CN1922986A - Method for obtaining Brassica oleracea var. acephala DC. small spore regenerated plants and special culture medium - Google Patents
Method for obtaining Brassica oleracea var. acephala DC. small spore regenerated plants and special culture medium Download PDFInfo
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- CN1922986A CN1922986A CNA2006101129975A CN200610112997A CN1922986A CN 1922986 A CN1922986 A CN 1922986A CN A2006101129975 A CNA2006101129975 A CN A2006101129975A CN 200610112997 A CN200610112997 A CN 200610112997A CN 1922986 A CN1922986 A CN 1922986A
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Abstract
The invention relates to a method for obtaining collard regenerated plant and special cultivation base. Wherein, said method comprises (1), planting the collard into induced cultivation base, until the density is 5*10<4-5*10<5n/mL, at 30-35Deg.C and in dark, treating it at high temperature for 24-72hours, and under 24-26Deg. C and in dark, inducing idiosome; (2), when the idiosome has been formed, under 24-26Deg. C, 1500-2500Lux, cultivating for 5-10days while each day lights 14-18hours; then grafting the seed into differentiation cultivation base, to be cultivated in same conditions; (3) when grows out branch and leaf, transplanting it into rooting cultivation base, under 24-26Deg. C, 1500-2500Lux, lighting 14-18hours each day, to root, to obtain the collard regenerated plant.
Description
Technical field
The present invention relates to microspores culture method and the special culture media thereof of plant, particularly relate to the method and the special culture media thereof that obtain the kale sporule regeneration plant.
Background technology
Plant has hybrid vigour, and therefore present crop breeding stresses in crossbreeding.Can utilize the growth vigor of hybrid to guarantee the crop varieties disease-resistant high yield so on the one hand, owing to the complex inheritance background of hybrid, make its use of should not reserving seed for planting, thereby can protect hybrid development person's interests on the other hand.In crossbreeding, the genetic background of used this material of father and mother must be isozygotied, and just can obtain the hybrid of proterties unanimity after breeding.And to obtain the parent material that genetic background is isozygotied, all must be in many crops by artificial auxiliary pollination self, just can reach relatively through the 6-10 year and to isozygoty, and per generation screening material because heterozygosity genetic background, the intensity of selecting can not too high (avoiding good recessive character to lose), thereby cause colony's amount of screening big, select reliability of material and stable low, therefore very time-consuming, the effort of this traditional crossbreeding means.Male sex-cell-pollen of plant, owing to only have a cover parent chromosome through postmeiotic, it is the haploidy cell, can obtain haplobiont by flower pesticide or unmature pollen (microspore) cultivation, can become the double haploid material by chromosome doubling again, can make the genetic background of this plant reach complete homozygotic state like this.In the time of can overcoming heterozygous state so on the one hand, dominant gene helps the expression of recessive mutation or genetic recombination proterties to the shielding action of recessive gene, obtains more material type; On the other hand can one the step obtain to isozygoty material, and then can be used as parent material and be applied in the crossbreeding of crop.Usually only need the 1-2 year by the microspores culture material that obtains to isozygoty, not only quickened breeding process greatly, make breeding efficiency also obtain significantly to improve, therefore in the world the haploid breeding research of crop is paid much attention to, and on various crop such as wheat, barley, rape and Chinese cabbage, set up good haploid breeding system.
View and admire the ornamental foliage plant that belongs to Cruciferae Brassicas broccoli class with kale (Brassica oleracea var.acephala), have another name called " leaf tree peony ", its painted leaf-head is prostrate, as tree peony in full bloom, elegant and poised, bright-coloured beautiful, and cold resistance is very strong, has become the main view ground cover plant in autumn and winter to early spring recently in many cities.This plant hybrid vigour is obvious, and growth potential is strong, and disease resistance is good, and as ornamental plants, the first generation of hybrid can guarantee the uniformity of population growth characteristic to the full extent, and regularity and ornamental value all are largely increased, at present, crossbreeding has become main breeding technique.But, because broccoli class plant is 2 years living plants, need long-time strict low temperature vernalization just can bloom, adopt the method for selfing repeatedly to obtain to isozygoty the material require 8-10 year in the traditional breeding method, not only take length, efficient is low, and is difficult to catch for the recessive character with ornamental value.Up to now, the isolated microspore culture technique of relevant kale and the application in breeding thereof do not appear in the newspapers both at home and abroad as yet.
