CN103155869A - Sweet cherry rootstock Colt tissue culture method - Google Patents
Sweet cherry rootstock Colt tissue culture method Download PDFInfo
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- CN103155869A CN103155869A CN2011104561675A CN201110456167A CN103155869A CN 103155869 A CN103155869 A CN 103155869A CN 2011104561675 A CN2011104561675 A CN 2011104561675A CN 201110456167 A CN201110456167 A CN 201110456167A CN 103155869 A CN103155869 A CN 103155869A
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Abstract
The invention discloses a sweet cherry rootstock Colt tissue culture method. The method comprises the following steps that (1) an annual sweet cherry rootstock Colt is selected for primary culture, an overwintering bud phosphorus slice of the annual sweet cherry rootstock Colt is striped off to cut and take a stem apex to be inoculated on a primary culture medium for culturing, the culture medium is made by the fact that raw materials are added into a 1/2 MS, and the raw materials and proportions of the raw materials are white granulated sugar of 25g, agar powder of 5g, 6-benayl aminopurine of 0.5-2.0mg, and indolebutyric acid of 0.01-0.10mg; (2) subculturing is transferred into an enrichment medium to be cultured, the enrichment medium is made by the fact that raw materials are added into a 1/2 MS enrichment medium of one liter, and the raw materials and proportions of the raw materials are white granulated sugar of 25g, agar powder of 5g, 6-benayl aminopurine 1.0-1.5mg, and indolebutyric acid of 0.15-0.20mg; (3) rooting culture is transferred into a rooting medium to be cultured, the rooting medium is made by the fact that raw materials are added into a 1/2 MS minimal medium, and the raw materials and proportions of the raw materials are white granulated sugar of 25g, agar powder of 5g, indolebutyric acid of 0.25-0.75mg; and (4) 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 sweet cherry rootstock Colt by plant tissue culture technique.
Background technology
sweet cherry rootstock Colt introduces from luxuriant woods experiment station, Britain east, by campstool moral and cherry cross breeding, be the good cherry rootstock of Britain, be fit to the garden high-yield cultivating use of large tracts of land cherry, this stock well developed root system, strong adaptability, drought resisting, stationarity is good, the plant branch is many, be clump shape growth, grafting affinity is strong, seedling growth is healthy and strong, field planting Jian Yuan garden is mutually neat, the plant strain growth character of fruitage is good, have yielding ability and yield stability preferably, by investigating from different cherry variety grafting growths, each kind all shows good affinity and proterties is stable, disease resistance in cultivation management, the tree bulk-growth is vigorous, the early bearing high yield, it is neat etc. that the garden resembles, show good cultivation effect, has wide developing market prospect.
In recent decades, along with the continuous progress of domestic and international biotechnology and perfect, its application aspect the fruit tree industry of flowers and plants is also more and more extensive, and this not only greatly promotes the flourish of the fruit tree industry of flowers and plants, has injected life and vigor also for the various places related industry.Particularly utilize the Plant Tissue Breeding quick propagating technology of one of biotechnology method, breeding to famous, excellent and new excellent fruit tree flower plants and nursery stock, shown powerful development prospect, many famous and precious, good, rare and kinds of newly breeding, usually because be difficult to rapid amount reproduction with conventional method, perhaps reproduction rate is extremely low, can not in time satisfy the demand of production ﹠ marketing, is perplexing to a certain extent the lifting with level of industry of developing rapidly of the fruit tree industry of flowers and plants.And use biotechnology to carry out Fast-propagation, and have the characteristics such as produce in the anniversary, reproduction coefficient is high, genetic character is relatively stable, to preserving clone germ plasm resource, produce virus-free nursery stock in enormous quantities, reduce nursery stock production cost etc. and have realistic meaning.
In the prior art, sweet cherry rootstock Colt group cultivating seedling and propagating coefficient is low, the difficulty of taking root, and transplanting survival rate is low makes it can't realize large-scale industrialized production always.
Summary of the invention
Technical problem to be solved by this invention is to provide the method for tissue culture of a kind of sweet cherry rootstock Colt.
