Method for efficiently creating brassica rapa and diplotaxis intergeneric distant hybrid

Abstract

The application relates to a method for efficiently creating a far-cross hybrid between Chinese cabbage and the genus Eruca, which comprises the following steps: taking the selfing line 'ASTRO' of the genus Eruca as a male parent, wherein the selfing line 'ASTRO' has excellent properties such as drought and flood resistance, disease resistance, salt and alkali tolerance, and the like; taking an excellent Chinese cabbage material '72049' as a female parent; spraying magnesium chloride hexahydrate; and then performing intergeneric hybridization. After embryo rescue and colchicine doubling, a doubled material is obtained, and the doubled material is transplanted into a field. Through cytological identification, morphological observation and pollen fertility identification, the doubled material is finally determined as a true hybrid plant.

Description

Technical Field

[0001] This invention belongs to the field of plant distant hybridization breeding, specifically involving a method for creating new germplasm from distant hybridization between Chinese cabbage and Arugula. Background Technology

[0002] Chinese cabbage (Brassica rapa, AA, 2n=20) belongs to the cultivated variety group of Brassica rapa L. in the genus Brassica of the family Cruciferae. Chinese cabbage ranks among the top vegetables in my country in terms of planting area and yield, occupying a pivotal position in agricultural production. Its genetics and molecular biology research have always received high attention.

[0003] Arugula (Eruca sativa Mill, EE, 2n=22) is named for its strong sesame aroma and is an annual herbaceous plant belonging to the genus Eruca Mill in the Brassicaceae family. Arugula is an important oilseed crop in arid and semi-arid regions of the world. It can also be used as a condiment and medicinal plant. It has a strong ability to adapt to the environment and has excellent traits such as drought resistance, tolerance to poor soil, disease resistance, strong cold resistance, salt and alkali resistance, drought and waterlogging resistance, and aphid resistance (Guijarro-Real et al., Large scale phenotyping and molecular analysis in a germplasm collection of rocket salad (Eruca vesicaria) reveal adifferentiation of the gene pool by geographical origin, Euphaitica, 2020, 216(3):53).

[0004] To create new high-quality Chinese cabbage germplasm with excellent traits, the aroma and other genes from arugula were introduced into Chinese cabbage. Distant hybridization is the most important means to solve the scarcity of Chinese cabbage germplasm resources. However, there are many reproductive obstacles in the process of distant hybridization. Embryo rescue is an effective method to overcome the incompatibility of distant hybridization after fertilization, including ovule culture, ovary culture and embryo culture. Hu Dayou et al. (2008) found that the time of ovary in vitro culture has a great influence on embryo development. Taking samples too early or too late is not conducive to embryo development and it is difficult to obtain seeds. If the samples are taken too early, the embryos are still dependent on the parent plant and it is difficult to obtain nutrition after in vitro culture. If the samples are taken too late, the embryos will abort and cannot obtain seeds (Chen Shuzhong, Yin Jiaming, Tang Zhanglin et al., Preliminary study on distant hybridization between Brassica napus and Kale, Journal of Southwest Agricultural University, 2000, (03): 208-210).

[0005] There are relatively few studies on distant hybridization between arugula and Brassica oleracea (Brassica oleracea family). In recent years, only a few studies have been conducted on the hybridization of rapeseed and arugula. When hybridizing Brassica napus and arugula, it was found that pollen has difficulties in several processes, including adhering to the stigma, germinating and growing pollen tubes, and pollen tubes entering the stigma. This confirms that the main obstacle in distant hybridization of Brassica species is the pre-fertilization barrier. At the same time, incompatibility after fertilization hinders the development of hybrid embryos and prevents the formation of normal seeds (Zhu Yuan, Lin Liangbin, Zhang Chuanli, Study on distant hybridization between Brassica napus and arugula, Journal of Yunnan Agricultural University, 2007, (01): 26-29). When rapeseed-radish cytoplasmic male sterile line (Ogu CMS) is crossed with arugula, the pollen tubes of arugula are blocked from entering the papillary cells (Wei Qin, Pan Yufeng, Zhou Lijun, et al. Study on compatibility of hybridization of six oil crops of the Brassica tribe with rapeseed-radish cytoplasmic male sterile line, Southwest China Journal of Agricultural Sciences, 2001, (01): 38-42). In addition, it is generally believed that hybrids obtained from intergeneric hybridization of Arugula and Brassica are generally maternal, and the proportion of true hybrids is very small. Pei Bingxue (2015) found that there are serious reproductive obstacles and high incompatibility in distant hybridization between Brassica napus and arugula. The overall morphology of the hybrid plants is maternal, and all of them are sterile (Pei Bingxue, Study on distant hybridization between Brassica napus and arugula, Hubei University, 2015). Matsuzawa et al. (1999) obtained CMS material with arugula cytoplasm by hybridizing arugula with Chinese rapeseed (Matsuzawa Y, Mekiganen S, Kaneko Y, et al. Male sterility alloplasmic Brassica rapa carrying Erucasativa cyto-plasm, Plant Breeding, 1999, 118(1):82-84).

