CN102577969A - Breeding method of tissue culture seedling of lonicera macranthoides Yulei No.1 - Google Patents
Breeding method of tissue culture seedling of lonicera macranthoides Yulei No.1 Download PDFInfo
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Abstract
The invention discloses a breeding method of tissue culture seedling of lonicera macranthoides Yulei No.1, comprising the following steps of: taking lamina extracted in the current year as explant, sterilizing the lamina and inoculating the lamina on an induced medium to culture callus in an inducing way, transplanting the callus into a differential medium to carry out the differential cultivation of adventitious bud, cutting the obtained primary cluster buds into single buds and transplanting the single buds into a subculture medium to carry out the subculture of adventitious buds, when the sub-cultured cluster buds grow to the height of 1.5-2cm, cutting the sub-cultured cluster buds into single buds and transplanting the single buds into a rooting medium to culture in a rooting way, and transplanting generated test-tube plantlet into a pearlite-peat soil mixed matrix with the volume ratio of 1:9 to culture, to obtain the tissue culture seedling of lonicera macranthoides Yulei No.1. According to the method, the induced medium, the differential medium, the subculture medium and the rooting medium can be optimized. The breeding method has the advantages of being high in callus induction rate, high in adventitious bud differentiation rate and multiplication coefficient, high in rooting rate, high in test-tube plantlet transplanting survival rate, robust in sprout growth, normal in lamina shape and color, subcutaneous in rooting, quick in the germination of new leaves and the like, so that the requirement of the large-area and large-scale cultivation of the lonicera macranthoides Yulei No.1 can be met.
Description
Technical field
The invention belongs to field of plant tissue culture technique, relate to a kind of mating system of plant tissue culture seedling.
Background technology
Largeflower-like honeysuckle flower (Lonicera m acranthoides Hand. Mazz) is perennial half evergreen winding undershrub of Caprifoliaceae Lonicera or upright undershrub plant; The chlorogenic acid content of its bud is the highest in the various honeysuckles in the whole nation, is one of principal item of southwest, ALFISOL IN CENTRAL commodity honeysuckle.
The authorization of the forest variety certification committee was new varieties of largeflower-like honeysuckle flower to Chongqing flower bud No. 1 through the Chongqing City in 2008.This kind per mu yield fresh flower reaches more than 400 kilograms; The raising the output of tradition plantation largeflower-like honeysuckle flower 30 ~ 80%, and also tradition man kind largeflower-like honeysuckle flower is strong for resistance and adaptability, and its outstanding feature is to reach 20 ~ 30 days the florescence and the whole florescence all is a bud; Basically do not bloom; Plant 7 ~ 10 days bud stage of largeflower-like honeysuckle flower with tradition man and compare, the picking period prolongs greatly, is convenient to pluck arrange recruitment arrangement and process time.This kind has been listed Chongqing " both wings " peasant household in and has been increased income in the works for ten thousand yuan.Because this kind tree characteristics variation is bigger; Usually adopt clone breeding method such as cuttage, propagation by grafiting fine individual plant clone at present, but the stock of grafting and scion resource are few, cuttage survival rate is lower than 40%; Cause reproduction coefficient lower, can not satisfy and produce and the market demand.Therefore, the vegetative propagation through tissue culture realizes that in a short time the seedling industrialization has market prospects very much.
Summary of the invention
In view of this; The object of the present invention is to provide No. 1 group of a kind of largeflower-like honeysuckle flower Chongqing flower bud to cultivate the mating system of seedling, have the callus induction rate height, differentiation adventitious buds rate and growth coefficient are high; Rooting rate is high; The test-tube seedling transplanting survival rate is high, and bud seedling robust growth, leaf leaf look normal be, subcutaneous takes root, young leaves is sprouted advantages such as fast, can satisfy the needs of No. 1 large tracts of land large-scale planting of largeflower-like honeysuckle flower Chongqing flower bud.
