CN101940161B - Method for inducing cluster buds of guoziman conifera - Google Patents
Method for inducing cluster buds of guoziman conifera Download PDFInfo
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Abstract
The invention provides a method for inducing cluster buds of guoziman conifera. Robust lateral buds of the guoziman conifera are explants; complete plantlets are formed by selecting the explants, sterilizing budlets, inducing and culturing the cluster buds, performing bud proliferation and subculture and performing root culture on buds; and the complete plantlets are acclimatized and transplanted in a seedling raising medium to grow normal guoziman conifera plants. The method for inducing the cluster buds of the guoziman conifera has the advantages that: the bud differentiation rate is 80.5 percent, each bud can be differentiated into 5.2 buds, the propagation coefficient of 4.0, bud is robust, the rooting rate is 83.0 percent and the transplantation survival rate is 90 percent. The method for inducing the cluster buds of the guoziman conifera realizes preservation of individual plants of the guoziman conifera, solves the problems of low explant survival rate, long seedling raising period, low propagation coefficient and poor planting consistency of the conventional breeding method and can be used for large-scale industrial production of seedlings to meet the requirements of the market on the guoziman conifera.
Description
Technical field
The present invention relates to a kind of grow thickly method of bud of torch pineapples of inducing, specifically is that the lateral bud with torch pineapples is an explant, on medium, cultivates, and obtains the method for a large amount of torch pineapples plant.
Background technology
Torch pineapples (Guoziman conifera) is the climing platymiscium of pineapple family fruit, originates in the torrid zone, the subtropical zone of Central and South America.Torch pineapples is the perennial evergreen ornamental foliage plant, plant height 30～50cm, and blade is wide linear, and leaf coloured light is bright, and quality is abundant, and leaf margin is stingless, and blade clusters on the cripetura stem, forms the leaf cup, in order to water storage and absorption nutrient.Leafage central authorities extract upright scape out, and style stands on the width of cloth and penetrates in well-balanced, the green sword-like leave clump, not branch; The spherical style top that is positioned at of inflorescence, the reddish yellow bract is arranged graceful, both like the and for example noble power rod of torch; Strain shape imposing manner is out of the ordinary; Whole strain attitude is graceful, lucuriant in design, and the florescence reaches 2～3 months, is that epochmaking indoor decorative flower and garden beautify plant.Torch pineapples adopts plant division or seed propagation, but the division propagation coefficient is low, and offspring's proterties of sowing is inconsistent.Main at present is explant with the terminal bud of virus-free maternal plant and the stem apex of lateral bud, and evoked callus and indefinite bud are bred in a large number.But adopt terminal bud and the lateral bud breeding of maternal plant, the explant limited amount, it is individual to be prone to the injury maternal plant; And explant sterilization success rate is extremely low, survival rate is low, and stem apex breeding start-up period is extremely long, and this has directly caused the torch pineapples growing-seedling period oversize; Reproduction coefficient is low; The market price is higher, is difficult to meet the need of market, and therefore presses for the method for quick torch pineapples in a large number that works out.
Tissue culture is an important channel of breeding the good plant kind fast; Existing some kind of the plant of the climing genus of pineapple family fruit has been carried out the relevant report of tissue culture; But the overwhelming majority is to utilize seed or callus approach to breed; Utilize bigger lateral bud as explant, breed the pineapple seedling in a large number through the bud approach of growing thickly and but do not see relevant report.The present invention utilizes lateral bud through the quick propagating torch pineapples of bud generation approach of growing thickly, and has not only overcome the problem that the stem apex sterilization is difficult, survival rate is low, startup is slow, and owing to without the callus stage, has simplified the flow process of obtaining of torch pineapples tissue cultivating seedling greatly.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, a kind of grow thickly method of bud of torch pineapples of inducing is provided.
