CN102919125A - Method for building efficient regeneration system of Yunnan rhododendron - Google Patents
Method for building efficient regeneration system of Yunnan rhododendron Download PDFInfo
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Abstract
The invention provides a method for building an efficient regeneration system of Yunnan rhododendrons and belongs to the field of a plant biotechnology. The method uses upper-middle portion leaves of Yunnan rhododendron aseptic seedlings as a material, one or more of hormones TDZ, ZT, 2,4-D, NAA, IAA and casein hydrolysate with appropriate concentration are mainly provided based on a woody plant medium (WPM) culture medium, and complete plants are obtained through a direct leaf regenerating way and an indirect leaf regenerating way. The method builds the efficient regeneration system of the Yunnan rhododendrons for the first time, provides a good theoretical basis and an experimental basis for genetic engineering breeding work of the Yunnan rhododendrons and simultaneously promotes biotechnology breeding progress of the rhododendrons effectively.
Description
Technical field
The invention belongs to azalea biotechnology breeding field, be specifically related to the method for building up of a kind of Yunnan cuckoo high-efficiency regeneration system, can be used for the screening of Yunnan cuckoo gene engineering and asexual mutantion line.
Background technology
Yunnan cuckoo (Rhododendron
Yunnanense) be Ericaceae, Genus Rhododendron shrub or dungarunga mainly are distributed in Yunnan (west, northwest, north, northeast), Southern Shaanxi, western Sichuan, northwest, Guizhou, Southeastern Tibet, and overseas also there is distribution the Burma northeast.Be born in hillside shaw, shrubbery, pine forest, pine-oakery, dragon spruce or fir border, 1600 meters-4000 meters above sea level.
The inventor begins in 2008 that pasture, Dali, master of great learning and integrity, Liang Wangshan introduce a fine variety the Yunnan cuckoo to academy of agricultural sciences, Yunnan Province flowers institute resource garden (Kunming) from Yunnan, find that the Yunnan cuckoo is the desirable gene expression acceptor material of azalea genetic engineering breeding, because Yunnan cuckoo plantlet of transplant is easy to survive, next year can bloom, the sowing seedling also can be bloomed after 3 years, as seen Yunnan cuckoo strong stress resistance has very strong adaptability and wider accommodation.
Genetic Transformation in Higher Plants is that transgenic technology is a biotechnology that grows up over nearly 40 years.Utilize transgenic technology can be under the prerequisite that keeps the original better genetic background of woody plant some proterties of directed change, simultaneously can also greatly shorten breeding time, this accelerates woody plant genetic breeding progress and provides convenience for overcoming woody plant traditional breeding method limitation.Transgenic technology has become the main research field of woody plant genetic breeding at present.Yet transgenic technology depends on the technology that the culture in vitro thing can efficient stable regeneration becomes whole plant greatly, and the genetic transformation system of setting up efficient stable is the basis of transgenic breeding.And woody plant tissure is cultivated and the difficulty of plant regeneration is larger.As lack suitable transformation receptor material, be difficult to set up the genetic conversion system of efficient stable.Next is that the genotype dependence is stronger, and transformation efficiency is not high.Therefore, system's further investigation affects the Yunnan cuckoo plant regeneration factor, induces the method for plant regeneration and obtains regeneration plant, set up specially the regenerating system for the efficient stable of Yunnan cuckoo, significant, can be Yunnan cuckoo transgenic breeding work and provide fundamental basis and experimental basis, will effectively advance azalea biotechnology breeding process simultaneously.The regenerating system of Yunnan cuckoo research so far there are no report.
Summary of the invention
The object of the invention provides a kind of specially for the method for building up of the high-efficiency regeneration system of Yunnan cuckoo.
