CN103704126A - Method for creating cymbidium resource for high-efficiency generation of 2n male gametes - Google Patents
Method for creating cymbidium resource for high-efficiency generation of 2n male gametes Download PDFInfo
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Abstract
The invention relates to the field of plant genetic breeding technology, and particularly discloses a method for creating a cymbidium resource for high-efficiency generation of 2n male gametes. The method includes parent matching, distant hybridization combination, hybrid colony construction, 2n male gamete identification, screening of the resource for high-efficiency generation of the 2n male gametes and the like. With utilization of the distant hybridization method, the cymbidium resource for high-efficiency generation of the 2n male gametes is obtained, and lays the foundation for high-efficiency creation of sexual polyploids by utilizing a sexual hybridization method, effective promotion of cymbidium polyploidy breeding, and cultivation of a breakthrough new cymbidium species in the absence of polyploids.
Description
Technical field
The invention belongs to plant genetics and breeding technical field, relate to particularly a kind of method that 2n andro gamete resource efficiently occurs orchid that creates.
Background technology
Orchid (
cymbidium) be world's famous flower, integrate view and admire, economy and cultureal value, be deeply subject to countries in the world people's favor.China is the abundantest country of orchid resource, known have 49 kinds, account for more than 2/3rds (Liu Zhongjian etc. of the world total, 2006), these resources of overseas utilization, have logined nearly 15000 of orchid collective hybrid in the Royal Society, and cultivate a large amount of commercialization kinds, but the hybrid of the domestic orchid new varieties of cultivating and login is all less, and it is just more rare to have the kind number of Commercial Prospect.Germ plasm resource is the material base of breeding, creating critical germ plasm resource is to cultivate the key with breakthrough kind, therefore study innovative approach Dui Geng good utilisation China orchid resource of germ plasm resource, cultivation has breakthrough orchid new varieties tool and is of great significance.
Polyploid breeding is the important means of orchid breeding, and current business-like Hybrid Cymbidium mostly is polyploid (Zhu Genfa etc., 2006), and create polyploid resource, is the key of carrying out orchid polyploid breeding.The establishment of orchid polyploid resource can be passed through the method for physics, chemistry and biology, create the method for polyploid compares with traditional physics and chemistry method, utilize biology (2n gamete approach) to create polyploid and there is unique superiority, it not only can directly obtain triploid, tetraploid resource, and can transmit the heterozygosity of parent Geng Gao and can overcome hybrid dysgenesis.Chinese scholars, utilizes 2n andro gamete approach successfully to obtain lily at present; (Barba-Gonzalez et al, 2006; Barba-Gonzalez et al, 2005), Chinese rose (Crespel et al, 2006), begonia (Dewitte et al, 2010) and calla lily (Wu Hongzhi etc., 2011) etc. have the polyploid resource of important ornamental value.
2n andro gamete can naturally-occurring also can artificial induction, the main method of artificial induction 2n andro gamete has physical method and chemical method.Physical method comprises temperature violent change, gas mutagenesis and ionizing radiation etc.Chemical method comprises colchicine, oryzalin (Oryzalin) and trefanocide (Trifluralin) and caffeine etc.At present in orchid breeding, the research of artificial induction 2n andro gamete report is less, and Fan Bin etc. (2012) utilize colchicine-induced spring sword 2n andro gamete, its highest inductivity is 2.65%, P WongprichAC(active carbon) han et al(2013) utilize N
2o successfully induces Moth orchid 2n andro gamete.Be no matter physical method or chemical method induction 2n andro gamete, all exist 2n andro gamete competitiveness to decline, the problem such as inductivity is on the low side and efficiency is unstable, this causes 2n andro gamete cannot effectively apply to breeding practice.
Therefore, create the orchid resource that 2n andro gamete efficiently occurs nature and become the key of carrying out orchid polyploid breeding.
