CN101147465A - Method for inducing plant embryo sac chromosome doubling to select and breed triploid - Google Patents
Method for inducing plant embryo sac chromosome doubling to select and breed triploid Download PDFInfo
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- CN101147465A CN101147465A CNA200710064712XA CN200710064712A CN101147465A CN 101147465 A CN101147465 A CN 101147465A CN A200710064712X A CNA200710064712X A CN A200710064712XA CN 200710064712 A CN200710064712 A CN 200710064712A CN 101147465 A CN101147465 A CN 101147465A
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Abstract
The present invention discloses a method capable of inducing plant embryonary sac chromosome to reduplicatively select and cultivate plant triploid. Said invention includes the following several procedures: pollination to plant female inflorescence, utilizing colchicine to make treatment, eliminating treatment, infructescence management, collecting seeds, sowing, seedling cultivating and chromosome detection. Said invention also provides the concrete steps of above-mentioned every procedure and its concrete requirement.
Description
Technical field
The present invention relates to a kind of method of seed selection plant polyploid, relate in particular to the method for a kind of inducing plant blastular (egagametophyte) chromosome doubling breed triploid, belong to the plant genetics and breeding field.
Background technology
The generation of natural plant polyploid all comes from genomic multiple variation in the cell nucleus, i.e. produce unreduced gamete in occurrent chromosome doubling and the reduction division process in the somatic cell division process.Artificial induction's chromosome doubling can be taked physics inducement methods such as chemical induction methods such as colchicine solution and abnormal temperature.And the approach that manually obtains polyploid comprises somatic double, Different Ploidy body intermolecular hybrid, the hybridization of natural or artificial unreduced gamete, endosperm cultivation and cytomixis etc.
Kang Xiangyang etc. (2004) are to silver-colored gland poplar (the Populus alba * P.glandnlosa) wait female inflorescence to carry out the colchicine solution processing of pollination regular period, obtained a collection of white poplar hybrid triploid, the highest yield of triploid induction reaches 57.1% (Kang Xiangyang `, Zhang Ping's winter, Gao Peng, etc. colchicin is induced the discovery of white poplar triploid new way. Beijing Forestry University's journal, 2004,26 (1): 1-4), for triploid induction has found a new approach.But this research only points out that under space-time condition at that time silver-colored gland poplar pollination back 24-36h applies the lasting 24h of processing of colchicine solution can obtain triploid, but wherein exists many key technical problems to be worth further research.What stage is oogamete formation and development as white poplar pollination back 24-36h in period actually? is which processing in period in the oogamete formation and development that triploid the best is induced period? how in practical operation, differentiate effective processing period of induced triploid at different phytobiology characteristics? in addition, the awarded phase of the most plant pistil stigma of cause is long, whether the different operating personnel have the criterion difference of awarding property to pistil stigma, causing pollination to have early has late, some plants that comprise silver-colored gland poplar are when concrete hybridization pollination even can differ 1-2d, the initial reference point of processing that this neither one is clear and definite, and the standard of only determining period as effective processing with the back 24-36h that pollinates etc. does not merely meet reality yet, there is the risk of failure, also needs from technological layer, to solve targetedly.
Because above-mentioned technical problem fails effectively to be solved, and the application of this method is restricted.Be that silver-colored gland poplar is applied same technical finesse flow process equally, sample time of concrete operations personnel difference, material the different or environmental condition of cultivating not simultaneously, there is significant difference in its result, show that only the risk by time control is bigger, more impossible poplar and the plant species in addition thereof of being extended to.Therefore, solve the after-applied colchicine solution of pollination and handle inducing plant triploid cytology period and correlation technique problem, form effective triploid induction technical support system, induce triploid stability of silver-colored gland poplar and high efficiency for realizing that the after-applied colchicin of pollination is handled, and the triploid induction etc. of this method being introduced other willow or other plant has important practice value.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, provides a kind of accuracy, stability and efficient the higher triploid method of seed selection plant.
