CN101803570B - Method for efficiently propagating torch pineapples by utilizing in-vitro leaves - Google Patents
Method for efficiently propagating torch pineapples by utilizing in-vitro leaves Download PDFInfo
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Abstract
The invention provides a method for efficiently propagating torch pineapples by utilizing in-vitro leaves, which comprises the following steps: using inner layer young leaves of strong side lateral buds of the torch pineapples as explants; carrying out explant selection, leaf disinfection, leaf inducement culture, adventitious bud multiplication successive transfer culture and adventitious bud rooting culture for forming complete plant seedlings; and domesticating and transplanting the complete plant seedlings into a seedling culture substrate for growing the plant seedlings into normal torch pineapple plants. The method for efficiently propagating the torch pineapples by utilizing the in-vitro leaves of the invention has the bud differentiation rate of 74.7 percent, each leaf has the differentiation bud number of 5.2, the multiplication coefficient is 3.6, in addition, the buds grow strongly, the rooting rate reaches 85.5 percent, and the transplanting survival rate reaches 95 percent. The method for efficiently propagating the torch pineapples by utilizing the in-vitro leaves of the invention realizes the storage on the single strains of the torch pineapples, overcomes the defects of overlong seedling culture period, low propagation coefficient and poor seedling consistency of a traditional propagating method, and can realize the factory large-scale production of the seedling for adapting to the requirements of the market on the torch pineapples.
Description
Technical field
The present invention relates to a kind of method of utilizing the excised leaf efficiently propagating torch pineapples, specifically is that tender of internal layer with the healthy and strong lateral bud of torch pineapples is an explant, on medium, cultivates, and obtains the method for torch pineapples plant fast.
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, and main at present is explant with the stem apex of virus-free maternal plant or the stem apex of lateral bud, and evoked callus and indefinite bud are bred in a large number.But adopt the breeding of maternal plant stem apex, the explant limited amount, and it is individual to be prone to the injury maternal plant; Adopt the breeding of lateral bud stem apex, lateral bud sterilization success rate is extremely low, and the bud start-up period reaches 90～150d; This has caused the torch pineapples growing-seedling period oversize, and reproduction coefficient is low, and the market price is higher; Be difficult to meet the need of market, therefore press 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 stem apex to breed, and utilizes the young leaflet tablet of lateral bud to carry out tissue culture and does not but see relevant report.The present invention utilizes the quick propagating torch pineapples of young leaflet tablet, directly obtains indefinite bud through organogenetic means, has not only overcome the difficult problem of stem apex sterilization, 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 method of utilizing the excised leaf efficiently propagating torch pineapples is provided.
A kind of method of excised leaf efficiently propagating torch pineapples of utilizing comprises 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 5～7cm; Continue water and rinse well, use scalpel to peel the long tender leaf of budlet internal layer 3～5cm, as explant;
2) blade sterilization: on the sterile working platform, earlier with 70% alcohol disinfecting, 8～12s, with 0.1% mercuric chloride sterilization, 6～8min, aseptic water washing 3～5 times blots blade surface moisture with blotting paper, and is subsequent use again with tender leaf;
3) blade inducing culture: the tender leaf after will sterilizing vertically inserts in the blade inducing culture, the blade tip of tender leaf up, leaf base is down; Induce the formation indefinite bud, every 30d subculture switching 1 time is transferred 3 times; Cultivate 90d altogether; Cultivation temperature is 20～25 ℃, and light application time is 8～12h/d, and intensity of illumination is 1000～1500lux;
4) adventitious bud proliferation successive transfer culture: the indefinite bud of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, are inoculated in the adventitious bud proliferation subculture medium; Every 25d subculture switching 1 time; Transfer 2 times, cultivate 50d altogether, cultivation temperature is 20～25 ℃; Light application time is 10～14h/d, and intensity of illumination is 2000～2500lux;
5) adventitious bud rooting is cultivated: the indefinite bud that 2.5～3.0cm in the step 4) is long downcuts; Be inoculated into and carry out culture of rootage in the adventitious bud rooting medium, incubation time is 40d, and cultivation temperature is 20～25 ℃; 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.
The basic source plant of described torch pineapples is the perennial evergreen herbaceous plant Guoziman conifera of the climing genus of pineapple family fruit.