Summary of the invention
The purpose of this invention is to provide a kind of medium that is used to cultivate the kale sporule regeneration plant.
The medium that is used to cultivate the kale sporule regeneration plant provided by the present invention, form by embryoid induction medium, differential medium and 3 kinds of medium of root media:
Wherein, the prescription of embryoid induction medium is: KNO
3100-150mg, Ca (NO
3)
24H
2O 450-550mg, MgSO
47H
2O 100-150mg, KH
2PO
4100-150mg, FeSO
47H
2O 27.5-28mg, EDTANa
237-37.5mg, KI 0.8-0.85mg, CoCl
26H
2O 0.02-0.03mg, H
3BO
36-6.5mg, Na
2MoO
42H
2O0.2-0.3mg, MnSO
44H
2O 22-22.5mg, CuSO
45H
2O 0.02-0.03mg, ZnSO
47H
2O 8.4-8.8mg, inositol 80-120mg, nicotinic acid 4-6mg, pyridoxine 0.4-0.6mg, thiamine 0.4-0.6mg, glycine 1.8-2.2mg, folic acid 0.4-0.6mg, vitamin h 0.04-0.06mg, glutamine 700-900mg, serine 80-120mg, glutathione 25-35mg, 6-benzyladenine (6-BA) 0.8-1.2mg, sucrose 100-150g, active carbon 0.10-0.30g, water is settled to 1L, pH 5.8-5.9;
The prescription of differential medium is: NH
4NO
31600-1700mg, KNO
31850-1950mg, CaCl
22H
2O400-480mg, MgSO
47H
2O 350-400mg, KH
2PO
4150-200mg, FeSO
47H
2O 27.5-28mg, EDTANa
237-37.5mg, KI 0.8-0.85mg, CoCl
26H
2O 0.02-0.03mg, H
3BO
36-6.5mg, Na
2MoO
42H
2O 0.2-0.3mg, MnSO
44H
2O 22-22.5mg, CuSO
45H
2O 0.02-0.03mg, ZnSO
47H
2O 8.4-8.8mg, inositol 80-120mg, nicotinic acid 0.4-0.6mg, pyridoxine 0.4-0.6mg, thiamine 0.08-0.12mg, glycine 1.8-2.2mg, sucrose 25-35g, agar 8-12g, water is settled to 1L, pH 5.8-5.9;
The prescription of root media is: NH
4NO
3800-850mg, KNO
3900-1000mg, CaCl
22H
2O200-240mg, MgSO
47H
2O 175-200mg, KH
2PO
475-100mg, FeSO
47H
2O 27.5-28mg, EDTANa
237-37.5mg, KI 0.8-0.85mg, CoCl
26H
2O 0.02-0.03mg, H
3BO
36-6.5mg, Na
2MoO
42H
2O 0.2-0.3mg, MnSO
44H
2O 22-22.5mg, CuSO
45H
2O 0.02-0.03mg, ZnSO
47H
2O 8.4-8.8mg, inositol 80-120mg, nicotinic acid 0.4-0.6mg, pyridoxine 0.4-0.6mg, thiamine 0.08-0.12mg, glycine 1.8-2.2mg, sucrose 10-20g, agar 8-10g, water is settled to 1L, pH 5.8-5.9.
Second purpose of the present invention provides a kind of method that obtains the kale sporule regeneration plant.
The method of acquisition kale sporule regeneration plant provided by the present invention may further comprise the steps:
1) microspore is inoculated in the embryoid induction medium, to final concentration be 5 * 10
4-5 * 10
5Individual/mL, after then high temperature stress is handled 24-72 hour under 30-35 ℃, dark condition, change inducing embryoid body under 24-26 ℃, dark condition again over to;
2) treat that torpedo stage to cotyledon period embryoid forms after, embryoid is earlier continued to cultivate 5-10 days under 24-26 ℃, 1500-2500Lux, 14-18 hour illumination/sky condition, again embryoid is inoculated in the differential medium, under the same conditions cultivation;
3) grow stem, leaf after, plant is transferred in the root media, under 24-26 ℃, 1500-2500Lux, 14-18 hour illumination/sky condition, carry out culture of rootage, obtain the kale regeneration plant.