[0006] Therefore, there is a need to further create new hybrid germplasm of Chinese cabbage and arugula with superior traits. Summary of the Invention

[0007] The first aspect of this invention provides a method for efficiently creating new superior Chinese cabbage germplasm with excellent traits such as tolerance to poor soil, disease resistance, cold resistance, salt stress resistance, drought and flood resistance, and aphid resistance. Specifically, it provides a method for creating a distant hybrid between Chinese cabbage and Arugula.

[0008] The method involves intergeneric hybridization of Chinese cabbage and Arugula; the method uses Chinese cabbage as the female parent and Arugula as the male parent. Preferably, the Chinese cabbage material '72049' is used as the female parent and the Arugula inbred line 'ASTRO' is used as the male parent.

[0009] Specifically, during the budding stage, the stigmas of Chinese cabbage were sprayed with a MgCl2·6H2O solution with a concentration of 62.3 mg / L. After the solution dried, hybridization pollination was carried out by smearing the stigmas. Seven days later, the ovary was removed, the ovules were peeled off for embryo rescue, and after the regenerated seedlings were formed, the treatment was doubled.

[0010] Furthermore, the air-drying time is 15-20 minutes;

[0011] Furthermore, the embryo rescue includes removing the ovules from the ovary, placing them on a sterile solid inoculation medium, and culturing them in the dark at room temperature for 20 days, wherein the room temperature is 25°C; preferably, the ovary is taken after 7 to 8 days; preferably, the ovary is taken after 7 days.

[0012] Furthermore, the solid inoculation medium is MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L + agar 7.2 g / L + sucrose 20 g / L + hydrolyzed casein 0.5% + activated charcoal 0.5%;

[0013] Furthermore, the embryo rescue includes removing the ovary, disinfecting and rinsing it clean, taking out the ovules from the ovary and placing them on a solid inoculation medium, with 30-50 ovules per dish, and culturing in the dark at room temperature (25°C) for 20 days. The culture dishes that have grown embryos are then placed on a light-lit culture rack for further cultivation. Once the embryos have recovered and turned green, they can be transferred to a solid subculture medium for subculture and propagation.

[0014] Furthermore, the solid subculture medium is B5+6-BA 1.0 mg / L + NAA 0.1 mg / L + agar 7.2 g / L + sucrose 20 g / L + activated carbon 0.5%;

[0015] Furthermore, the doubling treatment includes culturing in a doubling medium for 7 days, followed by transfer to a subculture medium to allow seedlings to recover and grow.

[0016] Furthermore, the doubling culture medium is a subculture medium supplemented with 0.02% colchicine;

[0017] Furthermore, the doubled culture medium is prepared by weighing 0.21g of colchicine (concentration of 0.02%) per liter of subculture medium, adding it to the medium, adjusting the pH to 5.8-5.9, sterilizing and solidifying it to obtain the doubled culture medium;

[0018] Furthermore, the subculture medium is B5+6-BA 1.0 mg / L + NAA 0.1 mg / L + agar 7.2 g / L + sucrose 20 g / L + activated carbon 0.5%.

[0019] The second aspect of this invention provides a method for improving the traits of Chinese cabbage and / or arugula, which is prepared by the method of creating intergeneric hybrids of Chinese cabbage and arugula in the first aspect;

[0020] In some embodiments, the traits include one or more of the following: plant height, plant width, sepal size, petal size, pistil length, stamen length, and leaf size; for example, sepal length, sepal width, petal length, petal width, pistil length, stamen length, maximum leaf length, and maximum leaf width.

[0021] The third aspect of this invention provides a method for introducing genes such as aroma from arugula into Chinese cabbage, which is prepared by the method of creating a distant hybrid between Chinese cabbage and arugula in the first aspect.