Induce the grow thickly method of bud of torch pineapples to comprise the steps:
1) selection of explant: cut the long healthy and strong lateral bud of 10～16cm that torch pineapples maternal plant base portion grows; Water is rinsed well, peels off outer Lao Ye layer by layer, cutting side bastem portion lignified tissue; Healthy and strong lateral bud is shelled into the long budlet of 1.5～3.0cm; Continue water and rinse well, as explant, subsequent use;
2) budlet sterilization: on the sterile working platform that budlet is first with 70% alcohol disinfecting, 20～30s, aseptic water washing 3～5 times, with 0.1% mercuric chloride sterilization, 8～10min, aseptic water washing 3～5 times blots the budlet surface moisture with blotting paper, and is subsequent use again;
3) inducing clumping bud is cultivated: divest the budlet outermost layer earlier because of contact 2 injured blades with thimerosal; Again budlet is vertically inserted in the inducing clumping bud culture medium; Induce and form the bud of growing thickly; Per 30～40d subculture switching 1 time; Transfer 5 times, cultivate 150～200d altogether, cultivation temperature is 25～28 ℃; Light application time is 8～12h/d, and intensity of illumination is 1000～1500lux;
4) the bud proliferation and subculture is cultivated: will induce the bud of growing thickly of formation to downcut in the step 3); Per 2～3 buds are one clump; Be inoculated in the bud proliferation and subculture culture medium, carry out proliferation and subculture and cultivate, form the bud of growing thickly; Per 30～40d subculture switching 1 time; Transfer 2 times, cultivate 60～80d altogether, cultivation temperature is 25～28 ℃; Light application time is 10～14h/d, and intensity of illumination is 2000～2500lux;
5) the blastogenesis root is cultivated: step 4) is grown thickly, and the long bud of 2.5～3.0cm downcuts in the bud; Be inoculated in the blastogenesis root medium and carry out culture of rootage; Obtain complete seedling, incubation time is 40～50d, and cultivation temperature is 25～28 ℃; Light application time is 10～14h/d, and intensity of illumination is 2000～2500lux;
6) domestication of plant and transplanting: the complete seedling that obtains in the step 5) is connected bottle place the refining seedling of remaining silent under the natural lighting; Take out behind 10～15d, clean the medium that adheres to, place 1000 times of carbendazim solutions to soak 20～30s; Transplant then in volume ratio is 5: 1 peat soil and vermiculite mixed-matrix; Spraying and moisturizing 80%～90% shades 75%～85%, is incubated 20～25 ℃ of cultivations.
Described torch pineapples is the perennial evergreen herbaceous plant Guoziman conifera of the climing genus of pineapple family fruit.
Inducing clumping bud medium in the described step 3) is 1/2MS+6-BA 1.5～2.5mg/l+Adenine 1.0mg/l+NAA 0.2～0.5mg/l; Wherein: MS is conventional minimal medium Murashige&Skoog 1962; Macroelement concentration in this routine minimal medium is reduced to 1/2MS; 6-BA is a 6-benzyladenine, and NAA is a methyl.
Bud proliferation and subculture medium in the described step 4) is MS+6-BA 0.8～1.2mg/l+NAA0.1～0.3mg/l, and wherein: MS is conventional minimal medium Murashige&Skoog 1962, and 6-BA is a 6-benzyladenine, and NAA is a methyl.
Blastogenesis root medium in the described step 5) is 1/3MS+NAA 0.1～0.4mg/l+ active carbon 1000mg/l; Wherein: MS is conventional minimal medium Murashige&Skoog 1962; Macroelement concentration in this routine minimal medium is reduced to 1/3MS, and NAA is a methyl.
All add the agar of 5.0～7.0g/l and the sucrose of 20～30g/l in described inducing clumping bud medium, bud proliferation and subculture medium, the blastogenesis root medium.
The pH value of described inducing clumping bud medium, bud proliferation and subculture medium and blastogenesis root medium is 5.6～5.8.
