NL2033132B1 - Method for formulating polyploid germplasm of bougainvillea and application thereof - Google Patents
Method for formulating polyploid germplasm of bougainvillea and application thereof Download PDFInfo
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- 241000219475 Bougainvillea Species 0.000 title claims abstract description 57
- 208000020584 Polyploidy Diseases 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 26
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 claims abstract description 64
- 229960001338 colchicine Drugs 0.000 claims abstract description 32
- 241000196324 Embryophyta Species 0.000 claims abstract description 23
- 210000000349 chromosome Anatomy 0.000 claims abstract description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 18
- 230000010152 pollination Effects 0.000 claims description 9
- 239000011159 matrix material Substances 0.000 claims description 5
- 238000009396 hybridization Methods 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 2
- 230000007226 seed germination Effects 0.000 claims description 2
- 208000036086 Chromosome Duplication Diseases 0.000 claims 1
- 230000006698 induction Effects 0.000 abstract description 17
- UUTKICFRNVKFRG-WDSKDSINSA-N (4R)-3-[oxo-[(2S)-5-oxo-2-pyrrolidinyl]methyl]-4-thiazolidinecarboxylic acid Chemical compound OC(=O)[C@@H]1CSCN1C(=O)[C@H]1NC(=O)CC1 UUTKICFRNVKFRG-WDSKDSINSA-N 0.000 abstract description 7
- 239000000203 mixture Substances 0.000 abstract description 7
- 210000001519 tissue Anatomy 0.000 abstract description 3
- 238000009472 formulation Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 208000035199 Tetraploidy Diseases 0.000 description 13
- 238000000684 flow cytometry Methods 0.000 description 11
- 208000026487 Triploidy Diseases 0.000 description 7
- 238000009395 breeding Methods 0.000 description 7
- 230000001488 breeding effect Effects 0.000 description 7
- 241000219477 Bougainvillea glabra Species 0.000 description 5
- 241001316288 Bougainvillea spectabilis Species 0.000 description 5
- 239000003415 peat Substances 0.000 description 5
- 210000003855 cell nucleus Anatomy 0.000 description 4
- 230000009089 cytolysis Effects 0.000 description 4
- 230000000717 retained effect Effects 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- 235000007575 Calluna vulgaris Nutrition 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000009331 sowing Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 230000021121 meiosis Effects 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241001388118 Anisotremus taeniatus Species 0.000 description 1
- 241000436234 Bougainvillea peruviana Species 0.000 description 1
- 241000257303 Hymenoptera Species 0.000 description 1
- 241000219469 Nyctaginaceae Species 0.000 description 1
- 235000011449 Rosa Nutrition 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 229940000425 combination drug Drugs 0.000 description 1
- 230000002380 cytological effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229940046546 flower allergenic extract Drugs 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/02—Flowers
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Physiology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A method for formulating a polyploid germplasm of Bougainvillea and an application thereof are provided, belonging to the technical field of plant polyploid induction. The present invention provides a method for formulating a polyploid germplasm of Bougainvillea; a colchicine solution is added dropwisely to leaf aXil of Bougainvillea seedlings for chromosome doubling, and the polyploid plant obtained after ploidy identification is the polyploid germplasm of Bougainvillea. The present invention avoids in vitro tissue culture and other complex technical links; the induction period is short, and it is only necessary to treat the leaf aXil of Bougainvillea seedlings with a colchicine solution, thus reducing the amount of colchicine used. Specific treatment modes and conditions of the colchicine solution are adjusted to achieve the formulation of the polyploid germplasm of Bougainvillea.
Description
P1597 /NL
METHOD FOR FORMULATING POLYPLOID GERMPLASM OF BOUGAINVILLEA AND
APPLICATION THEREOF
The present invention relates to the technical field of plant polyploid induction, and in particular to a method for formulating a polyploid germplasm of Bougainvillea and an application thereof.
Bougainvillea, also known as Bougainvillea willd, and the like. Bougainvillea was found in South America first, and is a common perennial woody plant of the Bougainvillea genus of Nycta- ginaceae. Bougainvillea has been widely used as an ornamental hor- ticultural plant and planted around the world, especially in trop- ical and subtropical areas at present. Bougainvillea has numerous varieties, strong adaptability, easy cultivation, multiple flower colors and long flowering period. Moreover, Bougainvillea has been widely used in garden landscaping, road and bridge beautification, festival flower exhibition, family potted plants, etc, which is one of the tropical landscape plants with largest industry scale.