[0022] Beneficial effects of the present invention

[0023] Through cytological identification, morphological observation, and pollen fertility assessment, it was finally determined that the method provided by this invention for creating intergeneric hybrids of Chinese cabbage and Arugula can produce true hybrid plants. This demonstrates the creation of intermediate material '72049 × Arugula', with the aim of cultivating new Chinese cabbage germplasm with sesame aroma, stress resistance, high quality, and high yield.

[0024] During the budding stage, the stigmas of Chinese cabbage were sprayed with a MgCl2·6H2O solution with a concentration of 62.3 mg / L. After the solution dried, pollination and hybridization were carried out by smearing. The germination rates of ovules after stigma treatment were 6.67%, 5.83%, and 9.17%, respectively, with an average germination rate of 7.22%, which was significantly higher than the 0.83% germination rate of the untreated control.

[0025] Morphological comparisons of the floral organs, leaves, and siliques of the allotetraploid and its parent plants revealed that the allotetraploid's traits fall between those of Chinese cabbage and arugula. Some traits are more similar to Chinese cabbage, while others are more similar to arugula. Except for the shorter stamens compared to arugula, the allotetraploid exhibited greater plant height, plant width, sepal length, sepal width, pistil length, maximum leaf length, and maximum leaf width than its parents, increasing by 243.27% and 40.39%; 122.14% and 20.42%; 103.82% and 15.90%; 107.79% and 36.17%; 138.87% and 41.10%; 74.37% and 22.66%; 96.54% and 18.98%; 148.65% and 118.93%; and 161.93% and 178.57%, respectively, demonstrating organ gigantism. The petal length of the allotetraploid increased by 83.37% compared to Chinese cabbage but decreased by 14.35% compared to arugula.

[0026] By observing and comparing the agronomic traits of the allotetraploids and their parents, such as plant height, plant width, leaves, and floral organ phenotypes, it was confirmed that the allotetraploids produced by the method of this invention are morphologically intermediate between the maternal parent (Chinese cabbage) and the paternal parent (arugula), with some organs exhibiting giant-like characteristics. This demonstrates that the method for creating distant hybrids provided by this invention can effectively obtain true hybrid plants.

[0027] Anther morphology and pollen fertility were observed and analyzed in the parents, allotetraploids, and alloteridoids. Intergeneric hybrids contain different chromosome sets from two parents, and the inability of chromosomes to pair and separate normally during meiosis, leading to reduced fertility or high sterility, is a common phenomenon in intergeneric hybrids. The allotetraploid pollen grains obtained by the method for creating distant hybrids provided in this invention have a fertility of 95.11%; while the fertility of the alloteridoid pollen grains in the control group is only 0.60%, with abnormal pollen grain morphology, extremely low staining rate, and severely abnormal meiotic behavior. This also indirectly proves that the method for creating distant hybrids provided in this invention can efficiently obtain true hybrids. Attached Figure Description

[0028] Figure 1 The morphology of the parent and doubled material plants is shown in Figure a, which is Chinese cabbage; Figure b is arugula; and Figures c1-c7 are F1 to F7. The scale bar is 10 cm.

[0029] Figure 2 The number of chromosomes in the root tip of the doubled material; d1-d7 represent F1 to F7.

[0030] Figure 3 Flow cytometry was used to identify the ploidy of the parent and allotetraploid; a is Chinese cabbage; b is arugula; c is the doubling material.

[0031] Figure 4 The leaf phenotypes of the allotetraploid and the parent were observed; e1 is Chinese cabbage; e2 is an allotetraploid; e3 is arugula; the scale bar is 10cm.

[0032] Figure 5 Phenotypic observation of the tetraploid and parental siliques; f1 is Chinese cabbage; f2 is allotetraploid; f3 is arugula; scale bar is 1 cm.

[0033] Figure 6 Phenotypic observation of flower organs between allotetraploids and parents; g1-j1 is Chinese cabbage; g2-j2 is allotetraploid; g3-j3 is arugula; g1-g3 is inflorescence; h1-h3 is complete flower; i1-i3 is pistil; j1-j3 is tetradynamous stamen; scale bar is 1cm.

[0034] Figure 7Alexander staining observation of pollen grains from the parent, allotetraploid, and allodiploid; a is Chinese cabbage; b is arugula; c is allotetraploid; k is allodiploid; scale bar is 200 μm. Detailed Implementation

[0035] 1. Materials

[0036] The female parent was Chinese cabbage '72049' (Brassica rapa; 2n=20; AA), the male parent was arugula (Eruca sativa Mill, EE, 2n=22), and a Chinese cabbage × arugula hybrid F1 was also used. These materials were provided by the Leafy Vegetable Research Group of the Vegetable Research Institute of Henan Academy of Agricultural Sciences and were planted and managed routinely at the Henan Modern Agricultural Research and Development Base.

