Embryo development culture medium and method for improving seedless grape embryo rescue breeding efficiency

Abstract

The invention relates to the technical field of grape embryo rescue, in particular to an embryo development culture medium and method for improving seedless grape embryo rescue breeding efficiency. The amino acid raw materials and the concentration of the amino acid raw materials in the basic culture medium are as follows: 0.5 mM to 4 mM of glutamine, 0.5 mM to 3 mM of arginine, 0.1 mM to 2 mM of glutamic acid, 0.1 mM to 1 mM of histidine, 0.1 mM to 1 mM of alanine, 0.1 mM to 0.6 mM of aspartic acid, 0.05 mM to 0.3 mM of leucine, 0.01 mM to 0.25 mM of methionine, 0.01 mM to 0.25 mM of isoleucine, 0.01 mM to 0.35 mM of gamma-aminobutyric acid and 0.01 mM to 0.1 mM of lysine. The embryo development culture medium disclosed by the invention can obviously improve the embryo development rate and the seedling rate of seedless grape embryo rescue by 8 percent and 5 percent respectively.

Description

Technical Field

[0001] The invention relates to the technical field of grape embryo rescue, and in particular to an embryo development medium and a method for improving the efficiency of seedless grape embryo rescue breeding. Background Art

[0002] Seedless grapes have become one of the important grape breeding targets due to their high economic value. The seedless grapes commonly used in production and breeding are pseudo-parthenocarpic seedless grapes, also known as seed-aborted seedless grapes. The main feature is that the ovules form zygotic embryos after normal pollination and fertilization, but the embryo development stops in the middle of the ovule development, forming fruits with seed scars. It is precisely because of this defect that it is difficult to form heritable mature seeds that hybrid breeding with seedless grapes as the mother parent is difficult to obtain hybrid offspring through conventional hybrid breeding technology. In order to break through this bottleneck, grape embryo rescue breeding technology removes ovules before embryo abortion and places them on a specific in vitro culture medium that can promote embryo development and seedling formation to obtain hybrid offspring, becoming the core means of seedless grape breeding. However, the efficiency of this technology is affected by many factors such as parental genotype, sampling time, type of culture medium and added ingredients, and culture conditions. Among them, the efficiency of the embryo development culture medium directly affects the development of the embryo and the quality of seedling formation. Therefore, optimizing key steps such as embryo development medium and added ingredients in embryo rescue breeding is of great significance to improving the breeding efficiency of seedless grapes.

[0003] At present, researchers have conducted a lot of exploration and research on the optimization of embryo development medium and achieved remarkable results. Among them, the media with better effects include Nitsch, NN, ER, MM3, and improved MM3 medium. The improved MM3 medium has been more widely used because of its higher embryo development rate, and the addition of exogenous substances such as hormones, amino acids, and polyamines in appropriate concentrations and proportions can better promote embryo development. In addition to learning from the experience of other crops, a more accurate and scientific method for adding exogenous substances to the culture medium is to make selections and adjustments based on the difference in content between seedless grapes and seeded grape ovules. Previous studies have clarified the differences between different hormones, polyamines and other substances in seedless grapes and seeded grape ovules, and optimized the embryo development medium based on these differences. It is believed that adding exogenous polyamines (putrescine 2mmol / L + spermine 2mmol / L + spermidine 2mmol / L) and exogenous hormones (IAA 1.0mg / L + 6-BA 2.0mg / L + GA3 2.0mg / L) are both beneficial to improving the embryo rescue seedling rate of seedless grapes. However, the changing patterns of amino acids in seedless grape ovules and seeded grape ovules are still unclear, so there is no culture medium for amino acids to improve the efficiency of seedless grape embryo rescue breeding. Summary of the invention

[0004] In order to solve the above problems, the present invention provides an embryo development medium and method for improving the efficiency of seedless grape embryo rescue breeding.

[0005] The invention discloses an embryo development medium for improving the efficiency of seedless grape embryo rescue breeding. The embryo development medium takes MM3 as a basic medium, and is composed of the basic medium and amino acid raw materials. The amino acid raw materials and their concentrations in the basic medium are as follows: 0.5 mmol to 4 mmol of glutamine, 0.5 mmol to 3 mmol of arginine, 0.1 mmol to 2 mmol of glutamic acid, 0.1 mmol to 1 mmol of histidine, 0.1 mmol to 1 mmol of alanine, 0.1 mmol to 0.6 mmol of aspartic acid, 0.05 mmol to 0.3 mmol of leucine, 0.01 mmol to 0.25 mmol of methionine, 0.01 mmol to 0.25 mmol of isoleucine, 0.01 mmol to 0.35 mmol of gamma-aminobutyric acid and 0.01 mmol to 0.1 mmol of lysine.

[0006] Preferably, the embryo development medium is composed of the basal medium and amino acid raw materials, and the amino acid raw materials and their concentrations in the basal medium are: glutamine 0.83mmol~3.34mmol, arginine 0.6mmol~2.42mmol, glutamate 0.37mmol~1.48mmol, histidine 0.14mmol~0.56mmol, alanine 0.2mmol~0.81mmol, aspartic acid 0.12mmol~0.47mmol, leucine 0.06mmol~0.23mmol, methionine 0.05mmol~0.19mmol, isoleucine 0.04mmol~0.18mmol, γ-aminobutyric acid 0.05mmol~0.22mmol and lysine 0.02mmol~0.09mmol.

