In-vitro induction method of trichosanthes kirilowii maxim autotetraploid

Abstract

The invention relates to the technical field of plant tissue culture, and discloses an in-vitro induction method of a trichosanthes kirilowii maxim autotetraploid, which successfully realizes in-vitro induction and high-speed propagation of the trichosanthes kirilowii maxim autotetraploid by vibrating and soaking a nodal stem segment of the trichosanthes kirilowii maxim through colchicine. After successful doubling is determined, optimizing a tetraploid plant artificial rapid propagation system; finally, the multiplication coefficient of the trichosanthes kirilowii maxim tetraploid can reach 6.43, the rooting rate is 100%, and the survival rate can reach 92% after regenerated seedlings are domesticated and transplanted. According to the method, the trichosanthes kirilowii maxim autotetraploid is successfully induced, an artificial efficient regeneration system of the trichosanthes kirilowii maxim autotetraploid is established, an excellent material is provided for new variety breeding and genetic research of trichosanthes kirilowii maxim, meanwhile, an experimental basis is provided for ploidy breeding of other species of cucurbitaceae, and the method has remarkable application and popularization value.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to an in vitro induction method for autotetraploids of Trichosanthes kirilowii. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Trichosanthes cucumerina (Trichosanthes cucumerina Linn.) is an annual climbing vine of the genus Trichosanthes in the Cucurbitaceae family. Because of its elongated, twisted fruit and soft, sweet, and sour taste, it is often called snake melon, snake tomato, snake gourd, or long tomato in many countries. It is primarily distributed in China, India, Pakistan, Sri Lanka, Nepal, and Bangladesh. As an important cultivated economic crop, Trichosanthes cucumerina has enormous potential for development and utilization. Its value system is diverse, encompassing edible, medicinal, and chemical uses. As a key specialty vegetable in Bangladesh and some African countries, it effectively fills the market demand during the vegetable off-season in late winter and early summer. Breeding, improving, and promoting Trichosanthes cucumerina Linn. can alleviate the tight supply and demand situation to a certain extent.

[0004] The ripe fruit of Trichosanthes kirilowii is often consumed as a vegetable. This is partly due to its rich content of essential nutrients, including vitamins, dietary fiber, and minerals, making it a viable alternative to milk and other expensive nutritional products. Furthermore, its unique taste and characteristics, such as its sweet flavor, deep red flesh when fully ripe, and resistance to sourness when heated, make it a popular alternative to tomatoes (Lycopersiconesculentum Mill.) from the Solanaceae family when prices are high or production is scarce. These qualities make Trichosanthes kirilowii fruit a popular alternative for stews and sauces. Studies have shown that Trichosanthes kirilowii pulp contains ascorbic acid, lycopene, phenols, and flavonoids, and its antioxidant capacity is comparable to that of tomatoes and even surpasses that of other edible vegetables in the Cucurbitaceae family. Furthermore, Bamidele et al. blended Trichosanthes kirilowii juice with pineapple juice, resulting in a juice blend with excellent color, flavor, and acceptability, suggesting that Trichosanthes kirilowii has potential for development in the fruit-flavored beverage market. In addition to its edible value, Trichosanthes kirilowii also possesses valuable medicinal properties, holding a prominent place in the Ayurvedic and Siddha systems of Indian medicine, as well as in Sri Lankan traditional medicine. The entire plant is a treasure trove, with its roots, leaves, fruit, and seeds all used as medicine. Modern medical research indicates that the fruit possesses anti-inflammatory, anti-tumor, anti-cancer, and hepatoprotective properties, and is used to treat conditions such as hypertension, heart disease, rheumatism, and psoriasis. Its roots have laxative, abortifacient, anthelmintic, diuretic, and emetic properties, and are used to treat constipation and skin edema. The leaves can be used to reduce fever or relieve spasms. Furthermore, research by Sandhya et al. has confirmed that an aqueous extract of Trichosanthes kirilowii leaves is an extremely effective natural hair growth promoter. Lectins can be extracted from its seeds, which are used for cell-cell adhesion reactions. Notably, in addition to its medicinal uses, the byproducts of its seed processing can be used as a biofuel feedstock, offering novel solutions for global energy sustainability and carbon emission reduction through biomass energy conversion technologies.

[0005] However, due to key challenges such as a scarcity of wild resources, a narrow genetic base, weak stress tolerance, and seasonal supply disruptions, Trichosanthes kirilowii has long received little attention from mainstream agriculture. However, systematic research on the agroecosystem services provided by Trichosanthes kirilowii, demonstrating its agricultural risk-buffering effects in enhancing farmland biodiversity and reducing the vulnerability of monoculture systems, has led to its inclusion in the global agricultural biodiversity conservation agenda.

