CN103704126B - A kind of method creating Cymbidium ensifolium (L.) Sw. generation 2n androgamete resource - Google Patents
A kind of method creating Cymbidium ensifolium (L.) Sw. generation 2n androgamete resource Download PDFInfo
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Abstract
The present invention relates to plant genetics and breeding technical field, specifically disclose a kind of method creating Cymbidium ensifolium (L.) Sw. generation 2n androgamete resource.Described method includes the steps such as parental apolegamy, Distant crossing combination, hybrid population's structure, the qualification of 2n androgamete, generation 2n androgamete Screening germplasm.The present invention utilizes distant hybridization means, it is thus achieved that the Cymbidium ensifolium (L.) Sw. resource of 2n androgamete occurs, for when not having polyploid, utilizing sexual hybridization method to create sexual polyploid, effectively facilitate Cymbidium ensifolium (L.) Sw. polyploid breeding, cultivate breakthrough Cymbidium ensifolium (L.) Sw. new varieties and lay the foundation.
Description
Technical field
The invention belongs to plant genetics and breeding technical field, create, more particularly to a kind of, the method that 2n androgamete resource occurs Cymbidium ensifolium (L.) Sw..
Background technology
Cymbidium ensifolium (L.) Sw. (Cymbidium) is world's famous flower, integrate view and admire, economy and cultureal value, the deep favor by countries in the world people.China is the country that Cymbidium ensifolium (L.) Sw. resource is the abundantest, known have 49 kinds, account for more than 2/3rds (Liu Zhongjian etc. of the world total, 2006), these resources of overseas utilization, have logged in nearly 15000 of Cymbidium ensifolium (L.) Sw. collective hybrid in the Royal Society, and have cultivated substantial amounts of commercial varieties, but the hybrid of the domestic Cymbidium ensifolium (L.) Sw. new varieties cultivated and login is all less, and the kind number with Commercial Prospect is just more rare.Germ plasm resource is the material base of breeding, creating critical germ plasm resource is the key cultivated and have breakthrough varieties, therefore studying the innovative approach of germ plasm resource to more good utilisation China Cymbidium ensifolium (L.) Sw. resource, cultivation has breakthrough Cymbidium ensifolium (L.) Sw. new varieties tool and is of great significance.
Polyploid breeding is the important means of Cymbidium ensifolium (L.) Sw. breeding, and current business-like Hybrid Cymbidium mostly is polyploid (Zhu Genfa etc., 2006), and creating polyploid resource is the key carrying out Cymbidium ensifolium (L.) Sw. polyploid breeding.The method that the establishment of Cymbidium ensifolium (L.) Sw. polyploid resource can pass through physics, chemistry and biology, compared with the method that traditional physics and chemical method create polyploid, utilize create biology (2n gamete approach) polyploid have uniqueness superiority, it not only can directly obtain triploid, tetraploid resource, and the heterozygosity of parent Geng Gao can be transmitted and hybrid dysgenesis can be overcome.Current Chinese scholars, utilizes 2n androgamete approach to be successfully obtained Bulbus Lilii；(Barba-Gonzalezetal, 2006；Barba-Gonzalezetal, 2005), Chinese rose (Crespeletal, 2006), Flos Begoniae Evansianae (Dewitteetal, 2010) and calla lily (Wu Hongzhi etc., 2011) etc. have the polyploid resource of important ornamental value.
2n androgamete can naturally-occurring can also artificial induction, the main method of artificial induction's 2n androgamete has physical method and chemical method.Physical method includes temperature violent change, gas mutation and ionizing radiation etc..Chemical method includes Colchicine, oryzalin (Oryzalin) and trefanocide (Trifluralin) and caffeine etc..At present in Cymbidium ensifolium (L.) Sw. breeding, the research report of artificial induction's 2n androgamete is less, Fan Bin etc. (2012) utilize colchicine-induced spring sword 2n androgamete, and its highest inductivity is 2.65%, PWongprichAC (activated carbon) hanetal (2013) utilizes N2O successfully induces iris 2n androgamete.No matter being physical method or chemical method induction 2n androgamete, all there is 2n androgamete competitiveness and decline, the problems such as it is unstable with efficiency that inductivity is on the low side, this causes that 2n androgamete cannot effectively apply to breeding practice.
Therefore, the Cymbidium ensifolium (L.) Sw. resource creating naturally-occurring 2n androgamete becomes the key carrying out Cymbidium ensifolium (L.) Sw. polyploid breeding.
