CN103155866A - Malus zumi tissue culture rapid propagation seedling raising method - Google Patents
Malus zumi tissue culture rapid propagation seedling raising method Download PDFInfo
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Abstract
The invention discloses a malus zumi tissue culture rapid propagation seedling raising method which comprises the following steps that (1) raw materials are processed: a terminal bud or a lateral bud of a field annual seedling malus zumi sapling is sheared; (2) primary culture is carried out: the terminal bud or the lateral bud is inoculated onto an induction medium to be cultured, the induction medium is made by the fact that raw materials are added into an MS minimal medium of one liter, and the raw materials and proportions of the raw materials are white granulated sugar of 10g, agar powder of 4.5g, and 6-benayl aminopurine of 0.2-0.3mg; (3) enrichment culture is carried out: the terminal bud or the lateral bud is transferred into an enrichment medium to be cultured, the enrichment medium is made by the fact that raw materials are added into an MS enrichment medium of one liter, and the raw materials and proportions of the raw materials are white granulated sugar of 10g, agar powder of 4.5g, and 6-benayl aminopurine 0.6-0.8mg; (4) rooting culture is carried out: the terminal bud or the lateral bud is placed into a root medium to be cultured, the root medium is made by the fact that raw materials are added into 1/2 MS minimal medium, and the raw materials and proportions of the raw materials are white granulated sugar of 10g, agar powder of 4.5g, and indolebutyric acid of 0.1-0.2mg; and (5) hardening-seedling and transplantation are carried out.
Description
Technical field
The invention belongs to field of plant tissue culture technique, specifically a kind of method of breeding the U.S. Malus spectabilis of pearl by plant tissue culture technique.
Background technology
The U.S. Malus spectabilis of pearl originates in Japanese Mountainous Area of North, it is the wild Natural hybrid of Siberian crabapple and toringo, has stronger saline-alkali tolerance, cold resistance, be also the time that China scientific worker went through 13 years, the anti-saline and alkaline new varieties that screening breeds are out observed through for many years and are found, the U.S. Malus spectabilis robust growth of pearl, tree crown circle are attractive in appearance, disease-free, be the desirable green tree species of varieties in saline-alkali areas, also can be used as in addition the stock grafting apple, apply in the area heavier in the saline land.
the plant tissue culture technology is to utilize the totipotency of plant cell, each cell that namely forms plant corpus has the potential ability that buds into a whole plant, take the individual cells on plant corpus, cell mass, the part of meristematic tissue or organ, cultivate and regulate and control its growth by the medium of artificial preparation Different Nutrition composition and hormone, make these cells, tissues etc. form thousands of plantlets and keep whole merits of maternal plant, this method can obtain a large amount of group training seedlings at short notice, and can carry out anniversary large-scale production under the manual control condition.Utilize the plant tissue culture technology to carry out the batch production production of fruit tree flowers good seed, become the foundation engineering of modern horticultural agricultural development.
In the prior art, the U.S. Malus spectabilis provenance of pearl lacks, the cuttage difficulty is taken root, and genetic variation easily occurs, and is difficult to realize the large-scale industrialized production of high-quality.
Summary of the invention
Technical problem to be solved by this invention is to provide the U.S. Malus spectabilis tissue-culturing rapid propagation of a kind of pearl seedling-cultivating method.