[0006] Plant polyploidization, as an important driving force for germplasm innovation, plays a key role in speciation and the evolution of cultivated crops. Plant polyploidization can be achieved through artificial induction. Its core inducer, colchicine, specifically binds to tubulin dimers, blocking the polymerization of spindle microtubules, leading to the arrest of metaphase chromosomes and nuclear recombination, ultimately forming polyploids. Polyploidized plants are often sterile and are an important way to produce seedless varieties, such as seedless grapes (Vitis vinifera L.) and seedless citrus (Citrus reticulata Blanco). In addition, a large number of studies have shown that after polyploidization, plants generally exhibit increased biological expression, enlargement of some organs, increased content of nutrients and secondary metabolites, and enhanced resistance to biotic and abiotic stresses. Although ploidy breeding has been successfully practiced in some species of the Cucurbitaceae family, Yashiro et al. successfully obtained doubled haploid plants by treating melon (Cucumis melo Linn.) haploids with colchicine; Hassan et al. obtained tetraploid plants from Trichosanthes dioica Roxb. seeds by the colchicine soaking method; Demirel et al. successfully obtained tetraploid plants from cucumber (Cucumis sativus L.) stem segments by in vitro induction with colchicine; however, the mainstream research on Trichosanthes cucurbitae is still focused on in vitro culture, active ingredients, pharmacological properties, etc., and no research related to ploidy breeding has been reported.

[0007] The inventors of this application innovatively used chromosome ploidy regulation as the core breakthrough point and overcame the problems of the scarcity of wild resources of Trichosanthes kirilowii through homologous polyploidy induction technology.

[0008] In the prior art, colchicine is used to induce tetraploids in Trichosanthes kirilowii by inducing the terminal bud and base of the cotyledon petiole of Trichosanthes kirilowii to generate tetraploids. The selected tissues are young and tender, have high cell division and regeneration capabilities, and are suitable for induction. Although the induction efficiency can reach 80%, a single plant has only one terminal bud, and the seed propagation polyploid expansion cycle is long, and the reproduction coefficient is low, only about 3 to 4. Summary of the Invention

[0009] The purpose of the present invention is to provide an in vitro induction method for autotetraploids of Trichosanthes kirilowii, in view of the current deficiencies such as the scarcity of wild resources, narrow genetic basis and weak stress resistance of Trichosanthes kirilowii. This study combines plant tissue culture with chemical mutagenesis technology, uses noded stem segments as materials, adopts colchicine solution immersion combined with oscillation treatment as an induction method, in order to obtain autotetraploid plants and carry out in vitro rapid propagation, aiming to create new germplasm with complex traits such as seedlessness, high yield and stress resistance.

[0010] The technical solutions of the present invention are as follows:

[0011] In one aspect, the present invention provides an in vitro method for inducing autotetraploids of Trichosanthes kirilowii, comprising the following steps:

[0012] Step (1): Explant acquisition: sterile seedlings are selected as induction raw materials, and the noded stem segments of the sterile seedlings are used as explants;

[0013] Step (2): Autotetraploid induction: The explants are placed in a liquid proliferation medium without adding agar for induction. After obvious regenerated buds appear at the nodes, they are transferred to a pre-sterilized colchicine solution with a concentration of 0.05%-0.10% for shaking and soaking for 24-108 hours. After the treatment time is reached, the node-bearing stem segments with regenerated buds are removed and rinsed with sterile water. Subsequently, the cleaned sterile node-bearing stem segments are inoculated into the first rooting medium for culture to induce the autotetraploids of Trichosanthes kirilowii.

[0014] According to a preferred embodiment, the concentration of the colchicine solution is 0.05%, and the treatment time is 72 hours.

[0015] According to a preferred embodiment, the node stem segments in step (1) are taken from the same sterile seedling. Affected by certain conditions (such as mutation, somatic variation, dedifferentiation, etc.), the genotype of the plant tissue culture may change, thereby affecting the polyploid induction efficiency. In order to reduce the influence of possible genotype differences on the polyploid induction effect, the induction materials (node ​​stem segments) used in the present invention are all from the same diploid sterile seedling, and subsequent experiments are carried out after reaching a certain number through 4 subcultures, thereby ensuring the consistency of the genotype of the induction materials to the greatest extent possible.

[0016] According to a preferred embodiment, the sterile seedlings in step (1) can be obtained as follows: a selected diploid test tube seedling is cut into a stem segment with at least one node (about 3 cm long) with scissors, which is inoculated into a proliferation culture medium, and sterile seedlings are obtained after four subcultures.

[0017] According to a preferred embodiment, the formula of the proliferation culture medium in step (2) is: MS + 2.5 mg·L -1 -3.5mg·L -1 6-BA+1.5mg·L -1 -2.5mg·L -1 KIN + 0.4mg·L -1 -0.6mg·L -1 NAA+360mg·L -1 -360.5mg·L -1 Ca 2+ .

[0018] According to a preferred embodiment, the formula of the proliferation culture medium in step (2) is: MS + 3.0 mg·L -1 6-BA+2.0mg·L -1 KIN + 0.5mg·L -1 NAA+360.36mg·L -1 Ca 2+ .

[0019] According to a preferred embodiment, the formula of the first rooting medium in step (2) is: MS + 0.4 mg·L -1 -0.6mg·L -1 NAA.

[0020] According to a preferred embodiment, the formula of the first rooting medium in step (2) is: MS + 0.5 mg·L -1 NAA.

[0021] According to a preferred embodiment, the length of the explant in step (2) is about 2 cm.

[0022] According to a preferred embodiment, the following steps are also included:

[0023] Step (3): Propagation of autotetraploids: The autotetraploid plants obtained in step (2) are transferred to a proliferation medium for continuous subculture, and then sterile node-bearing stem segments after subculture are taken for bud induction in a tetraploid bud induction medium and rooting induction in a second rooting medium;

[0024] Step (4): domestication and transplantation of autotetraploids: domestication, hardening and transplantation of the rooted autotetraploid plants to obtain the autotetraploids of Trichosanthes kirilowii.