Technical scheme of the present invention is: a kind of sweet cherry rootstock Colt method for tissue culture, it comprises the following steps: choose annual sweet cherry rootstock Colt secateurs before (1) just culture spring rudiment and become 2 centimeter length band bud sections, respectively with the vertical flat solution of evil, HgCl
2After solution disinfection, this band bud section of strip off bud phosphorus sheet of surviving the winter cuts stem apex, be inoculated in just and cultivate on the culture base, described just culture base is that 1/2MS adds following raw material and formula rate thereof: white granulated sugar 25g agar powder 5g 6-benzyl aminopurine 0.5～2.0mg indolebutyric acid 0.01～0.10mg, condition of culture: 20～28 ℃ of temperature, intensity of illumination 2000～2800Lx, light application time 10～12 hours/day, humidity 40～70%; (2) subculture is cultivated the stem apex of growing thickly of first culture in step (1) 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 1/2MS minimal medium: white granulated sugar 25g agar powder 5g 6-benzyl aminopurine 1.0～1.5mg indolebutyric acid 0.15～0.20mg, condition of culture is: temperature is 20～28 ℃, intensity of illumination 2000～2800Lx, light application time 10～12 hours/day, humidity are 40%～70%; (3) cultivate in the stem apex access root media of culture of rootage with propagation cultivation in step (2), this root media is for to add following raw material and proportioning thereof to make in 1 liter of 1/2MS minimal medium: white granulated sugar 25g agar powder 5g indolebutyric acid 0.25～0.75mg, condition of culture is: 20～28 ℃ of temperature, humidity 40～70%, intensity of illumination 2000～2800Lx, light application time 10～12 hours/day; (4) hardening cultivation and transplanting comprise following link: close bottle exercise, uncork exercise, be transplanted to vermiculite matrix, be transplanted in the Nutrition Soil that is comprised of grail, rural area soil, cow dung.
Preferably, described just culture base is for to add following raw material and proportioning thereof to make in 1 liter of 1/2MS minimal medium: white granulated sugar 25g agar powder 5g 6-benzyl aminopurine 1.0mg indolebutyric acid 0.1mg; Described proliferated culture medium is for to add following raw material and proportioning thereof to make in 1 liter of 1/2MS minimal medium: white granulated sugar 25g agar powder 5g 6-benzyl aminopurine 1.0mg 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 25g agar powder 5g indolebutyric acid 0.5mg.
Set up, expand relation or the impact of the stage culture effect such as numerous subculture cultivations, culture of rootage for medium composition and proportioning thereof and aseptic strain, the inventor has done experimental study, for further elaboration the present invention, experimental data and result of study is described below:
1, different minimal mediums are on the just impact of culture of test-tube plantlet.
The examination material is chosen 1 year livings resting shoot, clip branch section (about 2cm long, be with a bud), and detergent water soaks that after 3 hours, flowing water rinsed 10 minutes, uses mercuric chloride (HgCL
2) sterilized 8 minutes, aseptic water washing 4 times, then peel off outer field scale with the ophthalmology tweezers on superclean bench, stem apex by the 1.0mm-2.0mm size is positioned on different culture media, all adds 25g sucrose, 5g agar, add on this basis the growth regulator of variety classes and concentration, cultivation temperature is 25 ± 2 ℃, and intensity of illumination is 2000-3000lx, light application time 12 hours.
Shown by table 1, there is certain difference in three kinds of dissimilar medium to the sprouting of the little stem apex of sweet cherry rootstock Colt, and the medium of three types all reaches significant difference, all can induce stem apex to sprout, its inductivity of MS, 1/2MS is higher, be respectively 77% and 86.3%, inoculate and began in about 5 days to sprout, be grown in the stem apex performance of MS better, blade is light green, on the 1/2Ms medium, the seedling growth gesture is stronger, and L cultivates substantially, and stem apex is sprouted slower.After inoculating a week, just begin to sprout, blade is little yellowish green, little volume, and growth potential is general.
