Method for inducing dandelion chromosome doubling by colchicine

Abstract

The invention relates to the technical field of plant polyploidy breeding, and provides a method for inducing dandelion chromosome doubling by colchicine, which comprises the following steps: sowing and germinating to obtain seedlings, and coating plant growing points of the seedlings with a colchicine solution for induction treatment to obtain dandelion with doubled chromosomes. According to the technical scheme, the problems that traditional breeding of the alkaline land dandelions is difficult, novel breeding technologies are deficient, good varieties are few, biomass of some high-quality varieties is small, and industrial development of the alkaline land dandelions is affected in the prior art are solved.

Description

Technical Field

[0001] The invention relates to the technical field of plant polyploidy breeding, and in particular to a method for inducing dandelion chromosome doubling by using colchicine. Background Art

[0002] Alkali dandelion is a plant of the genus Taraxacum in the Asteraceae family. It is a traditional perennial plant with both medicinal and edible properties, a commonly used Chinese medicinal plant for clearing heat and detoxifying, and is known as the "natural antibiotic" and "Queen of Herbs" in traditional Chinese medicine. Not only can the whole plant be used as medicine, but it can also be used as a source of food, health food, livestock feed, etc., and is consumed in huge amounts every year. Alkaline soil dandelion resources are relatively rich, but due to long-term asexual reproduction, hybrid breeding is impossible, and the genetic and biological diversity of alkaline soil dandelions cannot be fully utilized. At the same time, some excellent germplasms are also endangered due to species degeneration. There are only four high-yield dandelion varieties reported and approved, including the new polyploid variety "Mingxian No. 1" bred by Professor Zhao Xiaoming of Shanxi Agricultural University from the wild population of medicinal dandelions after many years of systematic selection; Yang Guozheng used the method of systematic domestication and selection to select a new material with basically stable traits, good yield (yield is more than 3 times that before domestication), and strong stress resistance from wild dandelion resources, temporarily named "Huapu No. 1"; the Binhai Agricultural Research Institute of Hebei Academy of Agricultural and Forestry Sciences used a method combining salt-tolerant mutants with salt-tolerant identification to select salt-tolerant high-yield dandelion varieties "Binpu No. 1" and "Binpu No. 3". Therefore, the innovation of dandelion germplasm resources and the cultivation of new varieties are imperative.

[0003] Polyploid plants have a superposition effect due to the doubling of chromosomes, and are relatively "giant", with high plant biomass and secondary metabolites, strong adaptability and stress resistance. Therefore, polyploid breeding can increase crop yields, improve crop product quality, and enhance crop stress resistance. It is an important way to improve crop varieties. Polyploid breeding can also improve the medicinal value and commercial value of Chinese medicinal plants, providing possibilities for the creation of new germplasm and the cultivation of excellent new varieties.

[0004] Yang Yushuang et al. published a study titled "Comparative Study on the Effect of Amsulfuron and Colchicine on Inducing Tetraploidy in Rubber Grass" (Chinese Agricultural Science Bulletin 2021, 37(20): 35-41). They preliminarily explored the induction effects of two mutagens, amsulfuron and colchicine, on chromosome doubling in rubber grass. The results showed that amsulfuron was more suitable for inducing polyploidy in rubber grass than colchicine. The seedling rate of rubber grass treated with amsulfuron was 60%~75% and the highest mutagenesis rate was 14.08%, both higher than the seedling rate of 0~59% and the highest mutagenesis rate of 5.56% in colchicine treatment. Its chimera rate was 27.27%, lower than the 75.00% in colchicine treatment. The seedling rate was 71.00% and the mutation rate was 14.08% in 0.04mmol / L amsulfuron treatment for 30min, which was the best mutagenesis condition.