Technical scheme of the present invention is: the U.S. Malus spectabilis tissue-culturing rapid propagation of a kind of pearl seedling-cultivating method, it comprises the following steps: the U.S. Malus spectabilis treelet terminal bud of the annual pearl in (1) raw-material processing clip land for growing field crops or lateral bud, respectively with stand-by as explant after alcohol, mercuric chloride solution, hydrogen peroxide surface sterilization sterilization; (2) first culture is seeded in described explant on inducing culture and cultivates, this inducing culture is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.2～0.3mg, condition of culture is: 23 ± 2 ℃ of temperature, intensity of illumination 2000～2500Lx, light application time 10～13 hours/day, humidity 40%～50%; (3) propagation is cultivated the bud seedling after grow thickly stem apex or the propagation of first culture is transferred in proliferated culture medium and is cultivated, this proliferated culture medium is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.6～0.8mg indolebutyric acid 0.1～0.2mg, condition of culture is: 23 ± 2 ℃ of temperature, intensity of illumination 2500～3000Lx, light application time 10～13 hours/day, humidity 40%～50%; (4) culture of rootage will be bred 1.5～3cm stem apex of cultivating and be put into root media and cultivate, this root media is for to add following raw material and proportioning thereof to make in the 1/2MS minimal medium: white granulated sugar 10g agar powder 4.5g indolebutyric acid 0.1～0.2mg, condition of culture is: 23 ± 2 ℃ of temperature, humidity 40～50%, intensity of illumination 2500～3000Lx, light application time 24 hours/day; (5) acclimatization and transplants comprises following link: close bottle exercise, uncork exercise, be transplanted to vermiculite matrix, be transplanted in the Nutrition Soil that is comprised of soil, sand, decomposed manure.
Preferably, described inducing culture is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.2mg; Described proliferated culture medium is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium: white granulated sugar 10g agar powder 4.5g6-benayl aminopurine 0.8mg indolebutyric acid 0.2mg; Described root media is for to add following raw material and proportioning thereof to make in the 1/2MS minimal medium: white granulated sugar 10g agar powder 4.5g indolebutyric acid 0.2mg.
For medium composition and proportioning, condition of culture and just relation or the impact of the stage culture effect such as culture, propagation cultivation, culture of rootage, the inventor has carried out experimental study, in order further to set forth the present invention, experimental data and result of study are described below:
1, different culture media survives and affects on the growth bud
The young shoot of this test after with the sterilization of stalwartness is inoculated in different minimal medium MS, 1/2MS and F, on the variable concentrations of same hormone 6-benzyl aminopurine (6-BA), checks the survival rate of bud after 1 week.
Experiment conclusion: all there is certain facilitation in different minimal mediums to the sprouting of bud, but all sproutings of evoking adventive bud, but show obvious otherness, its inductivity of MS, 1/2MS is higher, inoculates to begin in about 5 days to sprout, be grown in the stem apex performance of MS better, blade is dark green, on the 1/2MS medium stem apex growth potential a little less than, on the L medium, bud is sprouted slower, one Zhou Houcai induces the part callus, forms a small amount of indefinite bud.In the MS medium, 0.2～0.3 the time, bud not only survival rate is high, and robust growth when 6-BA concentration, sprouts neat, and with the increase of concentration, the callus degree increases the weight of, and the seedling abnormal rate increases, and when concentration when above, only forms the bulk tissue 1.0, sprouts less.
The same hormone different culture media of table 1 survives and affects on the growth bud
2, the impact of hormone on bud propagation
1) impact of 6-benzyl aminopurine (6-BA) on bud propagation
Add the 6-BA of variable concentrations to carry out the cultivation of bud propagation in the MS minimal medium, experimental result sees Table 2.
The impact of table 2 6-BA on bud propagation
Experiment conclusion: when 6-BA concentration was low, blastogenesis was long healthy and strong, and the leaf look dark green, and along with the increase propagation quantity of concentration strengthens, but 1.0 and when above, the pumping bud is grown thickly thin and delicate, can't be used for propagation next time, and callus produces in a large number, was unfavorable for taking root; The long stalwartness of blastogenesis when concentration is too low, but the germination negligible amounts can not satisfy fast-propagation.Be used for so 0.6～0.8mg is 6-BA the suitable concentration that bud is bred, 0.8mg/l is optium concentration.
2) 6-benzyl aminopurine (6-BA) is used in combination with indolebutyric acid (IBA) impact that bud is bred
Add 6-BA and the IBA of variable concentrations to carry out the cultivation of bud propagation in the MS minimal medium, experimental result sees Table 3.
Table 3 6-BA and the IBA impact on bud propagation
Experiment conclusion: 6-BA0.6mg/l+IBA0.2mg/l or 6-BA0.8mg/l+IBA0.2mg/l are used in conjunction with, and the bud seedling of pumping is healthy and strong, growth is neat, more are conducive to follow-on expansion numerous and take root, and growth coefficient is up to 6.80 and 7.68.Find in test that 6-BA is that the cultivation of the U.S. Malus spectabilis stem apex propagation of pearl is necessary, it can break apical dominance, promotes axillary bud sprouting.
