CN116965333A - Method for inducing cymbidium plants to produce polyploids - Google Patents
Method for inducing cymbidium plants to produce polyploids Download PDFInfo
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
-
- 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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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Abstract
The present invention provides a method of inducing a plant of the genus cymbidium to produce polyploids comprising: (a) Germinating the seeds of the cymbidium on the first medium to form protocorms; and (b) inoculating the cymbidium protocorm onto a second medium containing colchicine at a concentration of 0.01% -0.2% for 1-15 d. The colchicine is used for obtaining the cymbidium polyploid with higher mutation rate by a co-culture method, and the identification method of the cymbidium polyploid is discussed, which comprises flow cytometry, chromosome tabletting, morphological observation, stomata identification or any combination thereof, and has important significance for cultivating new germplasm with high ornamental value.
Description
Technical Field
The invention belongs to the technical field of polyploid breeding, and particularly relates to a method for inducing protocorms of a cymbidium plant (such as cymbidium floridum) to generate polyploid by using colchicine.
Background
Cymbidium multiflorum (alides rose lodd.ex lindl. Et paxt.) belongs to the family of Orchidaceae (Orchidaceae), genus cymbidium, a uniaxially branched orchid. The flower color is tender, the flower quantity is large, the flower type is peculiar, the flower fragrance is strong, and the orchid has extremely high ornamental value. The white color of the flower is purple spots, and the flower shape is a pigeon; flowering period is 7 months, and fruit period is 8 months to 5 months of the next year. Produced in Guangxi, guizhou, yunnan. In addition to the unique flower shape and gorgeous flower color, unique aromaticity is also one of the important values of the cymbidium floribundum. Can hybridize with the related genus, and is a good strain for cultivating butterfly orchid with fragrance and large flower quantity.
Polyploid induction technology is an important means of orchid breeding at present. The plant is thick, the flower is larger, the stress resistance is stronger, and the like, which are excellent characters possessed by polyploid plants, and are more and more paid attention by breeding specialists. The induction and identification of orchid polyploids are greatly studied by domestic and foreign experts, and the tissue culture technology of tropical orchid such as dendrobium, butterfly orchid, big flower Huilan and the like and the tissue culture technology of cymbidium and the like is mature at present, and the polyploid induction is usually carried out by adopting the tissue culture technology in combination with chromosome doubling, and can also be called in vitro doubling induction. The in vitro doubling induction material is selected from seed, protocorm-like, stem tip, top bud, root tip, cluster bud, leaf, etc. and is used for chromosome doubling induction under the condition of tissue culture. Chemical induction is the most commonly used method for polyploid breeding, inducing plant cells to mutate, and usually uses auxin, anesthetic, various plant alkaloids and the like to induce the plant cells, and colchicine is the most commonly used mutagen. The damage to the plants caused by the concentration of colchicine is in direct proportion to the variation degree, and the damage to the plants and the variation degree are smaller when the concentration is lower. The research shows that compared with other explants, the protocorm has higher propagation coefficient and can be rapidly propagated to obtain a large number of plants, so that the protocorm is mostly selected as an in-vitro doubling induction material. Compared with the soaking method, the co-cultivation method has the advantages of low pollution rate, less damage to the protocorm and the like (Fan Wubo, 2007).
Although there have been successful cases of inducing polyploids by co-cultivation using colchicine in polyploid breeding of orchid plants, no study on polyploid induction has been reported in cymbidium plants. In addition, orchid has extremely rich species, extremely wide distribution and high species diversity, and the research results among different species and varieties often have huge differences, so that the existing research results are difficult to be referred to. For example, li Han (2005) induced by colchicine on agalloch eaglewood (c.iridioides) showed that colchicine treated for 72h and at a concentration of 0.05% gave a better induction. Yang Lijuan (2009) the co-cultivation method is used, colchicine with the concentration of 3% is added into a culture medium to induce the Huilan 'ruby' of big flowers, and experiments show that the effect of treating 15d is optimal, and the induction rate is as high as 30%. Therefore, when colchicine is used for co-cultivation, the requirements of different species of orchids on the conditions of colchicine concentration, induction time and the like are greatly different, and no rule can be followed.
