CN102550405B - Breeding method of poplar haploid - Google Patents
Breeding method of poplar haploid Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/04—Stems
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Botany (AREA)
- Genetics & Genomics (AREA)
- Physiology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a breeding method of poplar haploid and belongs to the field of selective breeding of poplar haploid. The breeding method comprises the following steps: (1) inoculating sterilized poplar anthers to a callus induction culture medium for callus induction culture to obtain poplar calli; (2) inoculating the poplar calli to an adventitious bud differentiation culture medium for differentiation culture of adventitious buds to obtain poplar adventitious buds; (3) inoculating the poplar adventitious buds to a rooting culture medium for rooting culture to obtain test-tube plantlets; and (4) performing ploidy identification to obtain poplar haploid plants. The breeding method of poplar haploid solves the problems occurring in poplar haploid breeding, has the advantages of high callus induction rate, high callus organ differentiation rate, high breeding rate and simple induced breeding operation, can produce high-quality poplar test-tube plantlets with high yield during a short time, achieves the purpose of large-scale industrial production, and provides a material basis for breeding, quality improvement and genetic research of poplar.
Description
Technical field
The present invention relates to a kind of breeding method of haplophyte, particularly a kind of breeding method that adopts Plant Tissue Breeding to obtain Haploid Poplar Plantlets, belong to the seed selection field of Haploid Poplar Plantlets.
Background technology
Beijing poplar (Populus beijingensis) is the filial generation of Cathay poplar and Lombardy poplar, it is the new varieties of poplar that after founding of New, forestry circle is bred as with artificial cross breeding method for the first time, it has cold-resistant, happiness liquid manure, characteristics such as tree state U.S. etc., good timber forest, shelter forest, street tree and " four is other " green tree species, mainly be distributed in the areas such as Chinese North China and northwest, liked by the masses.
Poplar is dioecism, and self-pollination is impossible realize basically.Obtain by conventional crossbreeding the breeding cycle that the strain of isozygotying need to be very long.In order to obtain the willow strain of isozygotying, the researcher adopted self-pollination or the method backcrossed in the past, but the breeding time limit that this breeding process needs is very long.Can first obtain the monoploid of target strain by anther culture, after it is doubled to become double haploid (doubled haploids, DHs), so significantly shorten breeding cycle.In addition, along with developing rapidly of modern biomolecule technology, plant haploid and product thereof are in many-sided successful Application such as the excavation of ploidy breeding, gene order-checking, map construction and functional gene and evaluations and obtained very large achievement, and haplophyte material and product thereof are favored by increasing researcher.
At present, the ways of regeneration of plant haploid comprises flower pesticide and pollen cultivation, parthenogenesis, chromosome elimination etc., and wherein anther culture is a kind of comparatively effectively technology that obtains plant haploid.At in vitro, by vitro method, cultivate flower pesticide, the development pathway of artificial change microspore, its Development of Gametophytes approach is stopped, turn to sporophyte to grow, by the approach of Organ Differentiation, obtain complete haplobiont, for artificial a large amount of production monoploid provides means effectively.Haplobiont by this approach regeneration, double through spontaneous doubling or artificial induction, obtains pure and mild liploid plant, thereby be the material that further breeding and genetic research provide use.Utilizing flower pesticide pollen cultivation acquisition monoploid to carry out breeding is the technical major reform of plant breeding, it can be combined with methods such as currently used crossbreeding, induced mutations breeding, distant hybridization, in order to overcome the deficiency in these methods, also can directly by the method, create new type.But, there are the problems such as callus induction rate is low, callus Organ Differentiation rate is low in the Poplar anthers of take in the incubation of explant seed selection Haploid Poplar Plantlets always, cause the breeding method of existing Haploid Poplar Plantlets can not meet the demand of research and production practices, urgently improvement far away.
Summary of the invention
Main purpose of the present invention is existing deficiency in the breeding method for existing Haploid Poplar Plantlets; a kind of breeding method of new Haploid Poplar Plantlets is provided; the Poplar anthers callus induction rate of the method is high, the rooting rate of the Organ Differentiation rate of callus and indefinite bud is high; a large amount of good Haploid Poplar Plantlets test-tube plantlets can be formed within a short period of time, scale, batch production production can be carried out.