Technical problem to be solved by this invention is achieved through the following technical solutions:
The triploid method of a kind of inducing plant embryo sac chromosome doubling to select and breed plant, comprise and pollinating growing to the plant female inflorescence of suitable time, female flower to the back regular period of pollinating applies colchicine solution and handles, remove processing with flushing with clean water, infructescence management, results seed, seeding and seedling raising, steps such as chromosome detection, wherein, described pollination is meant when the plant female inflorescence is in best pollinated stage and pollinates; Described processing is meant when the plant oogamete and is in two nuclears and four nuclear blastulars in period of occupying the majority (in the period that four nuclear blastulars occupy the majority more preferably), applies 0.1～0.6% colchicine solution and handles female flower 6-30 hour.
The triploid method of above-mentioned seed selection plant, described plant optimization are Populus (Populus) plant.
As a kind of embodiment preferred of the present invention, specific as follows:
A kind of method of inducing poplar embryo sac chromosome doubling to select and breed triploid, comprise and pollinating growing to the willow female inflorescence of suitable time, female flower to the back regular period of pollinating applies colchicine solution and handles, remove processing with flushing with clean water, the infructescence management, the results seed, seeding and seedling raising, steps such as chromosome detection, wherein, described pollination is to pollinate when the willow female inflorescence meets following condition: observe the pistil stigma surface and fully launch the papilla cell prosperity under stereomicroscope; Become blue and have a large amount of bubbles to produce with benzidine-hydrogen peroxide dyeing back column cap; Described processing is meant to grow period of being in two nuclears and four nuclear blastulars and occupying the majority when the blastular of willow oogamete and (that is: during apart from best pollinated stage in the pollination back 12～96h), applies 0.1～0.6% colchicine solution and handled female flower 6-30 hour.
In the said method, preferably, described processing is meant that to be in four nuclear blastulars be main period (that is: during apart from best pollinated stage in the pollination back 54～66h), to apply 0.3～0.5% colchicine solution and handled female flower 6-30 hour when the blastular of willow oogamete is grown.Wherein, described four nuclear blastulars are that the main quantity of four nuclear blastulars that is meant is higher than the quantity of monokaryon, two nuclears or eight nuclear blastulars.
Described willow female parent is preferably Mao Xinyang, and (((P.alba * P.tomentosa), Populus adenopoda (P.adenlpodn Mazim), false simon poplar bark (P.pseudo-simonii Kitag.) or wise man draw No. 3 poplars (P.pseudo-simonii * P.nigra var.lica), are more preferably the wise man and draw No. 3 poplars (P.pseudo-simonii * P.nigra var.lica) and be the willow female parent for P.alla * P.glandulosa), silver hair poplar for Populus tomentosa * P.balleana), silver-colored gland poplar; Described willow male parent is a male parent with Chinese white poplar (P.tomentosa), Populus adenopoda (P.adenlpodn Mazim), Beijing poplar (P.pyramidalis * P.cathayana cv.Beijingensis '), false simon poplar bark (P.pseudo-simonii Kitag.) etc. preferably, and more preferably Beijing poplar (P.pyramidalis * P.cathayana cv.Beijingensis ') is the willow male parent.
For how determining whether the plant female inflorescence is in best pollinated stage, thereby those of ordinary skills can judge in conjunction with the conventional method of some this areas whether the female inflorescence of target plant is in best pollinated stage again by the form of observing column cap.
Grow whether to be in the monokaryon blastular in the period that periods that eight nuclear mature embryo sac are grown or four nuclear blastulars occupy the majority as for blastular how to determine the plant oogamete, can judge or confirm that it is known or on top of that these methods or technology are those skilled in the art institute by methods such as routine sections.
It is the initial reference point of implementing to induce the embryo sac chromosome doubling to select processing that the triploid method of seed selection plant of the present invention clearly is provided with best pollinated stage, when stigma development arrives best pollinated stage, pollination in good time, and further definite female flower development to four nuclear blastular is apart from the time difference of best pollinated stage, control the processing time of embryo sac chromosome doubling to select more accurately, the after-applied colchicin of pollination be can increase and the accuracy and the stability of inducing plant embryo sac chromosome doubling to select handled, improve the triploid efficient of inducing, and can save the chemical reagent of handling usefulness and experiment material etc.
