CN1593107A - Yellow moccasin flower seed preparation and quick screening method - Google Patents
Yellow moccasin flower seed preparation and quick screening method Download PDFInfo
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- CN1593107A CN1593107A CN 200410040180 CN200410040180A CN1593107A CN 1593107 A CN1593107 A CN 1593107A CN 200410040180 CN200410040180 CN 200410040180 CN 200410040180 A CN200410040180 A CN 200410040180A CN 1593107 A CN1593107 A CN 1593107A
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- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000012216 screening Methods 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims description 14
- 241001516470 Cypripedium acaule Species 0.000 title 1
- 210000001161 mammalian embryo Anatomy 0.000 claims description 43
- 241001516485 Cypripedium Species 0.000 claims description 35
- 241000196324 Embryophyta Species 0.000 claims description 19
- 235000013399 edible fruits Nutrition 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 9
- 238000011534 incubation Methods 0.000 claims description 8
- FPCWRPBDOPARID-UHFFFAOYSA-N 2,3,4-triphenyl-1H-tetrazol-4-ium dichloride Chemical compound [Cl-].[Cl-].C1(=CC=CC=C1)N1N(N([NH+]=C1)C1=CC=CC=C1)C1=CC=CC=C1.C1(=CC=CC=C1)N1N(N([NH+]=C1)C1=CC=CC=C1)C1=CC=CC=C1 FPCWRPBDOPARID-UHFFFAOYSA-N 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 238000011010 flushing procedure Methods 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 238000007654 immersion Methods 0.000 claims description 4
- 239000012467 final product Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 10
- 241000519442 Cypripedium flavum Species 0.000 abstract description 4
- 238000004500 asepsis Methods 0.000 abstract description 4
- 238000009395 breeding Methods 0.000 abstract description 3
- 230000001488 breeding effect Effects 0.000 abstract description 3
- 230000006641 stabilisation Effects 0.000 abstract 1
- 238000011105 stabilization Methods 0.000 abstract 1
- 230000010152 pollination Effects 0.000 description 24
- 239000002775 capsule Substances 0.000 description 9
- 230000007226 seed germination Effects 0.000 description 9
- 230000035784 germination Effects 0.000 description 8
- 230000005070 ripening Effects 0.000 description 8
- 241000233855 Orchidaceae Species 0.000 description 4
- 230000003816 axenic effect Effects 0.000 description 3
- 230000010154 cross-pollination Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000000205 computational method Methods 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000012869 germination medium Substances 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000010153 self-pollination Effects 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The unique Cypripedium flavum peculiar to China adopts seed asepsis growing as the main approach for fast young sprout breeding. The present invention provides a Cyripedium flavum high grade seed cultivation and rapid screening method, is designed for sufficient and stabilization production.
Description
Technical field: the present invention relates to plant biotechnology field, relate in particular to the preparation and the rapid screening method of plant yellow lady's-slipper (Cypripedium flavum) seed.
Background technology: Cypripedium (Cypripedium) be the orchid family important view and admire one of genus, the lady's slipper pattern is gorgeous, the flower type is peculiar, its lip is calceoloid, so the title of " slippers orchid " is arranged, has the very high value of viewing and admiring and develop.Yellow lady's-slipper (C.flavum) is Chinese distinctive the orchid family flower plant, is chrysanthemum kind unique in the Chinese Cypripedium flower plant.Yellow lady's-slipper has yellowish or flavous flower, and the purple fleck is arranged in the lip, spends greatly, and delicate fragrance is arranged, and ornamental value is high, firmly gets liking of American-European countries orchid fan.In the international market, the about 5-30 dollar of annual seedling price of lady's slipper, the adult plant that blooms is the 25-300 dollar.Beautiful peculiar fancy points and high market value caused immoderate coyoting to be dug excessively, adds self-reproduction and poky physiological property, and Cypripedium plant wild resource is closely exhausted.American-European countries obtains the seedling of some lady's slipper kinds by the seed germination technology in recent years, but the production main body mostly is the amateur, and the seedling of cultivating and breeding is limited, does not form the scale breeding.This and high quality seed technology of preparing, seed asepsis sprouting rate are low, sprout that technical bottleneck such as regularity difference is still unresolved a direct relation.The lady's slipper seedling breeds the fruit of the wild plant of normal in the world employing as seed source at present; owing to be subjected to the influence of growing environment factor such as pollination medium, damage by disease and insect or plant physiological status etc. and usually suffer artificial collection, herd brokenly the influence of ring factor; lady's slipper ripening rate and seed vitality stability are very poor; sometimes possible ripening rate height; seed vitality might as well; but be that No kernels or seeds are gathered, as in a year of scarcity in the time of most of, can not ensure to obtain the required high-quality seed of large-scale production at all.