Blade inducing culture in the described step 3) is MS+6-BA 1.0～1.5mg/l+NAA 0.1～0.3mg/l, and wherein: MS is conventional minimal medium Murashige&Skoog 1962, and 6-BA is a 6-benzyladenine, and NAA is a methyl.
Adventitious bud proliferation subculture medium in the described step 4) is MS+6-BA 0.3～1.0mg/l+NAA0.02～0.1mg/l, and wherein: MS is conventional minimal medium Murashige&Skoog 1962, and 6-BA is a 6-benzyladenine, and NAA is a methyl.
Adventitious bud rooting 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 blade inducing culture, adventitious bud proliferation subculture medium, the root media.
The pH value of described blade inducing culture, adventitious bud proliferation subculture medium and root media 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 excised leaf 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, blade is directly induced the formation indefinite bud, has 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.
The basic source plant of described torch pineapples is the perennial evergreen herbaceous plant Guoziman conifera of the climing genus of pineapple family fruit in the present invention.
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; Healthy and strong lateral bud is shelled into the long budlet of 5cm; Continue water and rinse well, use scalpel to peel the long tender leaf of budlet internal layer 3cm, as explant;
(2) blade sterilization: on the sterile working platform, earlier with 70% alcohol disinfecting 8s, with 0.1% mercuric chloride sterilization 6min, aseptic water washing 3 times blots blade surface moisture with blotting paper, and is subsequent use again with tender leaf;
(3) blade inducing culture: the tender leaf after will sterilizing vertically inserts in the blade inducing culture, and this medium is: MS+6-BA 1.0mg/l+NAA 0.1mg/l, the agar of adding 5.0g/l in the medium, the sucrose of 20g/l; The pH value is 5.6, the blade tip of tender leaf up, leaf base is induced the formation indefinite bud down; Every 30d subculture switching 1 time is transferred 3 times, cultivates 90d altogether; Cultivation temperature is 20 ℃, and light application time is 8h/d, and intensity of illumination is 1000lux.Cultivate about 15d rear blade and become bottle green by light green, cultivate the 50d blade base and begin to expand, induce the little gemmule that produces white, continue to cultivate 28d, bud is stretched to about 1.0cm, and bud induction rate is 40.5%, and each blade induced bud number is 2.2.
(4) adventitious bud proliferation successive transfer culture: the indefinite bud of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, are inoculated in the adventitious bud proliferation subculture medium, and this medium is: MS+6-BA 0.3mg/l+NAA 0.02mg/l; The agar that adds 5.0g/l in the medium, the sucrose of 20g/l, the pH value is 5.6; Every 25d subculture switching 1 time is transferred 2 times, cultivates 50d altogether; Cultivation temperature is 20 ℃, and light application time is 10h/d, and intensity of illumination is 2000lux.Proliferation and subculture is cultivated 18d, and the bastem portion of growing thickly begins to sprout the sprouting point, and proliferation and subculture is cultivated 22d, and the sprouting blade begins to stretch grows, and the adventitious buds proliferation coefficient reaches 1.6 times, and the bud height is 2.1cm, and bud seedling robust growth, mounted blade are better.
(5) adventitious bud rooting is cultivated: the indefinite bud that 2.5～3.0cm in the step 4) is long downcuts, and is inoculated into and carries out culture of rootage in the adventitious bud rooting medium, 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; Incubation time is 40d; Cultivation temperature is 20 ℃, and 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 28d of culture of rootage, and grows new root gradually, and rooting rate is 52.6%, and the number of on average taking root is 1.8, and average root is long to be 1.5cm.
(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 85%.
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; Healthy and strong lateral bud is shelled into the long budlet of 5.5cm; Continue water and rinse well, use scalpel to peel the long tender leaf of budlet internal layer 3.5cm, as explant;
(2) blade sterilization: on the sterile working platform, earlier with 70% alcohol disinfecting 9s, with 0.1% mercuric chloride sterilization 7min, aseptic water washing 3 times blots blade surface moisture with blotting paper, and is subsequent use again with tender leaf;
(3) blade inducing culture: the tender leaf after will sterilizing vertically inserts in the blade inducing culture, and this medium is: MS+6-BA 1.2mg/l+NAA 0.15mg/l, the agar of adding 5.5g/l in the medium, the sucrose of 23g/l; The pH value is 5.6, the blade tip of tender leaf up, leaf base is induced the formation indefinite bud down; Every 30d subculture switching 1 time is transferred 3 times, cultivates 90d altogether; Cultivation temperature is 22 ℃, and light application time is 9h/d, and intensity of illumination is 1200lux.Cultivate about 12d rear blade and become bottle green by light green, cultivate the 46d blade base and begin to expand, induce the little gemmule that produces white, continue to cultivate 24d, bud is stretched to about 1.2cm, and bud induction rate is 48.3%, and each blade induced bud number is 3.0.