[0007] Preferably, the embryo development medium is composed of the basal medium and amino acid raw materials, and the amino acid raw materials and their concentrations in the basal medium are: glutamine 1.67mmol, arginine 1.21mmol, glutamate 0.74mmol, histidine 0.28mmol, alanine 0.41mmol, aspartic acid 0.23mmol, leucine 0.12mmol, methionine 0.09mmol, isoleucine 0.09mmol, γ-aminobutyric acid 0.11mmol and lysine 0.05mmol.

[0008] Preferably, the formula of the basal culture medium is: 762.6 mg / L potassium nitrate, 293.6 mg / L ammonium nitrate, 1254.6 mg / L magnesium sulfate, 780 mg / L sodium dihydrogen phosphate, 236 mg / L calcium nitrate, 3 mg / L manganese sulfate, 0.5 mg / L boric acid, 0.025 mg / L cobalt chloride, 0.025 mg / L copper sulfate, 0.5 mg / L zinc sulfate, 0.025 mg / L sodium molybdate, mg / L ferric citrate, 3 mg / L glycine, 0.25 mg / L vitamin B6, 0.25 mg / L vitamin B1, 0.25 mg / L calcium pantothenate, 500 mg / L acid hydrolyzed casein, 50 mg / L inositol, 60000 mg / L sucrose, 3000 mg / L activated carbon, 7000 mg / L agar and 1 mg / L indoleacetic acid, and the solvent is water.

[0009] Preferably, the method for preparing the embryo development medium is to add the amino acid raw material to the basal medium.

[0010] A method for improving seedless grape embryo rescue breeding efficiency comprises the following steps: inoculating a material to be improved into the embryo development medium for dark culture to improve the seedless grape embryo rescue breeding efficiency.

[0011] Preferably, the dark culture time is 7 to 9 weeks.

[0012] Preferably, the dark culture further includes embryo germination culture after embryo peeling, and subculture after seedling formation.

[0013] Preferably, embryo germination medium is used for embryo germination culture, and subculture medium is used for subculture.

[0014] Preferably, the embryo germination medium consists of a WPM medium and a first raw material, wherein the first raw material and its concentration in the WPM medium are 20 g / L sucrose, 1.5 g / L activated carbon, 7 g / L agar, 0.2 mg / L 6-BA and 0.1 g / L inositol;

[0015] The subculture medium consists of a WPM medium and a second raw material, wherein the second raw material and its concentration in the WPM medium are 30 g / L sucrose, 1.5 g / L activated carbon, 7 g / L agar, and 0.2 mg / L IBA.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The embryo development medium of the present invention can significantly increase the embryo development rate and seedling rate of seedless grape embryo rescue by 8% and 5% respectively when the amino acid components and contents are respectively 0.5mM-4mM glutamine, 0.5mM-3mM arginine, 0.1mM-2mM glutamic acid, 0.1mM-1mM histidine, 0.1mM-1mM alanine, 0.1mM-0.6mM aspartic acid, 0.05mM-0.3mM leucine, 0.01mM-0.25mM methionine, 0.01mM-0.25mM isoleucine, 0.01mM-0.35mM gamma-aminobutyric acid and 0.01mM-0.1mM lysine, and has important significance for improving the efficiency of seedless grape embryo rescue breeding and accelerating the breeding process of seedless grape new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The ovule phenotypes of different grape varieties at different developmental stages are shown in Figure 1. FS stands for 'Flame Seedless'; RS stands for 'Ruby Seedless'; MH stands for 'Rose Fragrance'; PN stands for 'Pinot Noir'; S1-S6 correspond to the EL-32 to EL-37 periods of the EL system for identifying grape growth and development.

[0019] Figure 2 The main process of embryo rescue of seedless grapes is as follows: (AB) artificial emasculation and pollination; (C) collection of hybrid fruits; (D) ovule extraction; (E) ovule culture; (FG) embryo cutting; (HJ) embryo development into seedlings; (KL) seedling hardening and transplanting.

[0020] Figure 3 Changes in total free amino acid content in grape ovules at different developmental stages in 2022 (A) and 2023 (B). FS stands for 'Flame Seedless'; RS stands for 'Ruby Seedless'; MH stands for 'Rose Fragrance'; PN stands for 'Pinot Noir'; S1-S6 correspond to the EL-32 to EL-37 periods of the EL system for grape growth and development identification.

[0021] Figure 4 The free amino acid content changes of grape ovules at different developmental stages in 2022. FS stands for 'Flame Seedless'; RS stands for 'Ruby Seedless'; MH stands for 'Rose Fragrance'; and PN stands for 'Pinot Noir'.

[0022] Figure 5 Changes in free amino acid content in grape ovules at different developmental stages in 2023. FS stands for 'Flame Seedless'; RS stands for 'Ruby Seedless'; MH stands for 'Rose Fragrance'; PN stands for 'Pinot Noir'; S1-S6 correspond to the EL-32 to EL-37 periods of the EL system for grape growth and development identification.

[0023] Figure 6Changes in mineral element content in grape ovules at different developmental stages in 2022.

[0024] Figure 7 These are the changes in mineral element contents of grape ovules at different developmental stages in 2023. FS stands for 'Flame Seedless'; RS stands for 'Ruby Seedless'; MH stands for 'Rose Fragrance'; PN stands for 'Pinot Noir'; S1-S6 correspond to the EL-32 to EL-37 periods of the EL system for grape growth and development identification.