The different minimal mediums of table 1 are on the just impact of culture of Colt test-tube plantlet
| Medium | Inoculation number (individual) | Survive bud number (individual) | Survival rate (%) |
| MS | 1000 | 770 | 77 |
| 1/2MS | 1000 | 863 | 86.3 |
| L | 1000 | 310 | 31.0 |
2, the impact of hormon on the test-tube plantlet cultivation
(1) hormon and concentration are on the just impact of culture of Colt.by finding out in table 2, there is significant difference in little stem apex germination rate on the medium of hormon concentration combination, when the concentration of 6-benzyl aminopurine (6-BA) is 0.5-1.0mg/L, along with the increase germination rate of 6-BA concentration is increase trend, concentration one timing as 6-BA, the germination rate of indolebutyric acid (IBA) under variable concentrations do not have certain rule, this explanation stem apex germination rate depends on both synergies, when 6-BA is 1.0mg/l, when IBA is 0.1mg/l, the germination rate of stem apex is higher than reaching 85.0%, the shoot growth speed, highly on average in the 4-5cm left and right, plant is also very healthy and strong, when 6-BA reaches 2.0mg/l, germination rate is also higher, but the plant growing way is bad, young sprout is of low quality.
| 6-BA(mg/l) | IBA(mg/l) | Inoculation bud number | Sprout germinative number |
| 0.50 | 0.01 | 100 | 34 |
| 0.50 | 0.10 | 100 | 30 |
| 1.00 | 0.01 | 100 | 74 |
| 1.00 | 0.10 | 100 | 85 |
| 2.00 | 0.01 | 100 | 50 |
| 2.00 | 0.10 | 100 | 41 |
[0017]The impact on the just culture bud sprouting of Colt test-tube plantlet of table 2 hormone kind and concentration
(2) hormon and the concentration impact on the cultivation of test-tube plantlet propagation
After one month that the Colt test-tube plantlet is cultivated in first culture base, being cut into length with scissors is that the stem with bud of about 1cm is inoculated in 9 kinds of proliferated culture mediums, cultivate each processing of investigation in 30 days to the cultivation effect of test-tube plantlet, found out by table 3, maximum hormone ratio to Colt stem apex propagation is 6-BA1.0mg/l, IBA0.2mg/l, and growth coefficient reaches 6.00, finds in test that 6-BA is that Colt stem apex propagation is cultivated necessary, it can break apical dominance, promotes axillalry bud to send out.
Table 3 6-BA and the IBA impact on bud propagation
| 6-BA(mg/l) | IBA(mg/l) | Bud propagation multiple |
| 0.50 | 0.05 | 2.92 |
| 0.50 | 0.15 | 2.89 |
| 0.50 | 0.20 | 3.92 |
| 1.00 | 0.05 | 4.67 |
| 1.00 | 0.15 | 5.57 |
| 1.00 | 0.20 | 6.00 |
| 1.50 | 0.05 | 4.76 |
| 1.50 | 0.15 | 4.50 |
| 1.50 | 0.20 | 3.64 |
(3) impact of sugared concentration on Colt test-tube plantlet propagation
The impact of the sugared concentration of table 4 on Colt test-tube plantlet propagation
| Sugar concentration g/l | 10 | 15 | 20 | 25 | 30 | 35 |
| The rate of increase (%) | 7.6 | 12.8 | 27.0 | 35.0 | 28.3 | 14.1 |
| Height of seedling (cm) | 2.0 | 3.7 | 4.2 | 5.2 | 5.3 | 3.4 |
[0025]Play an important role as the sucrose of the energy and the osmotic adjustment propagation to test-tube plantlet in Plant Tissue Breeding, as can be seen from Table 4, when sucrose contains 10g/l, average plant height 2.0cm, the symptoms such as poor growth appears in test-tube plantlet, and partial blade turns to be yellow, and seedling is thinner and more delicate, the rate of increase is 7.6%, illustrates that sugar contains quantity not sufficient.When cane sugar content was 20-30g/l, average plant height surpassed 6.0cm, and growth potential is strong, leaf green, and stem is sturdy, medium altitude, growth coefficient reaches more than 35.0%, and the growth coefficient of test-tube plantlet increases with the rising of sucrose concentration.When adding the sucrose of 35g/l in medium, average plant height is 3.4cm, and growth coefficient is that 14.1% blade is yellow, growth potential is weakened, illustrate that the osmotic pressure in medium reduces, be unfavorable for that plant absorbs the very nutriment of formula, so the sucrose of Colt Shoot Tip Culture choice for use 20-30g/l is for well.