[0005] Zhong Shumei published a master's thesis titled "Study on Artificial Doubling of Dandelion and Its Application Value" (2010, College of Plant Science and Technology, Huazhong Agricultural University). The leaves of dandelion were used as explants and inoculated into induction medium with different concentrations of colchicine. The results showed that the induction medium with a concentration of 75 mg / L had the highest induction rate, reaching 13.73%. However, its main disadvantages are as follows: 1) It is difficult to obtain homozygous plants in dandelion callus induction. Callus, as embryonic cells, accumulates together, and it cannot be guaranteed that all cells have been doubled. It is difficult to separate homozygous plants after induction doubling; 2) The construction of the induction system takes a long time, is easy to be contaminated, and has a low success rate; the induction system takes 5 to 6 months from explant inoculation to callus, bud formation, rooting, seedling hardening, transplanting, and identification of induced plants, and the technology is cumbersome; 3) The regenerated plant test tube seedlings are planted in the field, which requires a lot of manpower and material resources. They are thin and fragile during the growth process, and the survival rate of transplanting is low, which limits the dandelion variety improvement technology and makes it difficult to apply it in practice. Summary of the invention

[0006] The present invention provides a method for inducing dandelion chromosome doubling with colchicine, which solves the problems in the related art of difficulty in traditional breeding of dandelions in alkaline soil, lack of new breeding technology, few excellent varieties, small biomass of some high-quality varieties, and influence on the development of the dandelion industry in alkaline soil.

[0007] The technical solution of the present invention is as follows: The invention provides a method for inducing dandelion chromosome doubling by using colchicine, which comprises the following steps: sowing, obtaining seedlings after germination, applying colchicine solution to the plant growth points of the seedlings for induction treatment, and obtaining dandelions with doubled chromosomes.

[0008] As a further technical solution, the seedling period includes one or both of the two cotyledon period and the two-leaf-one-heart period.

[0009] As a further technical solution, the seedling is in the two-cotyledon period.

[0010] As a further technical solution, the mass concentration of the colchicine solution is 0.05%~0.15%.

[0011] As a further technical solution, the mass concentration of the colchicine solution is 0.05%~0.12%.

[0012] As a further technical solution, the mass concentration of the colchicine solution is 0.10%.

[0013] When the present invention uses a colchicine solution with a concentration of 0.10 (w / v)% and the treatment period is the two-cotyledon period, the survival rate of dandelion seedlings is 47.82%, and the tetraploid rate is as high as 38.66%. This method not only ensures a high tetraploid induction rate, but also takes into account a high seedling survival rate, provides a basis for polyploid breeding of dandelions in alkaline soils, and is of great significance to the creation of new germplasm and polyploid breeding of dandelions in alkaline soils.

[0014] As a further technical solution, the sowing method is hole sowing.

[0015] As a further technical solution, primary screening is performed after the induction treatment to obtain diploid and tetraploid chimeric plants and pure tetraploid plants, and the diploid and tetraploid chimeric plants are purified. The purification includes the following steps: continuing to plant the diploid and tetraploid chimeric plants until diploid and tetraploid chimeric seeds are obtained, sowing the diploid and tetraploid chimeric seeds, performing a chromosome ploidy test after phenotypic separation, and planting when determined to be tetraploid to obtain pure tetraploid plants.

[0016] The screening in the present invention is specifically as follows: three situations may occur in the new leaves after doubling, one is the diploid material that has not been doubling, one is the material of the chimera of diploid and tetraploid, and the other is the pure tetraploid material; after culturing for 30 days, seedlings with the characteristics of thickened and fleshy leaves, reduced leaf area, more leaves, reduced lateral roots, etc. are preliminarily screened from the plants, and the chromosome ploidy is measured by flow cytometry. In the measurement process, the new leaves of the undoubled material are used as a control, and plants with different ploidy are preliminarily screened and marked so as to facilitate subsequent ploidy determination.

[0017] As a further technical solution, the plant growth point is the position where true leaves grow.

[0018] As a further technical solution, alkaline soil dandelion seeds are used during sowing, and the alkaline soil dandelion seeds are diploid alkaline soil dandelion seeds.

[0019] The invention also provides a method for inducing dandelion chromosome doubling with colchicine and its application in inducing dandelion chromosome doubling.