In above-mentioned cultural method, in the step 1) preferred microspore separate self-contained 60% above monokaryon keep to the side phase microspore cell and 40% following dicaryotic phase the microspore cell bud; The microspore concentration that is inoculated in the embryoid induction medium is preferably 10
5Individual/mL; The high temperature stress treatment conditions are preferably under 33 ℃, dark condition and handled 24 hours; The embryoid induction temperature is preferably 25 ℃.
Step 2) in torpedo stage to cotyledon period embryoid is earlier continued cultivation 7 days under 25 ℃, 2000Lux, illumination in 16 hours/sky condition.
Culture of rootage condition in the step 3) is preferably 25 ℃, 2000Lux, 16 hours illumination/skies.
In addition, for making haplobiont obtain chromosome doubling as early as possible, can carry out Methods of Ploidy Identification to the kale regeneration plant that obtains in the step 3), and be that the colchicine of 0.15-2.5% is handled 36-60h continuously with volume ratio to the growing point position of haplobiont, be preferably 48h, so that chromosome doubling.
The invention provides a kind of method and special culture media thereof that obtains the kale sporule regeneration plant.This cultural method has the following advantages: 1) can obtain embryoid fast in 93% genotype, obtain monoploid and double haploid in 87% genotype, genotypic adaptation is wide, and germ extraction rate is higher; 2) acquisition time of the material that isozygotys in the conventional breeding can be reduced to 1-2 by 8-10, and genetic background is isozygotied fully; 3) in double haploid present age, recessive gene and recombination gene proterties all can obtain expressing, and greatly help the cultivation of the new proterties of ornamental plants; 4) it is preferred to carry out high strength to the present age and the fancy points in two generations such as pattern, plant type and cold resistance etc., have efficient, save time, laborsaving, stable, reliable advantage.Cultural method of the present invention and special culture media thereof will kale breed and breed improvement in play a significant role, have a extensive future.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is a kale Isolated microspore cell of cultivating division after 3 days in the embryoid induction medium
Fig. 2 is torpedo stage, the cotyledon period embryo that is formed by microspore
Fig. 3 is the kale whole plant of being grown by embryoid
Fig. 4 is the regeneration plant of different genotype
Fig. 5 is the different genotype regeneration plant The Characters in the present age
Fig. 6 is the good new material that isozygotys " ice is clear " that optimizes
Fig. 7 is the newtype material " powder plumage " that optimizes
Embodiment
Method therefor is conventional method if no special instructions among the following embodiment, and all medium are all prepared with distilled water.
The cultivation of embodiment 1, kale sporule regeneration plant
To view and admire with kale (Brassica oleracea var.acephala) donor maternal plant and grow in the plastic tunnel with fly net, genotype has contained various types of viewing and admiring uses kale, blade profile comprises full edge roundleaf, the wave leaf, the crape leaf, shallow decomposite leaf and drastic crack leaf etc., the leaf look comprises white, and is pale yellow, pink, bright red, purplish red etc., amount to 15 parts of materials, (note: draw materials season the first tenday period of a month month at 3-6 whole, in time beating blooms bear pods branch and weak branch guarantees the vigorous growth of new inflorescence) the collection microspore carries out culture in vitro with method of the present invention, and detailed process may further comprise the steps:
1) Isolated microspore is cultivated
By nuclear DAPI fluorescent staining (Colemen A W, Goff L J.Application offluorochrome to pollen biology I.Mithramycin and 4 ', 6-diamidino-2-phenylindole (DAPI) as vital stain and for quantification ofnuclear DNA.Stain Technol, 1985,60:145-154), microscope inspection, determine the developmental stage of microspore, get contain 70% monokaryon keep to the side phase microspore cell and 30% dicaryotic phase the microspore cell bud, with the bud volume ratio is liquor natrii hypochloritis's surface sterilizing of 2.5% 15 minutes, sterile water wash 3 times, with the pestle extrusion at B5 culture fluid (Gamborg et al.Plant tissue culture media.In vitro, 1976, discharge Isolated microspore 12:473-478), through 50 μ m aperture nylon net filter suspension, centrifugal 5 minutes of 1000rpm, abandon supernatant, with the B5 culture fluid precipitation that suspends again, again after the repeated washing 2 times, microspore is inoculated in the embryoid induction medium (prescription sees Table 1), to final concentration be 10
5Individual/mL, be sub-packed in the 6cm culture dish, every ware 2.5mL after then high temperature stress is handled 24 hours under 33 ℃, dark condition, changes 25 ℃, dark condition again over to and cultivates down, with inducing embryoid body.After 3 days, the microspore that is incubated in the embryoid induction medium promptly begins to occur once dividing (see figure 1), cell continues division in the incubation time afterwards, forms cell mass, proembryo, globular embryo and heart type embryo successively, forms torpedo stage, cotyledon period embryo (see figure 2) after about 25 days.