For achieving the above object, the present invention has adopted following technical scheme:
A kind of breeding method of Haploid Poplar Plantlets comprises:
1) Poplar anthers after disinfecting is seeded on callus inducing medium and carries out induction of callus, obtain Poplar Callus;
2) Poplar Callus is inoculated in to the differentiation of carrying out indefinite bud on the differentiation adventitious buds medium and cultivates, obtain the willow indefinite bud;
3) the willow indefinite bud is inoculated on root media and carries out culture of rootage, obtain the test tube seedling;
4), through Ploidy Identification, obtain the Haploid Poplar Plantlets plant.
Wherein, described willow is preferably Beijing poplar (Populus beijingensis).
In order to reach better cultivation effect, when monokaryon keeps to the side the phase, now flower pesticide is wherein taken out to the cultivation of inducing of carrying out callus when microspore development period of willow inflorescence, that can effectively improve callus induces the cultivation rate; Wherein, the willow microspore development period can be by the form of observing bud and the developmental stage that diameter judges roughly microspore.Comparatively effectively detection method is the developmental stage of dyeing Observation of Microspore, and these methods or means are those skilled in the art and understand thoroughly; The present invention dyes and observes the developmental stage of determining microspore by aceto-camine.
Because poplar flower is catkin, the scale on bud surface is micro-by felt hair, step 1) described in disinfect and adopt following processing mode to there is better aseptic result:
1-A) use the aseptic water washing bud after gently brushing bud scale surface with writing brush;
1-B) use alcohol-pickled bud;
1-C) with the liquor natrii hypochloritis, bud is carried out to surface sterilizing, use aseptic water washing;
1-D) blot after the bud surface moisture and take out flower pesticide from inflorescence, obtain.
Preferably, step 1-A), the aseptic water washing number of times is 4-5 time; The concentration of volume percent of alcohol step 1-B) is 70.0%; Soak time is 30s; Step 1-C) in, the mass percent concentration of clorox is 7.0%; The surface sterilizing time is 5min; The aseptic water washing number of times is 3-5 time.
Step 1) callus inducing medium described in is preferably: H minimal medium+naa (NAA) 0.5-1.0mg/L+6-chaff aminopurine (KT) 0.5-1.0mg/L+ sucrose 30g/L+ agar 5.5g/L; More preferably: H minimal medium+naa 1.0mg/L+6-chaff aminopurine 0.5-1.0mg/L+ sucrose 30g/L+ agar 5.5g/L; Most preferably be: H minimal medium+naa 1.0mg/L+6-chaff aminopurine 1.0mg/L+ sucrose 30g/L+ agar 5.5g/L; Further preferably, step 1), induction of callus carries out under the following conditions: under dark condition, cultivation temperature is 25 ± 1 ℃;
Step 2) medium optimization of differentiation adventitious buds described in is: MS medium+6-benzyladenine (BAP) 0.6mg/L+ naa (NAA) 0.2mg/L+ gibberellin (GA
3) 0.02-0.2mg/L+ sucrose 20g/L+ agar 5.5g/L; More preferably: MS medium+6-benzyladenine 0.6mg/L+ naa 0.2mg/L+ gibberellin 0.02mg/L+ sucrose 20g/L+ agar 5.5g/L; Further preferably, step 2), differentiation adventitious buds is cultivated and carried out under the following conditions: cultivation temperature is 25 ± 1 ℃, illumination 1500～2000lx, light application time 16 hours/day;
Step 3) root media described in is preferably: 1/2MS medium+indolebutyric acid (IBA) 0.2-0.5mg/L+ sucrose 20g/L+ agar 5.5g/L; More preferably: 1/2MS medium+indolebutyric acid 0.3mg/L+ sucrose 20g/L+ agar 5.5g/L; Further preferably, step 3), culture of rootage is carried out under the following conditions: culturing room's temperature is 25 ± 1 ℃, illumination 1500～2000lx, light application time 16 hours/day.
Breeding method of poplar haploid of the present invention utilize Poplar anthers after evoked callus again Organ Differentiation be plant, thereby obtain the Haploid Poplar Plantlets plant.In order to improve the problems such as callus induction rate in incubation, callus Organ Differentiation rate and rooting rate, the present invention is optimized the formula of callus inducing medium, indefinite bud Organ Differentiation nutrient chemical and root media, the optimum medium that final screening has obtained different cultivation stages forms, callus induction rate, callus Organ Differentiation rate and rooting rate have been improved significantly, finally improved the yield of Haploid Poplar Plantlets, for the breeding of willow, quality-improving, genetic research etc. provide material base.