Summary of the invention
The technical problem to be solved in the present invention is to overcome physical method and chemical method induction 2n androgamete in prior art, all there is 2n androgamete competitiveness to decline, the defect that inductivity is on the low side and efficiency is unstable, it is provided that a kind of method creating Cymbidium ensifolium (L.) Sw. generation 2n androgamete resource.The present invention compared with conventional physical, chemical method, 2n androgamete occur stable, vigor high, can reuse for a long time.
The purpose of the present invention is achieved by the following technical programs:
A kind of method creating Cymbidium ensifolium (L.) Sw. generation 2n androgamete resource, comprises the steps:
S1. Distant crossing combination configuration: select suitable parent, configure cross combination, cultivate maternal plant under appropriate conditions, it is thus achieved that fruit；
S2. hybrid population builds: fruit is carried out disinfection, and takes its seed and carries out brood body in the middle of aseptic culture acquisition, middle brood body is carried out plant regeneration, it is thus achieved that test tube Seedling, after test tube transplantation of seedlings, cultivates to plant blossom；
The qualification of S3.2n androgamete: plant to be planted Post flowering, takes different strain plant fresh mature pollen, after adopting direct compression process film-making, observes 2n pollen, the occurrence frequency of statistics 2n pollen under an optical microscope；
S4., 2n androgamete Screening germplasm occurring: the incidence rate plant more than 1% is carried out division propagation, and carries out more than 2 years repetitive identified, filtering out the high and stable strain of incidence rate is that 2n androgamete resource occurs Cymbidium ensifolium (L.) Sw..
The method according to the invention, wherein, described in step S1, Distant crossing combination preparation technique comprises the following steps:
The matching principle of parent: distant hybridization is commonly present the problem of cross incompatibility, the sibship of parent can not be too remote, it should be noted that the kind that apolegamy affinity is of a relatively high, and the Pollen Activity of parent wants height；Next avoids the blue strain selecting first time to bloom to be parent；The kind that seed is prone to sprout finally is selected to be parent, to ensure to obtain hybrid generation.
Hybridization technique: select healthy and strong anosis plant to make Parent, the massula in flower of blooming the maternal same day is taken out with tweezers, then the massula of male parent is put in the stamen chamber of female parent, on playing cards, the title of cross combination is write exactly with pencil, hybridization time, hang on maternal plant, and be placed on the place of Warming ventilating, it is prevented that cold wave.
Described in step S2, hybrid population's constructing technology comprises the following steps:
S21. the fruit sterilization of pod and seed germination: by the fruit of freshly harvested tap water 2～3min, after drying on superclean bench alcohol-pickled 30s with 75%, proceed to the 15min that sterilizes in the mercuric chloride of 0.1%, standby after aseptic water washing 3～5 times.Cut fruit pod two ends with dissecting knife and be about 0.5cm, then that fruit is longitudinally slit, by seed uniform broadcasting on suitable seed germination medium, and carry out light culture at suitable temperature conditions.
The propagation of the middle brood body of S22 and plant regeneration: by sprouting the middle brood body stripping and slicing formed, be inoculated into suitable proliferated culture medium and breed, be transferred to after successive transfer culture in suitable division culture medium and break up.The bud differentiated and Seedling proceed in suitable Rooting and hardening-off culture base, carry out strong plantlets and rootage.
S23. test tube transplantation of seedlings: seedling exercising can be carried out when seedling length to 5～8cm and when there is more than 2 roots.During seedling exercising, culture bottle lid is unscrewed, be placed under greenhouse 10～20d or outdoor place 3～5d without direct light local after, taking-up test tube transplantation of seedlings.
Seed germination medium described in step S21 is 1/2MS culture medium+0.2～0.5mg/L6BA+0.2～0.5mg/LNAA+30g/L sucrose+7.5g/L OK a karaoke club powder+100ml/LCW (Sucus Cocois)+0.5g/LAC (activated carbon).
Proliferated culture medium described in S22 is 1/2MS+0.5～1.0mg/L6BA+0.2～0.5mg/LNAA+30g/L sucrose+7.5g/L OK a karaoke club powder+0.5g/LAC (activated carbon).
Division culture medium described in S22 is 1/2MS+1.5～2.0mg/L6BA+0.2～0.5mg/LNAA+30g/L sucrose+7.5g/L OK a karaoke club powder.
Rooting and hardening-off culture base described in S22 is 1/2MS+0.1～0.2mg/L6BA+0.5～1.0mg/LNAA+20g/L sucrose+7.5g/L OK a karaoke club powder+0.5g/LAC (activated carbon).