The present invention compared with prior art, the beneficial effect that has: the method with the quick propagating torch pineapples of lateral bud has been set up in (1), compares traditional division propagation method, has greatly improved reproduction coefficient, for research and production cultivations provides technical support; (2) compare traditional planting seed propagation method, breed the seedling that obtains fast through cultured in vitro, it is strong to grow, and the leaf look dark green, and uniformity is strong, becomes seedling easy, and adaptability is strong, and damage by disease and insect is few, is easy to management; (3) without the callus stage, directly induce to form the bud of growing thickly, simplified the program of obtaining of tissue cultivating seedling greatly; (4) utilize the quick propagating torch pineapples of cultured in vitro, removed the restriction of season and weather, thereby shortened growing-seedling period, increased breeding amount; (5) very important meaning is arranged aspect the good mutant or the good first generation of hybrid preserving and breed, but a strain has the torch pineapples mutant of clear superiority or offspring's large tracts of land after tissue culture of a tool clear superiority is promoted.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
Described in the present invention torch pineapples is the perennial evergreen herbaceous plant Guoziman conifera of the climing genus of pineapple family fruit.
Described in the present invention MS is conventional minimal medium Murashige&Skoog 1962, and 6-BA is a 6-benzyladenine, and NAA is a methyl.
Embodiment 1
(1) selection of explant: cut the long healthy and strong lateral bud of 10cm that torch pineapples maternal plant base portion grows, water is rinsed well, peels off outer Lao Ye layer by layer; Cutting side bastem portion lignified tissue is shelled into the long budlet of 1.5cm with healthy and strong lateral bud, continues water and rinses well; As explant, subsequent use;
(2) budlet sterilization: on the sterile working platform that budlet is first with 70% alcohol disinfecting 20s, aseptic water washing 3 times, with 0.1% mercuric chloride sterilization 8min, aseptic water washing 3 times blots the budlet surface moisture with blotting paper, and is subsequent use again;
(3) inducing clumping bud is cultivated: divest the budlet outermost layer because of contact 2 injured blades with thimerosal, budlet is vertically inserted in the inducing clumping bud medium, this medium is: 1/2MS+6-BA 1.5mg/l+Adenine 1.0mg/l+NAA 0.2mg/l; The agar that adds 5.0g/l in the medium, the sucrose of 20g/l, the pH value is 5.6; Induce to form the bud of growing thickly, every 30d subculture switching 1 time is transferred 5 times; Cultivate 150d altogether; Cultivation temperature is 25 ℃, and light application time is 8h/d, and intensity of illumination is 1000lux.Budlet becomes bottle green by light green after cultivating about 25d, cultivates 65d budlet base portion and begins to expand, and induces the little gemmule that produces white, continues successive transfer culture 85d, and bud is stretched to about 1.0cm, and bud induction rate is 35.6%, and each budlet induced bud number is 2.6.
(4) the bud proliferation and subculture is cultivated: the bud of growing thickly of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, are inoculated in the bud proliferation and subculture medium, and this medium is: MS+6-BA 0.8mg/l+NAA 0.1mg/l; The agar that adds 5.0g/l in the medium, the sucrose of 20g/l, the pH value is 5.6, proliferation and subculture forms the bud of growing thickly; Every 30d subculture switching 1 time is transferred 2 times, cultivates 60d altogether; Cultivation temperature is 25 ℃, and light application time is 10h/d, and intensity of illumination is 2000lux.Proliferation and subculture is cultivated 24d, and the bastem portion of growing thickly begins to sprout the sprouting point, and proliferation and subculture is cultivated 38d, and the sprouting blade begins to stretch grows, and the adventitious buds proliferation coefficient reaches 1.8 times, and the bud height is 2.5cm, and bud seedling robust growth, mounted blade are better.
(5) the blastogenesis root is cultivated: step 4) is grown thickly, and the long bud of 2.5～3.0cm downcuts in the bud, is inoculated in the blastogenesis root medium and carries out culture of rootage, and this medium is: 1/3MS+NAA 0.1mg/l+ active carbon 1000mg/l; The agar that adds 5.0g/l in the medium, the sucrose of 20g/l, the pH value is 5.6; Obtain complete seedling, incubation time is 40d, and cultivation temperature is 25 ℃; Light application time is 10h/d, and intensity of illumination is 2000lux.Bastem portion begins to occur white root original hase projection behind the about 25d of culture of rootage, and grows new root gradually, and rooting rate is 42.8%, and the number of on average taking root is 2.0, and average root is long to be 1.6cm.