Summary of the invention: the present invention is directed to above-mentioned the deficiencies in the prior art,, provide the high quality seed preparation and the screening technique that meet the fast numerous needs of yellow lady's-slipper seedling scale at the key issue of seed preparation.This method can obtain to produce the required high-quality seed of seedling, carries out earlier detection and screening to producing used seed quality simultaneously, avoids blindly producing the wasting of resources that causes, and saves production cost greatly, has important and practical meanings.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
Preparation of yellow lady's-slipper seed and rapid screening method, select in 3 years not solid, plant is sturdy, the growth situation good yellow lady's-slipper plant that blooms, the flowers are in blossom, and palmification was carried out in 2-3 days in the back, and the picking fruit taking-up seed after 120 days of pollinating is in the TTC solution of immersion 1%, 25 ℃ of dark incubations 72 hours are chosen the seed of embryo vigor more than 70% as the fast numerous material of seedling.
First weighing 1gTTC (triphenyltetrazolium chloride triphenyltetrazolium chloride) is adopted in the preparation of said method TTC solution, in distilled water, adds the Tween-20 of 0.5ml in dissolved simultaneously, constant volume is 100ml, be mixed with 1% TTC solution, shake up, keep in Dark Place.
Said method places in the middle of the filter paper after seed takes out, and is folding, filter paper immersed in 1% the TTC solution, and 25 ℃ of dark incubations 72 hours took out filter paper then with flushing with clean water about 1 minute.
Red number/the seed of said seed embryo vigor=embryo has embryo rate * 100%.
Particularly, method of the present invention is selected in 3 years not solid earlier, plant is sturdy, the growth situation good yellow lady's-slipper plant that blooms, the flowers are in blossom, and the back was carried out palmification in 2-3 days, pollinating, picking fruit takes out seed after 120 days, place in the middle of the filter paper, folding, in the TTC solution with filter paper immersion 1%, first weighing 1gTTC (triphenyltetrazolium chloride triphenyltetrazolium chloride) is adopted in the preparation of TTC solution, in distilled water, add the Tween-20 of 0.5ml in dissolved simultaneously, constant volume is 100ml, be mixed with 1% TTC solution, shake up and get final product, seed is at 25 ℃ of dark incubations after 72 hours, and filter paper was taken out with flushing with clean water about 1 minute, choose the seed of embryo vigor more than 70% as the fast numerous material of seedling, the computational methods of seed embryo vigor are that the red number/seed of embryo has embryo rate * 100%.
Description of drawings:
Fig. 1 is the correlation schematic diagram of embryo vigor and germination rate.
Embodiment: in order to understand the present invention better, further specify essence of the present invention with embodiments of the invention, but content of the present invention is not limited thereto below in conjunction with accompanying drawing.