The Haploid Poplar Plantlets plant strain growth that the inventive method is cultivated is healthy and strong, reproduction coefficient is high, obtains test tube seedling transplanting survival rate up to 92.0%, is the simple, fast technical system of batch production large-scale production Beijing poplar plant.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can modify or replace details and the form of technical solution of the present invention, but these modifications and replacement all fall within the scope of protection of the present invention.
Embodiment 1
One, test material
1, for the examination material: Beijing poplar (Populus beijingensis);
2, plant growth regulator
The plant growth regulating substance used in the present invention adopts domestic methyl α-naphthyl acetate (NAA), 6-benzyl aminoadenine (BAP), 6-Furfurylaminopurine (KT), gibberellin (GA
3), 2, the 4-dichlorophenoxyacetic acid (2,4-D), indolebutyric acid (IBA).
3, the preparation of medium:
(1) composition and the compound method of " MS minimal medium ":
Table 1 MS medium (Murashige and Skoog, 1962)
The consumption of above various nutrient components, except the mother liquor I is 20 times of concentrates, remaining is 200 times of concentrates.After above-mentioned MS medium mother liquor prepares, be stored in 4 ℃ of refrigerators stand-by.According to the total amount of preparation medium, the agar that weighing is required and sucrose, be poured into and want to join in the distilled water of culture volume 3/4, and the limit heating edge stirs with glass bar, the shape until liquid is translucent.Then take out respectively required consumption with graduated cylinder or pipette from various mother liquors, as need add hormone according to kind and the concentration of hormone in culture medium prescription, it is put into to enamel graduate together with various mother liquors.Often add a kind of all fully stirrings, last adding distil water is settled to cumulative volume, stirs.By pH meter test media acid-base value, the NaOH solution of drawing 1.0mol/L with dropper dropwise adds in the medium of thawing, while dripping, stirs, until the pH value of medium is adjusted to 5.8.
(2) H minimal medium " composition and compound method:
Table 2 H medium
The consumption of above various nutrient components, except the mother liquor I is 20 times of concentrates, remaining is 200 times of concentrates.After above-mentioned H medium mother liquor prepares, be stored in 4 ℃ of refrigerators stand-by.According to the total amount of preparation medium, the agar that weighing is required and sucrose, be poured into and want to join in the distilled water of culture volume 3/4, and the limit heating edge stirs with glass bar, the shape until liquid is translucent.Then take out respectively required consumption with graduated cylinder or pipette from various mother liquors, as need add hormone according to kind and the concentration of hormone in culture medium prescription, it is put into to enamel graduate together with various mother liquors.Often add a kind of all fully stirrings, last adding distil water is settled to cumulative volume, stirs.By pH meter test media acid-base value, the NaOH solution of drawing 1.0mol/L with dropper dropwise adds in the medium of thawing, while dripping, stirs, until the pH value of medium is adjusted to 5.5.
(3) N
6The composition of minimal medium and compound method:
Table 3 N
6Medium (Plants of Beijing institute, the Exploitation of Agriculture in Heilongjiang academy of sciences, 1974)
The consumption of above various nutrient components, except the mother liquor I is 20 times of concentrates, remaining is 200 times of concentrates.Above-mentioned N
6After the medium mother liquor prepares, be stored in 4 ℃ of refrigerators stand-by.According to the total amount of preparation medium, the agar that weighing is required and sucrose, be poured into and want to join in the distilled water of culture volume 3/4, and the limit heating edge stirs with glass bar, the shape until liquid is translucent.Then take out respectively required consumption with graduated cylinder or pipette from various mother liquors, as need add hormone according to kind and the concentration of hormone in culture medium prescription, it is put into to enamel graduate together with various mother liquors.Often add a kind of all fully stirrings, last adding distil water is settled to cumulative volume, stirs.By pH meter test media acid-base value, the NaOH solution of drawing 1.0mol/L with dropper dropwise adds in the medium of thawing, while dripping, stirs, until the pH value of medium is adjusted to 5.8.
(4) callus inducing medium: H minimal medium+NAA 1.0mg/L+KT 1.0mg/L+ sucrose 30g/L+ agar 5.5g/L, regulating the pH value is 5.5, medium was 121 ℃ of lower constant temperature sterilizings 20 minutes.