[0025] Figure 8 PCA analysis of grape samples based on the changes in free amino acids (A) and mineral elements (B) in grape ovules. FS stands for ‘Flame Seedless’; RS stands for ‘Ruby Seedless’; MH stands for ‘Rose Fragrance’; PN stands for ‘Pinot Noir’; each grape variety has samples from six periods, corresponding to the six periods from E-L32 to E-L37 of the grape sampling system.

[0026] Fig. 9 FC analysis of the differences in free amino acid (A) and mineral element (B) contents in ovules of seeded grapes compared with seedless grapes. The Up condition was log2(FC)>1, p<0.05; the Down condition was log2(FC)<-1, p<0.05; the NoDiff condition was -1≤log2(FC)≤1, p≥0.05.

[0027] Fig.10 PLS-DA analysis of the differences in free amino acid (AB) and mineral element (CD) contents in ovules of seeded grapes compared with seedless grapes.

[0028] Fig.11 Statistics of embryo development rates of ovules of different hybrid combinations after culture on different media types, A: 'Flame Seedless' × 'Flame Seedless'; B: 'Flame Seedless' × 'Sunshine Rose'; C: 'Ruby Seedless' × 'Ruby Seedless'; D: 'Ruby Seedless' × 'Sunshine Rose'.

[0029] Fig.12 The statistics of seedling rates of ovules of different hybrid combinations after culture on different culture media types, A: 'Flame Seedless' × 'Flame Seedless'; B: 'Flame Seedless' × 'Sunshine Rose'; C: 'Ruby Seedless' × 'Ruby Seedless'; D: 'Ruby Seedless' × 'Sunshine Rose'.

[0030] Fig.13 The seedling status of ovules of 'Flame Seedless' × 'Flame Seedless' after being cultured on different types of culture media.

[0031] Fig.14This is a comparison of different hybrid combinations after seedlings were cultured using MM3 and AM3 culture media, A: Flame Seedless × Rose Fragrance; B: China Red Rose × Sunshine Rose; C: China Red Rose × Xinyu; D: Purple Crunchy Seedless × Rose Fragrance; E: Purple Crunchy Seedless × Sunshine Rose.

[0032] Fig.15 Correlation analysis between free amino acids and mineral elements in grape ovules, A: free amino acids; B: mineral elements; * indicates p<0.05, significant difference, ** indicates p<0.01, extremely significant difference. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0034] The seedless grapes described in the present invention all belong to seed aborted seedless grapes.

[0035] The preliminary investigation of the present invention found that there are huge differences in the development of ovules between seedless grapes and seeded grapes. The ovules of seedless grapes grow and develop to the largest and fullest state at the EL-34 stage, which is considered to be the best period for embryo rescue of seedless grapes. After that, the seedless ovules begin to shrink and abort rapidly, and the efficiency of embryo rescue also decreases rapidly. The ovules of seeded grape varieties continue to develop and expand, and the seed coat gradually becomes lignified until maturity ( Figure 1 ). Therefore, by analyzing the changing patterns of free amino acids and mineral elements in seedless and seeded grape ovules before and after the EL-34 period, we can clarify the differences in free amino acids and mineral elements between seedless and seeded grape ovules, thereby guiding the optimization of embryo development medium.

[0036] The present invention detects the contents of 21 free amino acids and 9 mineral elements in ovules of four grape varieties, namely, 'Flame Seedless', 'Ruby Seedless', 'Rose Fragrance' and 'Pinot Noir', at six developmental stages, to clarify the changing rules of different free amino acids and mineral elements in ovules of seedless grapes and seeded grapes. Combined with multivariate statistical analysis methods, it is clarified which free amino acids and mineral elements are significantly different between ovules of seedless grapes and seeded grapes. Then, based on these differences, an optimization scheme for embryo development culture medium is designed, and an attempt is made to screen out a culture medium that can significantly improve embryo development rate and seedling rate. This provides important reference value for in-depth analysis of the mechanism of seedless grape abortion, and provides key technical support for improving the efficiency of seedless grape embryo rescue breeding.

[0037] The present invention was carried out from May 2022 to October 2024. Indoor experiments were carried out in the National Key Laboratory of Crop Resistance and Efficient Production of Northwest A&F University and the Horticultural Science Research Center of the College of Horticulture. Outdoor experiments were carried out in the Grape Germplasm Resource Garden of the Horticulture Farm of Northwest A&F University (108°4'31.404"E, 34°17'41.244"N), Weinan Lvsheng Agricultural Technology Co., Ltd. (109°32'9.445"E, 34°43'11.201"N), Xi'an Pu Xiaotao Original Ecological Grape Garden (108°52'29.330"E, 34°24'37.948"N), and Xingping Nongtuo Grape Professional Cooperative (108°24'11.876"E, 34°15'16.812"N). The experimental materials were all obtained from 5-year-old grape vines. All hybrid combinations used seedless grapes as female parents and seedless or seeded grapes as male parents. The characteristics of the grape varieties used are shown in Table 1:

[0038] Table 1 List of grape varieties and their characteristics

[0039]