[0025] According to a preferred embodiment, the formula of the tetraploid bud induction medium in step (3) is: MS + 360.36 mg·L -1 Ca 2+ +1mg·L -1 ~3mg·L -1 6-BA+1mg·L -1 ~3mg·L -1 KIN + 0.5mg·L -1 ~2mg·L -1 NAA.

[0026] According to a preferred embodiment, the formula of the tetraploid bud induction medium in step (3) is: MS + 360.36 mg·L -1 Ca 2+ +3mg·L -1 6-BA+3mg·L-1 KIN + 1.0 mg L -1 NAA.

[0027] According to a preferred embodiment, the formula of the second rooting medium in step (3) is: 1 / 2~1MS+0mg·L -1 ~1.0mg·L -1 NAA.

[0028] According to a preferred embodiment, the formula of the second rooting medium in step (3) is: MS + 1.0 mg·L -1 NAA.

[0029] Compared with the existing technology, the beneficial effects of the present invention are:

[0030] 1. A method for inducing autotetraploids of Trichosanthes kirilowii in vitro, which successfully achieves polyploid induction in vitro. Nodular stem segments are selected as inducing explants, and the method has a high proliferation coefficient and induction rate, with the proliferation coefficient reaching 6.43 and the tetraploid induction rate reaching 31.33%. Compared with existing methods for obtaining polyploids (seed or living plant stem tip immersion method), the method has a shorter time period, higher propagation efficiency and proliferation coefficient, reduces the occurrence of chimeras, and ensures the year-round reproduction and preservation of polyploid materials. While meeting the needs of large-scale production, it also maintains the excellent traits of polyploids, making it the most effective proliferation method for artificial rapid propagation and improving seedling quality.

[0031] 2. An in vitro method for inducing autotetraploids in Trichosanthes kirilowii, which optimizes the cluster bud induction medium and rooting medium to meet the different growth requirements of polyploid and diploid plants, improves the regeneration ability of tetraploid plants and increases the proliferation coefficient of tetraploid plants compared to the original diploid medium;

[0032] 3. A method for inducing autotetraploids of Trichosanthes kirilowii in vitro was developed. The results showed that the successfully induced autotetraploids had a superior transplant survival rate compared to diploids. The results not only significantly increased the vegetative organs (leaves, stems, roots), growth parameters (leaf width, stomatal length, stomatal width, root fresh and dry weight), and cell structure (palisade tissue, spongy tissue, vascular tissue, starch grains, parenchyma cells, and cork cells), but also improved the transplant survival rate of tissue culture seedlings and overcame the problem of stem tip necrosis that occurs during diploid rapid propagation, representing significant progress.

[0033] 4. A method for in vitro induction of autotetraploids of Trichosanthes kirilowii, which uses tissue culture technology to achieve in vitro induction and in vitro propagation in a culture room, allowing year-round production and avoiding seasonal restrictions. At the same time, it greatly increases the number of induced plants, improves economic benefits, shortens the culture cycle, and has considerable benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Comparison of flow cytometry, root tip chromosome number, and leaf morphology between diploid and autotetraploid Trichosanthes kirilowii. Figure: A: Flow cytometry histogram of a diploid plant; B: Flow cytometry histogram of an autotetraploid plant; C, D: Somatic chromosome number in root tips of diploid plants, 2n=2x=22; E, F: Somatic chromosome number in root tips of autotetraploid plants, 2n=4x=44; G: Leaf morphology of diploid, mosaic, and autotetraploid plants.

[0035] Figure 2 Figure 1 shows the bud cluster and rooting induction of autotetraploid Trichosanthes kirilowii. Figures A and D show the bud cluster induction of autotetraploid stem segments with nodes of Trichosanthes kirilowii. Specifically, A: cultured for 10 days; B: cultured for 20 days; C: cultured for 30 days; and D: cultured for 40 days. Figures E and J show the rooting induction of autotetraploid stem segments with nodes of Trichosanthes kirilowii. Specifically, E and F: 0.1 mg·L was added to 1 / 2 MS. -1 G, H: cultured in MS medium for 40 days; I, J: cultured in MS medium supplemented with 1.0 mg·L -1 Cultured in NAA medium for 40 days;

[0036] Figure 3 Comparison of morphology and growth parameters of diploid and autotetraploid plants of Trichosanthes kirilowii; A: diploid plant 30 days after transplantation; B: autotetraploid plant 30 days after transplantation; C: diploid plant 90 days after transplantation; D: autotetraploid plant 90 days after transplantation; E: stem tip of diploid plant 90 days after transplantation; F: stem tip of autotetraploid plant 90 days after transplantation; G: leaf of diploid plant 90 days after transplantation; H: leaf of autotetraploid plant 90 days after transplantation; I: root of diploid plant 90 days after transplantation; J: root of autotetraploid plant 90 days after transplantation; KH: growth parameters of diploid and autotetraploid plants 90 days after transplantation: specifically K: leaf length; L: leaf width; M: leaf shape ratio; N: stem diameter; O: root fresh weight; P: root dry weight;