Summary of the invention
The technical problem to be solved in the present invention is in order to overcome physical method and chemical method induction 2n andro gamete in prior art, all exist 2n andro gamete competitiveness to decline, inductivity is on the low side and the unsettled defect of efficiency, and a kind of method that 2n andro gamete resource efficiently occurs orchid that creates is provided.The present invention compares with conventional physical, chemical method, and that 2n andro gamete occurs is stable, vigor is high, can reuse for a long time.
Object of the present invention is achieved by the following technical programs:
Create the method that 2n andro gamete resource efficiently occurs orchid, it is characterized in that, comprise the steps:
S1. Distant crossing combination configuration: select suitable parent, configuration hybrid combination is cultivated maternal plant under suitable condition, obtains fruit;
S2. hybrid population builds: fruit is carried out disinfection, get its seed and carry out the middle brood body of aseptic culture acquisition, middle brood body is carried out to plant regeneration, obtain test-tube plantlet, after test-tube seedling transplanting, cultivate to plant blossom;
The evaluation of S3.2n andro gamete: after plant to be planted is bloomed, get the fresh mature pollen of different strain plant, adopt after direct compression process film-making, at optical microphotograph Microscopic observation 2n pollen, the occurrence frequency of statistics 2n pollen;
S4. efficiently there is 2n andro gamete resource screening: incidence is greater than to 1% plant and carries out division propagation, and carry out 2 years above repetitive identified, filtering out the strain that incidence is high and stable is that 2n andro gamete resource efficiently occurs orchid.
The method according to this invention, wherein, Distant crossing combination preparation technique comprises the following steps described in step S1:
Parent's matching principle: distant hybridization often exists the problem of cross incompatibility, parent's affiliation can not be too far away, should note matching the kind that compatibility is relatively high, and it is high that parent's Pollen Activity is wanted; Next avoids selecting the blue strain of blooming to be for the first time parent; The kind of finally selecting seed to be easy to sprout is parent, to guarantee obtaining hybrid generation.
Hybridization technique: select healthy and strong anosis plant to make Parent, with tweezers, take out the pollinium of blooming in flower the maternal same day, then the pollinium of male parent is put into maternal stamen chamber, with pencil, on playing cards, write the title of hybrid combination exactly, hybridization time, hang on maternal plant, and be placed on the place of Warming ventilating, prevent cold wave.
Described in step S2, hybrid population's constructing technology comprises the following steps:
S21. sterilization and the seed germination of fruit pod: running water for the fruit of freshly harvested is rinsed to 2～3 min, after drying on superclean bench with 75% alcohol-pickled 30s, proceed to the 15min that sterilizes in 0. 1% mercuric chloride, standby after aseptic water washing 3～5 times.With scalpel, cut fruit pod two ends approximately 0.5 cm, then fruit is longitudinally cut, seed uniform broadcasting, on suitable seed germination medium, and is secretly cultivated at suitable temperature condition.
Propagation and the plant regeneration of brood body in the middle of S22: by sprouting the middle brood body stripping and slicing forming, be inoculated into suitable proliferated culture medium and breed, after subculture is cultivated, be transferred in suitable differential medium and break up.The bud differentiating and seedling proceed in suitable Rooting and hardening-off culture base, carry out strong plantlets and rootage.
S23. test-tube seedling transplanting: can carry out hardening when seedling grows to 5～8 cm and have more than 2 root.During hardening, blake bottle lid is unscrewed, be placed under greenhouse 10～20 d or place after 3～5 d in the outdoor place without direct light, take out test-tube seedling transplanting.
Described in step S21, seed germination medium is 1/2 mg/L 6BA+0.2～0.5, MS medium+0.2～0.5 m g/L NAA+30 g/L sucrose+7.5g/L OK a karaoke club powder+100ml/L CW(coconut milk)+0.5 g/L AC(active carbon).
Described in S22, proliferated culture medium is 1/2 MS+ 0.5～1.0 mg/L NAA+30g/L sucrose+7.5g/L OK a karaoke club powder+0.5, mg/L 6BA+0.2～0.5 g/L AC(active carbon).
Described in S22, differential medium is 1/2MS+ 1.5～2.0 mg/L 6BA+0.2～0.5 mg/L NAA+30g/L sucrose+7.5g/L OK a karaoke club powder.