The invention will be further described below by embodiment.Should be pointed out that these embodiment only are illustrations of the present invention, should not be construed as limitation of the present invention.
Embodiment
Embodiment
With Mao Xinyang (Populus tomentosa * P.balleana), silver gland poplar (P.alla * P.glandulosa), silver hair poplar (P.alba * P.tomentosa), Populus adenopoda (P.adenlpodn Mazim), false simon poplar bark (P.pseudo-simoniiKitag.), the wise man draws No. 3 poplars (P.pseudo-simonii * P.nigra var.lica) etc. for maternal, with Chinese white poplar (P.tomentosa), Populus adenopoda (P.adenlpodn Mazim), (Beijing poplar (P.pyramidalis * P.cathayanacv.Beijingensis '), false simon poplar bark (P.pseudo-simonii Kitag.) etc. is a male parent, after employing is pollinated to the female inflorescence that is in best pollinated stage, blastular by conventional sections observation oogamete is grown, the blastular of oogamete be two nuclear blastulars with four nuclear blastulars in period of occupying the majority (during apart from best pollinated stage after the pollination 12～96h) with 0.3～0.5% colchicine solution immersion treatment female inflorescence, 6～30h, the triploid yield is between 30%～70%.
Drawing No. 3 poplar * Beijing poplars with the wise man is example (table 1), the best pollinated stage that the wise man draws No. 3 poplar female inflorescences is to observe the pistil stigma surface under the stereomicroscope fully to launch, the papilla cell prosperity becomes blue and a large amount of bubble generations etc. is arranged with benzidine-hydrogen peroxide dyeing back column cap; Draw the wise man and to pollinate when No. 3 poplars reach best pollinated stage, grow by the blastular of conventional sections observation oogamete pollination back, when the blastular of oogamete is positioned at two nuclear blastulars and four periods of examining blastulars, (24～96h) applied 0.3～0.5% colchicine solution immersion treatment female inflorescence, 6～30h; Because the wise man draws the asynchronism of No. 3 poplar blastular growths, each handles the triploid that all can obtain some experiment, wherein especially so that (effect that applies the processing induced triploid during apart from best pollinated stage after the pollination 54～66h) time is best apart from four nuclear blastular periods of best pollinated stage pollination back, account for acquisition the triploid sum 2/3, the triploid yield is up to 66.7%.
The after-applied colchicin of table 1 pollination is handled to induce the wise man to draw No. 3 poplar * Beijing poplar triploids and grow with blastular and is concerned statistical form
| Hybrid combination | Colchicin concentration (%) | The pollination post processing time (h) | The blastular developmental process | Triploid yield (%) | |||||
| Tetrad | Monokaryon | Two nuclears | Four nuclears | Eight nuclears | Zygote | ||||
| The wise man draws poplar * Beijing poplar No. 3 | 0.3 | 24 | 6.38 | 44.68 | 31.91 | 17.02 | 0 | 0 | 4.1 |
| 0.5 | 54 | 0 | 7.58 | 15.15 | 40.91 | 36.36 | 0 | 9.5 | |
| 0.5 | 60 | 0 | 7.94 | 12.70 | 46.03 | 33.33 | 0 | 66.7 | |
| 0.5 | 66 | 0 | 3.57 | 8.93 | 41.07 | 46.43 | 0 | 20.0 | |
| 0.5 | 72 | 0 | 2.22 | 13.33 | 28.89 | 55.56 | 0 | 14.3 | |
| 0.5 | 96 | 0 | 0 | 1.45 | 17.39 | 59.42 | 21.74 | 7.1 | |
Annotate: the pollination back regular period applies colchicin and continues to handle 6-30h.