2) differentiation culture
Earlier with torpedo stage to the cultivation 7 days under 25 ℃, 2000Lux, illumination in 16 hours/sky condition of cotyledon period embryoid, again embryoid is inoculated in the differential medium (prescription sees Table 1), carry out differentiation culture under the same conditions, grow stem, leaf after about 20 days.
3) culture of rootage
The plant that grows stem, leaf is transferred in the root media (prescription sees Table 1), under 25 ℃, 2000Lux, illumination in 16 hours/sky condition, carry out culture of rootage, obtain kale regeneration plant (see figure 3).
Through observing, regeneration plant can directly be formed by this embryoid, but most of hypocotyl by this embryoid earlier forms secondary embryo, further is differentiated to form by secondary embryo again, obtains whole plant in about 45 days.Can produce several plant from an embryoid, the embryoid that yet has does not have the bud point owing to grow imperfection, can not form regeneration strain.In addition, the ability of microspores culture embryoid formation ability and embryoid regeneration plant obviously is subjected to genotypic the influence.According to statistics, in 15 parts of different genotype materials that tried, there are 14 parts of materials to obtain the microspore embryoid, account for and tried to have obtained regeneration plant in genotypic 93%, 13 part of material, account for and tried genotypic 87%.
Table 1 is used for the culture medium prescription and the sterilization method of culture in vitro kale Isolated microspore
| Form | Composition | The embryoid induction medium | Differential medium | Root media |
| A great number of elements (mg/L) molysite (mg/L) trace element (mg/L) organic (mg/L) hormone (mg/L) sucrose (g/L) active carbon (g/L) agar (g/L) pH sterilization treatment | (NH 4) 2SO 4 NH 4NO 3 KNO 3 Ca(NO 3) 2.4H 2O CaCl 2.2H 2O MgSO 4.7H 2O KH 2PO 4 FeSO 4.7H 2O EDTA.Na 2 KI CoCl 2.6H 2O H 3BO 3 Na 2MoO 4.2H 2O MnSO 4.4H 2O CuSO 4.5H 2O ZnSO 4，7H 2The plain Ser-Gln glutathione of O inositol nicotinic acid pyridoxol thiamine glycine folic acid biological 6-BA | 125 500 125 125 27.8 37.3 0.83 0.025 6.2 0.25 22.3 0.025 8.6 100 5 0.5 0.5 2.0 0.5 0.05 800 100 30 1.0 130 0.2 5.9 filtration sterilizations | 1,650 1,900 440 370 170 27.8 37.3 0.83 0.025 6.2 0.25 22.3 0.025 8.6 100 0.5 0.5 0.1 2.0 30 10 5.8 autoclavings | 825 950 220 185 85 27.8 37.3 0.83 0.025 6.2 0.25 22.3 0.025 8.6 100 0.5 0.5 0.1 2.0 15 8.0 5.9 autoclavings |
The cultivation of embodiment 2, kale sporule regeneration plant
To view and admire with kale (Brassica oleracea var.acephala) donor maternal plant and grow in the plastic tunnel with fly net, genotype has contained various types of viewing and admiring uses kale, blade profile to comprise full edge roundleaf, the wave leaf, the crape leaf, shallow decomposite leaf and drastic crack leaf etc., the leaf look comprises white, pink, bright red, purplish red and yellowish etc., amount to 16 parts of materials, gather microspore the first tenday period of a month month at 3-6 and carry out culture in vitro with method of the present invention, detailed process may further comprise the steps:
1) Isolated microspore is cultivated