(6) domestication of plant and transplanting: the complete seedling that obtains in the step 5) is connected bottle place the refining seedling of remaining silent under the natural lighting, take out behind the 10d, clean the medium that adheres to; Place 1000 times of carbendazim solutions to soak 20s; Transplant then in volume ratio is 5: 1 peat soil and vermiculite mixed-matrix, spraying and moisturizing 80% shades 75%; Be incubated 20 ℃ of cultivations, survival rate reaches 80%.
Embodiment 2
(1) selection of explant: cut the long healthy and strong lateral bud of 12cm that torch pineapples maternal plant base portion grows, water is rinsed well, peels off outer Lao Ye layer by layer; Cutting side bastem portion lignified tissue is shelled into the long budlet of 1.8cm with healthy and strong lateral bud, continues water and rinses well; As explant, subsequent use;
(2) budlet sterilization: on the sterile working platform that budlet is first with 70% alcohol disinfecting 23s, aseptic water washing 4 times, with 0.1% mercuric chloride sterilization 8min, aseptic water washing 3 times blots the budlet surface moisture with blotting paper, and is subsequent use again;
(3) inducing clumping bud is cultivated: divest the budlet outermost layer because of contact 2 injured blades with thimerosal, budlet is vertically inserted in the inducing clumping bud medium, this medium is: 1/2MS+6-BA 1.8mg/l+Adenine 1.0mg/l+NAA 0.25mg/l; The agar that adds 5.5g/l in the medium, the sucrose of 23g/l, the pH value is 5.6; Induce to form the bud of growing thickly, every 32d subculture switching 1 time is transferred 5 times; Cultivate 160d altogether; Cultivation temperature is 26 ℃, and light application time is 9h/d, and intensity of illumination is 1200lux.Budlet becomes bottle green by light green after cultivating about 22d, cultivates the 50d blade base and begins to expand, and induces the little gemmule that produces white, continues to cultivate 110d, and bud is stretched to about 1.3cm, and bud induction rate is 42.5%, and each budlet induced bud number is 3.8.
(4) the bud proliferation and subculture is cultivated: the bud of growing thickly of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, are inoculated in the bud proliferation and subculture medium, and this medium is: MS+6-BA 0.9mg/l+NAA 0.15mg/l; The agar that adds 5.5g/l in the medium, the sucrose of 23g/l, the pH value is 5.6, proliferation and subculture forms the bud of growing thickly; Every 32d subculture switching 1 time is transferred 2 times, cultivates 64d altogether; Cultivation temperature is 26 ℃, and light application time is 11h/d, and intensity of illumination is 2100lux.Proliferation and subculture is cultivated 27d, and the bastem portion of growing thickly begins to sprout the sprouting point, and proliferation and subculture is cultivated 40d, and the sprouting blade begins to stretch grows, and the adventitious buds proliferation coefficient reaches 2.1 times, and the bud height is 2.3cm, and bud seedling robust growth, mounted blade are better.
(5) the blastogenesis root is cultivated: step 4) is grown thickly, and the long bud of 2.5～3.0cm downcuts in the bud, is inoculated in the blastogenesis root medium and carries out culture of rootage, and this medium is: 1/3MS+NAA 0.2mg/l+ active carbon 1000mg/l; The agar that adds 5.5g/l in the medium, the sucrose of 23g/l, the pH value is 5.6; Obtain complete seedling, incubation time is 42d, and cultivation temperature is 26 ℃; Light application time is 12h/d, and intensity of illumination is 2100lux.Bastem portion begins to occur white root original hase projection behind the about 28d of culture of rootage, and grows new root gradually, and rooting rate is 68.2%, and the number of on average taking root is 2.0, and average root is long to be 1.7cm.
(6) domestication of plant and transplanting: the complete seedling that obtains in the step 5) is connected bottle place the refining seedling of remaining silent under the natural lighting, take out behind the 12d, clean the medium that adheres to; Place 1000 times of carbendazim solutions to soak 23s; Transplant then in volume ratio is 5: 1 peat soil and vermiculite mixed-matrix, spraying and moisturizing 84% shades 78%; Be incubated 22 ℃ of cultivations, survival rate reaches 83%.