[0037] 2. Equipment and Reagents

[0038] The main experimental instruments include: flow cytometer, microscope, fluorescence microscope, pollen scanning electron microscope, centrifuge, sterilizer, etc. The main experimental tools include: centrifuge tubes, forceps, dissecting needles, coverslips, glass slides, pipettes, absorbent paper, scissors, blades, etc.

[0039] Main experimental reagents: Carnoy's fixative, distilled water, glacial acetic acid, anhydrous ethanol, 8-hydroxyquinoline, carbofuran stain, DAPI stain, etc.

[0040] Example 1: Distant hybridization and embryo rescue

[0041] A hybrid cross was constructed using Chinese cabbage '72049' (provided by the Vegetable Research Institute of Henan Academy of Agricultural Sciences) as the female parent and arugula inbred line 'ASTRO' (collected and preserved by the Vegetable Research Institute of Henan Academy of Agricultural Sciences) as the male parent. The cross was constructed using bagging at the bud stage and artificial pollination. Two to three days before hybridization, the opened flowers on the male parent inflorescence were removed, and the plant was isolated by bagging. On the day the bagged male parent opened, the anthers were collected and used to pollinate the appropriately sized buds on the female parent plant. During the bud stage, the stigmas of the Chinese cabbage were sprayed with a 62.3 mg / L MgCl2·6H2O solution. After the solution dried (approximately 15 to 20 minutes), pollination and hybridization were performed by smearing the stigmas on the stigmas, as well as in the untreated group. After pollination, the plants were isolated by bagging to strictly control contamination by pollen from sources other than the male parent. Seven days after pollination, the ovaries were harvested, and embryo rescue was performed under aseptic conditions.

[0042] The test materials were kept fresh at 4℃ to reduce moisture evaporation; solid inoculation medium (MS + 6-BA 1.0mg / L + NAA 0.1mg / L + hydrolyzed casein 0.5% + agar 7.2g / L + sucrose 20g / L + activated carbon 0.5%), distilled water, petri dishes with filter paper, and inoculation petri dishes were autoclaved at 121℃ for 25min and prepared for use. The petri dishes were then sterilized with ultraviolet light in a clean bench for 20min. Gloves, masks, laboratory gowns, 75% alcohol, 0.1% mercuric chloride, waste liquid container, tweezers, scalpels and blades, alcohol lamp, marker pen, and sealing film were also prepared.

[0043] Using scissors, remove plump, healthy, and pest-free ovaries from the selected test material and place them in the corresponding beakers, clearly labeling them with the material name or corresponding code. Place the selected test material in a clean bench, first surface disinfecting with 75% alcohol for 30 seconds, then sterilizing with 0.1% mercuric chloride for 8 minutes. Afterward, rinse 3-5 times with sterile distilled water, pouring the waste liquid into a prepared waste liquid container. After rinsing thoroughly, use tweezers to place the ovaries in sterilized petri dishes with filter paper. Slowly cut along the midline of the ovary with a scalpel, applying moderate pressure, and use a blade to remove the ovules from the ovary pod. Place them evenly on sterile solid inoculation medium, with 30-50 ovules per dish depending on their size. Finally, seal the petri dishes tightly with sealing film, label the lids with the material name and operation date, and then place them in an incubator for dark incubation at room temperature (25℃). After about a week of cultivation, check for any contaminated culture dishes. Remove any contaminated culture dishes promptly and check them every 5 days to prevent contamination of other normally growing culture dishes.

[0044] After about 20 days of cultivation, observe whether new embryos have grown. Place the culture dishes with grown embryos on a light-lit culture rack for further cultivation. Once the embryos have recovered and turned green, they can be transferred to a solid subculture medium (B5 + 6-BA 1.0 mg / L + NAA 0.1 mg / L + agar 7.2 g / L + sucrose 20 g / L + activated carbon 0.5%) for subculture propagation.

[0045] The germination rate of ovules was statistically analyzed by observing whether new embryonic buds grew. The results are shown in Table 1: the germination rates of ovules treated with MgCl2·6H2O solution at a concentration of 62.3 mg / L were 6.67%, 5.83%, and 9.17%, respectively, with an average germination rate of 7.22%, which was significantly higher than the 0.83% germination rate of the untreated control.