By nuclear DAPI fluorescent staining, microscope inspection, determine the developmental stage of microspore, get contain 80% monokaryon keep to the side phase microspore cell and 20% dicaryotic phase cell bud, with the bud volume ratio is liquor natrii hypochloritis's surface sterilizing of 2.5% 15 minutes, sterile water wash 3 times, in the B5 culture fluid, discharge Isolated microspore with the pestle extrusion, through 50 μ m aperture nylon net filter suspension, centrifugal 5 minutes of 1000rpm abandons supernatant, with the B5 culture fluid precipitation that suspends again, after the repeated washing 2 times, microspore is inoculated in the embryoid induction medium (prescription sees Table 3) again, to final concentration be 5 * 10
4Individual/mL, be sub-packed in the 6cm culture dish, every ware 2.5mL after then high temperature stress is handled 48 hours under 30 ℃, dark condition, changes 26 ℃, dark condition again over to and cultivates down, with inducing embryoid body.After about 3-4 days, the microspore that is incubated in the embryoid induction medium promptly begins to occur once division, and cell continues division in the incubation time afterwards, forms cell mass, proembryo, globular embryo and heart type embryo successively, forms torpedo stage, cotyledon period embryo after about 25 days.
2) differentiation culture
Earlier with torpedo stage to the cultivation 10 days under 22 ℃, 2500Lux, illumination in 14 hours/sky condition of cotyledon period embryoid, again embryoid is inoculated in the differential medium (prescription sees Table 3), carry out differentiation culture under the same conditions, grow stem, leaf after about 25 days.
3) culture of rootage
After growing stem, leaf, plant is transferred in the root media (prescription sees Table 3), under 25 ℃, 2500Lux, illumination in 16 hours/sky condition, carry out culture of rootage, obtain the kale regeneration plant.
Through observing, regeneration plant can directly be formed by this embryoid, but most of hypocotyl by this embryoid earlier forms secondary embryo, further is differentiated to form by secondary embryo again, obtains whole plant in about 45 days.Can produce several plant from an embryoid, the embryoid that yet has does not have the bud point owing to grow imperfection, can not form regeneration strain.In addition, the ability of microspores culture embryoid formation ability and embryoid regeneration plant obviously is subjected to genotypic the influence.According to statistics, in 16 parts of different genotype materials that tried, there are 15 parts of materials to obtain the microspore embryoid, account for and tried to have obtained regeneration plant in genotypic 94%, 14 part of material, account for and tried genotypic 88%.
4) chromosome doubling is handled
Get the spire of the 184 strain kale regeneration plants that obtain in the step 3), utilize flow cytometer (FACSCalibur, U.S. company BD) carries out the early stage Methods of Ploidy Identification of seedling, the chromosome of regeneration plant constitutes complicated as a result, contain 25% monoploid, 43% dliploid (double haploid), all the other also have segmental polypoid and mixoplod.Is that 0.2% colchicine is handled 48h continuously to the growing point position of haplobiont with mass percentage concentration, so that chromosome doubling.The pollen situation in flowering stage is observed and is shown that the multiplying power that adds of these monoploid materials is about 60%.In addition, the ability of the spontaneously doubled haploid of the kale regeneration plant that obtains with the inventive method is also variant in different genotype, is example with the double haploid, and the ratio in different genotype is 22-67%.As seen the double haploid regeneration strain can be obtained effectively by Isolated microspore cultural method of the present invention, isozygotying of material genetic background can be realized at short notice.