3, medium, hormone and the condition of culture facilitation to taking root
This test will long 1.5-3cm, the propagation seedling cut-grafting of robust growth in the IBA of different minimal medium MS, 1/2MS and variable concentrations, check the situation of taking root after 20d.
1) different culture media and the variable concentrations IBA impact on the bud seedling rooting
Experiment conclusion: the test-tube plantlet that different culture media and variable concentrations IBA process, its rooting rate and the amount of taking root have larger difference.The situation of taking root on the 1/2MS minimal medium is generally higher than the MS minimal medium; In two kinds of different minimal mediums, when IBA concentration was 0.2mg/l, rooting rate was all high, and along with concentration increases, rooting rate and root system number descend on the contrary, showed when IBA concentration during higher than 0.2mg/l, can suppress taking root of test-tube plantlet.In all were processed, the 1/2MS+IBA0.2mg/l rooting rate was the highest, is 100%, and the number of on average taking root is 4.48.
Table 4 different culture media and the variable concentrations IBA impact on the bud seedling rooting.Annotate: on average take root and count the mean of the stock number of making a living.
2) impact of different light on the bud seedling rooting
The impact of table 5 different illumination conditions on the bud seedling rooting
| Cultivate under culturing room's full exposure | Cultivate under culturing room's natural lighting | |
| Rooting rate (%) | 97.4 | 81.4 |
| The number of taking root (bar) | 4.43 | 3.61 |
Experiment conclusion: the test-tube plantlet of access 1/2MS+IBA0.2mg/l root media, (2500～3000lx) cultivations can effectively improve rooting rate and the number of taking root through the group training chamber full exposures in 24 hours of about 10 days, average rooting rate reaches 97.4%, and the number of on average taking root reaches 4.43.(2000～5000lx) times the rooting of vitro seedling rate also reaches 81.4% in Indoor Natural illumination, the number of on average taking root reaches 3.61, but the root that produces under the Indoor Natural optical condition is more sturdy, quality is crisp, and the root that produces under the illumination cultivation condition of group training chamber is thinner, pliability is good, is difficult for impairedly during transplanting, and planting survival rate is high.
3) impact of different cultivation temperature on the bud seedling rooting
Test-tube plantlet in the 1/2MS+IBA0.2mg/l root media, under temperature condition not, group training chamber full exposure in 24 hours (is cultivated 15 days its rooting rates of " Invest, Then Investigate " and the number of taking root under 2500～3000lx) conditions.
Table 6 different temperatures is to bud seedling rooting affects on the growth
Experiment conclusion: different cultivation temperature have significant impact to the culture of rootage of test-tube plantlet, and cultivate 15d in same medium after, rooting rate is the highest under 25 ℃ of conditions, and the new root number of sending is maximum; Next is 30 ℃, 35 ℃, and rooting rate is minimum under 20 ℃ of conditions.Show that temperature 25 ℃ of left and right, be conducive to growth and the elongation of new root, and lower temperature is unfavorable for root growth.
4, different relative moisture, the different substrates impact on test-tube seedling transplanting
In greenhouse by solar heat, the impact that different relative moisture, different substrates survive test-tube seedling transplanting.
The impact that table 7 different humidity, different substrates survive test-tube seedling transplanting
Experiment conclusion: three processing from table are found out, the success or failure that damp condition is made decision and transplanted in very large situation, the transplanting survival rate of three kinds of different substrates is only in 10% left and right under greenhouse natural humidity condition, and the survival rate of three kinds of matrix reaches all and reaches more than 70% under little shed is manually sprayed damp condition.In three kinds of matrix, the transplanting survival rate of vermiculite is all high, is issued to more than 98% at the artificial spray condition of little shed.