There are 18 species of Bougainvillea, among which B. glabra,
B. spectabilis and B. peruviana are diploid and important gene sources of cultivars applied in horticultural cultivation for higher ornamental values, . However, there exists a highly sterile phenomenon in the interspecific hybrid varieties of Bougainvillea and in a triploid hybrid F1 (2n=3x=51) hybridized by a diploid (2n=2x=34) and a tetraploid (2n=4x=68), which restricts the sus- tainable development of breeding. The main reasons are as follows: (1) chromosomes of Bougainvillea have low homology among different species, and the chromosomes of the hybrid Fl cannot be paired in- to normal bivalents in meiosis, and low-homology chromosomes exist in a monovalent form; (2) interspecific hybrids have structural variations such as, chromosome translocations and inversions, thus forming multivalent and univalent chromosomes; (3) homologous chromosome associations of the triploid hybrid in meiosis are dis-
ordered and cannot be separated equationally. These all lead to the morphological structure variation, cytological variation and high sterility of hybrid gametes.. By establishing a chromosome multiplication technology system, more fertile hybrid offsprings can be obtained, which will clear the cbstacles for the large- scale breeding of Bougainvillea. Moreover, polyploidization endows plants with new excellent characters, such as thick and strong stalks, huge flowers and leaves, and strong stress resistance, thus producing excellent new germplasms with breeding application values.
It has been known that polyploidization of Bougainvillea mainly results from two ways: (1) natural variation, most of vari- ants with low occurrence are chimeras, which is not easy to be found; (2) artificial induction, there are many disadvantages to the induced culture of in vitro tissues , such as long period, complicated technical steps, high dependence on facilities condi- tion, only specific to particular species, and the existing induc- tion efficiency of the Bougainvillea polyploid is very low, which is not enough to satisfy the demands for a large number of fertile parent materials for large-scale breeding, limiting the breeding research and development of Bougainvillea.
The objective of the present invention is to provide a method for formulating a polyploid germplasm of Bougainvillea. Bougain- villea seedlings are used as an induction material, which can im- prove the induction efficiency of the polyploid germplasm of Bou- gainvillea and reduce the use of inducers. Therefore, the present invention has low costs, simple operation and short treatment time.
To solve the above technical problem, the present invention provides the following technical solutions.
The present invention provides a method for formulating a polyploid germplasm of Bougainvillea, comprising the steps of: dropwisely adding a colchicine solution to leaf axils of seedlings for chromosome doubling; where a polyploid plant ob- tained after ploidy identification is the polyploidy germplasm of
Bougainvillea; the colchicine solution has a concentration of 100- 500 mg/L, and the colchicine solution has a treatment time of 1-5 d.
Preferably, the colchicine solution contains 1-23% DMSO.
Preferably, the leaf axils are cotyledon axils of Bougainvil- lea seedlings or a growing point in the middle of two cotyledons when true leaves are not grown.
Preferably, the dropwise addition frequency is twice a day.
Preferably, an object of the ploidy identification is a shoot germinated after the chromosome doubling treatment.
Preferably, the obtained polyploid plant is subjected to the ploidy identification for equal to or greater than 3 times.
Preferably, the Bougainvillea seedlings are germinated by seeds obtained by natural pollination or parental hybridization, pollination and seed setting of Bougainvillea.
Preferably, the pollinated flowers are flowers blooming on the same day.
Preferably, a matrix for the seed germination is a porous ma- terial.
The present invention further provides an application of the above method in Bougainvillea breeding.
The present invention provides a method for formulating a polyploid germplasm of Bougainvillea. Bougainvillea seedlings are used as an induced material, and the treatment method is simple, easy to be controlled and operated. The method of the present in- vention only needs to treat leaf axil of Bougainvillea seedlings with a colchicine solution, thus reducing the amount of colchicine used. Specific treatment modes and conditions of the colchicine solution are adjusted to achieve the formulation of the polyploid germplasm of Bougainvillea. It is verified by experiments that the method of the present invention can improve the induction effi- ciency of the Bougainvillea polyploid germplasm up to 46.43%.
Meanwhile, there is not need for tissue culture operation and aseptic environment in the method. Therefore, the method is simple in operation, low in cost and convenient for popularization.