[0020] The working principle and beneficial effects of the present invention are: 1. In the present invention, a colchicine treatment solution is used to treat high-quality alkaline soil dandelion seedlings to produce polyploids, so as to cultivate high-yield, high-quality, salt-tolerant alkaline soil dandelions, achieve variety improvement, promote the development of the alkaline soil dandelion industry, and adapt to saline-alkali soil planting. In addition, the colchicine-induced dandelion chromosome doubling method provided by the present invention takes only 3 months from seed seedling cultivation to induction treatment, mutagenesis plant, and identification, which is half the time of the prior art, simple to operate, and improves breeding efficiency.

[0021] 2. In the present invention, dandelion seeds are directly sown in the field, colchicine is used as an inducer, the chromosomes of dandelion seedlings in alkaline soil are induced to double, and tetraploid plants are obtained. The plant seedling growth point is used as the induction site, which is simple to operate, has high feasibility, is easy to manage, and can significantly improve the survival rate of seedlings. This method overcomes the problems of pollution when using leaves as explants to induce doubling in the traditional method, avoids the cumbersome processes such as germination, rooting, seedling hardening, and transplanting of induction tube seedlings by the traditional callus approach, saves cultivation time, reduces labor and production costs, improves breeding efficiency, and ensures the feasibility of inducing tetraploid plants for field planting. This method is easy to operate and has stable induction, and is a method suitable for inducing polyploid dandelion in alkaline soil.

[0022] 3. In the present invention, colchicine is used to directly induce the growth points of dandelion seedlings in alkaline soil, with a high induction rate (the highest induction rate is 38.66%, which is 13.73% higher than callus induction), doubling the breeding time and saving 50%, simple operation, high feasibility, and reduced production costs.

[0023] 4. Alkali dandelion and rubber grass both belong to the genus Taraxacum, but they are not the same species, and their genes and biological characteristics are different: the rubber content of the rubber grass root is high, and it is one of the three major rubber-producing plants. Compared with the wild species of alkaline dandelion, the individual is small and the growth is weak; the present invention uses alkaline dandelion as a medicinal plant, and the whole plant is used as medicine. The main parts used are stems and leaves containing phenolic acid components such as chicoric acid and caffeic acid, and the salt and alkali tolerance is good, and the biomass is large compared to rubber grass. In addition, the doubling part used in the present invention is the young shoot induction after seed germination. Since the rubber grass seeds themselves are difficult to germinate and the germination rate is low, amosulfuron is suitable for rubber grass polyploid induction, and the mutation rate is 14.08%, while the colchicine doubling method has a low mutation rate of only 5.56%; the inventor has verified through a large number of experiments that the young shoots after germination of alkaline dandelion seeds are treated with colchicine doubling, and the survival rate is extremely low, almost zero. Therefore, the possibility of replicability of this technology in doubling alkaline dandelion is small, and it is not suitable for mutagenesis.

[0024] 5. The present invention directly uses seedlings grown to the two-cotyledon stage in the soil as test materials. Compared with the traditional method of using dandelion callus tissue in alkaline soil as material or using embryos after seed germination in a culture dish as material and then soaking them and then transplanting them into seedlings, the steps of seed germination and transplanting and callus tissue seedling formation are omitted, the process is simpler and the survival rate is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0026] Figure 1 The photos of dandelion seedlings in alkaline soil with different induction treatments at the two cotyledon stages; In the figure: a is water treatment, b is 0.05 (w / v)% colchicine solution treatment, c is 0.10 (w / v)% colchicine solution treatment, d is 0.12 (w / v)% colchicine solution treatment, e is 0.15 (w / v)% colchicine solution treatment; Figure 2 The photos are of chromosome ploidy determination of common diploid plants and tetraploid plants; In the figure: a is a common diploid plant, b is a tetraploid plant; Figure 3 The photos are of phenotypes of plants induced by different concentrations of colchicine. In the figure: a is a common diploid young leaf, b~c are tetraploid plants treated with 0.1 (w / v)% colchicine solution; Figure 4 The photos are of the phenotype and chromosome number of diploid and tetraploid plants; In the figure: a is the phenotype of a diploid plant, b is the phenotype of a tetraploid plant, c is the number of chromosomes of a diploid plant, and d is the number of chromosomes of a tetraploid plant; Figure 5 The photos are of the phenotype and chromosome number of diploid and tetraploid regenerated plants; In the figure: a and e are the phenotypes of diploid plants, b~c and f are the phenotypes of tetraploid regenerated plants, g is the chromosome number of diploid plants, and h is the chromosome number of tetraploid regenerated plants. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The following examples and comparative examples: The diploid alkaline soil dandelion seeds are the highly salt-tolerant narrow-leaf alkaline soil dandelion material "Chongli 1" from Chongli County, Zhangjiakou City.