Embodiment 3
(1) selection of explant: cut the long healthy and strong lateral bud of 14cm that torch pineapples maternal plant base portion grows, water is rinsed well, peels off outer Lao Ye layer by layer; Cutting side bastem portion lignified tissue is shelled into the long budlet of 2.4cm with healthy and strong lateral bud, continues water and rinses well; As explant, subsequent use;
(2) budlet sterilization: on the sterile working platform that budlet is first with 70% alcohol disinfecting 25s, aseptic water washing 4 times, with 0.1% mercuric chloride sterilization 9min, aseptic water washing 4 times blots the budlet surface moisture with blotting paper, and is subsequent use again;
(3) inducing clumping bud is cultivated: divest the budlet outermost layer because of contact 2 injured blades with thimerosal, budlet is vertically inserted in the inducing clumping bud medium, this medium is: 1/2MS+6-BA 2.0mg/l+Adenine 1.0mg/l+NAA 0.3mg/l; The agar that adds 6.0g/l in the medium, the sucrose of 25g/l, the pH value is 5.7; Induce to form the bud of growing thickly, every 35d subculture switching 1 time is transferred 5 times; Cultivate 175d altogether; Cultivation temperature is 26 ℃, and light application time is 10h/d, and intensity of illumination is 1200lux.Budlet becomes bottle green by light green after cultivating about 20d, cultivates 58d budlet base portion and begins to expand, and induces the little gemmule that produces white, continues to cultivate 117d, and bud is stretched to about 1.7cm, and bud induction rate is 80.5%, and each budlet induced bud number is 5.2.
(4) the bud proliferation and subculture is cultivated: the bud of growing thickly of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, are inoculated in the bud proliferation and subculture medium, and this medium is: MS+6-BA 1.0mg/l+NAA 0.2mg/l; The agar that adds 6.0g/l in the medium, the sucrose of 25g/l, the pH value is 5.7, proliferation and subculture forms the bud of growing thickly; Every 35d subculture switching 1 time is transferred 2 times, cultivates 70d altogether; Cultivation temperature is 26 ℃, and light application time is 12h/d, and intensity of illumination is 2300lux.Proliferation and subculture is cultivated 22d, and the bastem portion of growing thickly begins to sprout the sprouting point, and proliferation and subculture is cultivated 35d, and the sprouting blade begins to stretch grows, and the adventitious buds proliferation coefficient reaches 4.0 times, and the bud height is 2.7cm, and bud seedling robust growth, mounted blade are better.
(5) the blastogenesis root is cultivated: step 4) is grown thickly, and the long bud of 2.5～3.0cm downcuts in the bud, is inoculated in the blastogenesis root medium and carries out culture of rootage, and this medium is: 1/3MS+NAA 0.25mg/l+ active carbon 1000mg/l; The agar that adds 6.0g/l in the medium, the sucrose of 25g/l, the pH value is 5.7; Obtain complete seedling, incubation time is 45d, and cultivation temperature is 27 ℃; Light application time is 12h/d, and intensity of illumination is 2300lux.Bastem portion begins to occur white root original hase projection behind the about 20d of culture of rootage, and grows new root gradually, and rooting rate is 83.0%, and the number of on average taking root is 2.5, and average root is long to be 2.0cm.
(6) domestication of plant and transplanting: the complete seedling that obtains in the step 5) is connected bottle place the refining seedling of remaining silent under the natural lighting, take out behind the 13d, clean the medium that adheres to; Place 1000 times of carbendazim solutions to soak 25s; Transplant then in volume ratio is 5: 1 peat soil and vermiculite mixed-matrix, spraying and moisturizing 85% shades 80%; Be incubated 23 ℃ of cultivations, survival rate reaches 90%.