(5) differentiation adventitious buds culture medium prescription: MS minimal medium+BAP 0.6mg/L+NAA 0.2mg/L+GA
30.02mg/L+ sucrose 20g/L+ agar 5.5g/L, regulating the pH value is 5.8, and medium was 121 ℃ of lower constant temperature sterilizings 20 minutes.
(6) the adventitious bud rooting culture medium prescription is: 1/2MS minimal medium+IBA0.2-0.5mg/L sucrose 20g/L+ agar 5.5g/L, and regulating the pH value is 5.8, medium was 121 ℃ of lower constant temperature sterilizings 20 minutes.
Embodiment 2
1, the suitable developmental stage of the flower pesticide of cultured in vitro and microspore
Clip with Beijing poplar bloassom branch of full bud in 25 ℃ of lower water planting a few days of room temperature.The willow bud is placed on slide, strips out flower pesticide, then adopt aceto-camine dyeing, the developmental stage of sediments microscope inspection Observation of Microspore, to determine Pollen stage.This test is chosen microspore and is inoculated in the keep to the side flower pesticide of phase of monokaryon, and result of study shows now anther cultural best period just.
2, explant sterilization
To take off from catkin in the keep to the side bud of phase of monokaryon, first with writing brush, gently brush the surface of Beijing poplar bloassom perula sheet, the dirt of appearance, bacterium etc. will be removed, improve Disinfection Effect, then with aseptic water washing 5 times, the filter paper suck dry moisture.
Explant is placed on superclean bench, the alcohol-pickled bud 30s that is 70.0% with concentration of volume percent, carry out surface sterilization 5 minutes in the liquor natrii hypochloritis who is then 7.0% with mass percent concentration, then use aseptic water washing 3-5 time, take out bud.
Under anatomical lens, the scale of bud is peelled off, got the flower pesticide at inflorescence middle part, be inoculated in culture dish, with the preservative film sealing, secretly cultivate, 30 flower pesticide of each culture dish inoculation, each is processed and repeats 5 times.Condition of culture: dark condition, 25 ± 1 ℃ of cultivation temperature.
3, the screening of callus inducing medium
To be inoculated in respectively MS, H and N in the keep to the side flower pesticide of phase of monokaryon
6In three kinds of minimal mediums, add 1.0mg/L 2 in every kind, 4-D and 1.0mg/L BAP, sucrose 30g/L, agar 5.5g/L, pH 5.8.30 flower pesticide of each culture dish inoculation, each processing arranges 5 repetitions.Condition of culture: dark condition, 25 ± 1 ℃ of cultivation temperature, cultivate after 35 days and start the callus number that each processing of statistics forms, and cultivates after 120 days and calculate callus induction rate, and statistics and result of calculation are as table 4.
The induce effect of table 4 different culture media to callus
| Minimal medium | Inoculation flower pesticide number (individual) | Produce the flower pesticide number (individual) of callus | Callus induction rate (%) |
| MS | 150 | 3 | 2.0 |
| H | 150 | 5 | 3.3 |
| N 6 | 150 | 0 | 0.0 |
The result of the test of table 4 shows:
1) adopt the H medium, the callus induction rate of Poplar anthers is high.
2) the flower pesticide inoculation is about 35 days, starts to have observed callus and forms.All observe the formation of callus in MS, H medium, this callus quality consolidation, faint yellow opaque, surface is graininess, poor growth.At N
6Do not observe the formation of callus in medium.
4, callus inducing medium hormone kind screening
H minimal medium according to screening, screen different hormone kinds.
The flower pesticide of the phase of keeping to the side in monokaryon is inoculated in respectively in the H medium that adds hormon kind and concentration, the H medium that adds hormon kind and concentration is as shown in table 5.
The H medium that table 5 contains the hormon kind
Add sucrose 30g/L, agar 5.5g/L, pH5.5 in every kind of medium.30 flower pesticide of each culture dish inoculation, each is processed and repeats 3 times.Condition of culture: dark condition, 25 ± 1 ℃ of cultivation temperature, cultivate after 35 days and start the callus number that each processing of statistics forms, and cultivates after 120 days and calculate callus induction rate, and statistics and result of calculation are as table 6.
The impact of table 6 hormon kind on callus induction
Result of the test shows: hormone kind NAA and KT combination are the highest to the induction of anther callus rate, and the average callus induction rate of all processing experiments of this hormone combinations reaches 9.3%, is secondly the hormone combinations of 2,4-D and BAP.In table, data show, the hormone combinations of NAA and KT induces effect more satisfactory to callus.
