Method for producing litchi mutants through mutagenesis of pingyangmycin and application of litchi mutants

Abstract

The invention relates to the technical field of plant breeding, and provides a method for producing a litchi mutant by adopting pingyangmycin mutagenesis and application thereof in order to solve the problems of long period, large investment, low efficiency and the like of a traditional litchi breeding mode. The method for producing the litchi mutants by adopting pingyangmycin mutagenesis comprises the following steps: mutagenizing embryogenic calluses of litchis for 7 days by using a liquid containing 20mg / L pingyangmycin, performing recovery culture on the obtained mutagenized embryogenic calluses, and performing differentiation and maturation to obtain mature somatic embryos; and performing germination culture on the mature somatic embryo in a germination culture medium to obtain a regenerated plant, wherein the obtained regenerated plant is or is a candidate lychee mutant. According to the method, pingyangmycin is utilized for mutagenesis, an efficient litchi mutagenesis technical system is established, obtaining of litchi mutants is accelerated, and good technical support is provided for improved breeding of litchi varieties.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to a method for producing litchi mutants using bleomycin mutagenesis and its application. Background Technology

[0002] Lychee belongs to the genus Litchi of the family Sapindaceae ( Litchi chinensis For fruit trees (Sonn.), the main breeding methods are seed selection and hybridization. However, these traditional breeding methods have drawbacks such as long cycles, high investment, and low efficiency. Using chemical mutagens can greatly increase the mutation frequency of plants and produce new individuals with new traits that do not exist in nature or are extremely rare, thus promoting germplasm resource innovation and accelerating the process of variety improvement. Summary of the Invention

[0003] This invention addresses the shortcomings of traditional litchi breeding methods, such as long cycles, high investment, and low efficiency, by providing a method for producing litchi mutants using bleomycin mutagenesis.

[0004] The present invention provides a method for producing litchi mutants by mutagenesis using bleomycin, comprising the following steps: inducing embryogenic callus of litchi with a liquid containing 20 mg / L bleomycin for 7 days; after the obtained mutagenesis, the embryogenic callus is cultured and then differentiated and matured to obtain mature embryos; the mature embryos are germinated and cultured in a germination medium until regenerated plants are obtained, and the obtained regenerated plants are or are candidates for litchi mutants; The germination medium is a solid medium obtained by adding any one of ornithine, spermine, and spermidine to WPM medium as the basic medium, wherein the concentration of ornithine, spermine, or spermidine is 2-8 mg / L.

[0005] In the above method, the liquid containing 20 mg / L bleomycin is a suspension medium containing 20 mg / L bleomycin; the suspension medium containing 20 mg / L bleomycin is obtained by adding bleomycin to a suspension medium to make the bleomycin concentration 20 mg / L; the suspension medium is obtained by adding 2,4-D, inositol, coconut milk and sucrose to MS liquid medium to make the 2,4-D concentration 1 mg / L, the inositol concentration 150 mg / L, the coconut milk concentration 50 mL / L, and the sucrose concentration 20 g / L.

[0006] In the above method, the litchi is the litchi variety Fei Zi Xiao, and the germination medium is a solid medium obtained by adding ornithine, sucrose and agar to WPM liquid medium, so that the concentration of ornithine is 8 mg / L, the concentration of sucrose is 30 g / L, and the concentration of agar is 7 g / L.

[0007] In the above method, the litchi is the litchi variety Shangshuhuai, and the germination culture medium is a solid culture medium obtained by adding spermine, sucrose and agar to WPM liquid culture medium, so that the concentration of spermine is 8 mg / L, the concentration of sucrose is 30 g / L and the concentration of agar is 7 g / L.

[0008] In the above method, the litchi is the litchi variety Lingnan 15, and the germination medium is a solid medium obtained by adding spermidine, sucrose and agar to WPM liquid medium, so that the concentration of spermidine is 2 mg / L, the concentration of sucrose is 30 g / L, and the concentration of agar is 7 g / L.

[0009] In the above method, the recovery culture is a step of restoring the induced embryogenic callus tissue to a proliferation medium; the proliferation medium is a solid medium obtained by adding 2,4-D, sucrose and gellan gum to MS liquid medium, so that the concentration of 2,4-D is 1 mg / L, the concentration of sucrose is 30 g / L, and the concentration of gellan gum is 3.5 g / L.

