the condition of culture of step (3) is: 25～28 ℃ of temperature, and illuminance 1000～2000 luxs, light application time 11～13h/d, cultivates 45 days.
Wherein, the described shoot proliferation medium of step (3) consists of: methyl α-naphthyl acetate 0.8mg/L, 6-benzyl aminopurine 2.5mg/L, sucrose 30g/L, MS medium, agar 6g/L, pH 5.8.
Wherein, the condition of culture of step (4) is: 25～28 ℃ of temperature, and illuminance 1000～2000 luxs, light application time 11～13h/d, cultivates 50 days.
Wherein, it is characterized in that, in step (4), described strengthening seedling and rooting medium consists of: methyl α-naphthyl acetate 0.2mg/L, sucrose 30g/L, 1/3MS medium, agar 6g/L, pH 5.8.
Beneficial effect: compared with prior art, tool has the following advantages in the present invention:
1, after the branch of green cloud tea tree sterilization, the segment being cut into 1-2 axillalry bud is inoculated in the solid culture medium in step (2), adopt MS and be equipped with suitable hormone, compare with existing tea tree axillary bud sprouting medium, the method axillary bud sprouting also grows to time shorten 10～15d of 1-2cm, and germination rate is up to more than 80%, and aberration rate is less than 1%, sprout neat.
2, shoot proliferation medium adopts MS and is equipped with suitable growth hormone and the basic element of cell division, compares with traditional shoot proliferation medium, and the method can promote the branch of seedling better faster, and growth coefficient can reach 2-3.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described content of embodiment is only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1:
A method for the green cloud tea tree of efficient Fast-propagation test-tube plantlet, the method comprises the following steps:
(1) branch of the green cloud tea tree of clip, removes blade, is cut into the long segment of 10cm, with washing powder, clean 10min, then rinse 10min with flowing water, bring the branch cleaning up into superclean bench, with 75% alcohol-pickled 45～60s, then be positioned over and in mercuric chloride solution, soak 8-10min;
(2) segment that the aseptic skill bar of green cloud tea tree step (1) being obtained is cut into 1-2 axillalry bud is inoculated in solid culture medium, under 25～28 ℃, the condition of illuminance 1000～2000 luxs, light application time 11～13h/d, cultivate 40～50d, axillary bud sprouting, when bud grows to 1-2cm, get axillalry bud standby; Wherein, described solid culture medium consists of: methyl α-naphthyl acetate 0.5mg/L, and sucrose 30g/L, MS medium, agar 6g/L, pH 5.8;
(3) axillalry bud step (2) being obtained is placed in proliferation and subculture medium, under 25～28 ℃, the condition of illuminance 1000～2000 luxs, light application time 11～13h/d, cultivates 45 days, and green cloud tea tree branch propagation, growth coefficient reaches 2-3; Wherein, described shoot proliferation medium consists of: methyl α-naphthyl acetate 0.8mg/L, and 6-benzyl aminopurine 2.5mg/L, sucrose 30g/L, MS medium, agar 6g/L, pH 5.8;
(4) the green cloud tea tree seedling cutting and separating on superclean bench of growing thickly obtaining in step (3) is become to individual plant, transfer in strengthening seedling and rooting medium, under 25～28 ℃, the condition of illuminance 1000～2000 luxs, light application time 11～13h/d, cultivate 50 days, it is high that green cloud tea seedling grows up to 4-5cm, has the seedling of 2-3 bar root, wherein, described strengthening seedling and rooting medium consists of: methyl α-naphthyl acetate 0.2mg/L, sucrose 30g/L, 1/3MS medium, agar 6g/L, pH 5.8;
(5) seedling step (4) being obtained is taken out with clear water and rinse to be removed medium in bottle, and dislocation is covered with on the seedbed of Nutrition Soil, and the moisturizing of sheltering from heat or light, and until seedling, grows to 10cm when high, can carry out field-transplanting.
The tender tip in green cloud tea tree top is after sterilization, be inoculated in the solid culture medium in step (2), adopt MS and be equipped with suitable hormone, compare with existing axillary bud sprouting medium, the method axillary bud sprouting time shorten 7～10d, and germination rate is up to more than 80%, sprout neatly and comparatively healthy and strong, while continuing to cultivate 40-50 days, axillalry bud can grow to 1-2cm, than the high about 0.5cm of former formula.In the shoot proliferation medium of step (3), adopt MS and be equipped with suitable growth hormone and the basic element of cell division, compare with traditional shoot proliferation medium, although the method could not improve growth coefficient, but can make to shorten the time of tillering and breeding, make to breed seedling more neat, cultivate 45 days, propagation seedling reaches 2-3cm.Step (4) is inoculated in seedling in strengthening seedling and rooting medium, adopt 1/3MS and coordinate suitable growth hormone, compare with existing strengthening seedling and rooting medium, the method can make seedling take root simultaneously and grow tall, cultivate 50 days, seedling grows 2-3 bar root, and root system is healthy and strong, simultaneously seedling to grow to 4-5cm high, and longly have a 4-5 sheet blade.