[0037] Figure 4Microscopic observation of stomata, leaf cross sections, stem cross sections, and root powder of diploid and autotetraploid plants of Trichosanthes kirilowii. Figure A: Stomata on a diploid plant leaf; B: Stomata on an autotetraploid plant leaf; C: Comparison of guard cell lengths between diploid and autotetraploid plants; D: Comparison of guard cell widths between diploid and autotetraploid plants; E: Comparison of stomatal number between diploid and autotetraploid plants; F: Cross-sectional section and partial magnification of a diploid leaf; G : Cross-sectional slices and local magnifications of autotetraploid leaves; H: Cross-sectional slices and local magnifications of diploid stems; I: Cross-sectional slices and local magnifications of autotetraploid stems; KQ are microscopic observations of root powder; specifically, K: autotetraploid starch granules; L: diploid parenchyma cells; M: autotetraploid parenchyma cells; N: diploid reticulate vessels; O: autotetraploid reticulate vessels; P: diploid cork cells; Q: autotetraploid cork cells. DETAILED DESCRIPTION

[0038] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without the manufacturer specified are conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present invention can also be implemented without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are International Standard Units, and the numerical values ​​and numerical ranges appearing in this invention should be understood to include the inevitable systematic errors in industrial production.

[0040] Colchicine, sucrose, agar, NaOH, and plant growth regulators such as kinetin (KIN), 1-naphthaleneacetic acid (NAA), and 6-benzylpurine [N-(phenylmethyl)-9Hpurin-6-amine, 6-BA] were purchased from Beijing Dingguo Biotechnology Co., Ltd. (Beijing, China). All chemical reagents were of analytical grade.

[0041] The culture medium was supplemented with 3% (w / v) sucrose and 0.47% (w / v) agar, and the pH was adjusted to 5.6-5.8 with 1N NaOH before sterilization. The culture medium was then dispensed into glass culture bottles (13 cm high × 9.5 cm in diameter, each containing 50 mL of culture medium) and sterilized in a high-pressure steam sterilizer (sterilization pressure: 1.06 kg / cm 2 Sterilize the colchicine solution under the same sterilization conditions as the culture medium. The incubation room temperature was 25 ± 1°C, the light intensity was 1500-1800 lux, and the photoperiod was 12 hours. Concentrations not otherwise specified are by mass.

[0042] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0043] Example 1 Method for inducing autotetraploids of Trichosanthes kirilowii in vitro

[0044] 1 Materials and Methods

[0045] 1.1 Material sources and culture conditions

[0046] The experimental materials were internoded stem segments. The in vitro rapid propagation system of Trichosanthes kirilowii diploid test tube seedlings has been successfully established at the Yunnan Provincial Endangered Chinese Medicinal Materials Propagation and Cultivation Engineering Technology Research Center. The proliferation and first rooting culture media were as follows: the proliferation culture medium was MS + 3.0 mg·L -1 6-BA+2.0mg·L -1 KIN + 0.5mg·L -1 NAA+360.36mg·L -1 Ca 2+ ; The first rooting medium is MS + 0.5mg·L -1 NAA. The sterile stem segments with nodes used were all from the same mother plant. The specific operation was as follows: On a clean bench, a selected diploid test tube plantlet was cut into stem segments with at least one node (about 3 cm long) with scissors. These segments were then inoculated into a proliferation medium. After four subcultures (a subculture cycle of 45 days), the sterile seedlings obtained were used as the autotetraploid induction material.

[0047] 1.2 Autotetraploid induction of Trichosanthes kirilowii

[0048] The sterile diploid seedlings obtained after subculture in Section 1.1 were cut into single-node stem segments (approximately 2 cm long) and transferred to liquid proliferation medium without agar. After visible regeneration buds appeared at the nodes (approximately 7 days), they were transferred to a pre-sterilized colchicine solution for shaking and soaking. Colchicine solutions were set at different concentrations (0.05% and 0.10%, w / v) and treated for 24, 48, 72, 84, and 108 hours. After the treatment period, the seedlings were removed and rinsed three times with sterile water for at least 3 minutes each time. The cleaned sterile, node-bearing stem segments were then inoculated into the first rooting medium. Ten bottles were inoculated per group, with five seedlings per bottle. After 30 days of incubation, the number of surviving seedlings in each group was counted. Each treatment group was replicated three times.

[0049] 1.3 Flow cytometry and root tip chromosome counting

[0050] Flow cytometry (FCM) was performed according to the study of Ren et al. (Ren et al. 2024). Fresh leaf tissue (0.5 cm) of the test plant (colchicine-treated plant) and the reference plant (rice) was collected. 2 ) were mixed in a 1:1 ratio and minced. The mixture was allowed to stand for 10 min in 1 mL of nuclear extraction buffer (pH 7.0) consisting of 45 mM MgCl2·6H2O, 20 mM MOPS, 30 mM sodium citrate, 1% (w / v) PVP, 0.2% (v / v) TritonX-100, and 10 mM Na2EDTA, and then filtered through a 30 μm nylon mesh to obtain a cell nuclear suspension. Subsequently, 500 μL of propidium iodide (PI, 50 mg·mL) was added to the cell nuclear suspension. -1 ) solution and placed on ice in the dark for 0.5-1 hour. Finally, the cell nuclei suspension was analyzed using a flow cytometer (BD FACScalibur), using a diploid Trichosanthes cucurbitae as a control. 10,000 nuclei were collected for each assay, and the coefficient of variation (CV) was kept within 5%.