the invention has the beneficial effects as follows, the present invention adopts light to cultivate the preparation of organizing the training seedling, cultivate strong sprout and hestening rooting, and at the natural daylight lower refining seedling, transplant the method for tissue culture that synchronously carries out, group training step has adopted proliferated culture medium and the root media of the U.S. Malus spectabilis Fast-propagation of suitable pearl, propagation and rooting efficiency are good, make the rooting rate of the U.S. Malus spectabilis of pearl reach 100%, and root system is sturdy neat, the seedling of group training simultaneously robust growth, the leaf look dark green, transplanting survival rate can reach 95% left and right, for batch production production is laid a good foundation, the inventive method is simple and easy to do, production efficiency is high, the Fast-propagation that can be used for the U.S. Malus spectabilis of pearl.
Embodiment
Embodiment 1
The U.S. Malus spectabilis tissue-culturing rapid propagation of a kind of pearl seedling-cultivating method, it comprises the following steps:
(1) raw-material processing
Raw-material processing: the U.S. annual treelet terminal bud of Malus spectabilis of the vigorous good pearl of clip grown in field or lateral bud, with running water flow wash 20 minutes, then with bud on superclean bench with 75% alcohol surface sterilization 30 seconds, 0.1% mercuric chloride sterilization 5 minutes, 15% hydrogen peroxide was processed 10 minutes, aseptic water washing 4 times blots with aseptic filter paper, and is stand-by as explant.
(2) the first culture of group training seedling
Explant after sterilization is seeded in induces differentiation to cultivate on inducing culture, this inducing culture is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium.
White granulated sugar 10g
Agar powder 4.5g
6-benzyl aminopurine 0.2mg
The first culture base that configures is divided in blake bottle, every bottled 25 milliliters, seal bottleneck, be to sterilize 25 minutes under 0.1MPa at pressure, explant material is inserted in the medium of the bacterium of having gone out, it is that 21 ℃, humidity are that 40% culturing room cultivates that blake bottle is placed in intensity of illumination 2000Lx, temperature, shines every day 10 hours; Newly connect material and be transferred in new medium every 3 days, repeat 2 times,, bud is changed in growth medium grow no longer during brown stain when medium.
(3) group training seedling proliferation is cultivated
The stem section of growing thickly of inducing cultivation is transferred to carries out Fast-propagation in proliferated culture medium, this proliferated culture medium is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium.
The proliferated culture medium that configures is divided in blake bottle, every bottled 25 milliliters, seal bottleneck, be to sterilize 25 minutes under 0.1MPa at pressure, induced bud is inserted in the medium of the bacterium of having gone out, it is that 21 ℃, humidity are that 40% culturing room cultivates that blake bottle is placed in intensity of illumination 2500Lx, temperature, shines illumination cultivation 20 angel's seedling and bud proliferations every day 10 hours; For expanding propagation quantity, the bud seedling after propagation repeatedly is transferred in the proliferated culture medium of new configuration, the subculture condition of culture is identical with first generation condition of culture, by production task, breed 8-20 for the time will breed the bud seedling and carry out culture of rootage.
(4) group training seedling rooting is cultivated
The stem apex of the 1.5cm of subculture incubation growth stalwartness is put into root media carry out culture of rootage, this root media is for to add following raw material and proportioning thereof to make in the 1/2MS minimal medium.
White granulated sugar 10g
Agar powder 4.5g
Indolebutyric acid 0.1mg
Root media is divided in blake bottle, 25 milliliters every bottle, seal bottleneck, be to sterilize 25 minutes under 0.1MPa at pressure, it is that 21 ℃ of left and right, humidity are that 40% culturing room cultivates that blake bottle is placed in intensity of illumination 2500Lx, temperature, illumination every day was cultivated in 24 hours, can induce the formation of root in 8～10 days.Can pull out culturing room and carry out the natural daylight acclimatization and transplants when bottle seedling of taking root grows to 25～30 days.