3, the impact of IBA concentration on rooting of vitro seedling
Found out by table 5, the Colt test-tube plantlet can be taken root under the IBA variable concentrations, cultivating 10 days on the 1/2MS+IBA0.5 medium is the differentiation of visible root, produce several the roots of penetrating shape, along with the root ramp, two Zhou Houke grow to 6-7cm, and rooting rate reaches 100%, leaf dark green, root system are thick and a small amount of lateral root arranged.When IBA reached 1.0mg/l, rhizome section brought out callus, has suppressed the growth of root system and stem apex, be unfavorable for taking root and the normal growth of stem apex, and not the communicating of the root that is produced by callus and stem, be unfavorable for transplant survival.
The impact of table 5 IBA variable concentrations on rooting of vitro seedling
| IBA concentration | Rooting rate (100%) |
| 0.1 | 43.2 |
| 0.25 | 86.4 |
| 0.50 | 100.0 |
| 0.75 | 79.8 |
| 1.0 | 54.7 |
4, different humidity, the matrix impact on the test-tube plantlet rooting culture
Under the 3000-5000lx illumination condition, the impact that different relative moisture, different substrates are grown test-tube plantlet growth.
The impact on test-tube seedling transplanting of table 6 humidity and matrix
Test shows, the environmental condition that suitable Colt test-tube plantlet domestication is transplanted is: illumination 3000-5000lx, and relative moisture 80-90%, optimal grafting matrix are vermiculite, transplanting mode can be selected pot planting or the transition of furrow face, and best transplant time is that March is to April.
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 suitable Fast-propagation, propagation and rooting efficiency are good, make the rooting rate of Colt test-tube plantlet 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 the 80.5-84.8% 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, can be used for Fast-propagation.
Embodiment
Embodiment 1
The tissue-culturing rapid propagation seedling-cultivating method of a kind of sweet cherry rootstock Colt, it comprises the following steps:
(1) raw-material processing
Choose robust growth, the full Colt annual branch of sprout before the rudiment in spring, be cut into 2 centimetres of stem with bud in the laboratory, cleaned 10-15 minute with the vertical flat solution of 1000 times of evils, then rinsed 10 minutes in flowing water, put into again the 150ml triangular flask, and add the 0.1%HgCL of new configuration on superclean bench
2Solution disinfection 8 minutes is used the sterile water soaking and washing 4 times at last, and each about 5 minutes, with the survive the winter phosphorus sheet of bud of sterilization scalpel strip off, cut fast stem apex in ultra-clean work, stand-by as explant.
(2) first culture
Described explant is seeded in just cultivates on the culture base, this medium is for to add following raw material and proportioning thereof to make in 1 liter of 1/2MS minimal medium.
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 40% culturing room cultivates that blake bottle is placed in intensity of illumination 2000Lx, 20 ℃ of temperature, humidity, shines cultivation in 10 hours every day.
(3) propagation is cultivated
The stem apex of growing thickly of first culture 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 1/2MS 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 20 ℃, humidity are that 40% culturing room cultivates that blake bottle is placed in intensity of illumination 2000Lx, temperature, shone every day 10 hours, for expanding propagation quantity, 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 propagation is carried out culture of rootage after needs quantity.
(4) culture of rootage
Cultivate in 2cm left and right stem apex access root media with the propagation cultivation, this root media is for to add following raw material and proportioning thereof to make in the 1/2MS minimal medium.