Described in S22, Rooting and hardening-off culture base is 1/2MS+ 0.1～0.2 mg/L NAA+20g/L sucrose+7.5g/L OK a karaoke club powder+0.5, mg/L 6BA+0.5～1.0 g/L AC(active carbon).
The method according to this invention, wherein, the authenticate technology of 2n andro gamete comprises the following steps described in step S3:
S31 film-making: take out fresh flowers powder agglomates, be placed on clean slide, dry for preventing pollen, splash into a small amount of sterile water, with tweezers, pollen is dispersed on slide.
S32. dyeing: after material moisture dries on slide, evenly spread one deck improvement carbol fuchsin dye liquor above material, pass through fast flame 2 ~ 3 times.The about 3-5 minute of dyeing time.
S33. compressing tablet: vertically rap cover glass with the pencil with rubber, drive bubble out of.
S34. observe: with Olympus-I * 71 inverted microscope, observe 2n pollen occurrence frequency, under 40 * object lens, mobile slide, selects 10 visuals field to take pictures at random, finally add up the frequency that dyad in these 10 visuals field and triad occur.During observation, take 100 microsporocytes as 1 repetition, repeat 3 times.
S35. add up: the frequency that 2n andro gamete occurs is added up according to following formula:
The frequency of 2n andro gamete (%)=(2D+Tr)/(2D+3Tr+4Te) * 100;
Wherein D represents dyad; Tr represents triad; Te generation～table tetrad.
The method according to this invention, wherein, division propagation technology comprises the following steps described in step S4: division propagation technology: orchid division propagation carried out in resting stage, is preferably in that sprouting has formed but before being not yet unearthed.Orchid strain belongs to bunch type, and different cultivars plant division method is difference slightly, pins the basin soil of base of the plant during plant division with a hand, another hand by basin overturning out, shake gently matrix separated with tub wall, then orchid clump is taken out, gently that matrix is broken, make it separated with blue root, then from gap larger part, with the scraper of having sterilized, orchid clump is cut and cleaned by 2～3 strain one root and stem of certain plants, remove old root, rotten, dead leaf, place about 1 day, until blue root, returning can above basin when white.After orchid plant division improving activity of root system relatively a little less than, should note insulation, moisturizing management, to improve plant viablity.
The invention has the beneficial effects as follows: the present invention, by the method for distant hybridization, can obtain the orchid resource that 2n andro gamete occurs stability and high efficiency, compared with physics or the method for chemistry, 2n andro gamete occurs stable, can reuse for a long time, and the 2n andro gamete vigor of formation is high.This is to promoting the development of orchid polyploid breeding and industry, and the core competitiveness tool that improves China's orchid industry is of great significance.
figure of description
Fig. 1. the qualification result of Chinese cymbidium 2n andro gamete; Wherein a～d later stage II a. mono-split; B. dyad; C. triad; D. normal tetrad; E～h mature pollen e. in period mono-split f. dyad; G. triad; H. normal tetrad pollen.
Embodiment
Below in conjunction with specific embodiment, further describe the present invention.Unless stated otherwise, reagent, the equipment that the present invention adopts is the conventional reagent of the art and equipment.
The establishment of the efficient 2n andro gamete of embodiment 1 Jin Zui * Cymbidium lancifolium hybrid resource
S1. Distant crossing combination configuration: the golden mouth Chinese cymbidium florescence, select healthy and strong anosis plant to do maternal, get Cymbidium lancifolium pollinium for its pollination, with pencil, in the bright hybrid combination title of plastics playing cards subscript, hybridization time, hang on maternal plant.Under suitable condition, cultivate maternal plant, obtain fruit.