[0029] Example 1 A method for inducing dandelion chromosome doubling with colchicine comprises the following steps: S1. Pre-experimental seedling tray cultivation: The seeds of the alkaline soil dandelion resource "Chongli 1" were cultivated in 32-hole seedling trays, 41 trays were cultivated, 3-4 seeds were sown in each hole (to ensure 1 seed germinated), the sowing depth was 1 cm, and conventional management was performed; S2. Induction treatment: retain a seedling with two leaves and one heart in each hole, place a 3mm diameter cotton ball tightly against the growth point of the seedling (the location where true leaves grow) and stick it in the middle of the plant, and drip 10μL of clean water, 10μL of colchicine solution with a concentration of 0.05(w / v)%, 0.10(w / v)%, 0.12(w / v)%, and 0.15(w / v)% onto the cotton ball for the plant to absorb. Keep 1 plate for the 10μL clean water treatment, and 10 plates for each of the other treatments. Drip once at 9:00 a.m. and 3:00 p.m. every day, and continue the treatment for 48 hours before routine management to obtain dandelions induced by colchicine.

[0030] Example 2 The only difference from Example 1 is that the seedlings in the two-leaf and one-heart stage are replaced by seedlings in the two-cotyledon stage, and the dandelion seedlings in the alkaline soil treated by different induction methods are as follows: Figure 1 shown.

[0031] Comparative Example 1 The bud immersion method was used for mutagenesis treatment, as follows: A method for inducing dandelion chromosome doubling with colchicine comprises the following steps: S1. Pre-experimental seedling tray cultivation: The seeds of the alkaline soil dandelion resource "Chongli 1" were cultivated in 32-hole seedling trays, 30 trays were cultivated, 3-4 seeds were sown in each hole (to ensure 1 seed germinated), the sowing depth was 1 cm, and conventional management was performed; S2. Induction treatment: When the seeds germinate and the radicle grows to about 5-10 mm, they are soaked in colchicine solution with concentrations of 0.05 (w / v)%, 0.10 (w / v)%, and 0.20 (w / v)% for 30 min, respectively. After the treatment, they are rinsed 3 times with sterile water and transplanted to seedling trays for routine management to obtain dandelions induced by colchicine.

[0032] Test Example 1: Initial screening of chromosome ploidy: The alkaline soil dandelions induced by the colchicine-induced dandelion chromosome doubling method of Examples 1-2 and Comparative Example 1 were cultured for 30 days, and seedlings with thickened and fleshy leaves, reduced leaf area, more leaves, and fewer lateral roots were preliminarily screened from the plants. The chromosome ploidy was determined by flow cytometry, specifically: a razor blade was used to double the 1 cm 2 The leaves of different sizes were placed in 1 mL of extraction buffer (15 mmol·L -1 Tris-HCl (pH=7.5), 80mmol·L -1 KCl (1.192 g), 20 mmol·L -1 NaCl, 20mmol·L -1 EDTA-Na2, 15mmol·L -1Before loading, take 100 mL of the filtrate and add 80 mL of staining solution (0.01 M PBS buffer, 50 μg / L -1 RAase, 50 μg L -1 PI, stain for 2 hours and mix well. Ordinary diploid young leaves were used as control ( Figure 2 ), the induction rates of different treatments were determined, and the results are shown in Table 1.