[0051] Root tips of autotetraploids (0.5-1.0 cm in length, as determined by flow cytometry) were excised and immersed in a 0.05% (w / v) colchicine solution in the dark at 4°C for 5 h. Subsequently, they were fixed with Carnoy's fixative (acetic acid:anhydrous ethanol, 1:3, prepared freshly) for 8 h. Following fixation, the root tips were washed three times with 70% (v / v) ethanol, transferred to a 1N HCl solution, and dissociated in a 60°C waterbath for 8 min. The root tips were removed, washed four times with distilled water, and then softened in a 45% (v / v) acetic acid solution for 10 min. The softened root tips were placed on a glass slide, crushed, and stained with one drop of Carbofuchsin for 10 min. Subsequently, a coverslip was applied and tapped gently with a pencil to spread the root tip cells into a thin layer. Finally, the slides were examined under an optical microscope (Olympus BH-2, 100× oil immersion lens). Cells with good staining and evenly dispersed chromosomes were photographed and the chromosome number recorded. Using the diploid chromosome number of Trichosanthes kirilowii as a control, the ploidy level of the plants was further confirmed, and the number of mixed ploids, the number of polyploids, the mixed ploidy rate, and the polyploidy induction rate were calculated. The 145 autotetraploid plants obtained were serially numbered: T-1, T-2, ..., T-145.

[0052] When flow cytometry and root tip chromosome counting were performed, 10 diploid rooted seedlings were randomly selected for testing, and no chromosome number variation was found.

[0053] 1.4 Autotetraploid bud clustering and rooting induction of Trichosanthes kirilowii

[0054] (1) The sterile stem segments with nodes obtained by transferring the autotetraploid T-1 to the proliferation medium for four consecutive subcultures were used as materials for the orthogonal experiment of bud induction and rooting experiment. -1 Ca 2+ As the basic culture medium, on this basis, the auxin and cytokinin were recombined to carry out L9(3 4 )Orthogonal experimental design (Table 1).

[0055] Table 1 Autotetraploid plant regeneration L9(3 4 ) Orthogonal experimental design

[0056]

[0057]

[0058] After 45 days of culture, the proliferation coefficient of each experimental group was calculated and the experimental data were analyzed to screen out the suitable culture medium for inducing cluster shoots of autotetraploids of Trichosanthes kirilowii. Each treatment group was inoculated with 10 bottles, each containing 10 materials.

[0059] (2) The autotetraploid T-1 stem segment explants were inoculated with MS+1.0 mg·L -1 NAA, 1 / 2MS + 0.1mg·L -1 Rooting was induced in NAA and MS medium without any plant growth regulators. Ten bottles were inoculated into each treatment group, each containing 10 materials. After 40 days of incubation, the rooting status of each treatment group was observed and recorded.

[0060] The bud induction and rooting experiments were repeated three times.

[0061] 1.5 Acclimation and transplantation of diploid and autotetraploid Trichosanthes kirilowii

[0062] Seventy-five diploid and tetraploid internoded stem segments were simultaneously inoculated into a diploid-appropriate rooting medium and a second rooting medium to induce rooting. Roots were acclimated when they reached approximately 3-4 cm in length. Rooted seedlings were first placed indoors for 5 days and then moved outdoors for 10 days for hardening, with the caps of the glass culture bottles remaining open. After hardening, the rooted seedlings were removed, the rooting medium rinsed under tap water, and then soaked in a 0.1% (w / v) carbendazim solution for 5 minutes. The seedlings were then transplanted into pots (10.0 cm diameter, 30.0 cm height) filled with sterilized soil, watered thoroughly, and placed in a greenhouse (temperature range: 20-28°C, humidity level: 40%-60%, light intensity: 2400 lx). Seedling survival rates were determined after 30 days. The soil matrix is ​​made by mixing organic fertilizer, sandy soil and humus soil in a ratio of 1:4:2 (v / v).

[0063] 1.6 Morphological and microscopic observations of diploid and autotetraploid Trichosanthes kirilowii

[0064] Three months after transplanting, 50 diploid and tetraploid plants (25 each) were measured for multiple phenotypic traits, including leaf length, leaf width, stem diameter, leaf shape index (LSI), and root fresh and dry weight. Leaf length and width were measured using a stainless steel ruler, with the third leaf from the plant base as the target. Stem diameter was measured 10 cm from the plant base using an electronic digital caliper (Fowler & NSK Max-Cal, China). For stomatal analysis, the third fully expanded leaf from the top of six diploid and tetraploid plants (3 each) was decolorized in Carnoy's fixative for 6 h, then rinsed in distilled water for 3 min, mounted on a glass slide with the lower epidermis facing up, flattened with a coverslip, and observed under an Olympus BH-2 microscope. Six randomly selected microscopic fields were captured on each leaf to determine the number of stomata; within each microscopic field, the length and width of six randomly selected stomata were recorded.