S2. hybrid population builds
S21. fruit pod sterilization and seed germination: the fruit of freshly harvested is removed to carpopodium with scalpel, dip after appropriate liquid detergent cleans and rinse 2～3 min with running water, after drying on superclean bench with 75% alcohol-pickled 30s, proceed to the 15min that sterilizes in 0. 1% mercuric chloride, standby after aseptic water washing 5～8 times.With scalpel, cut fruit pod two ends approximately 0.5 cm, then fruit is longitudinally cut, by seed uniform broadcasting in 1/2 mg/L 6BA+0.2～0.5, MS medium+0.2～0.5 m g/L NAA+30 g/L sucrose+7.5g/L OK a karaoke club powder+100ml/L CW(coconut milk)+0.5 g/L AC(active carbon)) in medium, and with 26 ± 1 ℃ of dark surrounds under cultivate.
S22. on proliferated culture medium propagation and the plant regeneration of brood body in the middle of: be inoculated into 1/2 MS+ 0.5～1.0 mg/L NAA+30g/L sucrose+7.5g/L OK a karaoke club powder+0.5, mg/L 6BA+0.2～0.5 g/L AC(active carbon by sprouting the middle brood body stripping and slicing forming), breed, after many cultures, inoculate in the differential medium of 1/2MS+ 1.5～2.0 mg/L 6BA+0.2～0.5 mg/L NAA+30g/L sucrose+7.5g/L OK a karaoke club powder and break up, the bud differentiating and seedling proceed in following Rooting and hardening-off culture base: 1/2MS+ 0.1～0.2 mg/L NAA+20g/L sucrose+7.5g/L+0.5, mg/L 6BA+0.5～1.0 g/L AC(active carbon).
More than test all 26 ± 1 ℃ of temperature intensity of illumination 17 μ molm
-2s
-1cultivate under 12h illumination every day.
S23. test-tube seedling transplanting: can carry out hardening when seedling grows to 5～8 cm and have more than 2 root.During hardening, blake bottle lid is unscrewed, be placed under greenhouse 10～20 d or place after 3～5 d in the outdoor place without direct light, take out test-tube seedling transplanting.
S3. the authenticate technology of 2n andro gamete comprises film-making, dyeing, compressing tablet, observation and statistics:
S31. film-making: take out fresh flowers powder agglomates, be placed on clean slide, dry for preventing pollen, splash into a small amount of sterile water, with tweezers, pollen is dispersed on slide.
S32. dyeing: after material moisture dries on slide, evenly spread one deck improvement carbol fuchsin dye liquor above material, pass through fast flame 2～3 times.About 3～5 minutes of dyeing time.
S33. compressing tablet: vertically rap cover glass with the pencil with rubber, drive bubble out of.
S34. observe: with Olympus-I * 71 inverted microscope, observe 2n pollen occurrence frequency, under 40 * object lens, mobile slide, selects 10 visuals field to take pictures at random, finally add up the frequency that dyad in these 10 visuals field and triad occur.During observation, take 100 microsporocytes as 1 repetition, repeat 3 times.Observed result is shown in Fig. 1.
S35. add up: the frequency that 2n andro gamete occurs is added up according to following formula:
The frequency of 2n andro gamete (%)=(2D+Tr)/(2D+3Tr+4Te) * 100;
Wherein D represents dyad; Tr represents triad; Te represents tetrad.
S4. efficiently there is 2n andro gamete resource screening: incidence is greater than to 1% plant and carries out division propagation, and carry out 2 years above repetitive identified, filtering out the strain that incidence is high and stable is that 2n andro gamete resource efficiently occurs orchid.Described division propagation technology comprises the steps: that orchid division propagation carried out in resting stage, is preferably in that sprouting has formed but before being not yet unearthed.Orchid strain belongs to bunch type, and different cultivars plant division method is difference slightly, pins the basin soil of base of the plant during plant division with a hand, another hand by basin overturning out, shake gently matrix separated with tub wall, then orchid clump is taken out, gently that matrix is broken, make it separated with blue root, then from gap larger part, with the scraper of having sterilized, orchid clump is cut and cleaned by 2～3 strain one root and stem of certain plants, remove old root, rotten, dead leaf, place about 1 day, until blue root, returning can above basin when white.After orchid plant division improving activity of root system relatively a little less than, should note insulation, moisturizing management, to improve plant viablity.