The method according to the invention, wherein, described in step S3, the identification technology of 2n androgamete comprises the following steps:
S31 film-making: take out fresh flowers powder agglomates, be placed on clean microscope slide, for preventing pollen from drying, instill a small amount of sterilized water, make pollen be dispersed on microscope slide with tweezers.
S32. dyeing: after material moisture dries on microscope slide, uniformly smears last layer improvement carbol fuchsin dye liquor above material, moves quickly through flame 2～3 times.About 3-5 minute of dyeing time.
S33. tabletting: vertically rap coverslip with the pencil with rubber, drive bubble out of.
S34. observe: observe 2n pollen occurrence frequency with Olympus-I × 71 inverted microscope, under 40 × object lens, mobile microscope slide, randomly choose 10 visuals field and take pictures, finally add up the dyad in these 10 visuals field and the frequency of triad generation.During observation, repeat for 1 time with 100 microsporocytes, repeat 3 times.
S35. statistics: the frequency that 2n androgamete occurs is added up according to equation below:
Frequency (%)=(2D+Tr)/(2D+3Tr+4Te) × 100 of 2n androgamete；
Wherein D represents dyad；Tr represents triad；Te generation～table tetrad.
The method according to the invention, wherein, described in step S4, division propagation technology comprises the following steps: division propagation technology: Cymbidium ensifolium (L.) Sw. division propagation carries out at rest period, it is desirable to before sprouting has been formed but has not yet been unearthed.Cymbidium ensifolium (L.) Sw. strain belongs to bunch type, different cultivars plant division method slightly difference, pins the basin soil of base of the plant during plant division with a hands, another hands is by basin overturning out, it is shaken gently for substrate to separate with tub wall, then orchid clump is taken out, gently substrate is crushed, so as to separate with Cymbidium ensifolium (L.) Sw. root, then from gap larger part, with sterilized scraper, orchid clump is cut by 2～3 strain one root and stem of certain plants clean, remove old root, rotten, dead leaf, place about 1 day, can above basin when Cymbidium ensifolium (L.) Sw. root returns white.After Cymbidium ensifolium (L.) Sw. plant division, improving activity of root system is relatively weak, it should be noted that insulation, moisturizing management, to improve plant viablity.
The invention has the beneficial effects as follows: the present invention method by distant hybridization, can obtaining stability and high efficiency and the Cymbidium ensifolium (L.) Sw. resource of 2n androgamete, relatively physics or the method for chemistry occur, 2n androgamete occurs stable, can reuse for a long time, and the 2n androgamete vigor of formation is high.This development to promoting Cymbidium ensifolium (L.) Sw. polyploid breeding and industry, the core competitiveness tool improving China's orchid industry is of great significance.
Figure of description
Fig. 1. the qualification result of Chinese cymbidium 2n androgamete；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.
Detailed description of the invention
The present invention is further described below in conjunction with specific embodiment.Unless stated otherwise, the present invention adopts reagent, equipment are the art conventional reagent and equipment.
The establishment of the efficient 2n androgamete resource of embodiment 1 Jin Zui × Cymbidium lancifolium hybrid
S1. Distant crossing combination configuration: gold mouth Chinese cymbidium florescence, selects healthy and strong anosis plant to make female parent, takes Cymbidium lancifolium massula for its pollination, with pencil in the bright cross combination title of plastics playing cards subscript, hybridization time, hangs on maternal plant.Cultivate maternal plant under appropriate conditions, it is thus achieved that fruit.
S2. hybrid population builds
S21. the fruit sterilization of pod and seed germination: the fruit dissecting knife of freshly harvested is removed fruit stem, dip after appropriate liquid detergent cleans with tap water 2～3min, after drying on superclean bench alcohol-pickled 30s with 75%, proceed in the mercuric chloride of 0.1% sterilization 15min, standby after aseptic water washing 5～8 times.Cut fruit pod two ends with dissecting knife and be about 0.5cm, then fruit is longitudinally slit, by seed uniform broadcasting in 1/2MS culture medium+0.2～0.5mg/L6BA+0.2～0.5mg/LNAA+30g/L sucrose+7.5g/L OK a karaoke club powder+100ml/LCW (Sucus Cocois)+0.5g/LAC (activated carbon)) in culture medium, and with 26 ± 1 DEG C of dark surrounds under cultivate.