(4) adventitious bud proliferation successive transfer culture: the indefinite bud of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, be inoculated into the adventitious bud proliferation successive transfer culture basically in, this medium is: MS+6-BA 0.5mg/l+NAA 0.04mg/1; The agar that adds 5.5g/l in the medium, the sucrose of 23g/l, the pH value is 5.6; Every 25d subculture switching 1 time is transferred 2 times, cultivates 50d altogether; Cultivation temperature is 22 ℃, and light application time is 12h/d, and intensity of illumination is 2100lux.Proliferation and subculture is cultivated 15d, and the bastem portion of growing thickly begins to sprout the sprouting point, and proliferation and subculture is cultivated 20d, and the sprouting blade begins to stretch grows, and the adventitious buds proliferation coefficient reaches 2.2 times, and the bud height is 2.3cm, and bud seedling robust growth, mounted blade are better.
(5) adventitious bud rooting is cultivated: the indefinite bud that 2.5～3.0cm in the step 4) is long downcuts, and is inoculated into and carries out culture of rootage in the adventitious bud rooting medium, 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; Incubation time is 40d; Cultivation temperature is 22 ℃, and 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 25d 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 87%.
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; Healthy and strong lateral bud is shelled into the long budlet of 6cm; Continue water and rinse well, use scalpel to peel the long tender leaf of budlet internal layer 4cm, as explant;
(2) blade sterilization: on the sterile working platform, earlier with 70% alcohol disinfecting 10s, with 0.1% mercuric chloride sterilization 7min, aseptic water washing 4 times blots blade surface moisture with blotting paper, and is subsequent use again with tender leaf;
(3) blade inducing culture: the tender leaf after will sterilizing vertically inserts in the blade inducing culture, and this medium is: MS+6-BA 1.3mg/l+NAA 0.2mg/l, the agar of adding 6.0g/l in the medium, the sucrose of 25g/l; The pH value is 5.7, the blade tip of tender leaf up, leaf base is induced the formation indefinite bud down; Every 30d subculture switching 1 time is transferred 3 times, cultivates 90d altogether; Cultivation temperature is 23 ℃, and light application time is 10h/d, and intensity of illumination is 1200lux.Cultivate about 10d rear blade and become bottle green by light green, cultivate the 38d blade base and begin to expand, induce the little gemmule that produces white, continue to cultivate 20d, bud is stretched to about 1.4cm, and bud induction rate is 74.7%, and each blade induced bud number is 5.2.
(4) adventitious bud proliferation successive transfer culture: the indefinite bud of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, are inoculated in the adventitious bud proliferation subculture medium, and this medium is: MS+6-BA 0.6mg/l+NAA 0.05mg/l; The agar that adds 6.0g/l in the medium, the sucrose of 25g/l, the pH value is 5.7; Every 25d subculture switching 1 time is transferred 2 times, cultivates 50d altogether; Cultivation temperature is 23 ℃, and light application time is 12h/d, and intensity of illumination is 2300lux.Proliferation and subculture is cultivated 12d, and the bastem portion of growing thickly begins to sprout the sprouting point, and proliferation and subculture is cultivated 18d, and the sprouting blade begins to stretch grows, and the adventitious buds proliferation coefficient reaches 3.2 times, and the bud height is 2.8cm, and bud seedling robust growth, mounted blade are better.
(5) adventitious bud rooting is cultivated: the indefinite bud that 2.5～3.0cm in the step 4) is long downcuts, and is inoculated into and carries out culture of rootage in the adventitious bud rooting medium, 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; Incubation time is 40d; Cultivation temperature is 23 ℃, and 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 21d of culture of rootage, and grows new root gradually, and rooting rate is 85.5%, and the number of on average taking root is 2.5, and average root is long to be 2.3cm.
(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 92%.