[0033] Table 1 Effects of different colchicine treatments on the survival rate and tetraploid rate of dandelions in alkaline soil

[0034] Note: The comparative example is the survival rate and induction rate of radicle induced by immersion in different concentrations of colchicine at the optimal treatment time.

[0035] from Figure 1 As can be seen from Table 1, the survival rate gradually decreases with the increase of colchicine treatment concentration, and the tetraploid rate first increases and then decreases with the colchicine treatment concentration; when the treatment concentration is above 0.10 (w / v)%, at the same treatment concentration, the earlier the treatment period, the lower the survival rate, and at the same treatment concentration, the earlier the treatment period, the higher the tetraploid rate, and vice versa, the lower the tetraploid rate; too high colchicine concentration and too late treatment period cannot continuously improve the cell tetraploid rate, but will lead to a decrease in survival rate. In summary, the tetraploid rate of seedlings in the two cotyledon stage treated with 0.10 (w / v)% colchicine reached a maximum of 38.66%, and the difference reached an extremely significant level (p<0.01), which is the most suitable induction system.

[0036] Test Example 2: (1) Land preparation: Rotary tillage is performed on 2 mu of alkaline land for dandelion planting.

[0037] (2) Making ridges: Make ridges and beds. Each bed is 5m long and 1.2m wide. Sow three rows in each bed.

[0038] (3) Sowing: Select diploid alkaline dandelion seeds with full grains, sow them in holes, sow 3 to 4 seeds in each hole (to ensure that one seed germinates), sow at a depth of 1 cm, and perform routine management.

[0039] (4) Induction treatment: According to the above preliminary experiments, the seedling treatment period is determined to be the two-cotyledon stage. The optimal concentration of colchicine induction is 0.10 (w / v)%. A 3-mm-diameter cotton ball is tightly attached to the seedling growth point and stuck in the middle of the plant. 10 μL of colchicine solution is dripped onto the cotton ball for the plant to absorb. The solution is dripped once at 9:00 a.m. and 3:00 p.m. every day. The treatment is continued for 48 hours and then routine management is performed.

[0040] (5) Preliminary screening of induced plants: Preliminary screening of induced plants with slow growth process, thickened and fleshy leaves, reduced leaf area, more leaves, and fewer lateral roots was performed from the induced plants obtained in step (4). Young leaves were selected for ploidy identification using flow cytometry. The operation process was the same as that in Experimental Example 1, see Figure 3 .

[0041] (6) Plant ploidy identification: Select the root tips with vigorous growth and use the conventional chromosome preparation method to identify the chromosome number. When the number of new lateral roots of the seedlings grown in the soil for 30 days is vigorous, cut the root tips between 9:30 and 10:00, wash with water, pre-treat in 0.20 (w / v)% colchicine for 4 h, fix in Carnoy's fixative (ethanol: glacial acetic acid volume ratio of 3:1) for 24 h, and then place the root tips in a constant temperature water bath at 60°C and 1 mol / L HCl for 8 min. Pour away the HCl, rinse the material repeatedly with distilled water, stain with Carbofuxin for 15 min, press into slides as usual, and finally examine under an optical microscope.

[0042] The results are as follows Figure 4 and Figure 5 As shown: The leaves of some plants treated with colchicine are more rounded, more stretched and thicker than those of diploid plants ( Figure 4 a, b), after chromosome number identification, it was found that the number of chromosomes was 2n=48, which is twice that of the diploid control (2n=24) ( Figure 4 c, d), confirmed as tetraploid plants, indicating that the colchicine solution with a concentration of 0.1 (w / v)% induced tetraploid plants; through screening of induced plants, it was found that some plants showed thickened and fleshy leaves, reduced leaf area, and peculiar leaf shape ( Figure 5 a~c, e~f), indicating that some induced plants have variant phenotypes, which can be identified by the chromosome number in the root apical meristem ( Figure 5 g, h), confirming that these mutant plants are tetraploid. The above results show that tetraploid plants can be obtained by adding colchicine to the growth points of dandelion seedlings at the two cotyledon stage in alkaline soil.