The invention uses the plant of the genus Lawsonia (such as Lawsonia inermis) as an induction material, adopts a co-cultivation method, and discusses the proper condition for doubling colchicine-induced protocorm and a culture medium suitable for cultivating Lawsonia inermis by treating the colchicine with different concentrations for different times, and also discusses a method for identifying the polyploid of Lawsonia inermis, including flow cytometry, chromosome tabletting, morphological observation, stomatal identification and the like, so as to provide references for improving the orchid polyploid induction technology and cultivating new germplasm with high ornamental value.
Disclosure of Invention
In view of the problems of the prior art, the present invention provides a method for inducing the production of polyploids in cymbidium henryi, comprising the steps of:
(a) Germinating the seeds of the cymbidium on the first medium to form protocorms; and
(b) The cymbidium protocorm is inoculated onto a second medium containing colchicine at a concentration of 0.01% -0.2% (e.g., 0.01% -0.1%, 0.02% -0.09%, 0.03% -0.08%, 0.04% -0.07%, 0.05% -0.06%, 0.04%, 0.05%, 0.06%, or any value or range therebetween) for 1-15d (e.g., 1-10d, 2-10d, 3-10d, 4-10d, 5-10d, 6d, 10d, 5-8d, 6-9d, or any value or range therebetween).
In some embodiments, step (b) comprises culturing the cymbidium raw bulb on the second medium for 5-15d, such as 5-10d. In some embodiments, the second medium contains colchicine at a concentration of 0.05% -0.2%, for example 0.05% -0.1%, 0.05% -0.09%, 0.05% -0.08%, 0.05% -0.07%, 0.05% -0.06%. In some embodiments, step (b) comprises culturing the cymbidium protocorm on a second medium for 5-10d, and the second medium contains colchicine at a concentration of 0.05% or more, less than 0.1%, such as 0.05% to 0.07%, 0.05% to 0.06%.
In some embodiments, the first medium is a liquid medium. In some embodiments, the first medium comprises, or consists of, 1/2MS+0.5g.L-1 agar+20g.L-1 sucrose+100deg.C.L-1 coconut juice. In some embodiments, the first medium comprises or consists of 1/2MS+5g.L-1 carrageenan+100 g.L-1 banana puree+20g.L-1 sucrose. In some embodiments, the first medium comprises or consists of 1/2MS+0.5g.L-1 agar+100 g.L-1 banana puree+20g.L-1 sucrose.
In some embodiments, the second medium is a solid medium. In some embodiments, the second medium comprises or consists of 1/2MS+1.0 mg.L-1 6-benzyladenine+0.2 mg.L-1 agar+20 g.L-1 sucrose.
In some embodiments, the method further comprises: (c) Transferring the protocorm obtained in the step (b) into a third culture medium for subculture. In some embodiments, the third medium is a secondary medium, such as those well known in the art (e.g., 1/2MS+1.0 mg/L6-BA+0.2 mg. Multidot.L-1NAA+5.0 g. Multidot.L-1 agar+20 g. Multidot.L-1 sucrose). In some embodiments, step (c) is repeated at least one time or two or more times. In some embodiments, the step (c) is performed under the following conditions: illumination intensity: 2000lx, daily light time: 12-14 h, temperature: 25.+ -. 2 ℃.
In some embodiments, the method further comprises determining ploidy of the post-induction plant by flow cytometry, chromosome tabletting, morphological observation, stomatal identification, or any combination thereof. In some embodiments, the morphological observations include, but are not limited to, observations of the following traits: plant height, stem thickness, growth rate, leaf color, stomatal shape, or any combination thereof.
In some embodiments, colchicine-induced plant ploidy is identified using, for example, a flow cytometer.
In some embodiments, the variant plant exhibits one or more or all of the following morphological shapes compared to a control plant that is not colchicine-induced: plant dwarfing, strong rootstock, slow growth, dark green leaves, nearly circular stomata, or any combination thereof.
In some embodiments, the variability after colchicine induction is about 15-45%, e.g., about 20-45%, 23-43%, 25-40%, 29%, 30%, 35%, 36%, 37%, 36-40%, or any value or range therebetween.
In some embodiments, the survival rate after colchicine induction is about 16-65%, such as about 16.5-61%, 18-61%, 20-61%, 30-61%, 34-61%, 45-61%, 47-61%, or any number or range therebetween.