Embodiment 4
(1) selection of explant: cut the long healthy and strong lateral bud of 15cm that torch pineapples maternal plant base portion grows, water is rinsed well, peels off outer Lao Ye layer by layer; Cutting side bastem portion lignified tissue is shelled into the long budlet of 2.5cm with healthy and strong lateral bud, continues water and rinses well; As explant, subsequent use;
(2) budlet sterilization: on the sterile working platform that budlet is first with 70% alcohol disinfecting 27s, aseptic water washing 4 times, with 0.1% mercuric chloride sterilization 9min, aseptic water washing 4 times blots the budlet surface moisture with blotting paper, and is subsequent use again;
(3) inducing clumping bud is cultivated: divest the budlet outermost layer because of contact 2 injured blades with thimerosal, budlet is vertically inserted in the inducing clumping bud medium, this medium is: 1/2MS+6-BA 2.3mg/l+Adenine 1.0mg/l+NAA 0.4mg/l; The agar that adds 6.5g/l in the medium, the sucrose of 28g/l, the pH value is 5.6; Induce to form the bud of growing thickly, every 38d subculture switching 1 time is transferred 5 times; Cultivate 190d altogether; Cultivation temperature is 27 ℃, and light application time is 11h/d, and intensity of illumination is 1400lux.Budlet becomes bottle green by light green after cultivating about 30d, cultivates 65d budlet base portion and begins to expand, and induces the little gemmule that produces white, continues to cultivate 125d, and bud is stretched to about 1.4cm, and bud induction rate is 68.0%, and each budlet induced bud number is 3.8.
(4) the bud proliferation and subculture is cultivated: the bud of growing thickly of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, are inoculated in the bud proliferation and subculture medium, and this medium is: MS+6-BA 1.1mg/l+NAA 0.25mg/l; The agar that adds 6.5g/l in the medium, the sucrose of 28g/l, the pH value is 5.6, proliferation and subculture forms the bud of growing thickly; Every 38d subculture switching 1 time is transferred 2 times, cultivates 76d altogether; Cultivation temperature is 27 ℃, and light application time is 13h/d, and intensity of illumination is 2400lux.Proliferation and subculture is cultivated 28d, and the bastem portion of growing thickly begins to sprout the sprouting point, and proliferation and subculture is cultivated 42d, and the sprouting blade begins to stretch grows, and the adventitious buds proliferation coefficient reaches 3.0 times, and the bud height is 2.5cm, and bud seedling robust growth, mounted blade are better.
(5) the blastogenesis root is cultivated: step 4) is grown thickly, and the long bud of 2.5～3.0cm downcuts in the bud, is inoculated in the blastogenesis root medium and carries out culture of rootage, and this medium is: 1/3MS+NAA 0.3mg/l+ active carbon 1000mg/l; The agar that adds 6.5g/l in the medium, the sucrose of 28g/l, the pH value is 5.6; Obtain complete seedling, incubation time is 48d, and cultivation temperature is 24 ℃; Light application time is 13h/d, and intensity of illumination is 2400lux.Bastem portion begins to occur white root original hase projection behind the about 22d of culture of rootage, and grows new root gradually, and rooting rate is 70.0%, and the number of on average taking root is 2.5, and average root is long to be 1.8cm.
(6) domestication of plant and transplanting: the complete seedling that obtains in the step 5) is connected bottle place the refining seedling of remaining silent under the natural lighting, take out behind the 13d, clean the medium that adheres to; Place 1000 times of carbendazim solutions to soak 28s; Transplant then in volume ratio is 5: 1 peat soil and vermiculite mixed-matrix, spraying and moisturizing 87% shades 82%; Be incubated 24 ℃ of cultivations, survival rate reaches 87%.
Embodiment 5
(1) selection of explant: cut the long healthy and strong lateral bud of 16cm that torch pineapples maternal plant base portion grows, water is rinsed well, peels off outer Lao Ye layer by layer; Cutting side bastem portion lignified tissue is shelled into the long budlet of 3.0cm with healthy and strong lateral bud, continues water and rinses well; As explant, subsequent use;
(2) budlet sterilization: on the sterile working platform that budlet is first with 70% alcohol disinfecting 30s, aseptic water washing 5 times, with 0.1% mercuric chloride sterilization 10min, aseptic water washing 5 times blots the budlet surface moisture with blotting paper, and is subsequent use again;
(3) inducing clumping bud is cultivated: divest the budlet outermost layer because of contact 2 injured blades with thimerosal, budlet is vertically inserted in the inducing clumping bud medium, this medium is: 1/2MS+6-BA 2.5mg/l+Adenine 1.0mg/l+NAA 0.5mg/l; The agar that adds 7.0g/l in the medium, the sucrose of 30g/l, the pH value is 5.8; Induce to form the bud of growing thickly, every 40d subculture switching 1 time is transferred 5 times; Cultivate 200d altogether; Cultivation temperature is 28 ℃, and light application time is 12h/d, and intensity of illumination is 1500lux.Budlet becomes bottle green by light green after cultivating about 24d, cultivates 70d budlet base portion and begins to expand, and induces the little gemmule that produces white, continues to cultivate 130d, and bud is stretched to about 1.5cm, and bud induction rate is 55.2%, and each budlet induced bud number is 3.6.