5, callus inducing medium hormone concentration screening
According to minimal medium and the hormone kind of screening, the proportioning of screening hormone.
The flower pesticide of the phase of keeping to the side in monokaryon is inoculated in respectively in the H medium of the NAA, the KT that add variable concentrations, and adds sucrose 30g/L in every kind of medium, agar 5.5g/L, pH 5.5.30 flower pesticide of each culture dish inoculation, each processing arranges 3 repetitions.Condition of culture: dark condition, 25 ± 1 ℃ of cultivation temperature, cultivate after 35 days and start the callus number that each processing of statistics forms, and cultivates after 120 days and calculate callus induction rate, and statistics and result of calculation are as shown in table 7.
The impact of table 7 different hormone combinations on callus induction
Result of the test shows:
1) adopt medium to be: the callus induction rate of H+NAA1.0mg/L+KT1.0mg/L Poplar anthers is high, reaches 30.0%.
2) the present invention carries out in During Callus Induction Poplar anthers, and in the H medium of interpolation 1.0mg/LNAA and 1.0mg/LKT, the inductivity of callus is the highest, reaches 30.0%, is secondly to add 1.0mg/LNAA and 0.5mg/L KT in the H medium.To sum up, in the H medium, NAA and the KT of the appropriate concentration of interpolation are more favourable to the dedifferentiation of microspore.
6, the screening of callus differentiation adventitious buds medium
Callus differentiation adventitious buds medium be take MS as minimal medium, will cultivate about 35 days, and the callus that diameter is about 3mm is inoculated in the differentiation adventitious buds medium that contains hormon and breaks up cultivation.And add sucrose 20g/L, agar 5.5g/L, pH5.8 in every kind of medium.1 callus of each blake bottle inoculation, each is processed and repeats 5 times.Condition of culture: cultivation temperature is 25 ± 1 ℃, illumination 1500～2000lx, light application time 16 hours/day.Cultivate after 20 days and start the indefinite bud number that each processing of statistics is differentiated to form, calculate Differentiation ration of adventitious buds, statistics and result of calculation are as shown in table 8.
The impact of table 8 different hormone combinations on the Calli Differentiation effect
Result of the test shows:
1) after callus is inoculated in differential medium, its color transfers yellow green to by yellow and then becomes green, within about one week, in the part differential medium, can see that the bud point of green projection appears in the callus surface, and then vane extension out; And in the differential medium of interpolation different hormone combinations, stem's developmental state difference of indefinite bud is larger.
2) adopt medium to be: MS+BAP 0.6mg/L+NAA0.2mg/L+GA
30.02-0.2mg/L Poplar anthers callus Organ Differentiation medium, the differentiation rate of indefinite bud is high, good without offspring morphological feature and growing state, reaches 100.0%, and GA is described
3In going out the process of indefinite bud, Calli Differentiation brought into play crucial effect, especially putting forth at indefinite bud, bud in the process without offspring has important using value, so the callus that will obtain in the later stage is transferred in this best medium of differentiation effect.
7, the screening of adventitious bud rooting medium
The adventitious bud rooting medium be take 1/2MS as minimal medium, and the indefinite bud that will have 3-5 sheet young leaflet tablet is inoculated in the adventitious bud rooting medium that contains hormon and carries out culture of rootage.And add sucrose 20g/L, agar 5.5g/L, pH5.8 in every kind of medium.3 indefinite buds of each blake bottle inoculation, each is processed and repeats 5 times.Condition of culture: cultivation temperature is 25 ± 1 ℃, illumination 1500～2000lx, and light application time 16 hours/day, cultivate to add up afterwards in 20 days and respectively process the situation of taking root, and calculates the adventitious bud rooting rate, and statistics and result of calculation are as shown in table 9.
The impact of table 9 different hormone combinations on the adventitious bud rooting effect
Result of the test shows:
1) the rootability difference of different root medias to the willow indefinite bud, but, according to the quality and quantity of rooting rate, root, determine 1/2MS+IBA 0.3mg/L+ sucrose 20g/L+ agar 5.5g/L, the root media that the medium of pH5.8 is the willow indefinite bud.
2) as can be seen from Table 5, IBA is bringing into play crucial effect in the rooting process of Beijing poplar group training without offspring, wherein add in the 1/2MS root media of 0.2-0.5mg/L IBA and all can reach 100.0% rooting rate, wherein with interpolation the best of 0.3mg/L, the root feature is normal, main root is sturdy, and fibrous root is more.