In some embodiments, step (a) comprises collecting seeds of cymbidium henryi and aseptically seeding them on the first medium, thereby germinating them to form protocorms. In some embodiments, the germinated protocorms are green.
In some embodiments, the methods of the invention further comprise: the data obtained are analyzed to determine the appropriate induction conditions. In some embodiments, the data analysis comprises data analysis using Excel 2016, analysis of variance using SPSS2.0, using LSD multiple comparison methods, or any combination thereof.
In some embodiments, the nail blue is a cymbidium multiflorum.
Definition of the definition
In the present invention, the terms "induced plants", "variant plants" and "tissue culture seedlings" are sometimes used interchangeably and generally refer to plants grown from seeds treated by the methods of the present invention, such as for example, from seeds treated by the methods of the present invention. Unless the context indicates otherwise.
In the present invention, when it is mentioned that the concentration of colchicine is, for example, 0.05%, 0.1%, 0.2%, it is meant the mass ratio, i.e. the colchicine weight is 0.05%, 0.1%, 0.2% of the total solution mass, respectively.
In the present invention, when referring to 5d, 10d, 15d, etc., the "d" refers to the number of days.
In the present invention, when referring to "cymbidium", it is generally meant that the cymbidium plant, such as any species, subspecies, or variety of cymbidium plant, etc., unless otherwise indicated.
The invention adopts the cymbidium as the material, adopts the co-culture method to induce polyploidy to explore the effect of colchicine treatment on the chromosome doubling of the cymbidium, and obtains the proper induction condition for the chromosome doubling of the cymbidium. The method can generate more polyploids and provide data support for optimizing polyploid breeding technology. The method has important value in breeding and wide application prospect.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the following brief description will be given of the drawings of the present invention. It should be understood that the drawings described below are for purposes of illustration only and are not intended to limit the scope of the present invention.
Fig. 1 shows a cymbidium multiflorum flower (a), a pod (B) and a protocorm (C) formed after aseptic seeding according to example 1.
FIG. 2 shows the effect of colchicine treatment concentration and time on the survival of the protocorm of the Phalaenopsis amabilis.
FIG. 3 shows morphological features of a colchicine-induced variant of Hupeh's nail polish according to an embodiment of the invention. A: a control strain; b: a variant; c: control (left) and post-mutation leaf (right) were compared.
Fig. 4 shows the stomatal characteristics of colchicine-induced cymbidium floribundum according to an embodiment of the invention. A: control strain stomata (×10); b: mutant stomata (. Times.10); c: control plant stomata (×40); d: mutant stomata (. Times.40).
FIG. 5 shows chromosome identification results of the colchicine-induced Hupeh's cymbidium, according to an embodiment of the invention. A: peak plot of control plants; b: peak plots for variant lines; c: control plant chromosome status (×60); d: chromosomal condition of variant strain (. Times.60).
Examples
The invention is further described below with reference to examples.
1. Material
Seed of cymbidium floridum obtained by selfing in 2020 is used as a material by sprouting protocorm after aseptic seeding. The experiment is carried out in vegetable and flower research institute of Chinese agricultural academy of sciences, and the phenotypic character is shown in figure 1.
2. Statistical method
Data analysis is carried out by Excel 2016, variance analysis is carried out by SPSS2.0, and the LSD multiple comparison method is adopted, wherein the data variation range values (+ -) in the table are all + -standard deviation
Example 1: colchicine induced polyploid plant produced by henna
1. Polyploid induction
The collected pods are subjected to sterile sowing, seeds are germinated on a culture medium of 1/2MS+0.5g.L-1 agar+20g.L-1 sucrose+100deg.g.L-1 coconut juice to form protocorms, and then the protocorms germinated to green are transferred to a solid culture medium containing 0.05%, 0.1% and 0.2% colchicine (1/2MS+1.0mg.L-1 6-benzyl adenine (6-BA) +0.2mg.L-1 agar+20g.L-1 sucrose) for 5d, 10d and 15d respectively, and each treatment is repeated for 5 times. The specific scheme is shown in Table 1 below.
TABLE 1 colchicine treatment protocol
The colchicine treated protocorm is transferred to a secondary culture medium for 2-3 times of secondary culture, and the survival rate is calculated. Culture conditions: the illumination intensity was 2000lx, the daily illumination time: 12-14 h, temperature: 25.+ -. 2 ℃ and pH5.8.