[0043] Plant biomass determination: homozygous tetraploid materials and diploids were planted and managed routinely. After obtaining the plants, their leaf length, leaf width and number of leaves, chlorogenic acid, caffeic acid and other phenolic acid components were measured (see Table 2). Under the same culture conditions, the morphological characteristics of tetraploid and diploid plants were compared and analyzed. Phenolic acid component detection method: 0.75g of alkaline dandelion leaf dry powder (solid-liquid ratio 1g:300mL) was taken and placed in a volumetric flask containing 30mL of 70% methanol solution for ultrasonic extraction for 30min. The extract was centrifuged at 4000rpm for 10min, the supernatant was taken, and the filtrate was filtered through a 0.45μm filter membrane and the content of caffeic acid and chicoric acid was detected by high performance liquid chromatography. The detection instrument was Agilent 1200HPLC, the chromatographic column was Mars ODS-AQ (4.6 mm × 250 mm, 5 µm), the injection volume was 0.5 μL, the flow rate was 1 mL / min, the volume ratio of anhydrous methanol: 0.2 wt % phosphoric acid was 40:60, and the detection wavelength was 327 nm.

[0044] Table 2 Biomass determination of plants with different ploidy

[0045] As shown in Table 2, under the same culture conditions, the leaf width of tetraploid plants is greater than that of diploid plants, the leaf length is basically the same, and the number of leaves of plants is less than that of diploid plants, but the leaves of tetraploid plants are thicker. Compared with diploid plants, the caffeic acid and chicoric acid phenolic acid components of tetraploid plants are significantly increased, and these characteristics can further identify tetraploid alkaline land dandelions.

[0046] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for inducing chromosome doubling in dandelion with colchicine, characterized in that: The following steps are involved: After sowing and germination, seedlings are obtained, and colchicine solution is applied to the plant growth points of the seedlings for induction treatment to obtain dandelions with doubled chromosomes.

2. The method for inducing dandelion chromosome doubling with colchicine according to claim 1, characterized in that: The seedling period includes one or both of the two-cotyledon period and the two-leaf-one-heart period.

3. The method for inducing dandelion chromosome doubling with colchicine according to claim 1, characterized in that: The mass concentration of the colchicine solution is 0.05% to 0.15%.

4. The method for inducing dandelion chromosome doubling with colchicine according to claim 3, characterized in that: The mass concentration of the colchicine solution is 0.05% to 0.12%.

5. The method for inducing dandelion chromosome doubling with colchicine according to claim 4, characterized in that: The mass concentration of the colchicine solution is 0.10%.

6. The method for inducing dandelion chromosome doubling with colchicine according to claim 1, characterized in that: The sowing method is hole sowing.

7. The method for inducing dandelion chromosome doubling with colchicine according to claim 1, characterized in that: After the induction treatment, primary screening is performed to obtain diploid and tetraploid mosaic plants and pure tetraploid plants, and the diploid and tetraploid mosaic plants are purified. The purification includes the following steps: the diploid and tetraploid mosaic plants are continuously planted until diploid and tetraploid mosaic seeds are obtained, the diploid and tetraploid mosaic seeds are sown, and after phenotypic separation, a chromosome ploidy test is performed, and when it is determined to be tetraploid, the seeds are planted to obtain pure tetraploid plants.

8. The method for inducing dandelion chromosome doubling with colchicine according to claim 1, characterized in that: The plant growth point is the position where true leaves grow.

9. The method for inducing dandelion chromosome doubling with colchicine according to claim 1, characterized in that: The alkaline soil dandelion seeds are used during sowing, and the alkaline soil dandelion seeds are diploid alkaline soil dandelion seeds.

10. Use of the method for inducing dandelion chromosome doubling with colchicine according to any one of claims 1 to 9 in inducing dandelion chromosome doubling.

Images