Embodiment 3
1, prepare aseptic flower pesticide
Get the keep to the side willow bud of phase of monokaryon, with aseptic water washing bud 3-5 time, with the alcohol-pickled bud 30s that is 70.0% with concentration of volume percent after the filter paper suck dry moisture, the liquor natrii hypochloritis who is then 7.0% with the mass percent concentration 5min that sterilizes, then use aseptic water washing bud 3-5 time.Peel off the scale of bud on superclean bench, take out the flower pesticide at inflorescence middle part, obtain.
2, induction of callus
Flower pesticide is inoculated in callus inducing medium and secretly cultivates, condition of culture: under dark condition, cultivation temperature is 25 ± 1 ℃, cultivate 120 days, statistics induction of anther callus rate is 30.0%, wherein, callus inducing medium is: H+NAA 1.0mg/L+KT 1.0mg/L+ sucrose 30g/L+ agar 5.5g/L, pH5.5.
3, differentiation adventitious buds is cultivated
The Poplar anthers callus that the diameter that cultivation is obtained is the 3mm left and right is inoculated in the differentiation adventitious buds medium and carries out the differentiation adventitious buds cultivation, condition of culture: temperature is 25 ± 1 ℃, illumination 1500～2000lx, light application time 16 hours/day.In the differentiation incubation, the Poplar anthers callus transfers yellow green to by yellow and then becomes green, can see the bud point of green projection after one week on the callus surface, and then vane extension out, then put forth, within 20 days, left and right indefinite bud growing height can reach 1.5～2cm.Cultivating the differentiation rate of adding up afterwards indefinite bud in 20 days is 100.0%, and wherein, the differentiation adventitious buds culture medium prescription is: MS+BAP 0.6mg/L+NAA 0.2mg/L+GA
30.02mg/L+ sucrose 20g/L+ agar 5.5g/L, pH5.8.
4, culture of rootage
Be inoculated in root media and carry out culture of rootage being cultured to the indefinite bud with 3-5 sheet young leaflet tablet, condition of culture: cultivation temperature is 25 ± 1 ℃, illumination 1500～2000lx, light application time 16 hours/day, cultivate the situation of taking root of adding up afterwards indefinite bud in 20 days, and rooting rate reaches 100.0%, obtain willow test tube seedling, wherein, the adventitious bud rooting culture medium prescription is: 1/2MS+IBA 0.3mg/L+ sucrose 20g/L+ agar 5.5g/L, pH5.8.
5, hardening, transplanting
Choose the test tube seedling preferably of taking root, be placed on hardening in the shade in greenhouse and cultivate 2-3 days, then take out seedling and clean the root medium, be transplanted in willow soilless culture substrate (mixture of perlite and vermiculite), soilless culture substrate is perlite and the ratio of the volume of the vermiculite mixture that is 1: 1.Water permeablely after transplanting, after 15 days, add up transplanting survival rate, survival rate is 92.0%.
The Ploidy Identification that embodiment 4 flower pesticide are induced plant
Get appropriate nucleic acid lysate in little culture dish (No. 11), the tender leaf of the appropriate seedling to be measured of clip is placed in above-mentioned culture dish, with blade, leaf tissue is cut into small pieces, it is fully contacted with lysate, then be filtered in the centrifuge tube of 1.5 μ L, add the DAPI dye liquor 65 μ L that concentration is 5.0 μ g/mL in filtrate, be placed in the ploidy detection that flow cytometer carries out the plant tender leaf.
Get the tip of a root that Preliminary screening is haploid plant, adopt chromosome routine techniques compressing tablet, with paracide saturated solution pretreatment 3.5h, and use distilled water flushing; After at Kano fixer (alcohol: fixing 24h glacial acetic acid=3: 1); After distilled water flushing, in dissociation solution, (hydrochloric acid: 15min dissociates alcohol=1: 1); Then distilled water flushing 3 times, each 12min; Observe the counting chromosome number after adopting carbolfuchsin dyeing under microscope (10 * 80).
The chromosome number of Beijing poplar haplobiont is 19, and the chromosome number of liploid plant is 38, and through microexamination, Beijing poplar bloassom medicine is induced in the plant of acquisition, and the ratio of haplobiont is 10.3%, and the microscopy result is as shown in table 10.