[0010] In the above method, the differentiation is the step of differentiating the embryogenic callus after recovery culture into a somatic embryo differentiation medium to obtain a somatic embryo; the somatic embryo differentiation medium is a solid medium obtained by adding NAA, KT, inositol, LH, coconut milk, sucrose and agar to MS liquid medium, so that the concentration of NAA is 0.1 mg / L, the concentration of KT is 5 mg / L, the concentration of inositol is 100 mg / L, the concentration of LH is 0.4 g / L, the concentration of coconut milk is 100 mL / L, the concentration of sucrose is 60 g / L and the concentration of agar is 10 g / L.

[0011] In the above method, the maturation is the step of culturing the somatic embryo in maturation medium C19 until a mature somatic embryo is obtained; the maturation medium C19 is a solid medium obtained by adding IAA, ABA, sucrose and agar to MS liquid medium, so that the concentration of IAA is 0.5 mg / L, the concentration of ABA is 1 mg / L, the concentration of sucrose is 60 g / L and the concentration of agar is 10 g / L.

[0012] In the above method, the embryogenic callus can be the embryogenic callus obtained by proliferation culture in the proliferation medium. The above method further includes a step of suspending the culture in the suspension medium after proliferation culture before inducing mutagenesis.

[0013] In the above method, the mutagenesis is carried out under dark conditions at 23℃-27℃.

[0014] In the above method, the proliferation culture is carried out in the dark at 23℃-27℃ for 18-25 days.

[0015] In the above method, the suspension culture is carried out in the dark at 23℃-27℃ for 10-15 days.

[0016] In the above method, the recovery culture is carried out in the dark at 23℃-27℃ for 20-30 days.

[0017] In the above method, the differentiation culture is carried out in the dark at 23℃-27℃ for 40-60 days.

[0018] In the above method, the maturation culture is carried out in the dark at 23℃-27℃ for 50-70 days.

[0019] In the above method, the germination culture is carried out for 30-50 days under normal light conditions at 23℃-27℃.

[0020] The normal lighting is 15-17 hours / day, with a light intensity of 2000-5000 Lux.

[0021] The method described above, specifically the method for producing litchi mutants using bleomycin mutagenesis, includes the following steps: S1. After proliferating and culturing the embryogenic callus of litchi in the aforementioned proliferation medium, it was then suspended and cultured in the aforementioned suspension medium, and then transferred to the suspension medium containing 20 mg / L bleomycin for 7 days to induce mutagenesis and obtain the mutagenized embryogenic callus. S2. The induced embryogenic callus is restored and cultured in the proliferation medium and then transferred to the somatic embryo differentiation medium for differentiation culture until a somatic embryo is obtained. S3. The embryonic cells are cultured in the C19 maturation medium until mature embryonic cells are obtained. S4. The mature embryo is germinated in germination medium or 4 until a regenerated plant is obtained. The regenerated plant is or is a candidate for litchi mutant.

[0022] In the above method, the lychee can be selected from any one of the following varieties: Fei Zi Xiao, Lingnan No. 15, and Shang Shu Huai.

[0023] The present invention also includes the application of the method for producing litchi mutants by mutagenesis using bleomycin in litchi breeding.

[0024] This invention utilizes litchi embryogenic callus as material, involving callus proliferation, suspension, mutagenesis, and recovery culture. Through somatic embryo induction, complete in vitro regenerated plants are developed, constructing a method for producing litchi mutants using bleomycin mutagenesis. This method establishes an efficient litchi mutagenesis technology system using bleomycin mutagenesis, accelerating the acquisition of litchi mutants and providing excellent technical support for litchi variety improvement breeding. Attached Figure Description

[0025] Figure 1 This illustrates the changes in callus cell density over treatment time induced by different concentrations of bleomycin in Example 1 of the present invention. Figure 1 A represents the density of a single cell. Figure 1 B represents the density of small cell clusters. Figure 1 C represents the total cell density.