[0046] Table 1. Germination of Chinese cabbage (♀) × arugula (♂) under normal and untreated conditions and after treatment with MgCl2 and 6H2O.

[0047]

[0048] Example 2: Colchicine Double Treatment

[0049] Weigh out the required amounts of agar, sucrose, and B5 medium to prepare the subculture medium (B5 + 6-BA 1.0 mg / L + NAA 0.1 mg / L + agar 7.2 g / L + sucrose 20 g / L + activated carbon 0.5%). Weigh out 0.21 g of colchicine (concentration 0.02%) per liter of subculture medium and add it to the medium. Stir until completely dissolved. Adjust the pH to 5.8-5.9, then dispense into culture flasks. Autoclave at 121℃ for 25 minutes. After sterilization, allow the medium to solidify to obtain double the medium for later use.

[0050] Prepare all necessary experimental tools and doubled culture medium. Select healthy, vigorous plants with normal green leaves from the regenerated seedlings that need to be doubled and double-treat them for 7 days.

[0051] After the treatment period, the seedlings were transferred to normal subculture medium (B5 + 6-BA 1.0 mg / L + NAA 0.1 mg / L + agar 7.2 g / L + sucrose 20 g / L + activated carbon 0.5%) to allow for acclimatization. The material name and the number of days for the double treatment were clearly labeled on the outside of the culture bottles. If a culture bottle contained both strong and weak seedlings, the strong seedlings were transferred to the double treatment medium, while the weak seedlings were transferred to subculture medium without colchicine to continue growth. During the double treatment and subsequent acclimatization stages, close observation of plant growth, plant shape, and leaf changes was necessary. Timely adjustments and records were made based on these observations.

[0052] After embryo rescue and colchicine treatment, seven ovules formed doubled tissue culture seedlings. Then, through in vitro propagation, seven progeny populations were obtained, designated F1, F2, ... F7, with six plants in each population. The plants were then transplanted into the field and managed using standard methods, such as... Figure 1 As shown. However, it is currently uncertain whether the obtained material is a distant hybrid.

[0053] Example 3: Confirmation of Distant Hybrids

[0054] 1. Chromosome observation in root tips

[0055] Rinse the root tips of the parent plants and hybrid offspring multiple times with tap water until clean. Use scissors to cut fresh lateral roots with a diameter of 0.8-1.2 mm and a length of 10-15 mm, and rinse the collected lateral root samples with distilled water to further remove impurities. Place the clean lateral root samples into centrifuge tubes, fill them with 8-hydroxyquinoline, and incubate them in a 25°C incubator in the dark for 3.5 hours.

[0056] After incubation in the dark, remove the lateral roots from the centrifuge tubes, blot the residual liquid at the root tips with filter paper, fix them in centrifuge tubes containing Carnot fixative (alcohol: glacial acetic acid = 3:1), and store them in a -20°C freezer.

[0057] Remove the root tip, rinse it with distilled water, and then place it in 2 ml of 1 mol / L hydrochloric acid solution. Dissociate it in a water bath at 55-60℃ for 15 minutes.

[0058] After dissociation, the stem tip was removed, rinsed, placed on a glass slide, crushed, and then stained with Carbofuran stain for 5 minutes.

[0059] Cover with a coverslip, press the slide, and observe and photograph it under a microscope.

[0060] Chromosome number determination using root tip chromosome counting in seven groups of doubled chromosome materials can accurately determine whether a hybrid is a true hybrid. The expected true hybrid (AAEE) should have 42 chromosomes (AA+EE=42). Figure 2 As shown, the number of chromosomes in the root tips of all 7 groups of doubled materials was 42.

[0061] 2. Flow cytometry ploidy determination

[0062] Select healthy Chinese cabbage, arugula, and first-generation hybrids of Chinese cabbage and arugula, and examine the chromosome ploidy of each sample using flow cytometry. The specific steps are as follows:

[0063] Young, fresh leaves from the parent plants and the first generation of hybrids were selected as sample materials, and parts of the leaves with high fiber and vascular bundle content were removed as much as possible. A 3cm² area was used as the sample material. 2 The sample leaves were placed in a 350mm diameter glass culture dish. The tender, fresh leaves were chopped into small pieces with a blade, and 1ml of lysis buffer was added to allow the mesophyll cells to be released.

[0064] The prepared cell homogenate was filtered through a 30µm microporous membrane into PE centrifuge tubes, and each tube was labeled. After balancing the samples in lysis buffer, they were centrifuged at 1400 rpm and 6°C for 10 minutes. The supernatant was removed, and 500 pL of the fluorescent reagent PI (propidium iodide) was added to the samples in a fume hood. After mixing with a liquid mixer, the samples were placed in a refrigerator to allow for successful staining. Flow cytometry was then performed.