For achieving the above object, the present invention provides following technical scheme:
Largeflower-like honeysuckle flower Chongqing No. 1 group of flower bud is cultivated the mating system of seedling, may further comprise the steps:
A. callus induction:,, after the blade sterilization, be inoculated in and carry out callus induction on the inducing culture and cultivate as explant with the blade extracted out then, obtain callus; Said inducing culture is to be minimal medium with B5, and adds 6-benzyl aminopurine (6-BA) 2mg/L, kinetin (KT) 2mg/L, methyl (NAA) 0.1mg/L, carragheen 5.5 ~ 6.0g/L and sucrose 30g/L, regulates pH to 5.6 ~ 5.8;
B. differentiation adventitious buds: step a gained callus is forwarded to carries out differentiation adventitious buds in the differential medium and cultivate, obtain just the generation bud of growing thickly; Said differential medium is to be minimal medium with B5, and adds 6-BA 1mg/L, KT 2mg/L, NAA 0.1mg/L, carragheen 5.5 ~ 6.0g/L and sucrose 30g/L, regulates pH to 5.6 ~ 5.8;
C. indefinite bud subculture: with step b gained just generation grow thickly after bud cuts into simple bud, be forwarded to and carry out the indefinite bud successive transfer culture in the subculture medium, obtain the subculture bud of growing thickly; Said subculture medium is to be minimal medium with MB, and adds 6-BA 1mg/L, heteroauxin (IAA) 0.8mg/L, carragheen 5.5 ~ 6.0g/L and sucrose 30g/L, regulates pH to 5.6 ~ 5.8;
D. take root: treat that the blastogenesis of growing thickly of step c gained subculture grows to 1.5 ~ 2cm when high, after the bud of will growing thickly cuts into simple bud, be forwarded to and carry out culture of rootage in the root media, obtain test-tube plantlet; Said root media is to be minimal medium with the 1/3MB medium, and adds indolebutyric acid (IBA) 1.5mg/L, IAA 0.1mg/L, carragheen 5.5 ~ 6.0g/L and sucrose 30g/L, adjusting pH to 5.6 ~ 5.8;
E. test-tube seedling transplanting: steps d gained test-tube seedling transplanting is gone in the perlite that volume ratio is 1:9-peat soil mixed-matrix to cultivate, promptly get largeflower-like honeysuckle flower Chongqing No. 1 group of flower bud and cultivate seedling.
Further, the said callus induction of step a is cultivated, the said differentiation adventitious buds of step b is cultivated and the condition of the said culture of rootage of steps d is: be that 25 ± 1 ℃, intensity of illumination be the condition of 2000 ~ 3000lx under illumination cultivation 10 ~ 12 hour in temperature every day; The condition of the said indefinite bud successive transfer culture of step c is: be that 25 ± 1 ℃, intensity of illumination be the condition of 2000 ~ 3000lx under illumination cultivation 10 ~ 12 hour in temperature every day, and day and night temperature is controlled at 8 ~ 12 ℃; The said culture condition of step e is: illumination is 10 ~ 12 hours under the condition that be 75% ~ 85% at soil humidity every day, intensity of illumination is 1500 ~ 3000lx, uses 1 nitrogen quality percentage composition in per 10 days and be 15%, phosphorus and potassium quality percentage composition are 5% nitrogen phosphorus potassium ternary compound fertilizer.
The B5 medium that relates in the technique scheme is the medium of design such as nineteen sixty-eight Gamborg, is the medium commonly used in the Plant Tissue Breeding; The MS medium be Murashige in 1962 and Skoog for cultivating the medium that tobacco cell designs, also be the medium commonly used in the Plant Tissue Breeding; The MB medium is that the inventor improves on the basis of MS medium and gets; Mainly be that the kind and the concentration thereof of macroelement in the MS medium are adjusted; Kind but each component concentrations identical with the MS medium of trace element is equivalent to 2/3 of concentration in the MS medium approximately, and molysite is identical with the MS medium with organic kind and concentration thereof; 1/2,1/3, the 1/4MB medium is meant that each component concentrations of macroelement in the medium is equivalent to 1/2,1/3,1/4 of concentration in the MB medium respectively, trace element, molysite are identical with the MB medium with organic concentration.The concrete prescription of B5, MS and MB medium is as shown in the table.