[0026] Figure 2 The images show the callus cell states induced by different concentrations of bleomycin in Example 1 of this invention. Figure 2 A represents the single-cell survival rate. Figure 2 B represents the survival rate of small cell clusters. Figure 2 C represents the overall survival rate. Figure 2 D represents the cell compaction volume. The results of the significance analysis of the differences between treatments with different lowercase letters for bleomycin concentrations are as follows: P <0.05.

[0027] Figure 3 The SNP mutation spectrum after mutagenesis with different concentrations of bleomycin in Example 1 of this invention.

[0028] Figure 4 The InDel length distribution after mutagenesis with different concentrations of bleomycin in Example 1 of this invention.

[0029] Figure 5 This describes the somatic embryogenesis and germination of callus tissue treated with different concentrations of bleomycin in Example 1 of the present invention. Figure 5 Somatic embryogenesis in patients treated with 0 mg / L bleomycin (A) Figure 5 The somatic embryogenesis of B was observed in patients treated with 5 mg / L bleomycin. Figure 5 C represents the somatic embryogenesis of patients treated with 10 mg / L bleomycin. Figure 5 The somatic embryogenesis of patients treated with 20 mg / L bleomycin (D) was studied. Figure 5 The germination of somatic embryos treated with 0 mg / L bleomycin (E) was measured. Figure 5 F represents the germination of somatic embryos treated with 5 mg / L bleomycin. Figure 5 The germination of somatic embryos treated with 10 mg / L bleomycin (G) was observed. Figure 5 The germination of somatic embryos treated with 20 mg / L bleomycin (H) was observed.

[0030] Figure 6 These are germinating embryos on different germination media in Example 2 of the present invention. Figure 6 A: Concubine's Smile; Figure 6 B: Lingnan No. 15; Figure 6 C: Shangshu Huai.

[0031] Figure 7 The number of SNP mutations in the 20 *Euphorbia hirta* mutagenesis regenerated plants in Example 2 of this invention.

[0032] Figure 8 The number of InDel mutations in the 20 *Euphorbia hirta* mutagenesis regenerated plants in Example 2 of this invention.

[0033] Figure 9 The number of SNP mutations in the 20 Lingnan 15 mutagenesis regenerated plants in Example 2 of this invention.

[0034] Figure 10 The number of InDel mutations in the 20 Lingnan 15 mutagenesis regenerated plants in Example 2 of this invention. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] The sources of the reagents and culture media involved in the following examples are as follows: 2,4-Dichlorophenoxyacetic acid (2,4-D) is a product of Shanghai Yuanju Biotechnology Co., Ltd., with batch number 081223.

[0038] Inositol is a product of Shanghai Yuanju Biotechnology Co., Ltd., with batch number 071013.

[0039] α-Naphthaleneacetic acid (NAA) is a product of Shanghai Bio-Tech Co., Ltd., with batch number 090219.

[0040] 6-Furfural aminopurine (KT, kinetin) is a product of Shanghai Bio-Tech Co., Ltd., batch number 080623. Abscisic acid (ABA) is a product of Shanghai Yuanye Biotechnology Co., Ltd., with batch number S21F8J29658.

[0041] Bleocin (PYM, specifically the hydrochloride salt of bleocin, i.e., bleocin hydrochloride) is a product of Shanghai Yuanye Biotechnology Co., Ltd., with batch number M301S211062.

[0042] Indole-3-acetic acid (IAA) is a product of Sangon Biotech (Shanghai) Co., Ltd., with batch number CA13BA0040.

[0043] Hydrolyzed milk protein (Lactalbumin Hydrolysate, LH) is a product of Sangon Biotech (Shanghai) Co., Ltd., with batch number BB07BA0326.

[0044] MS medium is a product of Beijing Ximeijie Technology Co., Ltd., with batch number AAK0519209A.

[0045] Ornithine (L-Ornithine, orn) is a product of GlpBio Biotechnology Co., Ltd., with the product code GC30044.

[0046] Coconut juice is the coconut water obtained by filtering the juice from fresh coconuts through a double layer of gauze.

[0047] M3 medium is a solid medium obtained by adding 2,4-D at a concentration of 1 mg / L, sucrose at 30 g / L, and agar at 7 g / L to MS medium.

[0048] M4 medium is a solid medium obtained by adding 2,4-D at a concentration of 1 mg / L, KT at a concentration of 0.5 mg / L, AgNO3 at a concentration of 5 mg / L, sucrose at a concentration of 30 g / L, and agar at a concentration of 7 g / L to MS medium.