(5) training tissue culture seedling is transplanted
take root and to pull out when a bottle seedling grows to 25～30 days in culturing room's immigration greenhouse by solar heat, first hardening 5 days in the greenhouse, open the running water hardening 2 days that adds 0.5cm after sealing, when offspring leaf color to be generated slightly has intensification, take out from blake bottle, wash away the medium of shoot root section, with 600 times of carbendazim libation at an ancient wedding ceremony roots, transplant to the vermiculite seedling-raising cup of processing with bactericidal agent, 3 strains of planting of each seedling-raising cup, spread and sink in after cultivation permeable, be placed in the little shed of building in the greenhouse, 20 ℃ of temperature, the spraying of sheltering from heat or light in early stage, humidity remains on 80%, taking off gradually film after 10 days ventilates, throw off little shed canopy film after 15 days, spray during this period 1000 times of carbendazim solution spray nursery stocks, when nursery stock obviously grows, the nursery stock individual plant is moved into soil, husky, the decomposed manure volume ratio is in the nutrient matrix of 1: 1: 0.5, move into outside the greenhouse in shade, make nursery stock adapt to gradually the open country growing environment, when growing to the 10cm left and right Deng seedling in nutritive cube, root system band soil mud enters the land for growing field crops, cultivation management is with reference to the field seedling raising method.
Embodiment 2
The U.S. Malus spectabilis tissue-culturing rapid propagation of a kind of pearl seedling-cultivating method, it comprises the following steps:
(1) raw-material processing
Raw-material processing: the U.S. annual treelet terminal bud of Malus spectabilis of the vigorous good pearl of clip grown in field or lateral bud, with running water flow wash 10 minutes, then with bud on superclean bench with 75% alcohol surface sterilization 30 seconds, 0.1% mercuric chloride sterilization 10 minutes, 15% hydrogen peroxide was processed 15 minutes, aseptic water washing 4 times blots with aseptic filter paper, and is stand-by as explant.
(2) the first culture of group training seedling
Explant after sterilization is seeded in induces differentiation to cultivate on inducing culture, this inducing culture is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium.
White granulated sugar 10g
Agar powder 4.5g
6-benzyl aminopurine 0.3mg
The first culture base that configures is divided in blake bottle, every bottled 35 milliliters, seal bottleneck, be to sterilize 25 minutes under 0.15MPa at pressure, explant material is inserted in the medium of the bacterium of having gone out, it is that 25 ℃, humidity are that 50% culturing room cultivates that blake bottle is placed in intensity of illumination 2500Lx, temperature, shines every day 13 hours; Newly connect material and be transferred in new medium every 5 days, repeat 3 times,, bud is changed in growth medium grow no longer during brown stain when medium.
(3) group training seedling proliferation is cultivated
The stem section of growing thickly of inducing cultivation is transferred to carries out Fast-propagation in proliferated culture medium, this proliferated culture medium is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium.
The proliferated culture medium that configures is divided in blake bottle, every bottled 35 milliliters, seal bottleneck, be to sterilize 25 minutes under 0.15MPa at pressure, induced bud is inserted in the medium of the bacterium of having gone out, it is that 25 ℃, humidity are that 50% culturing room cultivates that blake bottle is placed in intensity of illumination 3000Lx, temperature, shines illumination cultivation 25 angel's seedling and bud proliferations every day 13 hours; For expanding propagation quantity, the bud seedling after propagation repeatedly is transferred in the proliferated culture medium of new configuration, the subculture condition of culture is identical with first generation condition of culture, and by production task, breeding will be bred the bud seedling during 8～20 generation and be carried out culture of rootage.
(4) group training seedling rooting is cultivated
The stem apex of the 3cm of subculture incubation growth stalwartness is put into root media carry out culture of rootage, this root media is for to add following raw material and proportioning thereof to make in the 1/2MS minimal medium.
White granulated sugar 10g
Agar powder 4.5g
Indolebutyric acid 0.2mg
Root media is divided in blake bottle, 35 milliliters every bottle, seals bottleneck, be to sterilize 25 minutes under 0.15MPa at pressure, it is that 25 ℃, humidity are that 50% culturing room cultivates that blake bottle is placed in intensity of illumination 3000Lx, temperature, and illumination every day was cultivated in 24 hours, can induce the formation of root in 8～0 days.Can pull out culturing room and carry out the natural daylight acclimatization and transplants when bottle seedling of taking root grows to 25～30 days.