FIG. 1 shows an interspecific crossing progeny Fl of B. spectabilis cv. 'Rosa Catalina' (2x) and B. glabra cv. 'Bixi' (2x) provided in Example 1 of the present invention;
FIG. 2 is flow cytometric analysis of cell nucleus DNA of the female parent B. spectabilis cv. 'Rosa Catalina' (2x) provided in
Example 1 of the present invention;
FIG. 3 is flow cytometric analysis of cell nucleus DNA of the male parent B. glabra cv. 'Bixi' (2x) provided in Example 1 of the present invention;
FIG. 4 is flow cytometric analysis of tetraploid cell nucleus
DNA of chromosome-doubled 'Rosa Catalina’! x 'Bixi' progeny seed- lings provided in Example 1 of the present invention;
FIG. 5 is flow cytometric analysis of a diploid/tetraploid mix of chromosome-doubled 'Rosa Catalina! x 'Bixi' progeny seed- lings provided in Example 1 of the present invention;
FIG. 6 shows a successfully doubled tetraploid plant of the 'Rosa Catalina’ x 'Bixi' progeny provided in Example 1 of the pre- sent invention;
FIG. 7 is a successfully doubled 2x/4x mixoploidy plant of the 'Rosa Catalina! x 'Bixi' progeny provided in Example 1 of the present invention;
FIG. 8 is flow cytometric analysis of a chromosome-doubled hexaploid of a hybridized progeny seedling with "Rosa Catalina’ (2x) as a female parent and ‘Tetra Miss Manila’ (4x) as a male parent provided in Example 2 of the present invention;
FIG. 9 is flow cytometric analysis of a chromosome-doubled 3x/6x mixoploidy of a hybridized progeny seedling with 'Rosa Cata- lina’ (2x) as a female parent and 'Tetra Miss Manila’ (4x) as a male parent provided in Example 2 of the present invention;
FIG. 10 shows a successfully doubled hexaploid plant with 'Rosa Catalina' (2x) as a female parent and 'Tetra Miss Manila! (4x) as a male parent provided in Example 2 of the present inven- tion.
The present invention provides a method for formulating a polyploid germplasm of Bougainvillea, comprising the steps of: dropwisely adding a colchicine sclution to eaf axils of seed- lings for chromosome doubling; a polyploid plant obtained after ploidy identification is the polyploidy germplasm of Bougainvil- 5 lea; the colchicine solution has a concentration of 100-500 mg/L, and the colchicine solution has a treatment time of 1-5 d.
In the present invention, the concentration of the colchicine solution is preferably 200-400 mg/L, more preferably 300 mg/L; the treatment time of the colchicine solution is preferably 2-4 d, more preferably 3 d. The colchicine solution of the present inven- tion preferably contains 1-2% DMSO, and the DMSO is capable of in- creasing osmosis and improving the induction efficiency of colchi- cine.
In the present invention, after Bougainvillea seeds germinate seedlings and grow true leaves, the true leaves are removed, and then the colchicine solution is dropwisely added onto leaf axil.
In the present invention, the leaf axils are preferably cotyledon axil of Bougainvillea seedlings or a growing point in the middle of two cotyledons when true leaves are not grown. In the present invention, the dropwise addition frequency is preferably twice a day, more preferably once every morning and evening, respectively.
The dropwise addition amount in the present invention is not par- ticularly limited as long as axillary buds are wrapped by the lig- uid drops of the colchicine solution without slipping. The mode of the dropwise addition is not particularly limited in the present invention, and in detailed embodiments of the present invention, the dropwise addition is preferably performed with a pipette.
Chromosome-doubled Bougainvillea is subjected to ploidy iden- tification in the present invention. In the present invention, the object of the ploidy identification is preferably a shoot germi- nated after the chromosome doubling treatment, and the subject of the ploidy identification is more preferably a tender leaf newly grown on shoots. The reason for choosing tender leaves in the pre- sent invention is as follows: tender leaves are easy to cut, and the prepared cell nucleus suspension has a higher concentration; there are less DNA fragments and less impurity peaks during the identification of flow cytometry. The specific mode of the ploidy identification in the present invention is not particularly lim- ited, and in detailed embodiments of the present invention, a flow cytometer is preferably used in the ploidy identification. In the present invention, prior to the detection of the flow cytometer, the object of the ploidy identification is preferably treated with a WPB lysis solution and a DAPI dyeing solution; the WEB lysis so- lution preferably includes the following components: 0.2 mol/L
Tris-HCL, 86 mmol/L Nacl, 10 mmol/L sodium pyrosulfite, 4 mmol/L
MCL; -6H.0, 2 mmol/L EDTA-Na, :2H:0, 1% PVP-10, 1% (V/V) Triton X- 100, pH=7.5. In the present invention, the obtained polyploid plant is subjected to the ploidy identification for equal to or greater than 3 times, more preferably equal to or greater than 5 times, and the result of each identification remains the same.