The method for building up of Yunnan of the present invention cuckoo high-efficiency regeneration system may further comprise the steps:
(1) acquisition of aseptic seedling
Get the full Yunnan cuckoo semi-lignified stem section of lateral bud, be cut into 1～2 lateral bud
The stem section, clean with washing powder water, afterwards clear water rinsing is clean, under aseptic condition, be that 0.12% mercuric chloride solution soaks 15min with mass fraction successively, mass fraction is that 2% liquor natrii hypochloritis soaks 8min, aseptic water washing 3～5 times, cutting stem section two is inoculated in and induces sprouting of lateral bud on the inducing culture, treat to downcut when indefinite bud grows to 2～3cm indefinite bud and change cultivation acquisition aseptic seedling on the proliferated culture medium over to, described inducing culture is MS+6-BA 1mg/L+ NAA 0.1 mg/L, and the pH value is 5.4; Described proliferated culture medium is: WPM+ZT 2 mg/L, and the pH value is 5.4;
(2) Establish of regeneration
1. blade Direct Regeneration indefinite bud
Middle and upper part blade with 28～32 days seedling age aseptic seedling of acquisition in the step (1) is cut into 0.5cm
2～0.8cm
2The leaf dish, and at leaf dish back side scuffing mouth, the leaf dish back side is inoculated among the leaf dish differential medium A down or among the leaf dish differential medium B, leaf dish differential medium A is: WPM+TDZ 0.4 mg/L+ NAA0.05 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4, leaf dish differential medium B is: WPM+ZT 2～4mg/L+NAA0.01mg/L+ sucrose 3%+ agar 0.6%, and the pH value is 5.4;
Or the 2. indirect regenerated adventitious bud of blade
A. with the middle and upper part blade of 28～32 days seedling age aseptic seedling of acquisition in the step (1), be cut into approximately 0.5cm
2～0.8cm
2The leaf dish, and at leaf dish back side scuffing mouth, the leaf dish back side is inoculated in the callus inducing medium down cultivates, described callus inducing medium is: WPM+TDZ 0～0.5 mg/L+2,4-D1.0 mg/L+ caseinhydrolysate 0～500 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4;
B. the callus that step (2) is 2. obtained among the A evoking adventive bud in the Calli Differentiation medium of transferring, described Calli Differentiation medium is: WPM+ZT4.0mg/L+NAA 0.1 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4;
(3) strong seedling culture
With step (2) 1. the blade Direct Regeneration indefinite bud indefinite bud of turning out or with (2) 2. the indirect regenerated adventitious bud of the blade indefinite bud of turning out divide and cut or be cut into 3-5 and be one clump and put into the strong seedling culture base and cultivated 20～25 days, described strong seedling culture base is: WPM+ sucrose 3%+ agar 0.6%, and the pH value is 5.4;
(4) culture of rootage
To divide an incision through the seedling of step (3) strong seedling culture, and be inoculated in the root media and cultivate, described
Give birth toThe root medium is: 1/2 WPM+ IAA1.0～1.5mg/L+ sucrose 3%+ agar 0.6%, and the pH value is 5.4;
Above each step condition of culture is: temperature is 2 ℃ of 23 ℃ of scholars, and intensity of illumination is 2000lx, and light application time is 12 hd
-1, the percentage of sucrose and agar content is mass percent in each medium.
Step (2) 1. preferred leaf dish differential medium B is: WPM+ZT4mg/L+NAA0.01mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4.
Step (2) the 2. preferred callus inducing medium of A is: WPM+TDZ 0.5 mg/L+2, and 4-D 1.0 mg/L+ caseinhydrolysates 500 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4.
The preferred culture of rootage of step (4) is: 1/2WPM+IAA 1.5mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4.
The invention has the beneficial effects as follows:
1, the present invention induces plant regeneration to carry out the deep research of system for Yunnan cuckoo blade first, adopt blade directly to induce or two approach of indirect induction plant regeneration, and specific medium, make the inductivity of the direct evoking adventive bud of blade can reach 62～73%, average bud number on each leaf dish can reach 6.93, the inductivity of Callus of Leaf reaches 62～100%, callus is loose, volume is large, quality is good, Calli Differentiation becomes the differentiation rate of indefinite bud more than 70%, blade is directly induced or the rooting rate of the indefinite bud of indirect induction all reaches 69～89%, Fig. 1-Fig. 9 also shows, the Yunnan cuckoo high-efficiency regeneration system that the inventive method is set up can make Yunnan cuckoo blade at blade Direct Regeneration indefinite bud or the indirect regenerated adventitious bud of blade, the strong seedling culture of indefinite bud, the culture of rootage links is all grown performance well, the robust plant of cultivating, well developed root system, showing that the inventive method has been set up can high-frequency produce callus and indefinite bud, and can high-frequency takes root and obtain the Yunnan cuckoo regenerating system of whole plant.Show that it is practical and effective that the inventive method is set up Yunnan cuckoo high-efficiency regeneration system.