[0065] Young leaves of Chinese cabbage, arugula, and the diploid parent were used for chromosome ploidy determination. The diploid parent, Chinese cabbage (AA), was used as the control group, with its peak value adjusted to 50 on the x-axis. The ploidy of the diploid parent was then determined by the position of the peak value in arugula (EE). Figure 3As shown, the peak value of arugula is approximately at 50, while the peak value of doubled material is close to 100.

[0066] Therefore, through observation of root tip chromosomes and flow cytometry identification of chromosome ploidy, the doubled material can be basically identified as a distant hybrid, and the allodiploid was successfully doubled into an allotetraploid.

[0067] Example 4: Morphological Analysis

[0068] After several subcultures, the embryos obtained through experiments gradually grow from new embryos into regenerated seedlings, eventually forming robust regenerated plants. At this point, robust seedlings can be selected for rooting culture. After about 10-15 days, robust seedlings with strong root systems are selected for transplanting.

[0069] Prepare appropriately sized seedling trays, trays, protective covers, sterile substrate, a fungicide solution to prevent pests and diseases, a rooting agent of a certain concentration, and tweezers. Fill the seedling trays with the prepared moist sterile substrate. Carefully remove strong seedlings and their well-developed roots using tweezers. Dip the roots in the fungicide solution and rooting agent before planting them into the corresponding holes on the label. Transplant each subsequent tissue culture seedling into the seedling trays using this method. After each tray is full, water it, cover it with a protective cover, and place it in a pre-set light incubator to allow the seedlings to recover and grow. Once the seedlings have recovered and reached a certain size, they can be transplanted to the experimental field for normal growth in a natural environment. Transplanting should be done according to the type, quantity, and size of the material to facilitate later experimental investigations and record-keeping of plant characteristics.

[0070] Observe and record the morphological characteristics of Chinese cabbage (mother parent), arugula (father parent), and F1 generation plants at different growth and development stages, such as plant trunk, lateral branches, flower shape, number of petals, flower bud morphology, inflorescence, stamens, and flower color.

[0071] The morphological data of the allotetraploid and the parent are shown in Table 2.

[0072] Table 2. Statistical data on allotetraploid and parental phenotypes

[0073]

[0074]

[0075] The plant height and width of the allotetraploid and parent plants were compared, as shown in Table 2. The average plant heights of the allotetraploid, Chinese cabbage, and arugula were 163.14 cm, 47.29 cm, and 117.43 cm, respectively; the average plant widths were 121.00 cm, 60.57 cm, and 101.00 cm, respectively. Compared with Chinese cabbage and arugula, the plant height of the allotetraploid plants increased by 246.50% and 38.93%, respectively; the plant width increased by 99.76% and 19.80%, respectively.

[0076] Leaf morphology of the allotetraploid and its parent, such as Figure 4 As shown in Table 2, the leaves of the allotetraploid (e2) are dark green, while the leaves of Chinese cabbage and arugula are green or light green. The leaf margins of Chinese cabbage have indistinct notches; the leaf notches of arugula are pinnately divided; the leaf notches of the allotetraploid are between those of Chinese cabbage (e1) and arugula (e3), being shallowly lobed. Furthermore, the allotetraploid leaves exhibit the same folds as Chinese cabbage, and their overall leaf appearance is more similar to Chinese cabbage, showing a clear difference from arugula. Table 2 shows that the average maximum leaf lengths of the allotetraploid, Chinese cabbage, and arugula are 59.14 cm, 22.11 cm, and 27.01 cm, respectively; the average maximum leaf widths are 35.66 cm, 13.61 cm, and 12.80 cm, respectively. The maximum leaf length of the allotetraploid increased by 148.65% and 118.93% compared to Chinese cabbage and arugula, respectively; the maximum leaf width increased by 161.93% and 178.57% compared to Chinese cabbage and arugula, respectively.

[0077] One-way ANOVA was performed using SPSS 23.0 software. The results showed that the plant height, plant width, maximum leaf length, and maximum leaf width of the allotetraploid were significantly different from those of the parent plants.