Beneficial effect of the present invention is: the present invention has set up and has been applicable to that No. 1 group of largeflower-like honeysuckle flower Chongqing flower bud cultivates the method that seedling is bred; And the inducing culture, differential medium, subculture medium, root media and the test-tube seedling transplanting matrix that relate in the method carried out system optimization; Income approach has the callus induction rate height, and differentiation adventitious buds rate and growth coefficient are high, and rooting rate is high; The test-tube seedling transplanting survival rate is high; Bud seedling robust growth, leaf leaf look normal be, subcutaneous takes root, young leaves is sprouted advantages such as fast, can satisfy the needs of No. 1 large tracts of land large-scale planting of largeflower-like honeysuckle flower Chongqing flower bud, has a good application prospect.
Description of drawings
Fig. 1 is the callus that induces.
The indefinite bud that Fig. 2 differentiates for callus.
Fig. 3 is the successive transfer culture of indefinite bud.
Fig. 4 is a culture of rootage.
Fig. 5 cultivates seedling for largeflower-like honeysuckle flower Chongqing No. 1 group of flower bud.
Embodiment
In order to make the object of the invention, technical scheme and advantage clearer, will combine accompanying drawing that the present invention is made further detailed description below.No. 1 explant of largeflower-like honeysuckle flower Chongqing flower bud that uses among the embodiment is provided by flowers research institute of Institute Of Unity and Coherence In Writing Of Chongqing green house.
One, No. 1 group of largeflower-like honeysuckle flower Chongqing flower bud is cultivated the condition of culture optimization of seedling
1, the optimization of inducing culture
Largeflower-like honeysuckle flower No. 1 explant of flower bud (then extract out blade) that changes is rinsed well with running water, and using volume fraction is 70% ethanolic solution rinsing 30 seconds, and using mass fraction again is 0.1% HgCl
2Solution disinfection 8 minutes is used 4 ~ 5 remaining HgCl of thorough flush away of aseptic water washing at last
2Blade after the sterilization is cut into 1cm
3Size is inoculated on the inducing culture.Inducing culture respectively with MS, MB and B5 as minimal medium, and add 6-BA 1mg/L, KT 2mg/L, NAA 0.1mg/L, carragheen 6g/L and sucrose 30g/L, regulate pH to 5.8.Every kind of medium is handled 3 bottles, 30 explants of every bottle graft kind.Carry out callus induction afterwards and cultivate, be that 25 ± 1 ℃, intensity of illumination be the condition of 2000 ~ 3000lx under illumination cultivation 12 hour in temperature every day, induction time and the callus number of statistics callus, calculating inductivity.
The result sees table 1; The minimal medium of inducing culture is bigger to the inductivity influence of No. 1 blade callus of largeflower-like honeysuckle flower Chongqing flower bud; The inductivity significant difference of MS, three kinds of different minimal mediums of MB and B5, wherein the callus of the average inductivity the highest (66.7%) of B5 medium and formation is yellow green, densification, growth comparatively fast; And the minimal medium of inducing culture is less to the induction time influence of No. 1 blade callus of largeflower-like honeysuckle flower Chongqing flower bud, and the induction time difference of three kinds of different minimal mediums is not remarkable, is all inducing 2 Zhou Houcai to have callus to form.Therefore, the preferred B5 of minimal medium of No. 1 blade callus inducing medium of largeflower-like honeysuckle flower Chongqing flower bud.