The ratio of table 10 haplobiont
Practical application shows: the inventive method can repeatedly obtain the Haploid Poplar Plantlets plant in different fully tests, and reliability is strong.Haplobiont is doubled or manually uses the colchicine treatment growing point through natural, obtain pure and mild dliploid.
Claims (5)
1. Beijing poplar (Populus beijingensis) monoploid breeding method comprises as follows step in sequence:
1) disinfect
1-A) microspore is taken off from catkin in the keep to the side bud of phase of monokaryon, use the aseptic water washing bud after gently brushing bud scale surface with writing brush;
1-B) use alcohol-pickled bud;
1-C) with the liquor natrii hypochloritis, bud is carried out to surface sterilizing, use aseptic water washing;
1-D) under anatomical lens, the scale of bud is peelled off after blotting the bud surface moisture, taken out flower pesticide from inflorescence;
2) Poplar anthers after disinfecting is seeded on callus inducing medium and carries out induction of callus, obtain Poplar Callus, wherein, described callus medium is: H minimal medium+naa 1.0mg/L+6-chaff aminopurine 1.0mg/L+ sucrose 30g/L+ agar 5.5g/L;
3) Poplar Callus being inoculated in to the differentiation of carrying out indefinite bud on the differentiation adventitious buds medium cultivates, obtain the willow indefinite bud, wherein, described differentiation adventitious buds medium is: MS medium+6-benzyladenine 0.6mg/L+ naa 0.2mg/L+ gibberellin 0.02mg/L+ sucrose 20g/L+ agar 5.5g/L;
4) the willow indefinite bud is inoculated on root media and carries out culture of rootage, obtain the test tube seedling; Described root media is: 1/2MS medium+indolebutyric acid 0.2-0.5mg/L+ sucrose 20g/L+ agar 5.5g/L;
5), through Ploidy Identification, obtain the Haploid Poplar Plantlets plant.
2. Beijing as claimed in claim 1 poplar monoploid breeding method, is characterized in that step 2) described in induction of callus carry out under the following conditions: under dark condition, cultivation temperature is 25 ± 1 ℃.
3. Beijing as claimed in claim 1 poplar monoploid breeding method, it is characterized in that differentiation adventitious buds described in step 3) is cultivated carries out under the following conditions: cultivation temperature is 25 ± 1 ℃, illumination 1500～2000lx, light application time 16 hours/day.
4. Beijing as claimed in claim 1 poplar monoploid breeding method, is characterized in that described in step 4), root media is: 1/2MS medium+indolebutyric acid 0.3mg/L+ sucrose 20g/L+ agar 5.5g/L.
5. Beijing as claimed in claim 1 poplar monoploid breeding method, it is characterized in that culture of rootage described in step 4) carries out under the following conditions: cultivation temperature is 25 ± 1 ℃, illumination 1500～2000lx, light application time 16 hours/day.
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| CN106171997B (en) * | 2016-07-20 | 2018-11-06 | 中国林业科学研究院林业研究所 | A kind of while explant surface and endophyte when removing Plant Tissue Breeding method |
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| CN107125087A (en) * | 2017-05-25 | 2017-09-05 | 浙江师范大学 | Bi-membrane method cultured in vitro willow and the method for Laccaria bicolor Applying Ectomycorrhizal Fungi syntaxial system |
| CN109618930A (en) * | 2018-12-28 | 2019-04-16 | 武汉博立达农业科技发展有限公司 | The method for cultivating giantreed using flower training monoploid |
| CN110800613A (en) * | 2019-12-11 | 2020-02-18 | 阳山县三连阳生态农林开发有限公司 | Breeding method for obtaining rhizoma bletillae haploid |
| CN111194695A (en) * | 2020-03-05 | 2020-05-26 | 山东农业大学 | Tissue culture rapid propagation method of Populus deltoides Lu Lin No. 1 |
| CN114467754B (en) * | 2022-02-24 | 2023-04-28 | 吉林农业大学 | Method for obtaining poplar aneuploid plants in silver |
| CN114885833B (en) * | 2022-04-28 | 2023-03-24 | 北京林业大学 | Induction method of 2n pollen of poplar |
| CN118489561A (en) * | 2024-06-06 | 2024-08-16 | 中国林业科学研究院林业研究所 | Method for maintaining activity of long-term subculture poplar tissue culture seedlings by utilizing shuttle culture and application |
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