[0040] Collection and processing of grape ovules

[0041] The grape varieties of 'Flame Seedless', 'Ruby Seedless', 'Pinot Noir' and 'Rose Fragrance' from the grape germplasm resource garden of the horticultural field of Northwest Agriculture and Forestry University were used as test materials. The EL system sampling standard for grape growth and development (Coombe 1995) was strictly followed. Sampling was carried out in 6 periods from EL-32 to EL-37. Each sampling time was between 8:00 and 10:00 in the morning. 12 clusters of fruits with the same development were selected from 6 plants with basically the same growth for each variety. Random sampling was carried out from the upper, middle and lower parts of the clusters. About 500 seeds were taken from 'Flame Seedless' and 'Ruby Seedless', and about 200 seeds were taken from 'Pinot Noir' and 'Rose Fragrance'. They were placed in an ice box for preservation, and the ovules were immediately peeled on ice. The peeled ovules were immediately placed in a 10.0 mL centrifuge tube frozen with liquid nitrogen to ensure that each sample in each period had at least 5 g. The collected ovules were marked and stored in a -80℃ refrigerator. After all samples from all periods were collected, they were uniformly ground using a high-throughput tissue grinder (SCIENTZ-48, Xinzhi, China), with a frequency of 50 Hz and a grinding time of 120 s for each sample. The samples were ground into powder, and the sample temperature was kept below -40 °C during the grinding process to prevent the samples from thawing. The ground samples were kept in a -80 °C refrigerator for future use.

[0042] Determination of Free Amino Acids in Grape Ovules

[0043] 0.1 g of fresh grape ovule sample was ground and diluted with 1 mL of 50% ethanol aqueous solution (containing 0.1 mol·L-1 The mixture was homogenized with 1% hydrochloric acid and centrifuged in a high-speed refrigerated centrifuge (5810R, Eppendorf, Germany) at a speed of 12000 r / min for 10 min. -1 The supernatant was diluted 20 times and then filtered into a 1.5 mL sample bottle using a 0.22 μm organic filter, and then measured on the machine. Liquid chromatography-mass spectrometry (LC-MC) was used for detection (QTRAP 5500, AB SCHEX, USA), the chromatographic column was Intertsil OSD-4C18 (150 mm × 3.0 mm, 3.5 μm), the mobile phase A was 0.1% formic acid water, the mobile phase B was 100% acetonitrile, and the flow rate was 0.3 mL min -1 The injection volume was 10 μL and the column temperature was 25°C. The standard curve obtained by using Sigma amino acid standards was used to compare the calculation results. Each variety was tested three times in each period.

[0044] Determination of Mineral Elements in Grape Ovules

[0045] Determination of nitrogen (N) and phosphorus (P) content: Add 0.20g of ground fresh grape ovule sample to a digestion tube containing 5mL of concentrated sulfuric acid and let it stand overnight; the next day, adjust the digestion furnace to 370℃, boil the digestion tube containing the sample for 30min, add 10 drops of hydrogen peroxide to the digestion tube, digest for 20min, and do it step by step until the solution is clear. After cooling, dilute it to 100mL with distilled water. After shaking and diluting it 10 times, transfer it to a 10mL centrifuge tube, place it in the sample tray of the continuous flow analyzer (Flowsys, Systea, Italy) in order, and then set the program for automatic detection (Liang et al. 2018). The detection of each variety in each period was repeated 3 times.

[0046] Determination of potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), zinc (Zn), iron (Fe) and sodium (Na): 0.20 g of fresh grape ovules were ground into a clean digestion tank, 6 mL of nitric acid was added, and the samples were pretreated at 120 °C on an acid remover for about 20 min; after the yellow smoke was gone, the samples were removed and cooled appropriately, the lid was tightened, and then the samples were digested in a microwave digester (Multiwave PRO, Anton Paar Gmbh, Austria) in order; after the digestion was completed, the digestion tank was removed and transferred to a fume hood and slowly opened, and the samples were placed on an acid remover at 150 °C to remove the acid until a soybean-sized amount of liquid remained in the digestion tank, and the acid removal was stopped. After cooling, the samples were transferred and the volume was adjusted to 15 mL, and the samples were determined using an atomic absorption spectrophotometer (ZA3000, Hitachi, Japan) (Liang et al. 2018). The test was repeated 3 times for each variety in each period.

[0047] Grape embryo rescue technology operation process

[0048] (1) Hybrid pollination

[0049] The fruits obtained from the four hybrid combinations of 'Flame Seedless' × 'Flame Seedless', 'Flame Seedless' × 'Sunshine Rose', 'Ruby Seedless' × 'Ruby Seedless' and 'Ruby Seedless' × 'Sunshine Rose' were used to screen different embryo development culture media; the hybrid fruits obtained from the five hybrid combinations of 'Flame Seedless' × 'Rose Fragrance', 'China Red Rose' × 'Sunshine Rose', 'China Red Rose' × 'Xinyu', 'Purple Crisp Seedless' × 'Rose Fragrance' and 'Purple Crisp Seedless' × 'Sunshine Rose' were used to verify the efficiency of the optimal embryo development culture medium.

[0050] (2) Culture medium configuration

[0051] Improved MM3 culture medium: MM3 + sucrose 60 g / L + activated carbon 3 g / L + agar 7 g / L + IAA 1 mg / L + hydrolyzed casein 0.5 g / L + inositol 0.1 g / L, pH = 5.8-6.0.

[0052] Embryo germination medium: WPM + sucrose 20 g / L + activated carbon 1.5 g / L + agar 7 g / L + 6-BA 0.2 mg / L + inositol 0.1 g / L, pH = 5.8-6.0.