[0065] Paraffin cross-sections were prepared from the middle region of the fourth fully expanded leaf at the stem apex and from the stem 10 cm from the plant base, and cellular characteristics were observed under an optical microscope. Paraffin sections were prepared using the paraffin embedding method. Four samples were analyzed for each ploidy level, and five sections were randomly selected from each sample for observation. Powder micrographs were prepared as follows: roots of diploid and tetraploid plants were shade-dried and then pulverized. A small amount of powder (approximately 0.1 g) of each ploidy level was placed on a glass slide. 1-2 drops of chloral hydrate solution were added and heated over an alcohol lamp for 10 seconds. One drop of dilute glycerol was then added to fix the sample. The sample was covered with a coverslip and the powder was observed under an optical microscope for microscopic characteristics. For starch granules, only 1-2 drops of distilled water were added without heating. The sample was covered with a coverslip and directly observed under an optical microscope.

[0066] 1.7 Data processing and statistical indicator calculation

[0067] The collected data were processed and analyzed using SPSS 26.0 (IBM Corporation, Armonk, USA) and Excel software (MC Corporation, Redmond, USA). One-way analysis of variance (ANOVA) and Duncan's multiple comparison test were used to determine whether the means between different treatments were statistically significant, and the significance level was set at P ≤ 0.05. The results are expressed as mean ± standard error (SE). Images were produced using Adobe Photoshop (version 2021) and Origin (version 2021) software. All relevant experiments were repeated three times.

[0068] The calculation formulas for various statistical indicators are as follows:

[0069] Survival number = total treatment number - (death number + contamination number);

[0070] Survival rate = number of survivors / total number of treatments;

[0071] Mixed ploidy rate = number of mixed ploids / number of survivors;

[0072] Polyploidy rate = number of autotetraploids / number of survivors;

[0073] Multiplication coefficient = number of effective transferred materials / number of initial materials;

[0074] Transplant survival rate = number of transplanted surviving plants / total number of transplanted plants;

[0075] Leaf shape index (LSI) = leaf length / leaf width.

[0076] 2 Results and Analysis

[0077] 2.1 Evaluation of the effect of colchicine inducing autotetraploidy

[0078] Flow cytometry (FCM) was used to analyze 828 rooted seedlings that survived colchicine treatment, and a total of 145 autotetraploids and 208 chimeras were obtained. The flow cytometric analysis histograms and somatic cell chromosome count results of representative samples are shown in Figure 2. Figure 1 As shown in the figure, the horizontal axis value represents the fluorescence channel value, and the vertical axis value represents the relative value of the measured cell number; the left peak represents the internal reference plant rice (Oryza sativa L.), and the right peak represents the sample plant. The peak of the diploid plant appears at channel 50 ( Figure 1 A), while the peak of tetraploid plants appears at channel 100 ( Figure 1 B), the peak value of tetraploid plants is about twice that of diploid plants. Further somatic cell chromosome counting results show that the chromosome number of diploid plants is 2n=2x=22( Figure 1 C, D), the chromosome number of autotetraploid plants is 2n=4x=44( Figure 1 E, F). The FCM analysis results were consistent with the somatic cell chromosome count results. In addition, compared with diploids, the leaf morphology of chimeras and tetraploids was significantly different ( Figure 1 G) Chimeras have deformed leaves with malformations, notches, and wrinkles; tetraploid leaves, on the other hand, have normal morphology but are wider than diploid leaves.

[0079] Table 2 Effects of colchicine concentration and treatment time on autotetraploid induction in Trichosanthes kirilowii

[0080]

[0081] The data are expressed as mean ± standard error (SE), and different letters in the table indicate significant differences at the 0.05 level.

[0082] Table 2 above shows the effects of colchicine concentration and treatment time on the ploidy level of stem segments. The results showed that compared with the control group, colchicine concentration and treatment time had a significant effect on the survival rate of stem segments and the induction rate of autotetraploidy. The survival rate gradually decreased with the increase of treatment time and colchicine concentration. When the colchicine concentration was 0.05% and the treatment time was 24h, the survival rate reached 98%, which was no significant change compared with the control group; when the colchicine concentration was increased to 0.1% and the treatment time was extended to 108h, the survival rate dropped to the lowest (5.33%). The chimera rate and the induction rate of autotetraploidy first increased and then decreased with the increase of treatment time and colchicine concentration. When the treatment time was 84h, the chimera rates of the two concentrations (0.05% and 0.1%) dropped to the lowest, at 5.33% and 4.00%, respectively. Comparison of plant survival rate, chimera rate, and autotetraploid induction rate at different colchicine concentrations showed that 0.05% colchicine had a superior overall induction effect compared to 0.1%. Furthermore, the autotetraploid induction rate of regenerated plants reached a maximum of 31.33% when treated for 72 hours. In summary, soaking stem segments in a 0.05% colchicine solution for 72 hours is the optimal treatment for autotetraploid induction in Trichosanthes kirilowii.

[0083] 2.2 Establishment of an efficient and stable autotetraploid regeneration system in Trichosanthes kirilowii

[0084] From Table 3 below, we can see that R A >R B >R C >R D , indicating that 6-BA, KIN and NAA all have reliable effects on the induction of tetraploid buds of Trichosanthes kirilowii, and the order of the effect size is 6-BA>KIN>NAA.

[0085] Table 3 Autotetraploid bud induction of Trichosanthes kirilowii L9(3 4 )Orthogonal experimental results

[0086]

[0087] K represents the mean; R represents the range; the numbers in the proliferation coefficient column represent the mean ± standard error.