Claims (7)
1. triploid method of inducing plant embryo sac chromosome doubling to select and breed plant, comprise and pollinating growing to the plant female inflorescence of suitable time, female flower to the back regular period of pollinating applies colchicine solution and handles, remove processing with flushing with clean water, the infructescence management, results seed, seeding and seedling raising, steps such as chromosome detection is characterized in that: described pollination is meant when the plant female inflorescence is in best pollinated stage and pollinates; Described processing is meant to grow when the plant oogamete and is in the period that two nuclears and four nuclear blastulars occupy the majority, and applies 0.1～0.6% colchicine solution and handles female flower 6-30 hour.
2. according to the method for claim 1, it is characterized in that: described processing is meant that the growth when the plant oogamete was in the period that four nuclear blastulars occupy the majority, and applies 0.3～0.5% colchicine solution and handles female flower 6-30 hour.
3. according to the method for claim 1, it is characterized in that: described plant is Populus (Populus) plant.
4. the method for an inducing poplar embryo sac chromosome doubling to select and breed triploid, comprise and pollinating growing to the willow female inflorescence of suitable time, female flower to the back regular period of pollinating applies colchicine solution and handles, remove processing with flushing with clean water, the infructescence management, the results seed, seeding and seedling raising, steps such as chromosome detection, it is characterized in that: described pollination is to pollinate when the willow female inflorescence meets following condition: observe the pistil stigma surface and fully launch the papilla cell prosperity under stereomicroscope; Become blue and have a large amount of bubbles to produce with benzidine-hydrogen peroxide dyeing back column cap; Described processing is meant that the blastular of willow oogamete is grown and is in the period that two nuclears and four nuclear blastulars occupy the majority, and applies 0.1～0.6% colchicine solution and handles female flower 6-30 hour.
5. according to the method for claim 4, it is characterized in that: described processing is meant to grow when the willow oogamete and is in the period that four nuclear blastulars occupy the majority, and applies 0.3～0.5% colchicine solution and handles female flower 6-30 hour.
6. according to the method for claim 4, it is characterized in that: described willow female parent is selected from Mao Xinyang, and (((P.alba * P.tomentosa), Populus adenopoda (P.adenlpodn Mazim), false simon poplar bark (P.pseudo-simonii Kitag.) or wise man draw poplar No. 3 for P.alla * P.glandulosa), silver hair poplar for Populustomentosa * P.balleana), silver-colored gland poplar; Described willow male parent is selected from Chinese white poplar (P.tomentosa), Populus adenopoda (P.adenlpodn Mazim), Beijing poplar (P.pyramidalis * P.cathayana cv.Beijingensis ') or false simon poplar bark (P.pseudo-simonii Kitag.).
7. according to the method for claim 6, it is characterized in that: described willow is maternal for the wise man draws No. 3 poplars (P.pseudo-simonii * P.nigra var.lica), and the willow male parent is Beijing poplar (P.pyramidalis * P.cathayanacv.Beijingensis ').
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392074A (en) * | 2011-11-16 | 2012-03-28 | 北京林业大学 | Method for directly identifying homologous recombination of higher plant DNA |
| CN102524056A (en) * | 2011-12-07 | 2012-07-04 | 北京林业大学 | Method for breeding poplar tetraploid plants with colchicine |
| CN108611313A (en) * | 2018-05-09 | 2018-10-02 | 扬州大学 | A method of detaching papilla cell from turnip column cap |
| CN118256551A (en) * | 2023-10-09 | 2024-06-28 | 北京林业大学 | Method for carrying out efficient gene editing in triploid populus tomentosa |
-
2007
- 2007-03-23 CN CNB200710064712XA patent/CN100563430C/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392074A (en) * | 2011-11-16 | 2012-03-28 | 北京林业大学 | Method for directly identifying homologous recombination of higher plant DNA |
| CN102524056A (en) * | 2011-12-07 | 2012-07-04 | 北京林业大学 | Method for breeding poplar tetraploid plants with colchicine |
| CN108611313A (en) * | 2018-05-09 | 2018-10-02 | 扬州大学 | A method of detaching papilla cell from turnip column cap |
| CN118256551A (en) * | 2023-10-09 | 2024-06-28 | 北京林业大学 | Method for carrying out efficient gene editing in triploid populus tomentosa |
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| CN100563430C (en) | 2009-12-02 |
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