[0053] Subculture culture medium: WPM + sucrose 30 g / L + activated carbon 1.5 g / L + agar 7 g / L + IBA 0.2 mg / L, pH = 5.8-6.0.

[0054] (3) Embryo rescue includes artificial emasculation and pollination, collection of hybrid fruits, ovule extraction, ovule culture, embryo cutting, embryo development into seedlings, seedling hardening and transplanting ( Figure 2 ).

[0055] Data analysis

[0056] Embryo development rate (%) = (number of developed embryos / number of inoculated ovules) × 100, seedling rate (%) = (number of seedlings / number of inoculated ovules) × 100. Statistical analysis of basic data was performed using Excel 2021, and one-way analysis of variance and significance test analysis were performed using SPSS 26.0. P < 0.05 indicated statistical significance. Line graphs were drawn using Origin 2018, and principal component analysis (PCA) and fold change analysis (FC) were performed using ChiPlot (https: / / www.chiplot.online / ), and partial least squares discriminant analysis (PLS-DA) was performed using MetaboAnalyst (https: / / www.metaboanalyst.Ca / MetaboAna lyst / faces / home.xhtml).

[0057] 1. Changes in free amino acids in ovules of different grape varieties

[0058] After testing 21 free amino acids in ovules of four grape varieties at six stages for two consecutive years, it was found that as the grapes continued to develop, the total free amino acids (TAA) content in the ovules of the four grape varieties changed in a basically consistent pattern over the two years, all showing a trend of first increasing and then decreasing, and all reaching a peak at the EL-34 stage ( Figure 3 ), among which the total free amino acid content of seedless grape varieties ('Flame Seedless' and 'Ruby Seedless') between EL-32 and EL-34 was significantly lower than that of seeded grape varieties ('Rose Fragrance' and 'Pinot Noir'), indicating that the EL-34 period was the period with the highest total free amino acid content in grape ovules, and also the period with the greatest difference in total amino acid content between seedless and seeded grape ovules. The change pattern of the content of 21 kinds of free amino acids in the ovules of the four grape varieties during the two years was basically the same, basically showing a trend of first increasing and then decreasing or continuously decreasing. Among them, the contents of 8 kinds of free amino acids such as arginine, glutamine, glutamic acid, alanine, histidine, aspartic acid, and asparagine were relatively high in the ovules of the four grapes and the change difference was relatively large ( Figure 4 , Figure 5). Between EL-32 and EL-34, the contents of 12 free amino acids, including arginine, glutamine, glutamic acid, alanine, histidine, aspartic acid, glycine, leucine, isoleucine, lysine, methionine, and γ-aminobutyric acid, in the ovules of seeded varieties were significantly higher than those in seedless varieties, while the contents of asparagine and phenylalanine in the ovules of seedless grape varieties were significantly higher than those in seeded grape varieties, indicating that free amino acids such as arginine, glutamine, glutamic acid, alanine, histidine, and aspartic acid are mainly involved in the accumulation and synthesis of substances in the early stage of the development of seeded grape ovules, while asparagine plays a major role in the development of seedless grape ovules.

[0059] 2. Changes in mineral elements in ovules of different grape varieties

[0060] After testing the contents of 9 mineral elements (N, P, K, Mg, Ca, Cu, Zn, Na, Fe) in ovules of 4 grape varieties at 6 stages for 2 consecutive years, it was found that the contents of the 9 mineral elements in both seedless and seeded grape ovules were N>K>Ca>P>Mg>Fe>Na>Zn>Cu, among which the contents of N, P, K, Mg, and Ca were all greater than 10 mg / 100 g (FW) in all samples. The contents of N, P, K, Ca, and Mg in seeded grape ovules were always higher than those in seedless grapes, and were in a continuous accumulation process in seeded grape ovules, while there was a slight downward trend in seedless grape ovules ( Figure 6 , Figure 7 ). There was no significant difference in the content of trace elements Zn and Na in the ovules of seeded grapes and seedless grapes during the overall development process. The content of trace element Cu in the ovules of seeded grapes in 2022 was always higher than that in seedless grapes, while there was no significant difference in the overall development process of the ovules of seeded grapes and seedless grapes in 2023. Between EL-32 and EL-35, the content of trace element Fe in the ovules of seedless grapes was always higher than that in seeded grapes. The above shows that there are large differences in the mineral elements N, P, K, Ca, Mg, and Fe between the ovules of seeded grapes and seedless grapes, while there is little difference in the mineral elements Zn and Na between the ovules of seeded grapes and seedless grapes.

[0061] 3. Principal component analysis of different free amino acids and mineral elements

[0062] The principal component analysis was performed on the mean values ​​of 21 free amino acids and 9 mineral elements in ovules of 4 grape varieties at 6 different stages for 2 consecutive years. It was found that the 21 free amino acids contributed 60.15% of the total on principal component 1 (PC1) and 11.03% on principal component 2 (PC2). Figure 8-A), which can effectively divide the seeded grape samples and seedless grape samples into two categories, among which the seeded grape samples mainly contribute more on PC1, while the seedless grape samples mainly contribute more on PC2. It is worth noting that the third period (EL-34) samples of the two seedless grape varieties 'Flame Seedless' and 'Ruby Seedless' are most separated from the third period (EL-34) samples of the two seeded grape varieties 'Rose Fragrance' and 'Pinot Noir', indicating that the free amino acid content of the ovules of the seeded grape varieties in the EL-34 period is most different from that of the seedless grape varieties. The 9 mineral elements contribute 70.87% of the total on PC1 and 14.57% of the total on PC2 ( Figure 8 -B), which can completely divide the seeded grape samples and seedless grape samples into two categories, indicating that there are also significant differences in the mineral element content of the ovules of seeded grape varieties and seedless grape varieties.