[0088] According to the results of variance analysis (Table 4), KIN and NAA had no significant effect on bud induction (P>0.05), while 6-BA had a significant effect (P<0.05).

[0089] Table 4 Results of variance analysis of proliferation coefficient

[0090]

[0091]

[0092] Further Duncan test results (Table 5) showed that level 3 (3.0 mg·L -1 ) had the best bud induction effect, followed by level 2 (2.0 mg·L -1 ) and level 1 (1.0 mg·L -1 ).

[0093] Table 56-BA mass concentration Duncan test

[0094]

[0095] Different lowercase letters in the table indicate significant effects at the 5% level.

[0096] Comparing and analyzing the average values ​​of each group, it was found that the best plant growth regulator combination for stem bud induction was A3B3C2, i.e. 3.0 mg·L -1 6-BA+3.0mg·L -1 KIN + 1.0 mg L -1 After analyzing and integrating the results of the orthogonal experiment, the final composition of the culture medium for inducing cluster buds of the tetraploid stem segments of Trichosanthes kirilowii was determined to be: MS + 3.0 mg·L -1 6-BA+3.0mg·L -1 KIN + 1.0 mg L -1 NAA, the culture proliferation coefficient under this formula can reach 6.43.

[0097] The node stem segments were inoculated into the culture medium of the best plant growth regulator combination for bud induction and cultured for 10 days. Axillary buds began to germinate ( Figure 2 A). After 20 days of cultivation, a large number of new shoots regenerated from the stem nodes and new roots appeared at the base ( Figure 2 B); After 30 days of cultivation, the plants were strong, the regenerated buds further elongated, and the leaves expanded ( Figure 2 C). After 40 days of cultivation, the plants grew rapidly, with full leaves, strong stems and developed root systems. The proliferation coefficient could reach 6.43 ( Figure 2 D).

[0098] The rooting results of the node stem segments showed that there were significant differences in the rooting performance of the three second rooting media. -1 In NAA culture medium, after 40 days of culture, the buds at the base of the stems grew slowly, the base of the stems turned yellow-brown, and the rooting rate was extremely low ( Figure 2 E, F). In MS medium without any plant growth regulators, the buds at the base of the stems can grow normally, but the regenerated roots show an abnormal morphology of pale white, swollen, and spongy ( Figure 2 G, H). When the culture medium was MS supplemented with 1.0 mg·L -1Under NAA, the basal buds have strong growth potential, the regenerated roots are light yellow-white, short and thick in shape, the rooting rate is 100% and the number of roots is large ( Figure 2 I, J). In summary, the culture medium suitable for rooting of autotetraploid stem segments of Trichosanthes kirilowii is MS supplemented with 1.0 mg·L -1 NAA.

[0099] 2.3 Acclimation, transplantation, and morphological comparison of Trichosanthes kirilowii diploids and their autotetraploids

[0100] Under field conditions, a total of 300 pots of Trichosanthes kirilowii were transplanted, of which 150 were diploid and 150 were tetraploid. Three months after transplanting, the survival rate of tetraploid plants was 92% (138 plants), while the survival rate of diploid plants dropped to 84.67% (127 plants). Figure 3 ), there are obvious morphological differences between tetraploid plants and diploid plants. Preliminary observations found that 30 days after transplanting, the growth rate of diploid plants was significantly faster than that of tetraploid plants ( Figure 3 A and B). As the transplanting time extended to 90 days, the tetraploid plants showed significant growth advantages, with an average plant height of 105 cm, significantly higher than that of the diploid plants (93 cm), and a more compact plant shape ( Figure 3 C and D). More detailed magnified images of the stem tip and leaves show that the leaves of the diploid plant are dark yellow-green, while the leaves of the tetraploid plant are dark green and significantly larger than those of the diploid plant ( Figure 3 E, F, G and H). In addition, in the comparison of roots, tetraploid plants have thicker taproots and more developed fibrous roots, which are significantly better than diploid plants ( Figure 3 I and J). It is speculated that the poor morphological characteristics of tetraploids in the early stages of development may be due to the longer cell cycle, slower cell division, and greater material and energy consumption associated with high ploidy levels. However, as growth progresses, the tetraploid "giant effect" gradually emerges. Enlarged leaves and roots increase photosynthetic efficiency and nutrient accumulation, thus compensating for these early growth disadvantages.

[0101] There were also significant differences between diploid and tetraploid plants in terms of growth parameters (Table 6, Figure 3 Compared with diploids, tetraploid plants had greater leaf width (81.29 mm), thicker stems (7.73 mm), and higher root fresh weight (18.2 g) and root dry weight (12.6 g), but no significant differences were found in leaf length and leaf shape index.

[0102] Table 6 Growth parameters of Trichosanthes kirilowii diploid and autotetraploid plants

[0103]

[0104] 2.4 Microscopic comparison of diploid and autotetraploid Trichosanthes kirilowii

[0105] The results of microscopic observation of leaves showed that ( Figure 4 ), the stomatal morphology of the leaf epidermal cells of diploid and tetraploid plants is not much different, both are elliptical or nearly circular. However, the stomatal pores of tetraploid plants are significantly larger, and the stomatal density per unit area is significantly reduced ( Figure 4 A and B). In addition, guard cell size measurements ( Figure 4 Figures C, D, and E show that the average length (48.00±3.5μm) and width (13.40±2.1μm) of tetraploid guard cells are larger than those of diploids (32.00±2.3μm and 8.90±1.5μm, respectively). The opposite is true for the number of stomata: in the same field of view, the number of stomata in tetraploids is significantly lower than that in diploids (the average number of stomata in diploids is 22.60±1.5, while the average number of stomata in tetraploids is 14.3±2.1).