Survival = surviving plants/inoculated plants x 100%.
The results show that the survival rate of the protocorm of the henna is affected by the concentration of colchicine and the treatment time. As colchicine concentration increases or treatment time increases, the survival of the protocorm tends to decrease (see fig. 2). The damage to the protocorm is small at the concentration of 0.05%, and the survival rates are 61%,47% and 34.2% respectively; when the colchicine concentration is 0.2%, the damage to the protocorm is large, and the survival rate is 16.4%,10.6% and 6.6% respectively.
2. Morphological observation
Referring to the method of Zhuo Xiaokang (2015), when the plants differentiate into seedlings and the leaves are fully spread, selecting untreated control plants with consistent culture conditions and well grown untreated control plants and treated and identified and doubled variant strains, 15 plants each, and measuring the growth vigor, plant height, leaf size and observed color of the plants.
After colchicine treatment, the difference between the mutant strain and the control strain is larger (fig. 3A-B), and the mutant strain and the control strain are mainly characterized by dwarfing plants, deepening of leaf colors, rough leaf surfaces, widening of leaves and the like (fig. 3B). As can be seen from table 2: the aspect ratios of the control and variant strains were 3.67 and 1.88, respectively, and the leaves of the control strain were relatively long and narrow, with the leaves of most variants being relatively short and rounded due to inhibition (fig. 3C). The plant height, leaf length, and width tended to decrease by 1.94, 1.03, and 0.04, respectively, and the aspect ratio tended to decrease by 1.79, respectively, as compared with the control plant.
TABLE 2 morphological index comparison of colchicine-induced variant strains of Phalaenopsis
3. Air hole identification
Because the chromosome doubling plant and the plant which is not subjected to doubling treatment have differences in the size of the stomata, the comparison of the change of the size of the stomata can also be used as a method for initially identifying the doubling plant. Referring to the method of Xiqing Zhang (2020), leaves of the same parts of untreated and treated doubled plants were selected, 10 plants each were first dropped with a drop of water, in order to avoid curling after peeling off the epidermis, then the lower epidermis of the leaf was peeled off with forceps, placed on the slide, covered with a cover glass and flattened, and placed under a Leica inverted microscope (Beijing Oriental Australia scientific development Co., beijing Feng Tai Co., ltd.) for observation and photographing. And randomly selecting a visual field with uniform pore distribution under a microscope, and recording the number of pores in a unit area.
As can be seen from Table 3, the control plant of the Phalaenopsis amabilis had a dense distribution of air holes, and an average of 39 air holes were distributed per field of view, whereas the doubled plant had 21 air holes per field of view, and the air holes had a larger length and width than the control plant. The air holes of the control strain are oblong, while the air holes of the variant strain are nearly circular. The air holes of the control strain were 31 μm wide and 34 μm long, and the air holes of the variant strain were 39 μm wide and 42 μm long. Figure 4 shows a comparison of stomata between variant and control strains.
TABLE 3 comparison of pore morphology index of colchicine-induced variant strain of Phalaenopsis
4. Flow cytometer ploidy analysis
The colchicine-treated plants were subjected to ploidy identification with reference to plants of the henna that were not subjected to doubling treatment. Fresh leaves of the plants to be tested were placed in a petri dish using Sysmex CyStain UV Precise P kit (Beijing Deshida technologies Co., ltd., beijing sea lake area) and cut into 0.5cm pieces 2 The extraction liquid is dropped on the leaf blade, the dye liquid is dyed for 2min in a dark place, then the flow cytometry is used for identification, each sample is repeated for 2 times, the number of variant strains is counted according to the result of the flow cytometry, and the variation rate is calculated.
The results are shown in FIGS. 4A-B. Untreated control plants were identified as diploid and the induced plants became variant but with some amount of chimeras.
As can be seen from table 4, when the treatment times are identical, the concentration and the variation rate are in a direct proportion, and the variation rate increases with the increase of the concentration; colchicine 15d at a concentration of 0.1% and 15d at a concentration of 0.2% reduced the variability by 13.24% and 14.25% respectively compared to the 10d treatment. From Table 4, it was found that the mutation rate reached 40% at the concentration of 0.05% after 10d treatment and the survival rate reached 45%. Next, after 5d treatment at a concentration of 0.05%, the mutation rate reached 36.36%, but the survival rate was the highest, 61%.