[0078] The silique morphology of the allotetraploid and the parent, such as Figure 5 As shown. The shape of the allotetraploid silique (f2) is between that of the Chinese cabbage silique (f1) and the arugula silique (f3). The needle-like upper part of the allotetraploid silique is similar to that of the Chinese cabbage, and is clearly different from the shield-shaped upper part of the arugula silique. The short and thick lower part and pedicel of the allotetraploid silique are similar to those of the arugula, and are clearly different from the slender lower part and pedicel of the Chinese cabbage silique.

[0079] The morphology of floral organs in the allotetraploid and the parent, such as Figure 6As shown. The compactness of the allotetraploid inflorescence (g2) is between that of the Chinese cabbage inflorescence (g1) and the arugula inflorescence (g3), meaning that the allotetraploid pedicel is shorter than that of the Chinese cabbage pedicel but longer than that of the arugula. Both the allotetraploid and arugula have sparse short hairs on their sepals during the bud stage, which fall off after flowering; the Chinese cabbage sepals are smooth and hairless throughout the flowering period.

[0080] Table 2 shows that the average sepal lengths of the allotetraploid, Chinese cabbage, and arugula were 1.44 cm, 0.71 cm, and 1.24 cm, respectively; the average sepal widths were 0.46 cm, 0.22 cm, and 0.34 cm, respectively. The sepal length of the allotetraploid increased by 103.82% and 15.90% compared to Chinese cabbage and arugula, respectively; the sepal width increased by 107.79% and 36.17% compared to Chinese cabbage and arugula, respectively.

[0081] The petals of the allotetraploid (h2) are oblong-ovate in shape, wide and rounded at the top and narrow and pointed at the bottom, resembling the upper part of the Chinese cabbage petals (h1) and the lower part of the arugula petals (h3). The allotetraploid petals have the same reticulate stripes as the arugula petals, and the petals do not overlap, clearly distinguishing them from the Chinese cabbage petals. Table 2 shows that the average petal lengths of the allotetraploid, Chinese cabbage, and arugula are 2.17 cm, 1.19 cm, and 2.54 cm, respectively; the average petal widths are 1.21 cm, 0.51 cm, and 0.86 cm, respectively. The petal length of the allotetraploid increased by 83.37% compared to Chinese cabbage, and decreased by 14.35% compared to arugula; the petal width increased by 138.87% and 41.10% compared to Chinese cabbage and arugula, respectively.

[0082] The allotetraploid pistil (i2) clearly distinguishes between its stigma, style, and ovary, but the style is not red and the ovary lacks white hairs, making it distinctly different from the Chinese cabbage pistil (i1) and similar to the arugula pistil (i3). Table 2 shows that the average pistil lengths of the allotetraploid, Chinese cabbage, and arugula are 1.48 cm, 0.84 cm, and 1.20 cm, respectively. The pistil length of the allotetraploid pistil is 74.37% and 22.66% longer than that of Chinese cabbage and arugula, respectively.

[0083] The stamens of the allotetraploid (j2) and Chinese cabbage (j1) exhibit typical tetradynamous stamen characteristics, with four longer inner whorls and two shorter outer whorls. However, the six stamens of arugula (j3) are of roughly equal length and do not show obvious tetradynamous stamen characteristics. Table 2 shows that the average stamen lengths of the allotetraploid, Chinese cabbage, and arugula are 1.46 cm, 0.74 cm, and 1.23 cm, respectively. The stamen length of the allotetraploid is 96.54% and 18.98% longer than that of Chinese cabbage and arugula, respectively.

[0084] One-way ANOVA was performed using SPSS 23.0. The results showed that there were highly significant differences between the sepal length, sepal width, petal length, petal width, pistil length, and stamen length of the allotetraploid and the sepal length, sepal width, petal length, petal width, pistil length, and stamen length of the allotetraploid.

[0085] Example 5: Pollen morphology observation and fertility analysis

[0086] Pollen was collected from fresh flowers of Chinese cabbage, arugula, and a first-generation hybrid of Chinese cabbage and arugula. The pollen was placed on a glass slide, and 2-3 drops of Alexander's stain were added and thoroughly mixed. The slide was covered and stained for 8 hours. Afterward, excess liquid was blotted away with absorbent paper, and the results were observed and recorded under a microscope.

[0087] Under a microscope, pollen grains from the parent, allotetraploid, and allodiploid were observed using Alexander staining. Figure 7 As shown in the diagram, almost all pollen grains from Chinese cabbage (a), arugula (b), and allotetraploid (c) were stained purplish-red, with only a few unstained. This indicates that Chinese cabbage, arugula, and allotetraploid pollen have high stainability and good fertility. In contrast, allotetraploid pollen grains (k) exhibit abnormal development, producing little or no pollen, and even those that do not stain purplish-red with Alexander's stain, indicating extremely low stainability and poor fertility. Under the same microscope magnification, the pollen grain sizes are, in descending order: allotetraploid pollen grains are the largest, followed by Chinese cabbage pollen grains, then arugula pollen grains, and finally, allotetraploid pollen grains are the smallest.