The different minimal mediums of table 1 are to the influence of No. 1 blade callus induction of largeflower-like honeysuckle flower Chongqing flower bud
2, the optimization of differential medium
The largeflower-like honeysuckle flower No. 1 blade callus of flower bud that changes is forwarded in the differential medium.Differential medium be with B5 as minimal medium, add 6-BA, KT and the NAA of variable concentrations proportioning respectively, add carragheen 6g/L and sucrose 30g/L simultaneously, regulate pH to 5.8.Every kind of medium is handled 30 callus.Carry out differentiation adventitious buds afterwards and cultivate, be that 25 ± 1 ℃, intensity of illumination be the condition of 2000 ~ 3000lx under illumination cultivation 12 hour in temperature every day, and statistics differentiation bud number calculates differentiation rate after 30 days.
The result sees table 2; The hormone concentration and media of differential medium is bigger to the influence of No. 1 blade callus differentiation of largeflower-like honeysuckle flower Chongqing flower bud; The differentiation rate significant difference of 6-BA, KT and NAA variable concentrations proportioning; Wherein the leaf color with the differentiation rate the highest (56.7%) of 6-BA 1mg/L+KT 2mg/L+NAA 0.1mg/L and bud is dark green, robust growth; In addition, analyze the influence of the concentration of 6-BA and KT to the callus differentiation, the 6-BA of visible high concentration makes the inclined to one side Huang of bud color and the differentiation rate that derive relatively low, and the KT of high concentration helps the blade differentiation of calli and the bud look green that differentiates, and bud is stalwartness.Therefore, the preferred 6-BA 1mg/L+KT of the hormone concentration and media 2mg/L+NAA 0.1mg/L of No. 1 blade differentiation adventitious buds of largeflower-like honeysuckle flower Chongqing flower bud medium.
The different hormone concentration and media of table 2 are to the influence of No. 1 blade callus differentiation of largeflower-like honeysuckle flower Chongqing flower bud
3, the optimization of subculture medium
Change first generation that No. 1 blade callus of flower bud the differentiates bud of growing thickly of largeflower-like honeysuckle flower is cut into simple bud, be forwarded in the subculture medium.Subculture medium respectively with B5, MS and MB as minimal medium, every kind of minimal medium adds 6-BA, IAA and the NAA of variable concentrations proportioning respectively, adds carragheen 6g/L and sucrose 30g/L simultaneously, regulates pH to 5.8.30 buds of every kind of culture medium inoculated.Carry out the indefinite bud successive transfer culture afterwards, be that 25 ± 1 ℃, intensity of illumination be the condition of 2000 ~ 3000lx under illumination cultivation 12 hour in temperature every day, and the total bud number of statistics propagation calculates growth coefficient after 25 days.
The result sees table 3; The minimal medium of subculture medium is bigger to the influence of No. 1 blade indefinite bud of largeflower-like honeysuckle flower Chongqing flower bud subculture; In B5, three kinds of different minimal mediums of MS and MB; Inclined to one side Huang of leaf color and growth coefficient that B5 medium is handled are relatively low, and the leaf look that MS and MB medium are handled is normal, wherein the bud seedling growing state handled of MB medium better and growth coefficient the highest; In addition, the hormone concentration and media of indefinite bud subculture medium is also bigger to the influence of No. 1 blade indefinite bud of largeflower-like honeysuckle flower Chongqing flower bud subculture, when the NAA concentration fixed; Along with the reduction of 6-BA concentration, growth coefficient obviously descends, when the 6-BA concentration fixed; Plain IAA of comparison of growth and BAA, it is thus clear that the growth coefficient difference of the two is less, but the indefinite bud base portion callus that NAA handles is big; Be unfavorable for that plant absorbs nutrition, and there is not this problem in IAA.Therefore, the preferred MB of minimal medium of No. 1 blade indefinite bud of largeflower-like honeysuckle flower Chongqing flower bud subculture medium, the preferred 6-BA 1mg/L+IAA of hormone concentration and media 0.8mg/L.