Embodiment 1:
Select in 3 years not solid earlier, plant is sturdy, the growth situation good yellow lady's-slipper plant that blooms, the flowers are in blossom, and the back was carried out palmification in 2-3 days, pollinating, picking fruit takes out seed after 120 days, place in the middle of the filter paper, folding, filter paper immersed in 1% the TTC solution, first weighing 1gTTC (triphenyltetrazolium chloride triphenyltetrazolium chloride) is adopted in the preparation of TTC solution, in dissolved in distilled water, the Tween-20 that adds 0.5ml simultaneously, constant volume is 100ml, is mixed with 1% TTC solution, shakes up to get final product, seed is at 25 ℃ of dark incubations after 72 hours, filter paper was taken out with flushing with clean water about 1 minute, choose the seed of embryo vigor 70% or more as seedling numerous material soon, the computational methods of seed embryo vigor are that the red number/seed of embryo has embryo rate * 100%.Particularly, adopt following step and method:
1, materials and methods
1.1 high quality seed preparation
Select in 3 years not solidly, plant is sturdy, good yellow lady's-slipper (Cypripedium flavum) plant that blooms of growth situation, and the back was carried out illegitimate pollination and palmification on the 2nd day respectively in blooming.The picking fruit (fruit is full maturity) after 120 days of pollinating, embryo rate, seed vitality (TTC method) are arranged and carry out the quality that seed germination rate that axenic germination obtains etc. is weighed seed quality with capsule volume, single fruit seed amount, seed, and determine to cultivate the pollination mode of high quality seed the best with this.
Pollination time adopts the cross pollination mode in the flowers are in blossom preceding 3 days, the flowers are in blossom back 1-3 days, and the flowers are in blossom back 4-6 days, the flowers are in blossom back 7-9 days, the flowers are in blossom after 10 days, and five periods are carried out artificial pollination.The picking fruit (fruit is full maturity) after 120 days of pollinating, embryo rate, seed vitality (TTC method) are arranged and carry out the quality that seed germination rate that axenic germination obtains or the like is weighed seed quality with capsule volume, single fruit seed amount, seed, and determine to cultivate the pollination time of high quality seed the best with this.
1.2 high quality seed rapid screening (TTC rapid screening method)
Step 1: the preparation of TTC solution
Weighing 1gTTC (triphenyltetrazolium chloride triphenyltetrazolium chloride) in distilled water, adds the Tween-20 of 0.5ml in dissolved simultaneously, and constant volume is 100ml, is mixed with 1% TTC solution, shakes up, and it is standby to keep in Dark Place.
Step 2: seed embryo vitality test
Taking out about 300 seed in needs are measured the yellow lady's-slipper capsule of seed vitality places clean filter paper (in the middle of the 3cm * 3cm), to be folded into 1cm
2The square shape paper piece.Filter paper immersed in 1% the TTC solution 25 ℃ of dark incubations 72 hours.Filter paper taken out place running water flushing 1 minute, launch behind the filter paper under anatomical lens the statistics seed embryo situation that reddens: the red number/seed of seed embryo vigor=embryo has embryo rate * 100%
Step 3: the checking of embryo vigour-testing method
Picked at random is 25 in the yellow lady's-slipper capsule that detects seed vitality through the TTC method, take out the part aseptically sowing seeds in media surface (method is reported in a separate paper), after 3 months cultivation, add up seed germination rate, seed germination rate=sprouting number/seed has embryo rate * 100%, and the seed vitality that its result and TTC method detect compares.Verify the feasibility of TTC rapid screening method with this.
2, result and discussion:
2.1 the pollination mode is to the influence of yellow lady's-slipper seed quality
The flowers are in blossom that the back was carried out illegitimate pollination and palmification on the 2nd day respectively yellow lady's-slipper, and result and solid naturally statistical data are compared.Experimental result shows that the artificial pollination ripening rate reaches 100%, is significantly higher than spontaneous pollination rate (2.0%); The self-pollination mode is in ripening rate, capsule volume, single fruit seed number and have on the index such as embryo rate basic identically with cross pollination mode gained result, but seed embryo vigor is then far below the cross pollination mode; Indexs minimum (seeing Table one) such as solid naturally ripening rate and seed vitality.Seed embryo vigor and germination rate are important index very in the seed quality, and therefore, the palmification mode is better than the illegitimate pollination mode.