(4) the bud proliferation and subculture is cultivated: the bud of growing thickly of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, are inoculated in the bud proliferation and subculture medium, and this medium is: MS+6-BA 1.2mg/l+NAA 0.3mg/l; The agar that adds 7.0g/l in the medium, the sucrose of 30g/l, the pH value is 5.8, proliferation and subculture forms the bud of growing thickly; Every 40d subculture switching 1 time is transferred 2 times, cultivates 80d altogether; Cultivation temperature is 28 ℃, and light application time is 14h/d, and intensity of illumination is 2500lux.Proliferation and subculture is cultivated 34d, and the bastem portion of growing thickly begins to sprout the sprouting point, and proliferation and subculture is cultivated 50d, and the sprouting blade begins to stretch grows, and the adventitious buds proliferation coefficient reaches 2.6 times, and the bud height is 2.7cm, and bud seedling robust growth, mounted blade are better.
(5) the blastogenesis root is cultivated: step 4) is grown thickly, and the long bud of 2.5～3.0cm downcuts in the bud, is inoculated in the blastogenesis root medium and carries out culture of rootage, and this medium is: 1/3MS+NAA 0.4mg/l+ active carbon 1000mg/l; The agar that adds 7.0g/l in the medium, the sucrose of 30g/l, the pH value is 5.8; Obtain complete seedling, incubation time is 50d, and cultivation temperature is 28 ℃; Light application time is 14h/d, and intensity of illumination is 2500lux.Bastem portion begins to occur white root original hase projection behind the about 25d of culture of rootage, and grows new root gradually, and rooting rate is 68.0%, and the number of on average taking root is 2.0, and average root is long to be 1.6cm.
(6) domestication of plant and transplanting: the complete seedling that obtains in the step 5) is connected bottle place the refining seedling of remaining silent under the natural lighting, take out behind the 15d, clean the medium that adheres to; Place 1000 times of carbendazim solutions to soak 30s; Transplant then in volume ratio is 5: 1 peat soil and vermiculite mixed-matrix, spraying and moisturizing 90% shades 85%; Be incubated 25 ℃ of cultivations, survival rate reaches 85%.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do and improvement, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. induce the grow thickly method of bud of torch pineapples for one kind, it is characterized in that comprising the steps:
1) selection of explant: cut the long healthy and strong lateral bud of 10～16cm that torch pineapples maternal plant base portion grows; Water is rinsed well, peels off outer Lao Ye layer by layer, cutting side bastem portion lignified tissue; Healthy and strong lateral bud is shelled into the long budlet of 1.5～3.0cm; Continue water and rinse well, as explant, subsequent use;
2) budlet sterilization: on the sterile working platform that budlet is first with 70% alcohol disinfecting, 20～30s, aseptic water washing 3～5 times, with 0.1% mercuric chloride sterilization, 8～10min, aseptic water washing 3～5 times blots the budlet surface moisture with blotting paper, and is subsequent use again;
3) inducing clumping bud is cultivated: divest the budlet outermost layer earlier because of contact 2 injured blades with thimerosal; Again budlet is vertically inserted in the inducing clumping bud culture medium; Induce and form the bud of growing thickly; Per 30～40d subculture switching 1 time; Transfer 5 times, cultivate 150～200d altogether, cultivation temperature is 25～28 ℃; Light application time is 8～12h/d, and intensity of illumination is 1000～1500lux;