Different minimal mediums of table 3 and hormone concentration and media are to the influence of No. 1 blade indefinite bud of largeflower-like honeysuckle flower Chongqing flower bud subculture
4, the optimization of root media
Largeflower-like honeysuckle flower the grow thickly robust growth in the bud, leaf leaf look bud normal, the about 1.5 ~ 2cm of plant height of No. 1 subculture of flower bud that change cut down and be forwarded in the root media.Root media respectively with 1/4MB, 1/3MB and 1/2MB (be that each component concentrations of macroelement is equivalent to concentration in the MB medium respectively in the medium 1/4,1/3 and 1/2; All the other components and concentration thereof are identical with the MB medium) as minimal medium; Every kind of minimal medium adds the IBA and the IAA of variable concentrations proportioning respectively; Add carragheen 5.5g/L and sucrose 20g/L simultaneously, regulate pH to 5.8.90 buds of every kind of culture medium inoculated.Carry out culture of rootage afterwards, be that 25 ± 1 ℃, intensity of illumination be the condition of 2000 ~ 3000lx under illumination cultivation 10 hour in temperature every day, and the statistics number of taking root calculates rooting rate after 20 days.
The result sees table 4; The minimal medium of root media is bigger to the influence of No. 1 blade adventitious bud rooting of largeflower-like honeysuckle flower Chongqing flower bud; The inorganic salt concentration reduction helps taking root of indefinite bud; But can reduce rooting rate again when concentration is lower than 1/3MB, the growing state of bud seedling also changes with the variation of mineral concentration, when inorganic salt concentration is lower than 1/3MB, begins to occur bud seedling leaf phenomenons such as Huang, tip buds die partially; In addition, the hormone concentration and media of root media is also bigger to the influence of No. 1 blade adventitious bud rooting of largeflower-like honeysuckle flower Chongqing flower bud, is mainly reflected on rooting rate and the bud seedling rooting position; When IBA concentration was 0.5mg/L, the base portion callus was very big, and the part root grows from callus; Rooting rate is lower, and along with the increase of IBA concentration, base portion does not form callus; Directly take root from skin, rooting rate reaches the highest (74%) when IBA concentration is 1.5mg/L.Therefore, the preferential 1/3MB of the minimal medium of root media, the preferred IBA 1.5mg/L+IAA of hormone concentration and media 0.1mg/L.
Different minimal mediums of table 4 and hormone concentration and media are to the influence of No. 1 blade adventitious bud rooting of largeflower-like honeysuckle flower Chongqing flower bud
5, the optimization of test-tube seedling transplanting matrix
The test-tube plantlet that largeflower-like honeysuckle flower changes behind No. 1 blade adventitious bud rooting of flower bud is transplanted respectively to 5 kinds of different substrates.5 kinds of matrix are respectively: 1. perlite; 2. volume ratio is perlite-peat soil mixture of 1:9; 3. volume ratio is perlite-peat soil mixture of 3:7; 4. volume ratio is perlite-peat soil mixture of 5:5; 5. peat soil.Every kind of matrix is transplanted 300 strain test-tube plantlets.Illumination cultivation is 12 hours under the condition that every day is about 80% at soil humidity afterwards, intensity of illumination is 1500 ~ 3000lx; Using 1 nitrogen phosphorus potassium gross mass percentage composition in per 10 days is 25% (N15P5K5; The quality percentage composition that is nitrogen is 15%; The quality percentage composition of phosphorus and potassium respectively is 5%) nitrogen phosphorus potassium ternary compound fertilizer, application concentration is 1mg/mL, the statistics test-tube plantlet number of become living after 40 days.