[0063] 4. Analysis of the difference multiples of different free amino acids and mineral elements

[0064] Based on the changing rules of free amino acids and mineral elements in seeded grape and seedless grape ovules and the results of principal component analysis, combined with phenotypic changes, it can be clearly seen that at the optimal sampling period for seedless grape embryo rescue (EL-34), the free amino acids and mineral elements content between seeded grape and seedless grape ovules differed the most. Therefore, the present invention conducts more detailed data mining and analysis on the differences in free amino acids and mineral elements content in grape ovules during the EL-34 period. First, in order to clarify which specific free amino acid or mineral element has the greatest impact between seeded grape and seedless grape ovules, the present invention calculates the difference multiple (FC) and the difference significance p value (p-value) of the free amino acid or mineral element content in seeded grape ovules compared with seedless grape ovules. The volcano plot shows the distribution of the difference multiples of different free amino acids or mineral elements ( Fig. 9), where each point represents a free amino acid or mineral element. The horizontal axis of the volcano plot represents the logarithm of the difference multiple with base 2 (log2(FC)), and the vertical axis represents the negative value of the logarithm of the p-value with base 10 (-log10(p-value)). When the log2(FC) value is greater than 1 and the p-value is less than 0.05, it indicates that the free amino acids or mineral elements play a positive role in the formation and development of the ovule. On the contrary, when the log2(FC) value is less than -1 and the p-value is less than 0.05, it indicates that the free amino acids or mineral elements have a negative effect on the formation and development of the ovule. It can be seen that the log2(FC) values ​​of 11 amino acids, including alanine, arginine, glutamic acid, glutamine, lysine, methionine, histidine, aspartic acid, leucine, isoleucine, and γ-aminobutyric acid, are greater than 1, and the p value is less than 0.05, which means that these 11 amino acids may be the key amino acids required for the normal development of the ovule; the log2(FC) value of asparagine is less than -1, and the p value is less than 0.05, which means that asparagine may play a certain inhibitory role in the normal development of the ovule ( Fig. 9 -A). The log2(FC) values ​​of N, Mg, and Ca are greater than 1, and the p value is less than 0.05, which means that N, Mg, and Ca may be the most required mineral elements in the normal development of ovules; the log2(FC) value of Fe is less than -1, and the p value is less than 0.05, which means that Fe may have a negative impact on the normal development of ovules ( Fig. 9 -B).

[0065] 5. Partial least squares discriminant analysis of different free amino acids and mineral elements

[0066] The contribution of different free amino acids and mineral elements between seeded and seedless grape ovules was determined by partial least squares discriminant analysis (PLS-DA). Fig.10 ). In the PLS-DA model score diagram based on 21 free amino acids, principal component 1 explains 58.6% of the total variation, and principal component 2 explains 15.6% of the total variation ( Fig.10 -A). The seedless grape samples are located on the positive axis of the horizontal axis, while the seeded grape samples are located on the negative axis of the horizontal axis. The seedless grape and seeded grape samples are clustered into one category and are clearly separated from each other, indicating that the free amino acid content of seedless grape and seeded grape ovules is significantly different. In order to determine the types of free amino acids that affect the model separation results, the variable projection importance (VIP) score diagram of the PLS-DA model was drawn ( Fig.10-B), VIP score represents the contribution of each free amino acid to the ovule samples of seeded grapes and seedless grapes. The higher the VIP score, the greater the contribution of the free amino acid to the sample classification. Generally, variables with a VIP score greater than 1 are selected as marker substances. Figure 8 -B shows that alanine, glutamic acid, arginine, lysine, aspartic acid, methionine, histidine, γ-aminobutyric acid, glutamine, and leucine, a total of 10 free amino acids, contribute more to the samples of seeded grape ovules; asparagine contributes more to the samples of seedless grape ovules, indicating that alanine, glutamic acid, arginine, lysine, aspartic acid, methionine, histidine, γ-aminobutyric acid, glutamine, and leucine may be the most important free amino acids in the normal development of grape ovules. In the same analysis method, based on the PLS-DA model score diagram constructed based on 9 mineral elements, principal component 1 explains 55.4% of the total variation, and principal component 2 explains 20.4% of the total variation ( Fig.10 -C), seedless grape and seeded grape ovule samples can also be clearly divided into two categories, among which N, Mg, Ca, and K contribute more to the seeded grape ovule samples; Fe contributes more to the seedless grape ovule samples ( Fig.10 -D), indicating that N, Mg, Ca, and K may be the most important mineral elements in the normal development of grape ovules.

[0067] Fig.15 Correlation analysis between the contents of free amino acids (A) and mineral elements (B) in grape ovules.