[0049] The proliferation medium was a solid medium obtained by adding 2,4-D at a concentration of 1 mg / L, sucrose 30 g / L, and gellan gum 3.5 g / L to MS medium.

[0050] The suspension medium was prepared by adding 2,4-D at a concentration of 1 mg / L, inositol at a concentration of 150 mg / L, coconut milk at a concentration of 50 mL / L, and sucrose at a concentration of 20 g / L to MS medium.

[0051] The somatic embryo differentiation medium was a solid medium obtained by adding NAA at a concentration of 0.1 mg / L, KT at a concentration of 5 mg / L, inositol at a concentration of 100 mg / L, LH at a concentration of 0.4 g / L, coconut juice at a concentration of 100 mL / L, sucrose at a concentration of 60 g / L, and agar at a concentration of 10 g / L to MS medium.

[0052] The mature medium C19 is a solid medium obtained by adding IAA at a concentration of 0.5 mg / L, ABA at a concentration of 1 mg / L, sucrose at a concentration of 60 g / L, and agar at a concentration of 10 g / L to MS medium.

[0053] Germination medium OR4 is a solid medium obtained by adding orn to MS medium at a concentration of 8 mg / L, followed by the addition of 60 g / L sucrose and 10 g / L agar.

[0054] The litchi varieties Fei Zi Xiao, Ling Nan No. 15, and Shang Shu Huai mentioned in the following examples are all commercially available.

[0055] The data were processed using DPS Data Processing System 18.10 Advanced Edition statistical software. The experimental results are expressed as average values, and the Duncan's new multiple range test was used.

[0056] Example 1: Screening of treatment concentrations for producing litchi mutants using bleomycin mutagenesis The test material was embryogenic callus from the anthers of the litchi 'Feizixiao' variety. The callus was alternately subcultured on basal subculture media M3 (MS + 2,4-D 1 mg / L) and M4 (MS + 2,4-D 1 mg / L + KT 0.5 mg / L + AgNO3 5 mg / L). Both M3 and M4 media were supplemented with 30 g / L sucrose and 7 g / L agar.

[0057] 1. Statistical analysis of the survival rate of callus induced by different concentrations of bleomycin The callus tissue was transferred to proliferation medium (MS + 2,4-D 1 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L) and cultured in the dark at 23℃-27℃ for 18-25 days (20 days in this example). Then, 1.5 g of embryogenic callus tissue was inoculated into suspension medium (MS + 2,4-D 1 mg / L + sucrose 20 g / L + inositol 150 mg / L + coconut milk 50 mL / L) and cultured with shaking. Large pieces of callus tissue were filtered out using a 20-mesh sieve. After three subcultures, the cells were cultured in suspension at 23-27℃ in the dark for a total of 10-15 days (11 days in this example), continuously removing dead cells to obtain embryogenic suspension cells. After resuspending and culturing for 1-2 days, the cells were used for bleomycin-induced mutagenesis.

[0058] Mutagenic treatment with bleomycin at concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, or 40 mg / L for 3 and 7 days was conducted to explore the sensitive concentration and appropriate treatment time for callus response to bleomycin (PYM). Details are as follows: Embryogenic suspension cells were transferred using a 1 ml pipette tip with the tip removed to 50 ml of suspension medium containing different concentrations of bleomycin (bleomycin was added to the suspension medium to achieve concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, or 40 mg / L, respectively). The flasks were then placed in the dark on a shaker at 23℃-27℃ and 120 r / min for mutagenesis. The density and viability of single cells and cell clusters of each treatment suspension were compared at 0, 3, and 7 days to preliminarily explore the sensitive concentration of bleomycin and the appropriate treatment time for embryogenic callus.

[0059] Each concentration of bleomycin was used in suspension culture medium in triplicate.