White granulated sugar 25g
Agar powder 5g
Indolebutyric acid 0.25mg
Root media is divided in blake bottle, 25 milliliters every bottle, seals bottleneck, be to sterilize 25 minutes under 0.1MPa at pressure, it is that 20 ℃, humidity are that 40% culturing room cultivates that blake bottle is placed in intensity of illumination 2000Lx, temperature, and illumination every day was cultivated in 10 hours, can induce the formation of root in 8 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) acclimatization and transplants
it is 3000Lx that the test tube of taking root moves into intensity of illumination, took exercise 3-4 days in the plastic tunnel of 25 ℃ of left and right of temperature, then open bottle cap, fill with 0.5cm water in blake bottle, took exercise 2-3 days, treat that the test-tube plantlet blade color slightly has intensification, when obvious growth sign is arranged, take out from blake bottle, clean the medium on root, with 600 times of carbendazim immersion treatment seedling roots, move into the vermiculite nutrient matrix, and spray nursery stocks with 1000 times of carbendazim, air humidity remains on 70%, ventilate gradually after 3 days, light intensity increases to 15000Lx gradually, when nursery stock has obvious growth phenomenon, nursery stock is moved into matrix be soil: sand: the decomposed manure nutrient matrix of 1: 1: 0.5, make nursery stock adapt to the open country growing environment, grow to the 10cm left and right Deng seedling, and can adapt to the open country environment fully the time, 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 tissue-culturing rapid propagation seedling-cultivating method of a kind of sweet cherry rootstock Colt, it comprises the following steps:
(1) raw-material processing
Raw-material processing: choose robust growth, the full Colt annual branch of sprout before the rudiment in spring, be cut into 2 centimetres of stem with bud in the laboratory, cleaned 10-15 minute with the vertical flat solution of 1000 times of evils, then rinsed 10 minutes in flowing water, put into again the 150ml triangular flask, and add the 0.1%HgCl of new configuration on superclean bench
2Solution disinfection 8 minutes is used the sterile water soaking and washing 4 times at last, each about 5 minutes, with the survive the winter phosphorus sheet of bud of sterilization scalpel strip off, cuts fast stem apex in ultra-clean work, with being that explant is stand-by.
(2) first culture
Described explant is seeded in just cultivates on the culture base, this medium is for to add following raw material and proportioning thereof to make in 1 liter of 1/2MS minimal medium.
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 70% culturing room cultivates that blake bottle is placed in intensity of illumination 2800Lx, 28 ℃ of temperature, humidity, shines cultivation in 12 hours every day.
(3) propagation is cultivated
The stem apex of growing thickly of first culture 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 1/2MS 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 28 ℃, humidity are that 70% culturing room cultivates that blake bottle is placed in intensity of illumination 2800Lx, temperature, shone every day 12 hours, for expanding propagation quantity, 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 propagation is carried out culture of rootage after needs quantity.
(4) culture of rootage
Cultivate in 2cm left and right stem apex access root media with the propagation cultivation, this root media is for to add following raw material and proportioning thereof to make in the 1/2MS minimal medium.
White granulated sugar 25g
Agar powder 5g
IBA 0.75mg
Root media is divided in blake bottle, 25 milliliters every bottle, seals bottleneck, be to sterilize 25 minutes under 0.1MPa at pressure, it is that 28 ℃, humidity are that 70% culturing room cultivates that blake bottle is placed in intensity of illumination 2800Lx, temperature, and illumination every day was cultivated in 12 hours, can induce the formation of root in 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) acclimatization and transplants
it is 5000Lx that the test tube of taking root moves into intensity of illumination, took exercise 4 days in the plastic tunnel of 25 ℃ of left and right of temperature, then open bottle cap, fill with 1cm water in blake bottle, took exercise 2-3 days, treat that the test-tube plantlet blade color slightly has intensification, when obvious growth sign is arranged, take out from blake bottle, clean the medium on root, with 800 times of carbendazim immersion treatment seedling roots, move into the vermiculite nutrient matrix, and spray nursery stocks with 1000 times of carbendazim, air humidity remains on 90%, ventilate gradually after 3 days, light intensity increases to 15000Lx gradually, when nursery stock has obvious growth phenomenon, it is water that nursery stock is moved into matrix: sand: the decomposed manure nutrient matrix of 1: 1: 0.5, make nursery stock adapt to the open country growing environment, grow to the 10cm left and right Deng seedling, and can adapt to the open country environment fully the time, 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 tissue-culturing rapid propagation seedling-cultivating method of a kind of sweet cherry rootstock Colt, it comprises the following steps:
(1) raw-material processing
Choose robust growth, the full Colt annual branch of sprout before sweet rudiment in spring, be cut into 2 centimetres of stem with bud in the laboratory, cleaned 10-15 minute with the vertical flat solution of 1000 times of evils, then rinsed 10 minutes in flowing water, put into again the 150ml triangular flask, and add the 0.1%HgCL of new configuration on superclean bench
2Solution disinfection 8 minutes is used the sterile water soaking and washing 4 times at last, each about 5 minutes, with the survive the winter phosphorus sheet of bud of sterilization scalpel strip off, cuts fast stem apex in ultra-clean work, with being that explant is stand-by.