[0088] The pollen grains of the parents, allotetraploids, and allodiploids were statistically analyzed, as shown in Table 3. The fertility of the pollen grains of Chinese cabbage was 95.20% (n=1536); the fertility of the pollen grains of arugula was 95.61% (n=1642); the fertility of the allotetraploid pollen grains was 95.11% (n=1597); and the fertility of the allodiploid pollen grains was only 0.60% (n=360).

[0089] Table 3. Pollen fertility statistics of allotetraploids, parents, and allodiploids.

[0090]

[0091]

[0092] in conclusion

[0093] This experiment employed artificial hybridization and embryo rescue to obtain the F1 generation of intergeneric hybrids of 'Chinese cabbage × Arugula'. After colchicine doubling treatment, the doubled materials underwent cytological, morphological, and pollen fertility assessments, thus verifying the creation of a true distant hybrid.

[0094] After treating the stigmas with a MgCl2·6H2O solution at a concentration of 62.3 mg / L, the germination rates of ovules after distant hybridization pollination were 6.67%, 5.83%, and 9.17%, respectively, with an average germination rate of 7.22%, which was significantly higher than the 0.83% germination rate of the untreated control.

[0095] After embryo rescue and colchicine treatment, a total of 7 ovules successfully formed doubled tissue culture seedlings. Subsequently, through in vitro propagation, a total of 7 offspring populations were obtained, with 6 plants in each offspring population.

[0096] Chromosome observation of the root tip of the allotetraploid revealed that it had 42 chromosomes, consistent with the expected number. Flow cytometry analysis of the allotetraploid, using Chinese cabbage at position 50 as a control, showed that the peak value of the allotetraploid was at position 100, thus confirming that the allotetraploid had been obtained.

[0097] By observing and comparing the agronomic traits of allotetraploids with their parents, such as plant height, plant width, leaves, and floral organ phenotypes, it was found that the allotetraploids, in terms of overall morphology, fall between the male parent (arugula) and the female parent (Chinese cabbage), with some organs exhibiting gigantism. However, some phenotypes are more similar to the male parent, while others are more similar to the female parent.

[0098] Anther morphology and pollen fertility were observed and analyzed in the parents, allotetraploids, and allodiploids. Intergeneric hybrids contain different chromosome sets from the two parents. During meiosis, chromosomes cannot properly pair and separate, leading to reduced fertility or severe sterility, a common phenomenon in intergeneric hybrids. Allodiploid pollen grains exhibited abnormal morphology and extremely low staining rates, along with severely abnormal meiotic behavior, which further supports the finding of true hybrids.

Claims

1. A method for efficiently creating intergeneric hybrids between Chinese cabbage and Arugula, characterized in that, Using Chinese cabbage '72049' as the female parent and arugula inbred line 'ASTRO' as the male parent, the stigma was sprayed with a 62.3 mg / L MgCl2·6H2O solution during the budding stage. After 15-20 minutes, cross-pollination was performed by smearing. Seven days later, the ovary was harvested for embryo rescue. After regenerated seedlings, a doubling treatment was applied. The embryo rescue process includes removing the ovules from the ovary, placing them on a sterile solid inoculation medium, and culturing them in the dark at room temperature for 20 days. The solid inoculation medium is MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L + agar 7.2 g / L + sucrose 20 g / L + hydrolyzed casein 0.5% + activated carbon 0.5%.

2. The method of claim 1, wherein, Pollination during the bud stage includes spraying the stigma with a 62.3 mg / L MgCl2·6H2O solution before pollination, and then applying the solution after it has dried.

3. The method of claim 1, wherein, The doubling treatment included the addition of colchicine, followed by culturing for 7 days and then transferring the seedlings to a subculture medium to allow them to recover and grow.

4. The method of claim 3, wherein, The addition of colchicine involves weighing 0.21 g of colchicine per liter of subculture medium and adding it to the medium, adjusting the pH to 5.8-5.

9.

5. The method of claim 4, wherein, The subculture medium consisted of B5+6-BA 1.0 mg / L + NAA 0.1 mg / L + agar 7.2 g / L + sucrose 20 g / L + activated charcoal 0.5%.

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