Table one: the pollination mode is to the influence of yellow lady's-slipper seed quality
| The pollination mode | Pollination number (or statistical number) | Ripening rate (%) | Capsule volume (cm 3) | Single fruit seed amount (ten thousand) | Embryo rate (%) is on average arranged | Average embryo vigor (%) | Average germination rate (%) |
| Artificial from spending the artificial different nature of spending solid | ????20 ????20 ????60 | ????100 ????100 ????2.0 | ????3.8-5.5 ????4-5.5 ????2.0-4.0 | ???0.6-2.0 ???0.8-2.0 ???0.1-2.0 | ??82.2 ??90.5 ??78.6 | ??45.1 ??88.0 ??43.3 | ??34.0 ??61.4 ??37.0 |
Table two: pollination time is to the influence of yellow lady's-slipper seed quality
| Pollination time | The pollination number | Ripening rate (%) | Capsule volume (cm 3) | Single fruit seed amount (ten thousand) | Embryo rate (%) is on average arranged | Average embryo vigor (%) | Average germination rate (%) |
| The flowers are in blossom in 7-9 days for the flowers are in blossom in 4-6 days the flowers are in blossom in 1-3 days the flowers are in blossom the flowers are in blossom in preceding 3 days back back back after 10 days | ??20 ??20 ??20 ??20 ??20 | ???0 ??100 ???90 ???70 ???18 | ?????- ???4-5.5 ???4-5.5 ???3.8-5.5 ???2.0-3.5 | ????- ????0.8-2.0 ????0.4-1.5 ????0.2-1.5 ????0-0.5 | ??- ??90.5 ??90.3 ??89.8 ??68.7 | ??- ??88.0 ??57.2 ??23.5 ??15.6 | ??- ??61.4 ??33.7 ??27.8 ???2.1 |
2.2 pollination time is to the influence of yellow lady's-slipper seed quality
About 15 days of yellow lady's-slipper florescence.Respectively in that the flowers are in blossom preceding 3 days, the flowers are in blossom back 1-3 days, the flowers are in blossom back 4-6 days, the flowers are in blossom back 7-9 days, the flowers are in blossom after 10 days, and five periods are carried out palmification, and the florescence of pollen plant and pollination plant is synchronous.Experimental result shows, back pollination in 1-3 days mode that the flowers are in blossom in ripening rate, capsule volume, single fruit seed number, have be on every indexs such as embryo rate, seed embryo vigor and germination rate excellent; Plant did not possess the pollination ability before the flowers are in blossom; Back 10 days plant pollination abilities that the flowers are in blossom fall sharply (seeing Table two).Should after the flowers are in blossom, carry out palmification in 1-3 days in the actual production.
2.3 the TTC method records yellow lady's-slipper seed embryo vigor and seed germination rate tool positive correlation
Adopt the seed of 25 yellow lady's-slipper capsules to carry out test of TTC method seed embryo vigor and seed asepsis sprouting respectively at random.The result shows: the seed embryo vigor and the seed germination rate that adopt the TTC method to record have positive correlation, and the TTC method is measured the high seed of vigor, and germination rate is also high on the axenic germination medium, (see figure 1) that vice versa.The embryo vigor numerical value that measures is a little more than seed germination rate, show to have vigor but the weak seed of vigor not necessarily can both be sprouted, but this not overslaugh adopts TTC vigor method of testing to weigh the feasibility of seed germination rate.The employing of this method can be carried out earlier detection and screening to producing used yellow lady's-slipper seed quality, and the wasting of resources of avoiding unnecessary production to cause saves production cost greatly.
In the production of yellow lady's-slipper seed asepsis propagation in scale seedling, the seed that obtains to have stabilizing amount and quality is to produce to make demands before successfully vital. Above-mentioned experimental result shows that this result of study can satisfy this production requirement. To sum up, can find out that excellent beneficial effect of the present invention is:
1, adopts Pollination Modes of the present invention and pollination time, greatly improve the plant setting percentage, can reach 100%, can guarantee to obtain enough required high-quality seeds of production seedling. It is few to have overcome the naturally solid grain weight of employing, can not satisfy the shortcoming of large-scale production needs.
2, adopt seed activity screening method of the present invention to carry out earlier detection and screening to producing used seed quality, avoid blindly producing the wasting of resources that causes, greatly save production cost. In the middle of producing, the application of this technology will produce good economic benefit, have important practical significance.
3, the method for this research employing has easy operating, quick sensitive characteristics, makes fast numerous being achieved of yellow lady's-slipper seedling scale.