Results and analysis:
By pressed disc method, observed Jin Zui, the generation of Cymbidium lancifolium and hybrid strain 2n andro gamete thereof (in Table 1 and table 2).Result shows, Jin Zui and Cymbidium lancifolium 2n andro gamete occurrence frequency are respectively 0.45% and 0.56%, and in its hybrid generation, has occurrence frequency that the 2n andro gamete occurrence frequency of 3 parts of strains is greater than 2%, 8 part of strain between 1～2%.The occurrence frequency of 2n andro gamete is greater than to 1% strain and screens, in the different times, carry out repeated authentication (table 3).Result shows, most high-frequencies generation 2n andro gamete strains, though have fluctuation at the 2n of different year andro gamete occurrence frequency, amplitude of variation is little, and more than 1%, apparently higher than parents' 2n andro gamete occurrence frequency.It is feasible that this proof utilizes distant hybridization to create the efficient 2n of generation andro gamete resource.
The generation of table 1 2012 annuity mouth * Cymbidium lancifolium hybrid 2n andro gametes
Table 2 2013 annuity mouth * Cymbidium lancifolium hybrid strain 2n andro gametes occur
Efficiently there is 2n andro gamete resource 2n andro gamete in different year and occur in table 3 Jin Zui * Cymbidium lancifolium
Claims (9)
1. create the method that 2n andro gamete resource efficiently occurs orchid, it is characterized in that, comprise the steps:
S1. Distant crossing combination configuration: select suitable parent, configuration hybrid combination is cultivated maternal plant under suitable condition, obtains fruit;
S2. hybrid population builds: fruit is carried out disinfection, get its seed and carry out the middle brood body of aseptic culture acquisition, middle brood body is carried out to plant regeneration, obtain test-tube plantlet, after test-tube seedling transplanting, cultivate to plant blossom;
The evaluation of S3.2n andro gamete: after plant to be planted is bloomed, get the fresh mature pollen of different strain plant, adopt after direct compression process film-making, at optical microphotograph Microscopic observation 2n pollen, the occurrence frequency of statistics 2n pollen;
S4. efficiently there is 2n andro gamete resource screening: incidence is greater than to 1% plant and carries out division propagation, and carry out 2 years above repetitive identified, filtering out the strain that incidence is high and stable is that 2n andro gamete resource efficiently occurs orchid.
2. method according to claim 1, is characterized in that, first suitable parent's matching principle for matching the kind that compatibility is relatively high described in S1, and it is high that parent's Pollen Activity is wanted; Next avoids selecting the blue strain of blooming to be for the first time parent; The kind of finally selecting seed to be easy to sprout is parent, to guarantee obtaining hybrid generation.
3. method according to claim 1, it is characterized in that, described in S1, the cross method of hybrid combination is: with tweezers, take out the pollinium of blooming in flower the maternal same day, the pollinium of male parent is put into maternal stamen chamber, with pencil, on playing cards, write the title of hybrid combination exactly, hybridization time, hangs on maternal plant, and be placed on the place of Warming ventilating, prevent cold wave.
4. method according to claim 1, is characterized in that, hybrid population builds and comprises the steps: described in S2
S41. fruit pod sterilization and seed germination: after the fruit of freshly harvested is sterile-processed, with scalpel, cut fruit pod two ends 0.5 cm, then fruit is longitudinally cut to taking-up seed, seed uniform broadcasting, on seed germination medium, is carried out to dark cultivation at suitable temperature condition and obtained middle brood body;
S42. propagation and the plant regeneration of brood body in the middle of: the middle brood body stripping and slicing that seed germination is formed, being inoculated into proliferated culture medium breeds, after subculture is cultivated, be transferred in differential medium and break up, the bud differentiating and seedling proceed in Rooting and hardening-off culture base, carry out strong plantlets and rootage;
S43. test-tube seedling transplanting: can carry out hardening when seedling grows to 5～8 cm and have more than 2 root; During hardening, blake bottle lid is unscrewed, be placed under greenhouse 10～20 d or place after 3～5 d in the outdoor place without direct light, take out test-tube seedling transplanting.