(2) first culture
Described explant is seeded in just cultivates on the culture base, this medium is for to add following raw material and proportioning thereof to make in 1 liter of 1/2MS minimal medium.
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 55% culturing room cultivates that blake bottle is placed in intensity of illumination 2500Lx, 24 ℃ of temperature, humidity, shines cultivation in 11 hours every day.
(3) propagation is cultivated
The stem apex of growing thickly of first culture 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 1/2MS 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 24 ℃, humidity are that 55% culturing room cultivates that blake bottle is placed in intensity of illumination 2500Lx, temperature, shone every day 11 hours, for expanding propagation quantity, 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 propagation is carried out culture of rootage after needs quantity.
(4) culture of rootage
Cultivate in 2cm left and right stem apex access root media with the propagation cultivation, this root media is for to add following raw material and proportioning thereof to make in the 1/2MS minimal medium.
White granulated sugar 25g
Agar powder 5g
Indolebutyric acid 0.5mg
Root media is divided in blake bottle, 25 milliliters every bottle, seals bottleneck, be to sterilize 25 minutes under 0.1MPa at pressure, it is that 25 ℃, humidity are that 55% culturing room cultivates that blake bottle is placed in intensity of illumination 2500Lx, temperature, and illumination every day was cultivated in 11 hours, can induce the formation of root in 9 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) acclimatization and transplants
it is 4000Lx that the test tube of taking root moves into intensity of illumination, took exercise 3 days in the plastic tunnel of 25 ℃ of left and right of temperature, then open bottle cap, fill with 0.8cm water in blake bottle, took exercise 3 days, treat that the test-tube plantlet blade color slightly has intensification, when obvious growth sign is arranged, take out from blake bottle, clean the medium on root, with 700 times of carbendazim immersion treatment seedling roots, move into the vermiculite nutrient matrix, and spray nursery stocks with 1000 times of carbendazim, air humidity remains on 80%, ventilate gradually after 3 days, light intensity increases to 15000Lx gradually, when nursery stock has obvious growth phenomenon, it is water that nursery stock is moved into matrix: sand: the decomposed manure nutrient matrix of 1: 1: 0.5, make nursery stock adapt to the open country growing environment, grow to the 10cm left and right Deng seedling, and can adapt to the open country environment fully the time, 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. a sweet cherry rootstock Colt method for tissue culture, is characterized in that, it comprises the following steps: choose annual sweet cherry rootstock Colt secateurs before (1) just culture spring rudiment and become 2 centimeter length band bud sections, respectively with the vertical flat solution of evil, HgCl
2After solution disinfection, this band bud section of strip off bud phosphorus sheet of surviving the winter cuts stem apex, be inoculated in just and cultivate on the culture base, described just culture base is that 1/2MS adds following raw material and formula rate thereof: white granulated sugar 25g agar powder 5g 6-benzyl aminopurine 0.5～2.0mg indolebutyric acid 0.01～0.10mg, condition of culture: 20～28 ℃ of temperature, intensity of illumination 2000～2800Lx, light application time 10～12 hours/day, humidity 40～70%; (2) subculture is cultivated the stem apex of growing thickly of first culture in step (1) 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 1/2MS minimal medium: white granulated sugar 25g agar powder 5g 6-benzyl aminopurine 1.0～1.5mg indolebutyric acid 0.15～0.20mg, condition of culture is: temperature is 20～28 ℃, intensity of illumination 2000～2800Lx, light application time 10～12 hours/day, humidity are 40%～70%; (3) cultivate in the stem apex access root media of culture of rootage with propagation cultivation in step (2), this root media is for to add following raw material and proportioning thereof to make in 1 liter of 1/2MS minimal medium: white granulated sugar 25g agar powder 5g indolebutyric acid 0.25～0.75mg, condition of culture is: 20～28 ℃ of temperature, humidity 40～70%, intensity of illumination 2000～2800Lx, light application time 10～12 hours/day; (4) hardening cultivation and transplanting comprise following link: close bottle exercise, uncork exercise, be transplanted to vermiculite matrix, be transplanted in the Nutrition Soil that is comprised of grail, rural area soil, cow dung.