(5) training tissue culture seedling is transplanted
take root and to pull out when a bottle seedling grows to 25～30 days in culturing room's immigration greenhouse by solar heat, first hardening 7 days in the greenhouse, open the running water hardening 3 days that adds 1.0cm after sealing, when offspring leaf color to be generated slightly has intensification, take out from blake bottle, wash away the medium of shoot root section, with 800 times of carbendazim libation at an ancient wedding ceremony roots, transplant to the vermiculite seedling-raising cup of processing with bactericidal agent, 4 strains of planting of each seedling-raising cup, spread and sink in after cultivation permeable, be placed in the little shed of building in the greenhouse, 25 ℃ of temperature, the spraying of sheltering from heat or light in early stage, humidity remains on 100%, taking off gradually film after 10 days ventilates, throw off little shed canopy film after 15 days, spray during this period 1000 times of carbendazim solution spray nursery stocks, when nursery stock obviously grows, the nursery stock individual plant is moved into soil, husky, the decomposed manure volume ratio is in the nutrient matrix of 1: 1: 0.5, move into outside the greenhouse in shade, make nursery stock adapt to gradually the open country growing environment, grow to the 10cm left and right Deng seedling in nutritive cube, root system band soil mud enters the land for growing field crops, cultivation management is with reference to the field seedling raising method.
Embodiment 3
The U.S. Malus spectabilis tissue-culturing rapid propagation of a kind of pearl seedling-cultivating method, it comprises the following steps:
(1) raw-material processing
Raw-material processing: the U.S. annual treelet terminal bud of Malus spectabilis of the vigorous good pearl of clip grown in field or lateral bud, with running water flow wash 15 minutes, then with bud on superclean bench with 75% alcohol surface sterilization 20 seconds, 0.1% mercuric chloride sterilization 7 minutes, 15% hydrogen peroxide was processed 12 minutes, aseptic water washing 4 times blots with aseptic filter paper, and is stand-by as explant.
(2) the first culture of group training seedling
Explant after sterilization is seeded in induces differentiation to cultivate on inducing culture, this inducing culture is identical with embodiment 1.
The first culture base that configures is divided in blake bottle, every bottled 30 milliliters, seal bottleneck, be to sterilize 25 minutes under 0.12MPa at pressure, explant material is inserted in the medium of the bacterium of having gone out, it is that 23 ℃, humidity are that 45% culturing room cultivates that blake bottle is placed in intensity of illumination 2200Lx, temperature, shines every day 11 hours; Newly connect material and be transferred in new medium every 4 days, repeat 2 times,, bud is changed in growth medium grow no longer during brown stain when medium.
(3) group training seedling proliferation is cultivated
The stem section of growing thickly of inducing cultivation is transferred to carries out Fast-propagation in proliferated culture medium, this proliferated culture medium is identical with embodiment 2.
The proliferated culture medium that configures is divided in blake bottle, every bottled 30 milliliters, seal bottleneck, be to sterilize 25 minutes under 0.12MPa at pressure, induced bud is inserted in the medium of the bacterium of having gone out, it is that 23 ℃, humidity are that 45% culturing room cultivates that blake bottle is placed in intensity of illumination 2800Lx, temperature, shines illumination cultivation 23 angel's seedling and bud proliferations every day 12 hours; For expanding propagation quantity, the bud seedling after propagation repeatedly is transferred in the proliferated culture medium of new configuration, the subculture condition of culture is identical with first generation condition of culture, and by production task, breeding will be bred the bud seedling during 8～20 generation and be carried out culture of rootage.
(4) group training seedling rooting is cultivated
The stem apex of the 1.5cm of subculture incubation growth stalwartness is put into root media carry out culture of rootage, this inducing culture of this root media is identical with embodiment 2.
Root media is divided in blake bottle, 25 milliliters every bottle, seal bottleneck, be to sterilize 25 minutes under 0.1MPa at pressure, it is that 21 ℃ of left and right, humidity are that 45% culturing room cultivates that blake bottle is placed in intensity of illumination 2500Lx, temperature, illumination every day was cultivated in 24 hours, can induce the formation of root in 8～10 days.Can pull out culturing room and carry out the natural daylight acclimatization and transplants when bottle seedling of taking root grows to 25～30 days.