In the present invention, the Bougainvillea seedlings are preferably germinated by seeds obtained by natural pollination or parental hybridization, pollination and seed setting of Bougain- villea. In the present invention, flowers selected for the polli- nation are flowers blooming on the same day. The Bougainvillea seedlings obtained according to the present invention are sown in- to a matrix for germination. In the present invention, the seeds are preferably subjected to pericarp peeling before sowing, and the matrix is preferably a porous material, more preferably fine peat. The matrix of the present invention may ensure better germi- nation of the Bougainvillea seeds, thus obtaining fine seedlings of Bougainvillea and facilitating subsequent induction.
The present invention further provides an application of the above method in breeding Bougainvillea.
In the present invention, the raw materials, reagents and equipment used herein are known products, and conventional commer- cially available products can be used.
To further describe the present invention, the technical so- lutions provided by the present invention will be described in de- tail with reference to the following examples, but shall not be construed as limiting the protection scope of the present inven- tion.
Example 1 1. Seed harvesting: B. spectabilis cv. 'Rosa Catalina' (2x)
and B. glabra cv. 'BiXi' (2x) were respectively used as a female parent and a male parent, flowers blooming on the same day in the flowering period were selected for artificial pollination. Full seeds with consistent size were selected for further use after ma- ture. 2. Sowing: the seeds whose pericarp was peeled off were sown in a 32-well plug tray filled with fine peat, 1 grain per well, watered daily to keep the peat wet, thus obtaining the normal hy- brid progeny seedling, as shown in FIG. 1. 3. Chromosome doubling: after the seeds germinated and grew 1 true leaf, the true leaf was removed, and only cotyledon and axil- lary buds not germinated at leaf axil were retained. A pipette was used to drop 10 pL of 0 mg/L, 300 mg/L and 500 mg/L colchicine so- lution containing 2% DMSO on bud points such that the bud points were wrapped by the solution without sliding, and treated for once every morning and evening for 5 d. 4. Ploidy identification: ploidy identification was performed on all the newly grown shoots 2 months later. 2 tender leaves on each shoot were chosen and mixed, and rapidly cut up with a sharp blade in a WPB lysis solution, and filtered with a nylon mesh, then added with a DAPI dyeing solution, and detected by a flow cy- tometer to determine the ploidy of shoots. 5. Field management: the identified tetraploid, and diploid and tetraploid mix were detected repeatedly, and the plant with the same ploidy identification results was determined as poly- ploid. For the plants with multiple shoots, the unsuccessfully doubled shoots were removed and only the successfully doubled shoots were retained.
Results
The results of detecting the female parent B. spectabilis cv. 'Rosa Catalina! (2x) by a flow cytometer are shown in FIG. 2, and the results of detecting the male parent B. glabra cv. 'Bixi' (2x) are shown in FIG. 3. The flow cytometric analysis of the chromo- some-doubled tetraploid seedling is shown in FIG. 4, and the flow cytometric analysis of the chromosome-doubled diploid/tetraploid mix seedling is shown in FIG. 5. All the control plants were nor- mal diploid.
Based on the statistics on the test results in this example, 9 tetraploid shoots (as shown in FIG. 6) were obtained by the treatment with 300 mg/L colchicine, accounting for 32.14%; there were 4 diploid/tetraploid mixes (as shown in FIG. 7), accounting for 14.29%, and the overall polyploid induction rate was 46.43%. 11 tetraploid shoots were obtained by the treatment with 500 mg/L colchicine, accounting for 27.50%; there were 5 diploid/tetraploid mixes, accounting for 12.50%, and the overall polyploid induction rate was 40.00%.