2, leaf dish and callus bi-material are to carry out the common used material that genes of interest is contaminated, the present invention is evoking adventive bud and callus efficiently, be the follow-up transgenic breeding work of Yunnan cuckoo, realize that good regenerating system and the method for foundation thereof of providing is provided high frequency.
Description of drawings
Fig. 1 is the indefinite bud point that induces in the direct evoking adventive bud process of Yunnan rhododendron leaves dish.
Fig. 2 is that the indefinite bud point grows up to indefinite bud in the direct evoking adventive bud process of Yunnan rhododendron leaves dish.
Fig. 3 induces callus in the rhododendron leaves dish indirect induction indefinite bud process of Yunnan.
Fig. 4 is callus propagation in the rhododendron leaves dish indirect induction indefinite bud process of Yunnan.
Fig. 5 is Calli Differentiation indefinite bud in the rhododendron leaves dish indirect induction indefinite bud process of Yunnan.
Fig. 6 is that the indefinite bud of Calli Differentiation in the rhododendron leaves dish indirect induction indefinite bud process of Yunnan is grown up.
Fig. 7 is the growth performance of Yunnan cuckoo strong seedling culture.
Fig. 8 is the growth performance of Yunnan cuckoo culture of rootage.
Fig. 9 is the growth performance of Yunnan cuckoo culture of rootage.
Embodiment
Below the used material of each embodiment and reagent be commercially available, each embodiment is conventional method without specified otherwise.
The method for building up of a kind of Yunnan provided by the invention cuckoo high-efficiency regeneration system, the implementation step is as follows:
1, the acquisition of aseptic seedling
Get the full Yunnan cuckoo semi-lignified stem section of lateral bud, be cut into the segment with 1～2 lateral bud, clean with the clear water rinsing after the washing powder water oscillation cleaning, sterilize under the aseptic condition, be that 0.12% mercuric chloride solution soaks 15min with mass fraction successively, mass fraction is that 2% liquor natrii hypochloritis soaks 8min, constantly rock therebetween, aseptic water washing is 3～5 times afterwards, cut stem section two and be inoculated in MS+6-BA (6-benzyladenine) 1mg/L+ NAA(methyl α-naphthyl acetate) 0.1 mg/L, the pH value is to induce sprouting of lateral bud on 5.4 the inducing culture, treat to downcut when indefinite bud grows to 2～3cm indefinite bud behind the 20d and change WPM+ZT 2 mg/L over to, the pH value is to obtain aseptic seedling on 5.4 the proliferated culture medium, and the material that is used for Regeneration System is the middle and upper part blade of the aseptic seedling of 28～32 days seedling ages.
2, Establish of regeneration
(1) blade Direct Regeneration indefinite bud
Middle and upper part blade with the aseptic seedling of 30 days seedling ages in the step 1 is cut into 0.5cm
2～0.8cm
2The leaf dish, and draw " well " word wound at leaf dish leaf back, the leaf dish back side is inoculated in down in each leaf dish differential medium of the listed hormone combinations of table 1 and cultivates.
After the leaf dish was cultivated a week, visible notching edge shrinkage occurred, expands, and left and right sides incision began to have the bud point to emerge in 25 days, during 50 days left and right sides, formed a large amount of clump buds on some leaf dishes, and part bud point has grown up to normal plant, can be used for strong seedling culture.Hence one can see that, the TDZ(thidiazuron) or the ZT(zeatin) all be conducive to induce the leaf dish directly to break up indefinite bud, increase with concentration, two kinds of hormones show opposite variation tendency to the inducibility of indefinite bud: in TDZ is 0.4～1.2 mg/L concentration range, increase with TDZ concentration, adventitious bud induction frequency and average bud number average reduce, and in ZT is 2～4 mg/L concentration ranges, increase with ZT concentration, adventitious bud induction frequency and average bud number improve.Therefore, blade Direct Regeneration indefinite bud can be inoculated in respectively following two kinds of leaf dish differential mediums and obtain high inductivity and than the beneficial effect of polygerm number, two kinds of leaf dish differential mediums are:
Leaf dish differential medium A:WPM+TDZ 0.4 mg/L+ NAA0.05 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4.