[0106] The cross-section anatomy of the leaf shows that ( Figure 4 F and G), which is composed of the upper epidermis, palisade tissue, spongy tissue and lower epidermis, and the cell thickness of each tissue (upper epidermis, palisade tissue, veins, spongy tissue and lower epidermis) of tetraploid leaves is significantly greater than that of diploid leaves. Figure 4 H and I) showed that the stem is composed of epidermis, cortex and vascular tissue. There was no significant difference in the thickness of the stem epidermis between the two ploidy plants, and the main difference came from the cortex and vascular tissue. In the comparison of the thickness of the stem cortex tissue, the diploid plant was significantly larger than the tetraploid plant; the vascular tissue of the tetraploid plant was more developed than that of the diploid plant. Microscopic examination of the root powder of the two ploidy plants found that the root powder contained starch granules (including single granules and compound granules, Figure 4 J and K), parenchyma cells ( Figure 4 L and M), reticulated duct ( Figure 4 N and O) and cork cells ( Figure 4 There were no significant differences in starch granules between diploid and tetraploid root powders, but the parenchyma cells of tetraploids were larger, the texture of the reticulated vessels was tighter, and the cork cells were larger with significantly thicker cell walls.

[0107] It is worth noting that in the present application, stem tip necrosis was found in the in vitro rapid propagation study of Trichosanthes kirilowii diploids; however, this phenomenon was not observed in its autotetraploids. It is speculated that this difference may be related to the increase in ploidy level.

[0108] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. A method for inducing autotetraploids of Trichosanthes kirilowii in vitro, characterized in that: The steps include: Step (1): Explant acquisition: sterile seedlings are selected as induction raw materials, and the noded stem segments of the sterile seedlings are used as explants; Step (2): Autotetraploid induction: The explants are placed in a liquid proliferation medium without adding agar for induction. After obvious regenerated buds appear at the nodes, they are transferred to a pre-sterilized colchicine solution with a concentration of 0.05%-0.10% for shaking and soaking for 24-108 hours. After the treatment time is reached, the node-bearing stem segments with regenerated buds are removed and rinsed with sterile water. Subsequently, the cleaned sterile node-bearing stem segments are inoculated into the first rooting medium for culture to induce the autotetraploids of Trichosanthes kirilowii.

2. The in vitro induction method for autotetraploids of Trichosanthes kirilowii according to claim 1, characterized in that: The concentration of the colchicine solution is 0.05%, and the treatment time is 72 hours.

3. The in vitro induction method for autotetraploids of Trichosanthes kirilowii according to claim 1, characterized in that: The noded stem segments in step (1) are taken from the same sterile seedling.

4. The in vitro induction method for autotetraploids of Trichosanthes kirilowii according to claim 1, characterized in that: The formula of the proliferation culture medium in step (2) is: MS + 2.5 mg·L -1 -3.5mg·L -1 6-BA+1.5mg·L -1 -2.5mg·L - 1 KIN + 0.4mg·L -1 -0.6mg·L -1 NAA+360mg·L -1 -360.5mg·L -1 Ca 2+ .

5. The in vitro induction method for autotetraploids of Trichosanthes kirilowii according to claim 1, characterized in that: The formula of the first rooting medium in step (2) is: MS + 0.4 mg·L -1 -0.6mg·L -1 NAA.

6. The in vitro induction method for autotetraploids of Trichosanthes kirilowii according to claim 1, characterized in that: The following steps are also included: Step (3): Propagation of autotetraploids: The autotetraploid plants obtained in step (2) are transferred to a proliferation medium for continuous subculture, and then the sterile nodular stem segments after subculture are taken for bud induction in a bud induction medium and for root induction in a second rooting medium; Step (4): domestication and transplantation of autotetraploids: domestication, hardening and transplantation of the rooted autotetraploid plants to obtain the autotetraploids of Trichosanthes kirilowii.

7. The in vitro induction method for autotetraploids of Trichosanthes kirilowii according to claim 6, characterized in that: The formula of the bud induction medium in step (3) is: MS + 360.36 mg·L -1 Ca 2+ +1mg·L -1 ~3mg·L -1 6-BA+1mg·L -1 ~3mg·L -1 KIN + 0.5mg·L -1 ~2mg·L -1 NAA.

8. The in vitro induction method for autotetraploids of Trichosanthes kirilowii according to claim 7, characterized in that: The formula of the bud induction medium in step (3) is: MS + 360.36 mg·L -1 Ca 2+ +3mg·L -1 6-BA+3mg·L -1 KIN + 1.0 mg L -1 NAA.

9. The in vitro induction method for autotetraploids of Trichosanthes kirilowii according to claim 6, characterized in that: The formula of the second rooting medium in step (3) is: 1 / 2~1MS+0.0~1.0mg·L -1 NAA.

10. The in vitro induction method of autotetraploids of Trichosanthes kirilowii according to claim 9, characterized in that: The formula of the second rooting medium in step (3) is: 1MS + 1.0mg·L -1 NAA.

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