Embodiment 2:
A method for the green cloud tea tree of efficient Fast-propagation test-tube plantlet, the method comprises the following steps:
(1) branch of the green cloud tea tree of clip, removes blade, is cut into the long segment of 10cm, with detergent, clean 10min, then rinse 10min with flowing water, bring the branch cleaning up into superclean bench, with 75% alcohol-pickled 45～60s, then be positioned over and in mercuric chloride solution, soak 8-10min;
(2) segment that the aseptic skill bar of green cloud tea tree step (1) being obtained is cut into 1-2 axillalry bud is inoculated in solid culture medium, under 22～25 ℃, the condition of illuminance 300～1000 luxs, light application time 11～13h/d, cultivate 40～50d, axillary bud sprouting, when bud grows to 1-2cm, get axillalry bud standby; Wherein, described solid culture medium consists of: methyl α-naphthyl acetate 0.2～0.5mg/L, and sucrose 25～30g/L, MS medium, agar 4.5～6g/L, pH 5.6～5.8;
(3) axillalry bud step (2) being obtained is placed in proliferation and subculture medium, under 25～28 ℃, the condition of illuminance 1000～2000 luxs, light application time 11～13h/d, cultivates 45 days, and green cloud tea tree branch propagation, growth coefficient reaches 2-3; Wherein, described shoot proliferation medium consists of: methyl α-naphthyl acetate 0.5～1.0mg/L, and 6-benzyl aminopurine 2-3mg/L, sucrose 25～30g/L, MS medium, agar 4.5～6g/L, pH 5.6～5.8;
(4) the green cloud tea tree seedling cutting and separating on superclean bench of growing thickly obtaining in step (3) is become to individual plant, transfer in strengthening seedling and rooting medium, under 25～28 ℃, the condition of illuminance 1000～2000 luxs, light application time 11～13h/d, cultivate 50 days, it is high that green cloud tea seedling grows up to 4-5cm, the seedling that has 2-3 bar root, wherein, described strengthening seedling and rooting medium consists of: methyl α-naphthyl acetate 0.2～0.5mg/L, sucrose 25～30g/L, 1/3MS medium, agar 4.5～6g/L, pH 5.6～5.8;
(5) seedling step (4) being obtained is taken out with clear water and rinse to be removed medium in bottle, and dislocation is covered with on the seedbed of Nutrition Soil, and the moisturizing of sheltering from heat or light, and until seedling, grows to 10cm when high, can carry out field-transplanting.
Embodiment 3:
Substantially the same manner as Example 2, in step (2), step (3), step (4), axillary bud sprouting, shoot proliferation cultivation, strong seedling culture three phases are combined into row filter with the listed medium of table 1 respectively, with preferred optimum medium combination formula.Table 1 is described as follows:
A, culture medium prescription optimization are mainly that the different basal medium of design, hormone concentration etc. are optimized design to medium, in this example, be all designed to solid culture medium, in every kind of formula, agar concentration is 6g/L, sucrose is 30g/L, and culture environment is in 25 ℃ of temperature, illuminance 1000～2000 luxs, light application time 12h/d culturing room.
B, for production, different cultivation stages has designed three groups of culture medium prescriptions, whether the planting seed stage is mainly observed the index judgement formulas such as speed, growing way, color (emerald green for good) of seed germination reasonable; Whether the shoot proliferation stage is mainly observed the seedling index such as propagation (The more the better), the vitrifying degree judgement formula of tillering reasonable; In the strong seedling culture stage, the indexs such as the growth potential of the observation seedling of being mainly open to the custom, vitrifying degree (more low better), color judge the quality of formula.By observing and the mensuration of indices, and the quality of filling a prescription with how many representatives of " ★ " and " ☆ ", ★ more multilist shows better, and ☆ is than the low grade of ★.
Table 1 screening of medium formula
Test shows, in axillary bud sprouting stage (step 2) 9 kinds of formula combination, basal medium and hormone combinations scope are within the scope of methyl α-naphthyl acetate 0.2～0.5mg/L+ sucrose 25～30g/L+MS medium+agar 4.5～6g/L, axillary bud sprouting rate is high and growth is fast, be more suitable combination formula combination, after branch inoculation, 40-45d left and right grows the axillalry bud of 1-2cm; In shoot proliferation stage (step 3) 16 kinds of formula combination, basal medium, hormone combinations scope are within the scope of methyl α-naphthyl acetate 0.5～1.0mg/L+6-benayl aminopurine 2-3mg/L+ sucrose 25～30g/L+MS medium+agar 4.5～6g/L medium, the growth potential of seedling is good, growth rate fast, vitrifying degree is low, 45d left and right, the growth coefficient of seedling can reach 2～3; In the strengthening seedling and rooting stage (step 4), result shows, methyl α-naphthyl acetate 0.2～0.5mg/L+ sucrose 25～30g/L+1/3MS medium+agar 4.5～6g/L is preferred culture medium formula combination.