Table 1 Progeny doubling results of the Bougainvillea hybrid combination Rosa Catalina' x 'BiXi'
Colchicine Number of | Number Number of 2x/4x Number of 4x concentration | seedlings of 2x mixoploid induction | induction (rate) mg/L {rate} (rate) we fo |E a
Treatment 1 300 28 15 4 (14.29%) 9 (32.14%) le
Treatment 2 500 40 24 5 {12.50%) 11 (27.50%) ee
Example 2 1. Seed obtaining: a diploid variety 'Rosa Catalina! (2x) and a tetraploid variety 'Tetra Miss Manila' (4x) of Bougainvillea were respectively used as a female parent and a male parent, flow- ers blooming on the same day in the flowering period were selected for artificial pollination. Full seeds with consistent size were selected for further use after mature. 2. Sowing: 85 seeds whose floral envelope tubes were peeled off were sown in a 32-well plug tray filled with fine peat, 1 grain per well, watered daily to keep the peat wet, thus obtaining the normal hybrid progeny Fl, as shown in FIG. 7. 3. Chromosome doubling: after the seeds germinated and grew 2 true leaves, the true leaves were removed, and only cotyledon and axillary buds not germinated at leaf axil were retained. A pipette was used to drop 12 pL of 0 mg/L, 300 mg/L and 500 mg/L colchicine solution containing 2% DMSO on bud points such that the bud points were wrapped by the solution without sliding, and treated for once every morning and evening for 5 d.
4. Ploidy identification: ploidy identification was performed on all the newly grown shoots 2 months later. 3 tender leaves on each shoot were chosen and mixed, and rapidly cut up with a sharp blade in a WPB lysis solution, and filtered with a nylon mesh, then added with a DAPI dyeing solution, and detected by a flow cy- tometer to determine the ploidy of shoots. 5. Field management: the identified hexaploid, and trip- loid/hexaploid mix were detected repeatedly, and the plant with the same ploidy identification results was determined as poly- ploid. For the plants with multiple shoots, the unsuccessfully doubled shoots were removed and only the successfully doubled shoots were retained.
Results
The flow cytometric analysis of the chromosome-doubled hexa- ploid seedling is shown in FIG. 8, and the flow cytometric analy- sis of the chromosome-doubled triploid/hexaploid mix seedling is shown in FIG. 9. All the control plants were normal triploid.
Based on the statistics on the test results in this example, 4 hexaploid shoots (as shown in FIG. 6) were obtained by the treatment with 300 mg/L colchicine, accounting for 12.90%; there were 7 triploid/hexaploid mixes (as shown in FIG. 7), accounting for 22.58%, and the overall polyploid induction rate was 35.48%. ¢ hexaploid shoots were obtained by the treatment with 500 mg/L col- chicine, accounting for 11.11%; there were 9 triploid/hexaploid mixes, accounting for 16.67%, and the overall polyploid induction rate was 27.78%.
Table 2 Progeny doubling results of Bougainvillea hybrid com- bination 'Rosa Catalina' (2x) x "Tetra Miss Manila! (4x)
Colchicine con- | Number of Number of 3x | 3x/6x mixoploid 6x induction
Gwe vw eww Jo Jo
What is described above are merely examples of the present invention, but are not thus construed as limiting the scope of the present invention. Any equivalent structure or equivalent flow transformation which is made according to the description of the present invention, or directly or indirectly applied in other re- lated technical fields shall fall within the protection scope of the present invention patent similarly.
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| CN102499071B (en) * | 2011-12-15 | 2013-04-10 | 深圳市仙湖植物园管理处 | Chemical mutation breeding method for bougainvillea |
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|---|---|---|---|---|
| CN106035076A (en) * | 2016-06-20 | 2016-10-26 | 西南林业大学 | Method for inducing production of Rosa sterilis tetraploid through soaking method |
| CN108990793A (en) * | 2018-08-15 | 2018-12-14 | 江苏农林职业技术学院 | A kind of breeding method of tetraploid petunia |
| US20220117184A1 (en) * | 2020-10-16 | 2022-04-21 | Green Point Research Holdings LLC | Method for Producing Sterile Cannabis Sativa L. Cultivars |
| CN113519394A (en) * | 2021-07-15 | 2021-10-22 | 云南中医药大学 | Efficient artificial seedling raising method for red fruit ginseng polyploid induction and polyploid plants |
| CN114667927A (en) * | 2022-04-24 | 2022-06-28 | 中国热带农业科学院热带作物品种资源研究所 | Method for in vitro induction of bougainvillea spectabilis polyploidy |
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| CN115152626A (en) | 2022-10-11 |
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