Or leaf dish differential medium B:WPM+ZT 2～4mg/L+NAA0.01mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4.Inducing the best hormone combinations of leaf dish differentiation indefinite bud among the leaf dish differential medium B is ZT 4 mg/L+NAA 0.1 mg/L, its adventitious bud induction frequency and average bud number are the highest, be respectively 73.0% and 6.93, there were significant differences in 0.5% level with the inductivity of other hormone combinations.(Fig. 1, Fig. 2).
Except above-mentioned blade Direct Regeneration indefinite bud, can also adopt the cultivation of the indirect regenerated adventitious bud of blade, specifically:
Or carry out the indirect regenerated adventitious bud of (2) blade, formed by following steps A and step B:
A. with the middle and upper part blade of the aseptic seedling of 30 days seedling ages in the step 1, be cut into 0.5cm
2～0.8cm
2The leaf dish, and draw " well " word wound at leaf dish leaf back, the leaf dish back side is inoculated in the callus inducing medium of the listed hormone combinations of table 2 down.
After the leaf dish is cultivated a week, notching edge begins to expand, the blade progressive additive, behind the 15d, observe yellowish green callus at notching edge, callus continues to grow up afterwards, during by 50 days, have no and differentiate indefinite bud on the callus, the callus induction rate of each hormone combinations is significant difference on 5% level.TDZ and 2 in numbering 1 and 2 liang of medium of numbering, when 4-D concentration difference is identical, the processing ratio of interpolation caseinhydrolysate is adder callus induction rate high (callus induction rate 100%) not, illustrates that caseinhydrolysate has facilitation to evoked callus under this condition.Test data draws the middle and upper part blade of 28～32 days seedling age aseptic seedling that will obtain in the step 1 from table, is cut into approximately 0.5cm
2～0.8cm
2The leaf dish, and draw " well " word wound at the leaf dish back side, the leaf dish back side is inoculated in the following callus inducing medium down cultivates, its callus induction rate can reach the above good effect of 62 .4%, described callus inducing medium is: WPM+TDZ 0～0.5 mg/L+2,4-D1.0 mg/L+ caseinhydrolysate 0～500 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4.
The best of breed of described callus inducing medium is: WPM+TDZ0.5mg/L+2,4-D 1.0 mg/L+ caseinhydrolysates 500 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4, its callus induction rate has reached 100%, the callus white powder that derives, more loose, volume is large.
B. according to above-mentioned test, ZT is conducive to induce the leaf dish directly to break up the result of indefinite bud, with step 2(2) callus that obtains among the A transfers and cultivates in the Calli Differentiation medium, described Calli Differentiation medium is: WPM+ZT4.0mg/L+NAA 0.1 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4, visible callus is local behind the 30d turns green, begins to have clump bud differentiation, and 50d metaplexus bud grows up to plantlet.The ratio of Calli Differentiation Cheng Congya can reach more than 70%.(Fig. 3, Fig. 4, Fig. 5, Fig. 6)
3, strong seedling culture
With step 2(1) blade Direct Regeneration the indefinite bud indefinite bud or the step 2(2 that turn out) the indirect regenerated adventitious bud of the blade indefinite bud of turning out divides an incision, or be cut into a 3-5 Xiao Cong and put into the WPM strong seedling culture base that does not add any hormone and cultivated 20-25 days, described strong seedling culture base is: WPM+ sucrose 3%+ agar 0.6%, the pH value is 5.4.Owing to not adding any hormone, suppressed to a certain extent the propagation of indefinite bud, be conducive to indefinite bud and grow up to stalwartness, unfold plant.(Fig. 7)
4, culture of rootage
After strong seedling culture, divide an incision with seedling, be inoculated in the listed root media of table 3.
After cultivating a week, find that the seedling basal part of stem grows a small amount of callus, after two weeks, at first at the IAA(heteroauxin) concentration is to observe on seedling basal part of stem and callus the superfine fibrous root of adularescent in the root media of 1.5mg/L to emerge.30 rear statistics rooting rates increase with IAA concentration, and rooting rate improves, and the suitable root media of Yunnan cuckoo is: 1/2 WPM+ IAA1.0～1.5mg/L+ sucrose 3%+ agar 0.6%, and the pH value is 5.4; Its best root media is: 1/2WPM+IAA 1.5mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4.At this medium seedling root of hair at first, and rooting rate is the highest, reaches 88.7%(Fig. 8, Fig. 9).