S22. the propagation of brood body and plant regeneration in the middle of: be inoculated on the proliferated culture medium of 1/2MS+0.5～1.0mg/L6BA+0.2～0.5mg/LNAA+30g/L sucrose+7.5g/L OK a karaoke club powder+0.5g/LAC (activated carbon) breed sprouting the middle brood body stripping and slicing formed, inoculating after many cultures in the division culture medium of 1/2MS+1.5～2.0mg/L6BA+0.2～0.5mg/LNAA+30g/L sucrose+7.5g/L OK a karaoke club powder and break up, the bud differentiated and Seedling proceed in following Rooting and hardening-off culture base: 1/2MS+0.1～0.2mg/L6BA+0.5～1.0mg/LNAA+20g/L sucrose+7.5g/L+0.5g/LAC (activated carbon).
More than experiment is all at temperature 26 ± 1 DEG C, 17 μm of ol m of intensity of illumination-2·s-1Every day cultivates under 12h illumination.
S23. test tube transplantation of seedlings: seedling exercising can be carried out when seedling length to 5～8cm and when there is more than 2 roots.During seedling exercising, culture bottle lid is unscrewed, be placed under greenhouse 10～20d or outdoor place 3～5d without direct light local after, taking-up test tube transplantation of seedlings.
The identification technology of S3.2n androgamete includes film-making, dyeing, tabletting, observation and statistics:
S31. film-making: take out fresh flowers powder agglomates, be placed on clean microscope slide, for preventing pollen from drying, instill a small amount of sterilized water, make pollen be dispersed on microscope slide with tweezers.
S32. dyeing: after material moisture dries on microscope slide, uniformly smears last layer improvement carbol fuchsin dye liquor above material, moves quickly through flame 2～3 times.About 3～5 minutes of dyeing time.
S33. tabletting: vertically rap coverslip with the pencil with rubber, drive bubble out of.
S34. observe: observe 2n pollen occurrence frequency with Olympus-I × 71 inverted microscope, under 40 × object lens, mobile microscope slide, randomly choose 10 visuals field and take pictures, finally add up the dyad in these 10 visuals field and the frequency of triad generation.During observation, repeat for 1 time with 100 microsporocytes, repeat 3 times.Observed result is shown in Fig. 1.
S35. statistics: the frequency that 2n androgamete occurs is added up according to equation below:
Frequency (%)=(2D+Tr)/(2D+3Tr+4Te) × 100 of 2n androgamete；
Wherein D represents dyad；Tr represents triad；Te represents tetrad.
S4. efficiently there is 2n androgamete Screening germplasm: the incidence rate plant more than 1% being carried out division propagation, and carries out more than 2 years repetitive identified, filtering out the high and stable strain of incidence rate is that 2n androgamete resource efficiently occurs Cymbidium ensifolium (L.) Sw..Described division propagation technology comprises the steps: that Cymbidium ensifolium (L.) Sw. division propagation carries out at rest period, it is desirable to before sprouting has been formed but has not yet been unearthed.Cymbidium ensifolium (L.) Sw. strain belongs to bunch type, different cultivars plant division method slightly difference, pins the basin soil of base of the plant during plant division with a hands, another hands is by basin overturning out, it is shaken gently for substrate to separate with tub wall, then orchid clump is taken out, gently substrate is crushed, so as to separate with Cymbidium ensifolium (L.) Sw. root, then from gap larger part, with sterilized scraper, orchid clump is cut by 2～3 strain one root and stem of certain plants clean, remove old root, rotten, dead leaf, place about 1 day, can above basin when Cymbidium ensifolium (L.) Sw. root returns white.After Cymbidium ensifolium (L.) Sw. plant division, improving activity of root system is relatively weak, it should be noted that insulation, moisturizing management, to improve plant viablity.
Result and analysis:
Jin Zui is observed, the generation (see table 1 and table 2) of Cymbidium lancifolium and hybrid strain 2n androgamete thereof by pressed disc method.It is shown that Jin Zui and Cymbidium lancifolium 2n androgamete occurrence frequency respectively 0.45% and 0.56%, and having the 2n androgamete occurrence frequency of 3 parts of strains in its hybrid generation more than 2%, the occurrence frequency of 8 parts of strains is between 1～2%.The occurrence frequency of the 2n androgamete strain more than 1% is screened, the different times is repeated checking (table 3).It is shown that 2n androgamete strain occurs most altofrequencies, though the 2n androgamete occurrence frequency in different year has fluctuation, but amplitude of variation is little, and more than 1%, hence it is evident that higher than the 2n androgamete occurrence frequency of parents.This proves that it is feasible for utilizing distant hybridization to create efficiently generation 2n androgamete resource.