2. sweet cherry rootstock Colt method for tissue culture according to claim 1, it is characterized in that, described just culture base is for to add following raw material and proportioning thereof to make in 1 liter of 1/2MS minimal medium: white granulated sugar 25g agar powder 5g 6-benzyl aminopurine 1.0mg indolebutyric acid 0.1mg; Described proliferated culture medium is for to add following raw material and proportioning thereof to make in 1 liter of 1/2MS minimal medium: white granulated sugar 25g agar powder 5g6-benayl aminopurine 1.0mg 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 25g agar powder 5g indolebutyric acid 0.5mg.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103416308A (en) * | 2013-08-08 | 2013-12-04 | 巴中市光雾山植物研究所 | Tissue culture rapid propagation method for wild sweet cherry trees |
| CN103688852A (en) * | 2013-10-15 | 2014-04-02 | 陕西理工学院 | Method for rapidly breeding large cherry Gisela |
| CN104145814A (en) * | 2014-07-24 | 2014-11-19 | 四川农业大学 | Method for obtaining regeneration plants by stem tissue culture of cerasus cerasoides (var. cerasoides) |
| CN105359977A (en) * | 2015-12-01 | 2016-03-02 | 南京林业大学 | Tissue culture rapid propagation method for Cerasus xueluoensis C.H.Nan & X.R.Wang |
| CN109430062A (en) * | 2018-12-29 | 2019-03-08 | 南京大花滩生物科技有限公司 | A kind of tissue culture method that large cherry is quickly bred |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103416308A (en) * | 2013-08-08 | 2013-12-04 | 巴中市光雾山植物研究所 | Tissue culture rapid propagation method for wild sweet cherry trees |
| CN103416308B (en) * | 2013-08-08 | 2015-06-10 | 巴中七彩林业科技有限公司 | Tissue culture rapid propagation method for wild sweet cherry trees |
| CN103688852A (en) * | 2013-10-15 | 2014-04-02 | 陕西理工学院 | Method for rapidly breeding large cherry Gisela |
| CN103688852B (en) * | 2013-10-15 | 2016-08-31 | 陕西理工学院 | A kind of large cherry Gisela fast breeding method |
| CN104145814A (en) * | 2014-07-24 | 2014-11-19 | 四川农业大学 | Method for obtaining regeneration plants by stem tissue culture of cerasus cerasoides (var. cerasoides) |
| CN104145814B (en) * | 2014-07-24 | 2017-02-22 | 四川农业大学 | Method for obtaining regeneration plants by stem tissue culture of cerasus cerasoides (var. cerasoides) |
| CN105359977A (en) * | 2015-12-01 | 2016-03-02 | 南京林业大学 | Tissue culture rapid propagation method for Cerasus xueluoensis C.H.Nan & X.R.Wang |
| CN105359977B (en) * | 2015-12-01 | 2017-12-15 | 南京林业大学 | One kind snow falls cherry quick breeding by group culture method |
| CN109430062A (en) * | 2018-12-29 | 2019-03-08 | 南京大花滩生物科技有限公司 | A kind of tissue culture method that large cherry is quickly bred |
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