Above each step condition of culture is: temperature is 2 ℃ of 23 ℃ of scholars; Intensity of illumination is that the 2000lx light application time is 12 hd
-1, the percentage of sucrose and agar content is mass percent in each medium.The percentages such as all the other adventitious bud induction frequencies, callus induction rate, rooting rate are quantity percentage.
Fig. 1-Fig. 9 also shows, the Yunnan cuckoo high-efficiency regeneration system that the inventive method is set up can make Yunnan cuckoo blade at blade Direct Regeneration indefinite bud or the indirect regenerated adventitious bud of blade, the strong seedling culture of indefinite bud, culture of rootage links are all grown performance well, robust plant, the well developed root system cultivated show that the Yunnan cuckoo high-efficiency regeneration system that the inventive method is set up is practical and effective.
Claims (5)
1. the method for building up of Yunnan cuckoo high-efficiency regeneration system is characterized in that may further comprise the steps:
(1) acquisition of aseptic seedling
Get the full Yunnan cuckoo semi-lignified stem section of lateral bud, be cut into the stem section with 1～2 lateral bud, after the cleaning of washing powder water, the clear water rinsing is clean, under aseptic condition, be that 0.12% mercuric chloride solution soaks 15min with mass fraction successively, mass fraction is that 2% liquor natrii hypochloritis soaks 8min, aseptic water washing 3～5 times, cutting stem section two is inoculated in and induces sprouting of lateral bud on the inducing culture, treat to downcut when indefinite bud grows to 2～3cm indefinite bud and change cultivation acquisition aseptic seedling on the proliferated culture medium over to, described inducing culture is: MS+6-BA 1mg/L+ NAA 0.1 mg/L, and the pH value is 5.4; Described proliferated culture medium is: WPM+ZT 2 mg/L, and the pH value is 5.4;
(2) Establish of regeneration
1. blade Direct Regeneration indefinite bud
Middle and upper part blade with 28～32 days seedling age aseptic seedling of acquisition in the step (1) is cut into 0.5cm
2～0.8cm
2The leaf dish, and at leaf dish back side scuffing mouth, the leaf dish back side is inoculated among the leaf dish differential medium A down or among the leaf dish differential medium B, leaf dish differential medium A is: WPM+TDZ 0.4 mg/L+ NAA0.05 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4, leaf dish differential medium B is: WPM+ZT 2～4mg/L+NAA0.01mg/L+ sucrose 3%+ agar 0.6%, and the pH value is 5.4;
Or the 2. indirect regenerated adventitious bud of blade
A. with the middle and upper part blade of 28～32 days seedling age aseptic seedling of acquisition in the step (1), be cut into approximately 0.5cm
2～0.8cm
2The leaf dish, and at leaf dish back side scuffing mouth, the leaf dish back side is inoculated in the callus inducing medium down cultivates, described callus inducing medium is: WPM+TDZ 0～0.5 mg/L+2,4-D1.0 mg/L+ caseinhydrolysate 0～500 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4;
B. the callus that step (2) is 2. obtained among the A evoking adventive bud in the Calli Differentiation medium of transferring, described Calli Differentiation medium is: WPM+ZT4.0mg/L+NAA 0.1 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4;
(3) strong seedling culture
With step (2) 1. the blade Direct Regeneration indefinite bud indefinite bud of turning out or with (2) 2. the indirect regenerated adventitious bud of the blade indefinite bud of turning out divide and cut or be cut into 3-5 and be one clump and put into the strong seedling culture base and cultivated 20～25 days, described strong seedling culture base is: WPM+ sucrose 3%+ agar 0.6%, and the pH value is 5.4;
(4) culture of rootage
To divide an incision through the seedling of step (3) strong seedling culture, and be inoculated in the root media and cultivate, described root media is: 1/2 WPM+ IAA1.0～1.5mg/L+ sucrose 3%+ agar 0.6%, and the pH value is 5.4;
Above each step condition of culture is: temperature is 2 ℃ of 23 ℃ of scholars, and intensity of illumination is 2000 lx, light application time 12 hd
-1, the percentage of sucrose and agar content is mass percent in each medium.