The generation of table 12012 annuity mouth × Cymbidium lancifolium hybrid 2n androgamete
Table 22013 annuity mouth × Cymbidium lancifolium hybrid strain 2n androgamete occurs
Table 3 Jin Zui × Cymbidium lancifolium efficiently occurs 2n androgamete resource 2n androgamete in different year to occur
Claims (3)
1. one kind creates the method that 2n androgamete resource occurs Cymbidium ensifolium (L.) Sw., it is characterised in that comprise the steps:
S1. Distant crossing combination configuration: select suitable parent, configure cross combination, cultivate maternal plant under appropriate conditions, it is thus achieved that fruit；
S2. hybrid population builds: fruit is carried out disinfection, and takes its seed and carries out brood body in the middle of aseptic culture acquisition, middle brood body is carried out plant regeneration, it is thus achieved that test tube Seedling, after test tube transplantation of seedlings, cultivates to plant blossom；
The qualification of S3.2n androgamete: plant to be planted Post flowering, takes different strain plant fresh mature pollen, after adopting direct compression process film-making, observes 2n pollen, the occurrence frequency of statistics 2n pollen under an optical microscope；
S4., 2n androgamete Screening germplasm occurring: the incidence rate plant more than 1% is carried out division propagation, and carries out more than 2 years repetitive identified, filtering out the high and stable strain of incidence rate is that 2n androgamete resource occurs Cymbidium ensifolium (L.) Sw.；
Wherein, the hybridizing method of cross combination described in S1 is: take out the massula blooming in flower the maternal same day with tweezers, the massula of male parent is put in the stamen chamber of female parent, on playing cards, the title of cross combination is write exactly with pencil, hybridization time, hang on maternal plant, and be placed on the place of Warming ventilating, it is prevented that cold wave；
Hybrid population described in S2 builds and comprises the steps:
S21. the fruit sterilization of pod and seed germination: after sterile-processed for the fruit of freshly harvested, fruit pod two ends 0.5cm is cut with dissecting knife, then, by longitudinally slit for fruit taking-up seed, by seed uniform broadcasting on seed germination medium, carry out light culture at suitable temperature conditions and obtain middle brood body；
S22. the propagation of brood body and plant regeneration in the middle of: the middle brood body stripping and slicing that seed germination is formed, it is inoculated into proliferated culture medium to breed, being transferred in division culture medium after successive transfer culture and break up, the bud differentiated and Seedling proceed in Rooting and hardening-off culture base, carry out strong plantlets and rootage；
S23. test tube transplantation of seedlings: seedling exercising can be carried out when seedling length to 5～8cm and when there is more than 2 roots；During seedling exercising, culture bottle lid is unscrewed, be placed under greenhouse 10～20d or outdoor place 3～5d without direct light local after, taking-up test tube transplantation of seedlings；
Cross combination described in S1 is with gold mouth Chinese cymbidium for female parent, with Cymbidium lancifolium for male parent；
Seed germination medium described in S21 is 1/2MS culture medium+0.2～0.5mg/L6BA+0.2～0.5mg/LNAA+30g/L sucrose+7.5g/L OK a karaoke club powder+100ml/L Sucus Cocois+0.5g/L activated carbon；
Proliferated culture medium described in S22 is 1/2MS+0.5～1.0mg/L6BA+0.2～0.5mg/LNAA+30g/L sucrose+7.5g/L OK a karaoke club powder+0.5g/L activated carbon；
Division culture medium described in S22 is 1/2MS+1.5～2.0mg/L6BA+0.2～0.5mg/LNAA+30g/L sucrose+7.5g/L OK a karaoke club powder；
Rooting and hardening-off culture base described in S22 is 1/2MS+0.1～0.2mg/L6BA+0.5～1.0mg/LNAA+20g/L sucrose+7.5g/L OK a karaoke club powder+0.5g/L activated carbon.
2. method according to claim 1, it is characterised in that described in S1, the matching principle of suitable parent is the kind that first apolegamy affinity is of a relatively high, and the Pollen Activity of parent wants height；Next avoids the blue strain selecting first time to bloom to be parent；The kind that seed is prone to sprout finally is selected to be parent, to ensure to obtain hybrid generation.
3. method according to claim 1, it is characterized in that, disinfect as by the fruit tap water 2～3min of freshly harvested described in S21, with the alcohol-pickled 30s of 75% after drying, then fruit is proceeded in the mercuric chloride of 0.1% sterilization 15min, standby after aseptic water washing 3～5 times.
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| 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 |
| 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 |
| 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 |
| 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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