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; Healthy and strong lateral bud is shelled into the long budlet of 6.5cm; Continue water and rinse well, use scalpel to peel the long tender leaf of budlet internal layer 4.5cm, as explant;
(2) blade sterilization: on the sterile working platform, earlier with 70% alcohol disinfecting 11s, with 0.1% mercuric chloride sterilization 7min, aseptic water washing 5 times blots blade surface moisture with blotting paper, and is subsequent use again with tender leaf;
(3) blade inducing culture: the tender leaf after will sterilizing vertically inserts in the blade inducing culture, and this medium is: MS+6-BA 1.3mg/l+NAA 0.25mg/l, the agar of adding 6.5g/l in the medium, the sucrose of 28g/l; The pH value is 5.6, the blade tip of tender leaf up, leaf base is induced the formation indefinite bud down; Every 30d subculture switching 1 time is transferred 3 times, cultivates 90d altogether; Cultivation temperature is 24 ℃, and light application time is 11h/d, and intensity of illumination is 1400lux.Cultivate about 12d rear blade and become bottle green by light green, cultivate the 40d blade base and begin to expand, induce the little gemmule that produces white, continue to cultivate 23d, bud is stretched to about 1.7cm, and bud induction rate is 68.5%, and each blade induced bud number is 4.6.
(4) adventitious bud proliferation successive transfer culture: the indefinite bud of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, are inoculated in the adventitious bud proliferation subculture medium, and this medium is: MS+6-BA 0.8mg/l+NAA 0.08mg/l; The agar that adds 6.5g/l in the medium, the sucrose of 28g/l, the pH value is 5.6; Every 25d subculture switching 1 time is transferred 2 times, cultivates 50d altogether; Cultivation temperature is 24 ℃, and light application time is 13h/d, and intensity of illumination is 2400lux.Proliferation and subculture is cultivated 18d, and the bastem portion of growing thickly begins to sprout the sprouting point, and proliferation and subculture is cultivated 20d, and the sprouting blade begins to stretch grows, and the adventitious buds proliferation coefficient reaches 3.6 times, and the bud height is 2.4cm, and bud seedling robust growth, mounted blade are better.
(5) adventitious bud rooting is cultivated: the indefinite bud that 2.5～3.0cm in the step 4) is long downcuts, and is inoculated into and carries out culture of rootage in the adventitious bud rooting medium, 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; Incubation time is 40d; Cultivation temperature is 24 ℃, and 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 12d of culture of rootage, and grows new root gradually, and rooting rate is 82.6%, and the number of on average taking root is 2.4, 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 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 95%.
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; Healthy and strong lateral bud is shelled into the long budlet of 7cm; Continue water and rinse well, use scalpel to peel the long tender leaf of budlet internal layer 5cm, as explant;
(2) blade sterilization: on the sterile working platform, earlier with 70% alcohol disinfecting 12s, with 0.1% mercuric chloride sterilization 8min, aseptic water washing 5 times blots blade surface moisture with blotting paper, and is subsequent use again with tender leaf;
(3) blade inducing culture: the tender leaf after will sterilizing vertically inserts in the blade inducing culture, and this medium is: MS+6-BA 1.5mg/l+NAA 0.3mg/l, the agar of adding 7.0g/l in the medium, the sucrose of 30g/l; The pH value is 5.8, the blade tip of tender leaf up, leaf base is induced the formation indefinite bud down; Every 30d subculture switching 1 time is transferred 3 times, cultivates 90d altogether; Cultivation temperature is 25 ℃, and light application time is 12h/d, and intensity of illumination is 1500lux.Cultivate about 14d rear blade and become bottle green by light green, cultivate the 42d blade base and begin to expand, induce the little gemmule that produces white, continue to cultivate 28d, bud is stretched to about 1.5cm, and bud induction rate is 51.7%, and each blade induced bud number is 3.3.
(4) adventitious bud proliferation successive transfer culture: the indefinite bud of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, are inoculated in the adventitious bud proliferation subculture medium, and this medium is: MS+6-BA 1.0mg/l+NAA 0.1mg/l; The agar that adds 7.0g/l in the medium, the sucrose of 30g/l, the pH value is 5.8; Every 25d subculture switching 1 time is transferred 2 times, cultivates 50d altogether; Cultivation temperature is 25 ℃, and light application time is 14h/d, and intensity of illumination is 2500lux.Proliferation and subculture is cultivated 23d, and the bastem portion of growing thickly begins to sprout the sprouting point, and proliferation and subculture is cultivated 20d, and the sprouting blade begins to stretch grows, and the adventitious buds proliferation coefficient reaches 3.2 times, and the bud height is 2.0cm, and bud seedling robust growth, mounted blade are better.