[0060] The results are as follows Figure 1 As shown, when the PYM treatment time was 3 days, the survival rate of suspended cells under all concentrations was close to 100%, with almost no cell death observed. Although the density of single cells and cell clusters decreased slightly with increasing PYM concentration, they remained at a high level overall, indicating that short-term treatment had little impact on cell viability. However, as the treatment time was extended to 7 days, high concentrations of PYM (20 mg / L) led to a significant decrease in cell viability. At this concentration, the single-cell survival rate was 47.3%, the cell cluster survival rate was 53.3%, and the total cell survival rate was 68.09%, all close to the half-lethal dose. This indicates that a 7-day treatment time combined with 20 mg / L PYM can serve as a suitable mutagenic condition, capable of inducing mutations while maintaining a certain level of cell viability.

[0061] Further analysis results can be found in Figure 2 When the treatment time was fixed at 7 days and the PYM concentration was below 10 mg / L, the survival rate of single cells and cell clusters remained at a high level, and the cell growth state was relatively stable. This indicates that low concentrations of PYM have low cytotoxicity, but may not be sufficient to induce significant mutations. In contrast, 20 mg / L PYM treatment for 7 days effectively induced cell mutations while avoiding excessive lethality, providing suitable mutant material for subsequent somatic embryogenesis and plant regeneration. This indicates that PYM has strong cytotoxicity to litchi embryogenic callus in a concentration-dependent manner. Furthermore, by comparing the changes in cell density and sedimentation volume under different treatment times, it was found that 7-day treatment can more effectively screen cell populations with mutation potential, providing good starting material for subsequent somatic embryogenesis and plant regeneration.

[0062] 2. DNA variation in embryonic suspension cells treated with different concentrations of bleomycin Take 2 g of callus tissue treated with different concentrations of bleomycin 7 days after the above mutagenesis, centrifuge, flash freeze in liquid nitrogen, and extract DNA for resequencing; calculate the genomic mutation rate and molecular marker polymorphism ratio of each material under different treatments to determine the sensitive concentration of bleomycin to embryogenic callus.

[0063] SNP detection results showed that the total number of variants increased with increasing bleomycin concentration, reaching a peak at 20 mg / L (3,260,858), and slightly decreased at 40 mg / L (Table 1). Figure 3 SNPs are most commonly found in intergenic regions, with approximately twice the number found in intronic regions. Nonsynonymous mutations are most prevalent in exon regions, and mutations gaining stop codons are significantly more numerous than those losing them. Transition mutations are approximately twice the number found in transversion mutations, with C:G>T:A and T:A>A:T being the dominant types.

[0064] InDel analysis showed the same trend, with the highest total number of InDels (491,161) at 20 mg / L treatment, with intergenic regions and introns being the InDel-rich areas (Table 2). The InDel length was predominantly 1 bp (accounting for over 50%), with 2 bp accounting for approximately 15%, indicating that bleomycin mainly induces single / double base insertions or deletions. Figure 4 ).

[0065] Table 1 SNP Detection and Annotation

[0066] Table 2 InDel Detection and Annotations

[0067] In conclusion, treatment with 20 mg / L bleomycin for 7 days represents the optimal mutagenesis condition for litchi suspension cells. This treatment, while ensuring cell half-lethality and regeneration capacity, induced the highest number of genetic variations (over 3.26 million SNPs and 490,000 InDels, respectively), providing an efficient technical system for the construction of litchi mutant libraries and the discovery of functional genes.

[0068] 3. Somatic embryogenesis and germination of callus induced by bleomycin After treatment with bleomycin (0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, or 40 mg / L, 7 days), the callus tissue was rinsed 4-5 times with sterile water to eliminate the residual toxicity of bleomycin. The mutagenized embryogenic callus was then placed on proliferation medium and cultured in the dark at 23℃-27℃ for 20-30 days (25 days in this example) to allow the mutated genes to be expressed. After recovery culture, the callus tissues were transferred to somatic embryo differentiation medium (MS + NAA 0.1 mg / L + KT 5 mg / L + inositol 100 mg / L + LH 0.4 g / L + coconut milk 100 mL / L + sucrose 60 g / L + agar 10 g / L) and recovered cultured in the dark at 23℃-27℃ for 40-60 days. At 45 days, the number of milky somatic embryos, the proportion of dicotyledonous embryos, and the morphology of somatic embryos were recorded.