5. method according to claim 4, it is characterized in that, disinfect described in S41 as running water for the fruit of freshly harvested rinses 2～3 min, dry the alcohol-pickled 30s of rear use 75%, then fruit is proceeded to the 15min that sterilizes in 0. 1% mercuric chloride, standby after aseptic water washing 3～5 times.
6. method according to claim 4, it is characterized in that, seed germination medium is 1/2 m g/L NAA+30 g/L sucrose+7.5g/L OK a karaoke club powder+100ml/L coconut milk+0.5, mg/L 6BA+0.2～0.5, MS medium+0.2～0.5 g/L active carbon described in S41.
7. method according to claim 4, is characterized in that, proliferated culture medium is 1/2 MS+ 0.5～1.0 mg/L NAA+30g/L sucrose+7.5g/L OK a karaoke club powder+0.5, mg/L 6BA+0.2～0.5 g/L active carbon described in S42.
8. method according to claim 4, is characterized in that, differential medium is 1/2MS+ 1.5～2.0 mg/L 6BA+0.2～0.5 mg/L NAA+30g/L sucrose+7.5g/L OK a karaoke club powder described in S42.
9. method according to claim 4, is characterized in that, Rooting and hardening-off culture base is 1/2MS+ 0.1～0.2 mg/L NAA+20g/L sucrose+7.5g/L OK a karaoke club powder+0.5, mg/L 6BA+0.5～1.0 g/L active carbon described in S42.
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| CN105230488A (en) * | 2015-10-29 | 2016-01-13 | 广西大学 | Cymbidium lancifolium leaf tissue culture rapid propagation method |
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| CN107667848A (en) * | 2017-09-26 | 2018-02-09 | 华南农业大学 | A kind of method for creating easy industrialized propagation Chinese cymbidium resource |
| CN110313401A (en) * | 2019-07-12 | 2019-10-11 | 华南农业大学 | A method of promoting bud differentiation in enterprise's sword chalk Chinese cymbidium tissue culture procedures |
| CN113179948A (en) * | 2021-04-22 | 2021-07-30 | 广西壮族自治区农业科学院 | Formula of sterile seeding culture medium for cymbidium bicolor and tissue culture method |
| CN114467744A (en) * | 2022-03-21 | 2022-05-13 | 玉林师范学院 | Method for creating pluriploid resource of plukenetia volubilis linneo |
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|---|---|---|---|---|
| CN105230488A (en) * | 2015-10-29 | 2016-01-13 | 广西大学 | Cymbidium lancifolium leaf tissue culture rapid propagation method |
| CN107347627A (en) * | 2017-07-11 | 2017-11-17 | 华南农业大学 | A kind of method of the blue sexual polyploid of efficiently founder |
| CN107667848A (en) * | 2017-09-26 | 2018-02-09 | 华南农业大学 | A kind of method for creating easy industrialized propagation Chinese cymbidium resource |
| CN107667848B (en) * | 2017-09-26 | 2019-11-15 | 华南农业大学 | A method of creating easy industrialized propagation Chinese cymbidium resource |
| CN110313401A (en) * | 2019-07-12 | 2019-10-11 | 华南农业大学 | A method of promoting bud differentiation in enterprise's sword chalk Chinese cymbidium tissue culture procedures |
| CN113179948A (en) * | 2021-04-22 | 2021-07-30 | 广西壮族自治区农业科学院 | Formula of sterile seeding culture medium for cymbidium bicolor and tissue culture method |
| CN113179948B (en) * | 2021-04-22 | 2023-01-13 | 广西壮族自治区农业科学院 | Formula of sterile seeding culture medium for cymbidium bicolor and tissue culture method |
| CN114467744A (en) * | 2022-03-21 | 2022-05-13 | 玉林师范学院 | Method for creating pluriploid resource of plukenetia volubilis linneo |
| CN114467744B (en) * | 2022-03-21 | 2023-05-16 | 玉林师范学院 | Method for creating star-oil vine polyploid resource |
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