Claims (5)
1, preparation of yellow lady's-slipper seed and rapid screening method, it is characterized in that selecting in 3 years not solid, plant is sturdy, the growth situation good yellow lady's-slipper plant that blooms, the flowers are in blossom, and palmification was carried out in 2-3 days in the back, and the picking fruit taking-up seed after 120 days of pollinating is in the TTC solution of immersion 1%, 25 ℃ of dark incubations 72 hours are chosen the seed of embryo vigor more than 70% as the fast numerous material of seedling.
2, method according to claim 1, first weighing 1gTTC (triphenyltetrazolium chloride triphenyltetrazolium chloride) is adopted in the preparation that it is characterized in that TTC solution, in dissolved in distilled water, the Tween-20 that adds 0.5ml simultaneously, constant volume is 100ml, be mixed with 1% TTC solution, shake up, keep in Dark Place.
3, method according to claim 1, it is characterized in that seed takes out after, place in the middle of the filter paper, folding, filter paper immersed in 1% the TTC solution, 25 ℃ of dark incubations 72 hours took out filter paper then with flushing with clean water about 1 minute.
4, method according to claim 1 is characterized in that the red number/seed of seed embryo vigor=embryo has embryo rate * 100%.
5, according to claim 1 or 2 or 3 or 4 described methods, it is characterized in that selecting in 3 years not solid, plant is sturdy, the growth situation good yellow lady's-slipper plant that blooms, the flowers are in blossom, and the back was carried out palmification in 2-3 days, pollinating, picking fruit takes out seed after 120 days, place in the middle of the filter paper, folding, in the TTC solution with filter paper immersion 1%, first weighing 1gTTC (triphenyltetrazolium chloride triphenyltetrazolium chloride) is adopted in the preparation of TTC solution, in distilled water, add the Tween-20 of 0.5ml in dissolved simultaneously, constant volume is 100ml, be mixed with 1% TTC solution, shake up and get final product, seed is at 25 ℃ of dark incubations after 72 hours, and filter paper was taken out with flushing with clean water about 1 minute, choose the seed of embryo vigor more than 70% as the fast numerous material of seedling, the red number/seed of seed embryo vigor=embryo has embryo rate * 100%.
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| CN 200410040180 CN1287659C (en) | 2004-07-09 | 2004-07-09 | Yellow moccasin flower seed preparation and quick screening method |
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| CN1287659C CN1287659C (en) | 2006-12-06 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100358413C (en) * | 2005-04-22 | 2008-01-02 | 云南农业大学 | Cymbidium plant seed asepsis sprouting and plant cultivating method |
| CN102630464A (en) * | 2012-04-23 | 2012-08-15 | 中国科学院昆明植物研究所 | Method for reproducing paphiopedilum armeniacum |
| CN110786235A (en) * | 2018-08-02 | 2020-02-14 | 北京林业大学 | Method for aseptically germinating cypripedium guttatum seeds |
| CN113341075A (en) * | 2021-06-10 | 2021-09-03 | 中国林业科学研究院亚热带林业研究所 | Method for judging seed rapid germination type plant and application of seed rapid germination type plant in ecological restoration |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101536629B (en) * | 2009-03-17 | 2011-07-27 | 北京市植物园 | Method for causing seeds of cypripedium macranthum to sprout |
-
2004
- 2004-07-09 CN CN 200410040180 patent/CN1287659C/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100358413C (en) * | 2005-04-22 | 2008-01-02 | 云南农业大学 | Cymbidium plant seed asepsis sprouting and plant cultivating method |
| CN102630464A (en) * | 2012-04-23 | 2012-08-15 | 中国科学院昆明植物研究所 | Method for reproducing paphiopedilum armeniacum |
| CN102630464B (en) * | 2012-04-23 | 2013-04-24 | 中国科学院昆明植物研究所 | Method for reproducing paphiopedilum armeniacum |
| CN110786235A (en) * | 2018-08-02 | 2020-02-14 | 北京林业大学 | Method for aseptically germinating cypripedium guttatum seeds |
| CN113341075A (en) * | 2021-06-10 | 2021-09-03 | 中国林业科学研究院亚热带林业研究所 | Method for judging seed rapid germination type plant and application of seed rapid germination type plant in ecological restoration |
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