The result sees table 5, and test-tube seedling transplanting matrix becomes influence alive and growth bigger to No. 1 test-tube plantlet of largeflower-like honeysuckle flower Chongqing flower bud, the test-tube plantlet survival rate of different substrates and growing state significant difference; Its mesostroma transplanting survival rate 2. the highest (89.6%) and bud seedling are healthy and strong, the leaf look normal, young leaves is sprouted soon, infer that reason is that the perlite slit is big, permeability is good, certain water retention capacity is arranged, but nutrition are relatively poor; When being matrix with it separately, the test-tube plantlet nutrient absorption is less, causes new root to recover slower; Plant strain growth is relatively poor, and peat soil has certain permeability, good water-retaining property; Nutrition is abundanter, and when being matrix with it separately, the test-tube plantlet root is prone to ponding; Cause the mashed leaf phenomenon of mashed root to occur,, sufficient organic nutrient can be provided again when maintenance is penetrating, moistening perlite and peat soil rational proportion; Make the fast quick-recovery of Xin Gen and plant, improve survival rate.Therefore, test-tube seedling transplanting matrix preferred substrate 2..
The influence that the different transplanting mediums of table 5 become to live and grow to No. 1 test-tube plantlet of largeflower-like honeysuckle flower Chongqing flower bud
6, the optimization of other condition
Find also in the experiment that cutting technique and day and night temperature also have considerable influence to No. 1 blade indefinite bud of largeflower-like honeysuckle flower Chongqing flower bud subculture.When growing thickly the bud subculture, should the bud of growing thickly be divided into simple bud, the main bud that is about on each bud clump cuts down separately, in order to avoid competition nutrition causes main bud withered in the subculture process.Day and night temperature in the incubation should be controlled between 8 ~ 12 ℃, and the temperature difference can make the slow and stipes shortening of bud seedling growth less than above-mentioned scope, influences taking root of back; The temperature difference is greater than above-mentioned scope, and the old blade of bud seedling can the flavescence gradually with the prolongation in cycle.
Two, No. 1 group of largeflower-like honeysuckle flower Chongqing flower bud is cultivated the optimization mating system of seedling
Comprehensive aforementioned condition of culture Optimization result, the optimization mating system that largeflower-like honeysuckle flower Chongqing No. 1 group of flower bud is cultivated seedling may further comprise the steps:
A. callus induction: the blade of selecting to extract out then is as explant; Blade is rinsed well with running water, and using volume fraction is 70% ethanolic solution rinsing 30 seconds, and using mass fraction again is 0.1% HgCl
2Solution disinfection 8 minutes is used 4 ~ 5 remaining HgCl of thorough flush away of aseptic water washing at last
2Blade after the sterilization is cut into 1cm
3Size is inoculated in and carries out callus induction on the inducing culture and cultivate, and be that 25 ± 1 ℃, intensity of illumination be the condition of 2000 ~ 3000lx under illumination cultivation 12 hour in temperature every day, obtains callus (Fig. 1); Said inducing culture is to be minimal medium with B5, and adds 6-BA 2mg/L, KT 2mg/L, NAA 0.1mg/L, carragheen 6g/L and sucrose 30g/L, regulates pH to 5.8;
B. differentiation adventitious buds: step a gained callus is forwarded to carries out differentiation adventitious buds in the differential medium and cultivate, be that 25 ± 1 ℃, intensity of illumination be illumination cultivation 12 hours under the condition of 2000 ~ 3000lx in temperature every day, obtains just the generation bud (Fig. 2) of growing thickly; Said differential medium is to be minimal medium with B5, and adds 6-BA 1mg/L, KT 2mg/L, NAA 0.1mg/L, carragheen 6g/L and sucrose 30g/L, regulates pH to 5.8;