5) proterties of regeneration plant is preferred
The careful agar of cleaning the regeneration plant root is gone into (see figure 4) in the greenhouse flowerpot in transfer in October then, after the growth of preserving moisture in a week, throws off the plastic foil of covering, about one month of domestication growth, and be transplanted into ground December.Along with the reduction of autumn air temperature, blade is annesl progressively, and regeneration strain colony shows the diversity (see figure 5) on leaf look, blade profile, plant type etc.With reference to Vegetable Research method (the general teaching material of national AGRICULTURAL UNIVERSITIES AND COLLEGES, the chief editor of Agricultural University Of Southwest, 1986) relevant broccoli class crop character investigation index and investigation method in, to regeneration strain present age each individual plant carry out color, plant type, blade profile, painted phase sooner or later and to view and admire the high strength of characteristic preferred the bolting phase.On the one hand, because material is the genetic background of isozygotying fully, can not occur the separation of phenotype the offspring, so the reliability of selection result is very high; On the other hand, because recessive gene and recombination gene proterties can both be expressed, therefore some new types with ornamental value may appear in colony.Through preferred, in the regeneration colony that obtains with above-mentioned breeding method, the only extremely short living type of 10cm of plant height has appearred, only 9 of its green outer numbers of sheets, the binder number is greater than 30, white, red heart, painted morning, it is extremely even to paint, and does not have mottled blade, called after " ice is clear " (see figure 6).A in addition material blade profile is the pinniform checking, purple powder, and the newtype that does not have in the donor material has appearred in called after " powder plumage " (see figure 7).Preferred strain adopts strict bagging pollination self to reserve seed for planting.Preferable material carried out examining of proterties in 1 year, can enter the hybrid preparation after further preferred.The above results shows with cultural method of the present invention and special culture media thereof not only can obtain the material that isozygotys fast, and can formulate the type of viewing and admiring that makes new advances efficiently.
Table 2 is used for culture medium prescription and the sterilization method that the kale Isolated microspore is cultivated
| Form | Composition | The embryoid induction medium | Differential medium | Root media |
| A great number of elements (mg/L) molysite (mg/L) trace element (mg/L) | (NH 4) 2SO 4 NH 4NO 3 KNO 3 Ca(NO 3) 2.4H 2O CaCl 2.2H 2O MgSO 4.7H 2O KH 2PO 4 FeSO 4.7H 2O EDTA.Na 2 KI CoCl 2.6H 2O H 3BO 3 Na 2MoO 4.2H 2O | 150 550 150 150 27.5 37.5 0.8 0.03 6.5 0.3 | 1600 1850 400 350 150 28.0 37.0 0.80 0.02 6.0 0.2 | 800 900 200 180 100 27.8 37.3 0.83 0.025 6.2 0.25 |
| Organic (mg/L) hormone (mg/L) sucrose (g/L) active carbon (g/L) agar (g/L) pH sterilization treatment | MnSO 4.4H 2O CuSO 4.5H 2O ZnSO 4，7H 2The plain Ser-Gln glutathione of O inositol nicotinic acid pyridoxol thiamine glycine folic acid biological 6-BA | 22 0.03 8.4 120 6 0.6 0.6 2.2 0.6 0.06 900 120 35 0.8 140 0.22 5.9 filtration sterilizations | 22.0 0.02 8.4 80 0.4 0.4 0.08 1.8 35 12 5.8 autoclaving | 22.3 0.025 8.6 100 0.6 0.4 0.12 1.8 20 7.0 5.9 autoclaving |