2. the method for building up of Yunnan according to claim 1 cuckoo high-efficiency regeneration system, it is characterized in that: step (2) 1. described leaf dish differential medium B is: WPM+ZT4mg/L+NAA0.01mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4.
3. the method for building up of Yunnan according to claim 1 and 2 cuckoo high-efficiency regeneration system, it is characterized in that: step (2) the 2. described callus inducing medium of A is: WPM+TDZ 0.5 mg/L+2,4-D 1.0 mg/L+ caseinhydrolysates 500 mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4.
4. the method for building up of Yunnan according to claim 1 and 2 cuckoo high-efficiency regeneration system, it is characterized in that: the described culture of rootage of step (4) is: 1/2WPM+IAA 1.5mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4.
5. the method for building up of Yunnan according to claim 3 cuckoo high-efficiency regeneration system, it is characterized in that: the described culture of rootage of step (4) is: 1/2WPM+IAA 1.5mg/L+ sucrose 3%+ agar 0.6%, the pH value is 5.4.
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| CN103371103A (en) * | 2013-07-30 | 2013-10-30 | 杭州植物园 | Rapid propagation method for tissue culture of Rhododendron delavayi Franch |
| CN103371102A (en) * | 2013-07-30 | 2013-10-30 | 杭州植物园 | Rhododendron spiciferum Franch. tissue culture and rapid propagation method |
| CN103371102B (en) * | 2013-07-30 | 2015-04-22 | 杭州植物园 | Rapid propagation method for rhododendron spiciferum Franch tissue cultur |
| CN104221858A (en) * | 2014-08-29 | 2014-12-24 | 南京林业大学 | Culture medium for survival culture of wild rhododendron hybrid embryos and survival method of wild rhododendron hybrid embryos |
| CN104221858B (en) * | 2014-08-29 | 2016-05-18 | 南京林业大学 | A kind of culture medium that survives cultivation for Rhododendron simsii Planch hybridization embryo and one-tenth activating method thereof |
| CN104285819A (en) * | 2014-11-04 | 2015-01-21 | 中国科学院昆明植物研究所 | Tissue culture propagation method for rhododendron hancockii |
| CN104285819B (en) * | 2014-11-04 | 2015-11-18 | 中国科学院昆明植物研究所 | The tissue culture propagation method of a kind of the southern regions of the Yunnan Province cuckoo |
| CN104472354A (en) * | 2014-11-21 | 2015-04-01 | 广西中医药大学 | Method for promoting intermediate propagation of craibiodendron yunnanense |
| CN105660397A (en) * | 2016-01-13 | 2016-06-15 | 杭州市园林绿化股份有限公司 | High-frequency regeneration and rapid propagation method of Enkianthus chinensis tissue culture seedlings |
| CN105830917A (en) * | 2016-03-24 | 2016-08-10 | 江苏省农业科学院 | Method of regeneration plant from rhododendron molle leaves |
| CN105830917B (en) * | 2016-03-24 | 2017-11-17 | 江苏省农业科学院 | The method of Chinese azalea leaf regeneration plant |
| CN105850737A (en) * | 2016-04-08 | 2016-08-17 | 石家庄市农林科学研究院 | Rhododendron Delavayi 'Cosmopolitan' chromosome doubling method |
| CN107079815A (en) * | 2017-05-25 | 2017-08-22 | 贵州师范大学 | A kind of method for inducing charming cuckoo and Rhododendron delavayi cenospecies callus |
| CN107079815B (en) * | 2017-05-25 | 2019-06-11 | 贵州师范大学 | A method of inducing charming cuckoo and Rhododendron delavayi cenospecies callus |
| CN109349109A (en) * | 2018-11-23 | 2019-02-19 | 云南省农业科学院花卉研究所 | Shaping methods in the bottle of alpine rose young beauty |
| CN109937885A (en) * | 2019-05-07 | 2019-06-28 | 贵州师范大学 | A kind of sterile seedling fast reproducing method of wrinkle leaf cuckoo |
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