C. indefinite bud subculture: with step b gained just generation the bud of growing thickly cut into simple bud; Be forwarded to and carry out indefinite bud successive transfer culture (Fig. 3) in the subculture medium; Be that 25 ± 1 ℃, intensity of illumination be the condition of 2000 ~ 3000lx under illumination cultivation 12 hour in temperature every day; Day and night temperature is controlled at 8 ~ 12 ℃, obtains the subculture bud of growing thickly; Said indefinite bud subculture medium is to be minimal medium with MB, and adds 6-BA 1mg/L, IAA 0.8mg/L, carragheen 6g/L and sucrose 30g/L, regulates pH to 5.8;
D. take root: treat that the blastogenesis of growing thickly of step c gained subculture grows to 1.5 ~ 2cm when high; The bud of will growing thickly cuts into simple bud; Be forwarded to and carry out culture of rootage (Fig. 4) in the root media; Be that 25 ± 1 ℃, intensity of illumination be the condition of 2000 ~ 3000lx under illumination cultivation 10 hour in temperature every day, obtains test-tube plantlet; Said root media be with the 1/3MB medium as minimal medium, and add IBA 1.5mg/L, IAA 0.1mg/L, carragheen 5.5g/L and sucrose 20g/L, regulate pH to 5.8;
E. test-tube seedling transplanting: steps d gained test-tube seedling transplanting is gone in the perlite that volume ratio is 1:9-peat soil mixed-matrix; Illumination cultivation is 12 hours under the condition that every day is about 80% at soil humidity, intensity of illumination is 1500 ~ 3000lx; The nitrogen phosphorus potassium ternary compound fertilizer that to use 1 nitrogen phosphorus potassium gross mass percentage composition in per 10 days be 25% (N15P5K5); Application concentration is 1mg/mL, promptly gets largeflower-like honeysuckle flower Chongqing No. 1 group of flower bud and cultivates seedling (Fig. 5).
Explanation is that above embodiment only is used to explain technical scheme of the present invention, does not constitute the restriction to content of the present invention at last.Although the present invention has been done comparatively detailed giving an example through the foregoing description; But those skilled in the art still can be according to summary of the invention part and the described technology contents of embodiment part; In form with on the details it is made various changes, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Claims (2)
1. No. 1 group of largeflower-like honeysuckle flower Chongqing flower bud is cultivated the mating system of seedling, it is characterized in that, may further comprise the steps:
A. callus induction:,, after the blade sterilization, be inoculated in and carry out callus induction on the inducing culture and cultivate as explant with the blade extracted out then, obtain callus; Said inducing culture is to be minimal medium with B5, and adds 6-BA 2mg/L, KT 2mg/L, NAA 0.1mg/L, carragheen 5.5 ~ 6.0g/L and sucrose 30g/L, regulates pH to 5.6 ~ 5.8;
B. differentiation adventitious buds: step a gained callus is forwarded to carries out differentiation adventitious buds in the differential medium and cultivate, obtain just the generation bud of growing thickly; Said differential medium is to be minimal medium with B5, and adds 6-BA 1mg/L, KT 2mg/L, NAA 0.1mg/L, carragheen 5.5 ~ 6.0g/L and sucrose 30g/L, regulates pH to 5.6 ~ 5.8;
C. indefinite bud subculture: with step b gained just generation grow thickly after bud cuts into simple bud, be forwarded to and carry out the indefinite bud successive transfer culture in the subculture medium, obtain the subculture bud of growing thickly; Said subculture medium is to be minimal medium with MB, and adds 6-BA 1mg/L, IAA 0.8mg/L, carragheen 5.5 ~ 6.0g/L and sucrose 30g/L, regulates pH to 5.6 ~ 5.8;
D. take root: treat that the blastogenesis of growing thickly of step c gained subculture grows to 1.5 ~ 2cm when high, after the bud of will growing thickly cuts into simple bud, be forwarded to and carry out culture of rootage in the root media, obtain test-tube plantlet; Said root media is to be minimal medium with the 1/3MB medium, and adds IBA 1.5mg/L, IAA 0.1mg/L, carragheen 5.5 ~ 6.0g/L and sucrose 30g/L, adjusting pH to 5.6 ~ 5.8;
E. test-tube seedling transplanting: steps d gained test-tube seedling transplanting is gone in the perlite that volume ratio is 1:9-peat soil mixed-matrix to cultivate, promptly get largeflower-like honeysuckle flower Chongqing No. 1 group of flower bud and cultivate seedling.