Claims (7)
1, be used to cultivate the medium of kale sporule regeneration plant, form by embryoid induction medium, differential medium and 3 kinds of medium of root media:
Wherein, the prescription of embryoid induction medium is: KNO
3100-150mg, Ca (NO
3)
24H
2O 450-550mg, MgSO
47H
2O 100-150mg, KH
2PO
4100-150mg, FeSO
47H
2O 27.5-28mg, EDTANa
237-37.5mg, KI 0.8-0.85mg, CoCl
26H
2O 0.02-0.03mg, H
3BO
36-6.5mg, Na
2MoO
42H
2O0.2-0.3mg, MnSO
44H
2O 22-22.5mg, CuSO
45H
2O 0.02-0.03mg, ZnSO
47H
2O 8.4-8.8mg, inositol 80-120mg, nicotinic acid 4-6mg, pyridoxine 0.4-0.6mg, thiamine 0.4-0.6mg, glycine 1.8-2.2mg, folic acid 0.4-0.6mg, vitamin h 0.04-0.06mg, glutamine 700-900mg, serine 80-120mg, glutathione 25-35mg, 6-benzyladenine 0.8-1.2mg, sucrose 100-150g, active carbon 0.10-0.30g, water is settled to 1L, pH 5.8-5.9;
The prescription of differential medium is: NH
4NO
31600-1700mg, KNO
31850-1950mg, CaCl
22H
2O400-480mg, MgSO
47H
2O 350-400mg, KH
2PO
4150-200mg, FeSO
47H
2O 27.5-28mg, EDTANa
237-37.5mg, KI 0.8-0.85mg, CoCl
26H
2O 0.02-0.03mg, H
3BO
36-6.5mg, Na
2MoO
42H
2O 0.2-0.3mg, MnSO
44H
2O 22-22.5mg, CuSO
45H
2O 0.02-0.03mg, ZnSO
47H
2O 8.4-8.8mg, inositol 80-120mg, nicotinic acid 0.4-0.6mg, pyridoxine 0.4-0.6mg, thiamine 0.08-0.12mg, glycine 1.8-2.2mg, sucrose 25-35g, agar 8-12g, water is settled to 1L, pH 5.8-5.9;
The prescription of root media is: NH
4NO
3800-850mg, KNO
3900-1000mg, CaCl
22H
2O200-240mg, MgSO
47H
2O 175-200mg, KH
2PO
475-100mg, FeSO
47H
2O 27.5-28mg, EDTANa
237-37.5mg, KI 0.8-0.85mg, CoCl
26H
2O 0.02-0.03mg, H
3BO
36-6.5mg, Na
2MoO
42H
2O 0.2-0.3mg, MnSO
44H
2O 22-22.5mg, CuSO
45H
2O 0.02-0.03mg, ZnSO
47H
2O 8.4-8.8mg, inositol 80-120mg, nicotinic acid 0.4-0.6mg, pyridoxine 0.4-0.6mg, thiamine 0.08-0.12mg, glycine 1.8-2.2mg, sucrose 10-20g, agar 8-10g, water is settled to 1L, pH 5.8-5.9.
2, medium according to claim 1 is characterized in that: the prescription of described embryoid induction medium is: KNO
3125mg, Ca (NO
3)
24H
2O 500mg, MgSO
47H
2O 125mg, KH
2PO
4125mg, FeSO
47H
2O27.8mg, EDTANa
237.3mg, KI 0.83mg, CoCl
26H
2O 0.025mg, H
3BO
36.2mg, Na
2MoO
42H
2O0.25mg, MnSO
44H
2O 22.3mg, CuSO
45H
2O 0.025mg, ZnSO
47H
2O 8.6mg, inositol 100mg, nicotinic acid 5mg, pyridoxine 0.5mg, thiamine 0.5mg, glycine 2mg, folic acid 0.5mg, vitamin h 0.05mg, glutamine 800mg, serine 100mg, glutathione 30mg, 6-benzyladenine 1mg, sucrose 130g, active carbon 0.2g, water is settled to 1L, and pH 5.9; The prescription of differential medium is: NH
4NO
31650mg, KNO
31900mg, CaCl
22H
2O 440mg, MgSO
47H
2O 370mg, KH
2PO
4170mg, FeSO
47H
2O 27.8mg, EDTANa
237.3mg, KI 0.83mg, CoCl
26H
2O 0.025mg, H
3BO
36.2mg, Na
2MoO
42H
2O0.25mg, MnSO
44H
2O 22.3mg, CuSO
45H
2O 0.025mg, ZnSO
47H
2O 8.6mg, inositol 100mg, nicotinic acid 0.5mg, pyridoxine 0.5mg, thiamine 0.1mg, glycine 2mg, sucrose 30g, agar 10g, water is settled to 1L, and pH 5.8; The prescription of root media is: NH
4NO
3825mg, KNO
3950mg, CaCl
22H
2O220mg, MgSO
47H
2O 185mg, KH
2PO
485mg, FeSO
47H
2O 27.8mg, EDTANa
237.3mg, KI 0.83mg, CoCl
26H
2O 0.025mg, H
3BO
36.2mg, Na
2MoO
42H
2O 0.25mg, MnSO
44H
2O22.3mg, CuSO
45H
2O 0.025mg, ZnSO
47H
2O 8.6mg, inositol 100mg, nicotinic acid 0.5mg, pyridoxine 0.5mg, thiamine 0.1mg, glycine 2mg, sucrose 15g, agar 8g, water is settled to 1L, and pH 5.9.