[0068] 6. Optimization of embryo development medium

[0069] Based on the above analysis results, the present invention believes that compared with the nucleated grape ovules, the content of 11 free amino acids such as glutamine, arginine, glutamic acid, histidine, alanine, aspartic acid, leucine, methionine, isoleucine, γ-aminobutyric acid, and lysine, as well as 5 mineral elements such as N, P, K, Mg, and Ca in the seedless grape ovules is relatively low, that is, the lack of these substances may be one of the reasons for the abortion of the seedless grape ovules. In order to clarify whether increasing the content of the corresponding substances in the embryo development medium of the seedless grape embryo rescue breeding can better promote embryo development, the present invention first calculated the difference between the nucleated grape and the seedless grape ovules in the EL-34 period for all free amino acids and mineral elements (Table 2, Table 3). Then, four concentration gradients (A1-A4) were set based on the differences of 11 free amino acids, including glutamine, arginine, glutamic acid, histidine, alanine, aspartic acid, leucine, methionine, isoleucine, γ-aminobutyric acid, and lysine (Table 4); four concentration gradients (Y1-Y4) were set based on the differences of five mineral elements, including N, P, K, Mg, and Ca (Table 5); and four concentration gradients (H1-H4) were set by mixing 11 free amino acids and five mineral elements together (Table 6). A total of 12 treatment schemes were set to optimize the embryo development medium. All schemes used the modified MM3 medium as the control (Table 7, Table 8), and the solvent in the modified MM3 medium was water.

[0070] Table 2 Analysis of the differences in the contents of 21 free amino acids in ovules of seeded and seedless grapes at the EL-34 stage in different years

[0071]

[0072] Table 3 Differences in the contents of nine different mineral elements in seeded grape ovules, seedless grape ovules and MM3 medium at the EL-34 stage in different years

[0073]

[0074] Table 4 The contents of 11 amino acids added to different embryo development media

[0075]

[0076] Table 5 Contents of five mineral elements added to different embryo development media

[0077]

[0078] Table 6 Types and supplementary ingredients of 13 embryo development media

[0079]

[0080]

[0081] Table 7: Formula of improved MM3 medium (pH=5.8)

[0082]

[0083] Table 8 Types and specific additives of 13 embryo development culture media

[0084]

[0085]

[0086] The fruits obtained from the four hybrid combinations of 'Flame Seedless'×'Flame Seedless', 'Flame Seedless'×'Sunshine Rose', 'Ruby Seedless'×'Ruby Seedless' and 'Ruby Seedless'×'Sunshine Rose' were used as test materials and inoculated into 13 embryo development mediums including MM3 medium. After 8 weeks of dark culture, the embryos were peeled. The embryos were cultured into seedlings using embryo germination medium, and the seedlings were subcultured and preserved using subculture medium. The number of developed embryos and seedlings was counted.

[0087] The results showed that the embryo development rates of AM3 medium in the combinations of 'Flame Seedless'×'Flame Seedless', 'Ruby Seedless'×'Ruby Seedless', 'Flame Seedless'×'Sunshine Rose' and 'Ruby Seedless'×'Sunshine Rose' were increased by 9.17%, 7.56%, 5.68% and 11.02% respectively compared with the control MM3 medium, and the seedling rates were increased by 7.98%, 6.71%, 6.68% and 7.47% compared with the control MM3 medium, and there were significant differences compared with the control MM3 medium; followed by AM4 medium which also obtained higher embryo development rate and seedling rate in the four hybrid combinations, but in 'Flame Seedless'×' There was no significant difference in the embryo development rate obtained in the 'Sunshine Rose' combination compared with the control MM3 medium; the embryo development rate and seedling rate obtained by AM2 medium in the 'Ruby Seedless'×'Sunshine Rose' combination were significantly higher than those in the control MM3 medium, but there was no significant increase in the other three combinations compared with the control MM3 medium; the embryo development rate and seedling rate obtained by YM3, YM4, HM3, and HM4 medium in the four hybrid combinations were not significantly higher than those in the control MM3 medium; while the embryo development rate and seedling rate obtained by AM1, YM1, YM2, HM1, and HM2 medium in the four hybrid combinations were lower than those in the control MM3 medium ( Fig.11 , Fig.12 ).

[0088] It is generally believed that compared with the control MM3 medium, the AM3 medium initially screened out in the present invention has an average increase of 8.36% and 7.21% in embryo development rate and seedling rate in 4 hybrid combinations, and the medium has a good promoting effect on embryo development and seedling formation ( Fig.13 ), which can significantly improve the efficiency of embryo rescue breeding.

[0089] 7. Validation and application of embryo development medium

[0090] Based on the above research results, five different hybrid parent combinations of 'Flame Seedless' × 'Rose Fragrance', 'China Red Rose' × 'Sunshine Rose', 'China Red Rose' × 'Xinyu', 'Purple Crisp Seedless' × 'Rose Fragrance', and 'Purple Crisp Seedless' × 'Sunshine Rose' were added, and MM3 medium was used as a control to further verify the culture effect of AM3 medium.

[0091] Specifically, the seeds were inoculated into 13 embryo development mediums including MM3 medium, and the embryos were peeled after 8 weeks of dark culture. The embryos were cultured into seedlings using embryo germination medium, and the seedlings were subcultured using subculture medium, and the number of developed embryos and seedlings was counted.