(5) training tissue culture seedling is transplanted
take root and to pull out when a bottle seedling grows to 25～30 days in culturing room's immigration greenhouse by solar heat, the hardening 5d in the greenhouse of elder generation, open the running water hardening 2 days that adds 0.5cm after sealing, when offspring leaf color to be generated slightly has intensification, take out from blake bottle, wash away the medium of shoot root section, with 600 times of carbendazim libation at an ancient wedding ceremony roots, transplant to the vermiculite seedling-raising cup of processing with bactericidal agent, 3 strains of planting of each seedling-raising cup, spread and sink in after cultivation permeable, be placed in the little shed of building in the greenhouse, 20 ℃ of temperature, the spraying of sheltering from heat or light in early stage, humidity remains on 80%, taking off gradually film after 10 days ventilates, throw off little shed canopy film after 15 days, spray during this period 1000 times of carbendazim solution spray nursery stocks, when nursery stock obviously grows, the nursery stock individual plant is moved into water, husky, the decomposed manure volume ratio is in the nutrient matrix of 1: 1: 0.5, move into outside the greenhouse in shade, make nursery stock adapt to gradually the open country growing environment, grow to the 10cm left and right Deng seedling in nutritive cube, root system band soil mud enters the land for growing field crops, cultivation management is with reference to the field seedling raising method.
Claims (2)
1. the U.S. Malus spectabilis tissue-culturing rapid propagation of pearl seedling-cultivating method, it is characterized in that, it comprises the following steps: the U.S. Malus spectabilis treelet terminal bud of the annual pearl in (1) raw-material processing clip land for growing field crops or lateral bud, respectively with stand-by as explant after alcohol, mercuric chloride solution, hydrogen peroxide surface sterilization sterilization; (2) first culture is seeded in described explant on inducing culture and cultivates, this inducing culture is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.2～0.3mg, condition of culture is: temperature is 23 ± 2 ℃, intensity of illumination 2000～2500Lx, light application time 10～13 hours/day, humidity are 40%-50%; (3) propagation is cultivated the bud seedling after grow thickly stem apex or the propagation of first culture is transferred in proliferated culture medium and is cultivated, this proliferated culture medium is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.6～0.8mg indolebutyric acid 0.1～0.2mg, condition of culture is: temperature is 23 ± 2 ℃, intensity of illumination 2500～3000Lx, light application time 10～13 hours/day, humidity are 40%～50%; (4) culture of rootage will be bred 1.5～3cm stem section of cultivating and be put into root media and cultivate, this root media is for to add following raw material and proportioning thereof to make in the 1/2MS minimal medium: white granulated sugar 10g agar powder 4.5g indolebutyric acid 0.1～0.2mg, condition of culture is: 23 ± 2 ℃ of temperature, humidity 40～50%, intensity of illumination 2500～30000Lx, light application time 24 hours/day; (5) acclimatization and transplants comprises following link: close bottle exercise, uncork exercise, be transplanted to vermiculite matrix, be transplanted in the Nutrition Soil that is comprised of soil, sand, decomposed manure.
2. the U.S. Malus spectabilis tissue-culturing rapid propagation of pearl according to claim 1 seedling-cultivating method, is characterized in that, described inducing culture is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.2mg; Described proliferated culture medium is for to add following raw material and proportioning thereof to make in 1 liter of MS minimal medium: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.8mg indolebutyric acid 0.2mg; Described root media is for to add following raw material and proportioning thereof to make in the 1/2MS minimal medium: white granulated sugar 10g agar powder 4.5g indolebutyric acid 0.2mg.
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| CN106212274A (en) * | 2016-07-15 | 2016-12-14 | 武汉市农业科学技术研究院林业果树科学研究所 | The method for tissue culture of special beautiful Caulis et folium euphorbiae milii |
| CN111194693A (en) * | 2020-01-19 | 2020-05-26 | 南京林业大学 | Tissue culture rapid propagation method for new variety of Chinese flowering crabapple |
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