(5) adventitious bud rooting is cultivated: the indefinite bud that 2.5～3.0cm in the step 4) is long downcuts, and is inoculated into and carries out culture of rootage in the adventitious bud rooting medium, 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; Incubation time is 40d; Cultivation temperature is 25 ℃, and 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 15d of culture of rootage, and grows new root gradually, and rooting rate is 73.5%, and the number of on average taking root is 1.8, and average root is long to be 1.5cm.
(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 90%.
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 (2)
1. a method of utilizing the excised leaf efficiently propagating torch pineapples 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 5～7cm; Continue water and rinse well, use scalpel to peel the long tender leaf of budlet internal layer 3～5cm, as explant;
2) blade sterilization: on the sterile working platform, earlier with 70% alcohol disinfecting, 8～12s, with 0.1% mercuric chloride sterilization, 6～8min, aseptic water washing 3～5 times blots blade surface moisture with blotting paper, and is subsequent use again with tender leaf;
3) blade inducing culture: the tender leaf after will sterilizing vertically inserts in the blade inducing culture, the blade tip of tender leaf up, leaf base is down; Induce the formation indefinite bud, every 30d subculture switching 1 time is transferred 3 times; Cultivate 90d altogether; Cultivation temperature is 20～25 ℃, and light application time is 8～12h/d, and intensity of illumination is 1000～1500lux;
4) adventitious bud proliferation successive transfer culture: the indefinite bud of inducing formation in the step 3) is downcut, and per 2～3 buds are one clump, are inoculated in the adventitious bud proliferation subculture medium; Every 25d subculture switching 1 time; Transfer 2 times, cultivate 50d altogether, cultivation temperature is 20～25 ℃; Light application time is 10～14h/d, and intensity of illumination is 2000～2500lux;
5) adventitious bud rooting is cultivated: the indefinite bud that 2.5～3.0cm in the step 4) is long downcuts; Be inoculated into and carry out culture of rootage in the adventitious bud rooting medium, incubation time is 40d, and cultivation temperature is 20～25 ℃; 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;
Blade inducing culture in the described step 3) is MS+6-BA 1.0～1.5mg/l+NAA 0.1～0.3mg/l, and wherein: MS is conventional minimal medium Murashige&Skoog 1962, and 6-BA is a 6-benzyladenine, and NAA is a methyl;
Adventitious bud proliferation subculture medium in the described step 4) is MS+6-BA 0.3～1.0mg/l+NAA0.02～0.1mg/l, and wherein: MS is conventional minimal medium Murashige&Skoog 1962, and 6-BA is a 6-benzyladenine, and NAA is a methyl;
Adventitious bud rooting 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 blade inducing culture, adventitious bud proliferation subculture medium, the root media.
2. a kind of method of utilizing the excised leaf efficiently propagating torch pineapples according to claim 1 is characterized in that: the pH value of described blade inducing culture, adventitious bud proliferation subculture medium and root media is 5.6～5.8.
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| CN101940161B (en) * | 2010-08-27 | 2012-01-25 | 浙江传化生物技术有限公司 | Method for inducing cluster buds of guoziman conifera |
| CN102668984A (en) * | 2012-05-24 | 2012-09-19 | 南京农业大学 | Method for pear blade inducing adventitious buds to regenerate plant |
| CN103385169A (en) * | 2013-07-04 | 2013-11-13 | 中国热带农业科学院热带作物品种资源研究所 | Liquid cuttage method for pineapple single leaf |
| 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 |
| CN103461120B (en) * | 2013-08-22 | 2015-04-22 | 江苏农林职业技术学院 | Air plant tissue culture medium and method for tissue cultivating and quickly propagating air plant |
| 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 |
| CN104255321A (en) * | 2014-09-17 | 2015-01-07 | 江苏农林职业技术学院 | Tillandsia stricta rigid leaf tissue culture seedling strengthening method |
| 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 |
| CN109006488A (en) * | 2018-10-15 | 2018-12-18 | 四川农业大学 | A kind of red luxuriant pineapple breeding method based on using buds to propagate buds |
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