[0069] The somatic embryos were then placed on maturation medium C19 (MS + IAA 0.5 mg / L + ABA 1 mg / L + sucrose 60 g / L + agar 10 g / L) and cultured in the dark at 23℃-27℃ for 50-70 days (60 days in this example). After that, they were placed on germination medium OR4 (MS + orn 8 mg / L + sucrose 60 g / L + agar 10 g / L) and cultured under normal light (15-17 hours / day, light intensity 2000-5000 Lux) at 23℃-27℃. After 50 days, the embryogenesis and germination of the surviving subcultured callus tissue were observed.

[0070] Embryogenic suspension callus obtained by mutagenesis germinated after somatic embryonic induction, see Figure 5 It was found that materials treated with 0–20 mg / L could successfully induce somatic embryos and develop into seedlings, while treatment with 40 mg / L completely inhibited somatic embryo differentiation. This indicates that cell embryogenicity is lost at this concentration, but treatment with 20 mg / L and below does not affect regeneration capacity.

[0071] Example 2: Screening of germination medium for producing litchi mutants using bleomycin mutagenesis Embryogenic callus (derived from anthers) from three litchi varieties—Feizixiao, Lingnan 15, and Shangshuhuai—was used as materials. Following the method described in Example 1, embryogenic callus from each variety, after approximately 20 days of proliferation culture on a proliferation medium, was transferred to a suspension medium containing bleomycin. Mutagenesis was induced for 7 days with 20 mg / L bleomycin. The induced embryogenic callus was cultured on a proliferation medium for approximately 25 days and then transferred to a somatic embryo differentiation medium. Somatic embryos were obtained after 45 days of induction. These somatic embryos were placed on a maturation medium (C19) and cultured for approximately 60 days to obtain mature somatic embryos of Feizixiao, Lingnan 15, and Shangshuhuai, respectively. These mature somatic embryos were used as experimental materials for two rounds of germination medium screening.

[0072] 1. First round of germination medium screening Using WPM3 medium as a control, 12 treatment media were set up, with the specific formulations as follows: 1) Control culture medium WPM: WPM + 30 g / L sucrose + 7 g / L agar; WPM3: WPM + calcium nitrate tetrahydrate 11.12 g / L + sucrose 30 g / L + agar 7 g / L; 2) Treatment group culture medium sr1: WPM + Spd 1 mg / L + sucrose 30 g / L + agar 7 g / L; sr2: WPM + Spd 2 mg / L + sucrose 30 g / L + agar 7 g / L; sr3: WPM + Spd 4 mg / L + sucrose 30 g / L + agar 7 g / L; sr4: WPM + Spd 8mg / L + sucrose 30 g / L + agar 7 g / L; sr5: WPM + SA 0.5 mg / L + sucrose 30 g / L + agar 7 g / L; sr6: WPM + SA 1 mg / L + sucrose 30 g / L + agar 7 g / L; sr7: WPM + SA 2 mg / L + sucrose 30 g / L + agar 7 g / L; sr8: WPM + SA 4 mg / L + sucrose 30 g / L + agar 7 g / L; sr9: WPM + Spm 1 mg / L + sucrose 30 g / L + agar 7 g / L; sr10: WPM + Spm 2 mg / L + sucrose 30 g / L + agar 7 g / L; sr11: WPM + Spm 4 mg / L + sucrose 30 g / L + agar 7 g / L; sr12: WPM + Spm 8 mg / L + sucrose 30 g / L + agar 7 g / L.

[0073] In the above culture medium formulations, Spd represents spermidine, Spm represents spermine, and SA represents salicylic acid. For example, sr1 medium is a solid medium obtained by adding spermidine, sucrose, and agar to WPM medium as the basal medium. The concentration of spermidine in sr1 medium is 1 mg / L, the concentration of sucrose is 30 g / L, and the concentration of agar is 7 g / L. The culture media for other treatment groups follow the same principle.

[0074] Mature embryos of Lingnan 15 were inoculated onto the above 12 treatment groups and WPM3 medium (control), respectively, in 3 replicates, with 30 embryos treated per variety and per medium per replicate.

[0075] Approximately 50 days later, the results of somatic embryo germination for the three varieties are shown in the figure. Figure 6 .