TABLE 4 influence of colchicine treatment concentration and time on the variation of the protocorm of Phalaenopsis
2.5 chromosome tabletting analysis
With reference to the tabletting method of Zhou Jianjin, zhuang Donggong and the like, the selection of tissue culture root tips is carried out in the period of 9:00-11:00 am, and the specific operation method is as follows:
(1) The root tip of the tissue culture seedling is selected, cut to 1-2cm, the epidermis is scraped by a blade, and then the tissue culture seedling is placed in a prepared 0.002 mol/L8-hydroxyquinoline solution for soaking for 7-8h;
(2) Cleaning with sterile water, soaking in newly configured Carnot fixing solution for 24 hr, fixing, and directly tabletting or storing in 70% ethanol;
(3) Treating the treated material with 1mol/L hydrochloric acid at normal temperature for 5-6min, and soaking in a constant-temperature water bath at 60 ℃ for 7-8min;
(4) Dyeing the modified phenol fuchsin dye droplets on the treated root tips;
(5) Crushing the root tip with forceps after 10min to remove residues, and covering with a cover glass; tapping the cover glass by using a pencil with a rubber to ensure that chromosomes are uniformly distributed;
(6) And observing and counting the number of the chromosomes under a microscope, selecting chromosome cells with good visual field and uniform dispersion in 5 areas, counting the number of the chromosomes and taking pictures.
Flow cytometry allows for a large number of ploidy identifications of plants, but the results of chromosome bearing were the most intuitive and accurate (FIGS. 4C-D). After chromosome plating, the number of chromosomes of the control strain was found to be 2n=2x=36 (fig. 4C), while the number of chromosomes of the induced variant strain was found to be 2n=4x=74 (fig. 4D), and cells that were not doubled successfully remained in the variant strain.
Example 2: screening of additives in seed germination Medium
To verify the effect of different additives on seed germination rate, 1/2MS+5g.L-1 carrageenan+20g.L-1 sucrose was used as a basal medium to which 100 g.L-1 corn juice, banana puree, coconut juice were added, respectively, and medium without additives was used as a control, and the seeds of Phalaenopsis amabilis were observed after aseptic sowing. Under the condition of the same culture time, the seeds of the cymbidium floribundum have different development conditions due to different additives. Some seeds develop into healthy green protocorms and even grow into seedlings; some of the seed developed protocorms were mostly yellow in color. Wherein the germination rate is highest in the culture medium 1/2MS+5g.L-1 carrageenan+100deg.g.L-1 banana puree+20g.L-1 sucrose, and the culture medium is full in size (Table 5).
TABLE 5 Effect of additives on seed germination of Phalaenopsis amabilis
This study shows that a large number of variant plants can be obtained in a short time by combining chromosome doubling techniques with plant tissue culture. Because the research on the henna is less, the double induction test is carried out on the henna, and the henna has important research significance. Colchicine is used for in vitro doubling to induce protocorms and autumn-like protocorms, and the induction effect is optimal. In this study, the variability of 15d in the plant treatment was reduced at concentrations of 0.1% and 0.2%. Therefore, it is considered that the induction of the protocorm by the co-culture method is performed for a period of time less than 15d and the induction effect is excellent in a concentration range of < 0.1%.
On morphological characteristics, the variant plants all show the characteristics of dwarfing plants, thick and strong rootstock, slow growth, dark green leaves and the like. Meanwhile, the leaves of the control strain are long and narrow, and the leaves of the variant strain are short and round. In terms of stomata, the stomata of the variant were nearly circular, while the stomata of the control were nearly oblong. Therefore, the plant morphology and stomata characteristic can be used as an indirect method for identifying the henland ploidy.
Compared with the method of inducing polyploidy by the soaking method, the co-culture method has the advantages of high induction rate and lower pollution rate, but a certain amount of chimera exists after induction. The study uses a co-cultivation method to induce polyploid of the protocorm, compares the concentration of colchicine with the treatment time, obtains a better treatment combination and obtains a variant strain. Meanwhile, a flow cytometer can be used for screening a large number of treated protocorms to obtain polyploids, so that polyploid plants are obtained.
Reference to the literature
[1] Chinese plant Saint [ M ] Beijing, science Press, 1993, (19): 384.