4) the bud proliferation and subculture is cultivated: will induce the bud of growing thickly of formation to downcut in the step 3); Per 2～3 buds are one clump; Be inoculated in the bud proliferation and subculture culture medium, carry out proliferation and subculture and cultivate, form the bud of growing thickly; Per 30～40d subculture switching 1 time; Transfer 2 times, cultivate 60～80d altogether, cultivation temperature is 25～28 ℃; Light application time is 10～14h/d, and intensity of illumination is 2000～2500lux;
5) the blastogenesis root is cultivated: step 4) is grown thickly, and the long bud of 2.5～3.0cm downcuts in the bud; Be inoculated in the blastogenesis root medium and carry out culture of rootage; Obtain complete seedling, incubation time is 40～50d, and cultivation temperature is 25～28 ℃; Light application time is 10～14h/d, and intensity of illumination is 2000～2500lux;
6) domestication of plant and transplanting: the complete seedling that obtains in the step 5) is connected bottle place the refining seedling of remaining silent under the natural lighting; Take out behind 10～15d; Clean the culture medium that adheres to; Place 1000 times of carbendazim solutions to soak 20～30s; Transplant then in volume ratio is 5: 1 peat soil and vermiculite mixed-matrix; Spraying and moisturizing 80%～90% shades 75%～85%, is incubated 20～25 ℃ of cultivations;
Inducing clumping bud medium in the described step 3) is 1/2MS+6-BA 1.5～2.5mg/l+ adenine 1.0mg/l+NAA 0.2～0.5mg/l; Wherein: MS is conventional minimal medium Murashige&Skoog1962; Macroelement concentration in this routine minimal medium is reduced to 1/2MS; 6-BA is a 6-benzyladenine, and NAA is a methyl; Bud proliferation and subculture medium in the described step 4) is MS+6-BA 0.8～1.2mg/l+NAA 0.1～0.3mg/l, and wherein: MS is conventional minimal medium Murashige&Skoog1962, and 6-BA is a 6-benzyladenine, and NAA is a methyl; Blastogenesis root medium in the described step 5) is 1/3MS+NAA 0.1～0.4mg/l+ active carbon 1000mg/l; Wherein: MS is conventional minimal medium Murashige&Skoog 1962; Macroelement concentration in this routine minimal medium is reduced to 1/3MS, and NAA is a methyl.
2. a kind of grow thickly method of bud of torch pineapples of inducing according to claim 1, it is characterized in that: described torch pineapples is the perennial evergreen herbaceous plant Guoziman conifera of the climing genus of pineapple family fruit.
3. a kind of grow thickly method of bud of torch pineapples of inducing according to claim 1 is characterized in that: all add the agar of 5.0～7.0g/l and the sucrose of 20～30g/l in described inducing clumping bud medium, bud proliferation and subculture medium, the blastogenesis root medium.
4. a kind of grow thickly method of bud of torch pineapples of inducing according to claim 1, it is characterized in that: the pH value of described inducing clumping bud medium, bud proliferation and subculture medium and blastogenesis root medium is 5.6～5.8.
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| CN103477886A (en) * | 2013-08-22 | 2014-01-01 | 江苏农林职业技术学院 | Method for testing influence of different 6-BA concentration on development of air plant tiller bud |
| CN103493736B (en) * | 2013-09-29 | 2015-10-14 | 江苏农林职业技术学院 | The method of scale Fast-propagation Tillandsia-Si Chuikete test-tube plantlet |
| CN107251838A (en) * | 2017-07-13 | 2017-10-17 | 江苏省农业科学院 | A kind of birch-leaf pear tissue-cultured seedling root media |
| CN111937744A (en) * | 2019-05-14 | 2020-11-17 | 福建宝旺莱生态农业科技有限公司 | Tissue culture method for pineapple flowers |
| CN113796297B (en) * | 2020-07-30 | 2022-06-07 | 佛山市连艺生物科技有限公司 | Cultivation method for improving water culture quality of curcuma longa |
| CN113016618A (en) * | 2021-04-14 | 2021-06-25 | 苏州回文生物种业科技有限公司 | Tissue culture and rapid propagation method of Oringales and songbians |
| CN115039590B (en) * | 2022-07-15 | 2023-08-01 | 广西南亚热带农业科学研究所 | Pineapple in-vitro tissue induction culture method |
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