Claims (10)
1. a method for the green cloud tea tree test-tube plantlet of breeding, is characterized in that, the method comprises the following steps:
(1) branch of the green cloud tea tree of clip, is cut into the long segment of 10cm after removing blade, cleans 10min, then rinse 10min with flowing water with washing powder, soaks, then be positioned in mercuric chloride solution and soak with ethanol water;
(2) the aseptic skill bar of green cloud tea tree step (1) being obtained is cut into the segment with 1-2 axillalry bud, is inoculated in solid culture medium and cultivates, and after axillary bud sprouting, gets axillalry bud standby; Wherein, described solid culture medium consists of: methyl α-naphthyl acetate 0.5～1mg/L, sucrose 25～30g/L, MS medium, agar 4.5～6g/L, pH 5.6～5.8;
(3) axillalry bud step (2) being obtained is placed in proliferation and subculture medium and cultivates the green cloud tea tree seedling that obtains growing thickly; Wherein, shoot proliferation medium consists of: methyl α-naphthyl acetate 0.4～1.2mg/L, 6-benzyl aminopurine 1.5～3mg/L, sucrose 25～30g/L, MS medium, agar 4.5～6g/L, pH 5.6～5.8;
(4) the green cloud tea tree seedling cutting and separating on superclean bench of growing thickly obtaining in step (3) is become to individual plant, transfer in strengthening seedling and rooting medium and cultivate, green cloud tea seedling grows up to the seedling that 4-5cm is high, have 2-3 bar root; Wherein, strengthening seedling and rooting medium consists of: methyl α-naphthyl acetate 0.2～0.5mg/L, sucrose 25～30g/L, 1/3MS medium, agar 4.5～6g/L, pH 5.6～5.8;
(5) seedling step (4) being obtained is taken out with clear water and rinse to be removed medium in bottle, and dislocation is covered with on the seedbed of Nutrition Soil, and the moisturizing of sheltering from heat or light, and until seedling, grows to 10cm when high, carries out field-transplanting.
2. the method for the green cloud tea tree of breeding according to claim 1 test-tube plantlet, is characterized in that, the described branch of step (1) is for giving birth to then semi-lignified branch.
3. the method for the green cloud tea tree of breeding according to claim 1 test-tube plantlet, is characterized in that, the described ethanol of step (1) is water-soluble, and the volumn concentration of ethanol is 75%, and ethanol water soak time is 45～60s.
4. the method for the green cloud tea tree of breeding according to claim 1 test-tube plantlet, is characterized in that, in the described mercuric chloride solution of step (1), the quality percentage composition of mercury chloride is 0.1%～0.15%, and mercuric chloride solution soak time is 7min.
5. the method for the green cloud tea tree of breeding according to claim 1 test-tube plantlet, is characterized in that, the condition of culture of step (2) is: 22～25 ℃ of temperature, illuminance 300～1000 luxs, under light application time 11～13h/d condition, cultivate 40～50d.
6. the method for the green cloud tea tree of efficient Fast-propagation according to claim 1 test-tube plantlet, is characterized in that, the described solid culture medium of step (2) consists of: methyl α-naphthyl acetate 0.5mg/L, sucrose 30g/L, MS medium, agar 6g/L, pH 5.8.
7. the method for the green cloud tea tree of breeding according to claim 1 test-tube plantlet, is characterized in that, the condition of culture of step (3) is: 25～28 ℃ of temperature, and illuminance 1000～2000 luxs, light application time 11～13h/d, cultivates 45 days.
8. the method for the green cloud tea tree of efficient Fast-propagation according to claim 1 test-tube plantlet, it is characterized in that, the described shoot proliferation medium of step (3) consists of: methyl α-naphthyl acetate 0.8mg/L, 6-benzyl aminopurine 2.5mg/L, sucrose 30g/L, MS medium, agar 6g/L, pH 5.8.
9. the method for the green cloud tea tree of breeding according to claim 1 test-tube plantlet, is characterized in that, the condition of culture of step (4) is: 25～28 ℃ of temperature, and illuminance 1000～2000 luxs, light application time 11～13h/d, cultivates 50 days.
10. the method for the green cloud tea tree of efficient Fast-propagation according to claim 1 test-tube plantlet, it is characterized in that, in step (4), described strengthening seedling and rooting medium consists of: methyl α-naphthyl acetate 0.2mg/L, sucrose 30g/L, 1/3MS medium, agar 6g/L, pH 5.8.
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| CN109618926A (en) * | 2018-11-28 | 2019-04-16 | 西南林业大学 | A method of passing through test tube seedling continuous production tealeaves |
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| CN109618926A (en) * | 2018-11-28 | 2019-04-16 | 西南林业大学 | A method of passing through test tube seedling continuous production tealeaves |
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Application publication date: 20141210 |