[2] Li Han, zheng Saixiang, long Chunlin. Induced primordial detection of Dendrobium devonianum polyploid [ J ]. Yunnan plant research 2005, 27 (5): 552-556.
[3]Xiqing Zhang,Jiangyun Gao.Colchicine-induced tetrapoloidy in Dendrobium carniferum and ite effect on plantet morphology,anatomy and genome size[J].
Original Article,2020.
[4] Zhuo Xiaokang construction of a rapid-propagation system of Dendrobium macrocarpium Rolfe and polyploid induction [ D ]. Fujian university of agriculture and forestry 2015.
[5] Zhou Jianjin, zeng Ruizhen, liu Fang, yisheng, et al chromosome ploidy studies of hybrid progeny of different ploidy butterfly orchids [ J ]. Horticulture journal, 2009, 36 (10): 1491-1497.
[6] Zhuang Donggong, qu Ying, xu Daxiong, li Jun, chen Zhiling.2007 chromosome number and morphology analysis of several varieties (lines) of butterfly orchid, gardening journal, (05): 1257-1262.
[7] yang Lijuan, gao Suping, zonglan, chen fruit colchicine induced in vitro the Huilan polyploid experiment of big flowers [ J ].
Northern gardening, 2009 (6): 51-53.
Claims (10)
1. A method of inducing polyploid production in a nail orchid comprising the steps of:
(a) Germinating the seeds of the cymbidium on the first medium to form protocorms; and
(b) The cymbidium is inoculated on a second culture medium, and is cultured for 1-15d, wherein the second culture medium contains colchicine with the concentration of 0.01% -0.2%.
2. The method of claim 1, further comprising: (c) Transferring the protocorm obtained in the step (b) into a third culture medium for subculture.
3. The method of any one of claims 1-2, wherein the first medium is a liquid medium; and/or the first culture medium comprises 1/2MS+0.5g.L-1 agar+20g.L-1 sucrose+100deg.g.L-1 coconut juice, or the first culture medium comprises 1/2MS+5g.L-1 carrageenan+100deg.g.L-1 banana puree+20g.L-1 sucrose.
4. A method according to any one of claims 1-3, wherein the second medium is a solid medium and/or the second medium comprises 1/2ms+1.0 mg-L-1 6-benzyladenine+0.2 mg-L-1 agar+20 g-L-1 sucrose.
5. The method according to any one of claims 1-4, comprising repeating step (c) at least once or twice, and/or, said step (c) being performed under the following conditions: illumination intensity: 2,000 lx, daily light time: 12-14 h, temperature: 25.+ -. 2 ℃.
6. The method according to any one of claims 1-5, wherein step (b) comprises culturing the cymbidium protocorm on a second medium for 5-15d, such as 5-10d.
7. The method according to any one of claims 1-6, wherein the second medium contains colchicine at a concentration of 0.05% -0.2%, such as 0.05% -0.1%.
8. The method of any one of claims 1-7, wherein step (b) comprises culturing the cymbidium protocorm on a second medium for 5-10d, and the second medium contains colchicine at a concentration of 0.05% or more, less than 0.1%.
9. The method of any one of claims 1-8, further comprising determining ploidy of the post-induction plant by flow cytometry, chromosome tabletting, morphological observation, stomata identification, or any combination thereof.
10. The method of claim 9, wherein the morphological observations include, but are not limited to, observations of the following traits: plant height, stem thickness, growth rate, leaf color, stomatal shape, or any combination thereof.
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| CN112293257A (en) * | 2020-11-12 | 2021-02-02 | 中国农业科学院蔬菜花卉研究所 | Method for inducing phalaenopsis protocorm to generate polyploidy by colchicine |
| CN114568304A (en) * | 2021-11-12 | 2022-06-03 | 中国农业科学院蔬菜花卉研究所 | Method for inducing phalaenopsis seeds to generate polyploids and improving germination rate |
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| CN112293257A (en) * | 2020-11-12 | 2021-02-02 | 中国农业科学院蔬菜花卉研究所 | Method for inducing phalaenopsis protocorm to generate polyploidy by colchicine |
| CN114568304A (en) * | 2021-11-12 | 2022-06-03 | 中国农业科学院蔬菜花卉研究所 | Method for inducing phalaenopsis seeds to generate polyploids and improving germination rate |
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