2. No. 1 group of largeflower-like honeysuckle flower according to claim 1 Chongqing flower bud is cultivated the mating system of seedling; It is characterized in that the said callus induction of step a is cultivated, the said differentiation adventitious buds of step b is cultivated and the condition of the said culture of rootage of steps d is: be that 25 ± 1 ℃, intensity of illumination be the condition of 2000 ~ 3000lx under illumination cultivation 10 ~ 12 hour in temperature every day; The condition of the said indefinite bud successive transfer culture of step c is: be that 25 ± 1 ℃, intensity of illumination be the condition of 2000 ~ 3000lx under illumination cultivation 10 ~ 12 hour in temperature every day, and day and night temperature is controlled at 8 ~ 12 ℃; The said culture condition of step e is: illumination cultivation is 10 ~ 12 hours under the condition that be 75% ~ 85% at soil humidity every day, intensity of illumination is 1500 ~ 3000lx, uses 1 nitrogen quality percentage composition in per 10 days and be 15%, phosphorus and potassium quality percentage composition are 5% nitrogen phosphorus potassium ternary compound fertilizer.
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| CN105918125A (en) * | 2016-05-05 | 2016-09-07 | 中国科学院植物研究所 | Method and culture medium for obtaining lonicera macranthoides regeneration plants through tissue culture |
| CN107549014A (en) * | 2017-09-27 | 2018-01-09 | 重庆市天沛农业科技有限公司 | A kind of mating system of red magnificent Kiwifruit Tissue Culture seedling |
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| CN103548690A (en) * | 2013-11-01 | 2014-02-05 | 重庆文理学院 | Proliferation culture medium and proliferation method for large-flower-like honeysuckle flower Yulei #1 |
| CN103548687A (en) * | 2013-11-01 | 2014-02-05 | 重庆文理学院 | Rooting culture medium and rooting culture method for tissue culture seedling of lonicera macranthoides Hand.Mazz Yulei #1 |
| CN104304002A (en) * | 2014-08-29 | 2015-01-28 | 广西壮族自治区药用植物园 | Inducing method of lonicera confusa leaf adventitious bud |
| CN105123521A (en) * | 2015-09-06 | 2015-12-09 | 湖南省林业科学院 | Culture medium and method for honeysuckle direct somatic embryogenesis and plant regeneration |
| CN105123521B (en) * | 2015-09-06 | 2017-07-07 | 湖南省林业科学院 | There is the culture medium and method with plant regeneration in a kind of direct body embryo of honeysuckle |
| CN105918125A (en) * | 2016-05-05 | 2016-09-07 | 中国科学院植物研究所 | Method and culture medium for obtaining lonicera macranthoides regeneration plants through tissue culture |
| CN107549014A (en) * | 2017-09-27 | 2018-01-09 | 重庆市天沛农业科技有限公司 | A kind of mating system of red magnificent Kiwifruit Tissue Culture seedling |
| CN109757372A (en) * | 2017-11-15 | 2019-05-17 | 江西农业大学 | A kind of production method rich in chlorogenic acid substance honeysuckle cell |
| CN111248085A (en) * | 2020-03-05 | 2020-06-09 | 烟台市林业科学研究所 | Method for inducing callus formation by bitter candy overwintering flower buds |
| CN111248085B (en) * | 2020-03-05 | 2022-09-06 | 烟台市林业科学研究所 | Method for inducing callus formation by overwintering flower buds of bitter candies |
| CN114041422A (en) * | 2021-11-29 | 2022-02-15 | 浙江理工大学 | Honeysuckle tissue culture method based on low-mesh-number activated carbon culture medium |
| CN116267621A (en) * | 2023-04-24 | 2023-06-23 | 遵义申未辰农业科技开发有限公司 | Rapid seedling method and culture medium for lonicera macranthoides |
| CN116267621B (en) * | 2023-04-24 | 2024-04-16 | 遵义申未辰农业科技开发有限公司 | Rapid seedling method and culture medium for lonicera macranthoides |
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