[0092] The results showed that the embryo development rates of AM3 medium in the combinations of 'Flame Seedless' × 'Rose Fragrance', 'China Red Rose' × 'Sunshine Rose', 'China Red Rose' × 'Xinyu', 'Purple Crisp Seedless' × 'Rose Fragrance' and 'Purple Crisp Seedless' × 'Sunshine Rose' were increased by 8.24%, 7.21%, 9.49%, 8.42% and 6.86% compared with the control MM3 medium, and the seedling rates were increased by 4.93%, 4.15%, 7.13%, 3.35% and 3.13% compared with the control MM3 medium (Table 9, Fig.14 ), that is, compared with the control MM3 medium, the embryo development rate and seedling rate of AM3 medium in 5 hybrid combinations were increased by 8.04% and 4.54% on average, proving that AM3 medium (improved MM3 + glutamine 1.67mM + arginine 1.21mM + glutamate 0.74mM + histidine 0.28mM + alanine 0.41mM + aspartic acid 0.23mM + leucine 0.12mM + methionine 0.09mM + isoleucine 0.09mM + γ-aminobutyric acid 0.11mM + lysine 0.05mM + sucrose 60g / L + activated carbon 3g / L + agar 7g / L + IAA 1mg / L + hydrolyzed casein 0.5g / L + inositol 0.1g / L, pH=5.8-6.0) can be used as an optimized embryo development medium and has a good promoting effect on embryo development.

[0093] Table 9 Comparison of the effects of different hybrid combinations using MM3 and AM3 medium

[0094]

[0095] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0096] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0097] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An embryo development medium for improving the efficiency of seedless grape embryo rescue breeding, taking MM3 as a basic medium, wherein the embryo development medium is composed of the basic medium and amino acid raw materials, and the amino acid raw materials and their concentrations in the basic medium are: 0.5mmol-4mmol of glutamine, 0.5mmol-3mmol of arginine, 0.1mmol-2mmol of glutamic acid, 0.1mmol-1mmol of histidine, 0.1mmol-1mmol of alanine, 0.1mmol-0.6mmol of aspartic acid, 0.05mmol-0.3mmol of leucine, 0.01mmol-0.25mmol of methionine, 0.01mmol-0.25mmol of isoleucine, 0.01mmol-0.35mmol of gamma-aminobutyric acid and 0.01mmol-0.1mmol of lysine.

2. The embryo development medium according to claim 1 is characterized in that the embryo development medium is composed of the basal medium and amino acid raw materials, and the amino acid raw materials and their concentrations in the basal medium are: glutamine 0.83mmol~3.34mmol, arginine 0.6mmol~2.42mmol, glutamic acid 0.37mmol~1.48mmol, histidine 0.14mmol~0.56mmol, alanine 0.2mmol~0.81mmol, aspartic acid 0.12mmol~0.47mmol, leucine 0.06mmol~0.23mmol, methionine 0.05mmol~0.19mmol, isoleucine 0.04mmol~0.18mmol, γ-aminobutyric acid 0.05mmol~0.22mmol and lysine 0.02mmol~0.09mmol.

3. The embryo development medium according to claim 1 is characterized in that the embryo development medium is composed of the basal medium and amino acid raw materials, and the amino acid raw materials and their concentrations in the basal medium are: 1.67mmol glutamine, 1.21mmol arginine, 0.74mmol glutamic acid, 0.28mmol histidine, 0.41mmol alanine, 0.23mmol aspartic acid, 0.12mmol leucine, 0.09mmol methionine, 0.09mmol isoleucine, 0.11mmol γ-aminobutyric acid and 0.05mmol lysine.

4. The embryo development medium according to any one of claims 1 to 3, characterized in that the formula of the basic medium is: potassium nitrate 762.6 mg / L, ammonium nitrate 293.6 mg / L, magnesium sulfate 1254.6 mg / L, sodium dihydrogen phosphate 780 mg / L, calcium nitrate 236 mg / L, manganese sulfate 3 mg / L, boric acid 0.5 mg / L, cobalt chloride 0.025 mg / L, copper sulfate 0.025 mg / L, sulfuric acid Zinc 0.5 mg / L, sodium molybdate 0.025 mg / L, ferric citrate mg / L, glycine 3 mg / L, vitamin B6 0.25 mg / L, vitamin B1 0.25 mg / L, calcium pantothenate 0.25 mg / L, acid hydrolyzed casein 500 mg / L, inositol 50 mg / L, sucrose 60000 mg / L, activated carbon 3000 mg / L, agar 7000 mg / L and indoleacetic acid 1 mg / L. The solvent is water. 5 . The embryo development medium according to claim 1 , wherein the embryo development medium is prepared by adding the amino acid raw material to the basal medium.

6. A method for improving the efficiency of seedless grape embryo rescue breeding, characterized in that: The following steps are involved: The material to be improved is inoculated into the embryo development medium of claim 1 and cultured in the dark to improve the efficiency of seedless grape embryo rescue breeding.

7. The method according to claim 6, characterized in that The dark culture time is 7 to 9 weeks.

8. The method according to claim 6, characterized in that Dark culture also includes embryo germination culture after embryo peeling, and subculture after seedling formation.

9. The method according to claim 8, characterized in that Use embryo germination medium for embryo germination culture, and use subculture medium for subculture.

10. The method according to claim 8, characterized in that The embryo germination medium consists of a WPM medium and a first raw material, wherein the first raw material and its concentration in the WPM medium are 20 g / L sucrose, 1.5 g / L activated carbon, 7 g / L agar, 0.2 mg / L 6-BA and 0.1 g / L inositol; The subculture medium consists of a WPM medium and a second raw material, wherein the second raw material and its concentration in the WPM medium are 30 g / L sucrose, 1.5 g / L activated carbon, 7 g / L agar, and 0.2 mg / L IBA.

Images