3, a kind of method that obtains the kale sporule regeneration plant may further comprise the steps:
1) microspore is inoculated in claim 1 or the 2 described embryoid induction medium, to final concentration be 5 * 10
4-5 * 10
5Individual/mL, after then high temperature stress is handled 24-72 hour under 30-35 ℃, dark condition, change inducing embryoid body under 24-26 ℃, dark condition again over to;
2) treat that torpedo stage to cotyledon period embryoid forms after, embryoid is earlier continued cultivation 5-10 days under 24-26 ℃, 1500-2500Lux, 14-18 hour illumination/sky condition, again embryoid is inoculated in claim 1 or the 2 described differential mediums, cultivates under the same conditions;
3) grow stem, leaf after, plant is transferred in claim 1 or the 2 described root medias, under 24-26 ℃, 1500-2500Lux, 14-18 hour illumination/sky condition, carry out culture of rootage, obtain the kale regeneration plant.
4, cultural method according to claim 3 is characterized in that: the microspore in the described step 1) separate self-contained 60% above monokaryon keep to the side phase microspore cell and 40% following dicaryotic phase the microspore cell bud; The microspore concentration that is inoculated in the embryoid induction medium is 10
5Individual/mL; The high temperature stress treatment conditions are to handle 24 hours under 33 ℃, dark condition; The embryoid induction temperature is 25 ℃.
5, cultural method according to claim 3 is characterized in that: described step 2) torpedo stage to cotyledon period embryoid is earlier continued cultivation 7 days under 25 ℃, 2000Lux, illumination in 16 hours/sky condition.
6, cultural method according to claim 3 is characterized in that: the culture of rootage condition in the described step 3) is 25 ℃, 2000Lux, 16 hours illumination/skies.
7, cultural method according to claim 3, it is characterized in that: the kale regeneration plant that obtains in the step 3) is carried out Methods of Ploidy Identification, and be that the colchicine of 0.15-2.5% is handled 36-60h continuously with volume ratio the growing point position of haplobiont.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102228003A (en) * | 2011-05-03 | 2011-11-02 | 浙江大学 | Culture method for brassica oleracea L. var. acephala microspore regeneration plant |
| CN102860202A (en) * | 2012-09-13 | 2013-01-09 | 江苏丘陵地区镇江农业科学研究所 | Cultivation method of collard and other grass and flower combined year-around ornament |
| CN109662025A (en) * | 2018-12-29 | 2019-04-23 | 北京市农林科学院 | A kind of breeding method of colour cabbage new germ plasm |
-
2006
- 2006-09-14 CN CNB2006101129975A patent/CN100422314C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102228003A (en) * | 2011-05-03 | 2011-11-02 | 浙江大学 | Culture method for brassica oleracea L. var. acephala microspore regeneration plant |
| CN102228003B (en) * | 2011-05-03 | 2012-11-07 | 浙江大学 | Culture method for brassica oleracea L. var. acephala microspore regeneration plant |
| CN102860202A (en) * | 2012-09-13 | 2013-01-09 | 江苏丘陵地区镇江农业科学研究所 | Cultivation method of collard and other grass and flower combined year-around ornament |
| CN102860202B (en) * | 2012-09-13 | 2014-07-02 | 江苏丘陵地区镇江农业科学研究所 | Cultivation method of collard and other grass and flower combined year-around ornament |
| CN109662025A (en) * | 2018-12-29 | 2019-04-23 | 北京市农林科学院 | A kind of breeding method of colour cabbage new germ plasm |
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