[0076] Spermine, spermine, or salicylic acid can all promote the germination of Lingnan 15 (Table 3), especially Spd or Spm. The germination rates were higher at Spd of 2 mg / L (sr2 medium) and Spm of 8 mg / L (sr12 medium), at 87.5% and 77.27%, respectively. The obtained tissue culture seedlings were robust, with approximately 4 leaves and vigorous root systems. Therefore, sr2 (WPM + Spd 2 mg / L + sucrose 30 g / L + agar 7 g / L) was determined as the optimal medium for somatic embryo germination of Lingnan 15.

[0077] Table 3. Effects of spermidine, spermine, and salicylic acid on the germination of somatic embryos of Lingnan 15.

[0078] 2. Second round of germination medium screening Based on the SR2 and SR12 media selected in the first round of screening, the concentrations of spermidine and spermine were further screened, and the effect of ornithine was also screened. WPM and WPM3 media were used as controls, and 12 treatment media were set up. The specific formulations are shown in Table 4. In the media in Table 4, Spd represents spermidine, Spm represents spermine, and ornithine represents ornithine.

[0079] Mature embryos of Fei Zi Xiao and Shang Shu Huai were inoculated onto 12 treatment culture media and two control culture media, respectively, in 3 replicates. Each replicate contained 30 embryos of each variety and each culture medium.

[0080] The germination results of the somatic embryos were statistically analyzed after about 50 days, and the results are shown in Table 4.

[0081] Table 4. Effects of spermidine, spermine, and ornithine on embryo germination of *Feizixiao* and *Shangshuhuai* embryos.

[0082] The optimal germination rate of *Smilax china* was 31.65% on germination medium OR4 (WPM + orn 8 mg / L + sucrose 30 g / L + agar 7 g / L, i.e., a solid medium obtained by adding ornithine, sucrose, and agar to WPM medium as the basal medium; the concentrations of ornithine in OR4 medium were 8 mg / L, sucrose 30 g / L, and agar 7 g / L). (Table 4) Figure 6 Therefore, or4 (WPM + orn 8 mg / L + sucrose 30 g / L + agar 7 g / L) was determined as the embryo germination medium for *Euphorbia hirta*.

[0083] On germination medium SR12 (WPM + SPM 8 mg / L + sucrose 30 g / L + agar 7 g / L, i.e., a solid medium obtained by adding spermine, sucrose and agar to WPM medium as the basal medium, with spermine concentration of 8 mg / L, sucrose concentration of 30 g / L, and agar concentration of 7 g / L in SR12 medium), Shang Shuhuai achieved the best germination rate, 16.67% (Table 4). Figure 6 Therefore, sr12 (WPM + Spm 8 mg / L + sucrose 30 g / L + agar 7 g / L) was determined to be the medium for Shangshuhuai's embryo germination.

[0084] In summary, the suspension material treated with 20 mg / L bleomycin exhibited the greatest variation, with single-cell and cell cluster survival rates exceeding 50%. After mutagenesis, callus tissue developed normally into plants via somatic embryogenesis, yielding over 160,000 plant variants. Therefore, 20 mg / L bleomycin treatment for 7 days was determined to be the optimal mutagenic dose for *Smilax china* suspension material. Furthermore, SR2 was the most effective germination medium for *Lingnan 15*, OR4 for *Smilax china*, and SR12 for *Smilax china*.

[0085] In addition, to further evaluate the mutagenesis effect, 20 individual plants were selected from each of the mutagenesis and regeneration populations of the two varieties, Feizixiao and Lingnan 15, and leaf DNA was extracted for high-throughput sequencing. The analysis focused on single nucleotide polymorphisms (SNPs) and insertion / deletion (InDel) variations.

[0086] The results showed that ( Figure 7 – Figure 10 Treatment with bleomycin effectively induced widespread mutations in the litchi genome. SNP mapping revealed numerous point mutations on different chromosomes in the induced plants, including transitions and transversions, with significantly different distribution frequencies, indicating that bleomycin has a specific damaging effect on the DNA strand. InDel mapping further confirmed that, in addition to point mutations, the mutation also caused numerous insertion and deletion events. These variations were mostly concentrated in intergenic regions or intronic regions, but some were also located in exon regions, potentially affecting gene function.

[0087] The combined results of variation detection in the two varieties showed that bleomycin-induced mutagenesis produced abundant genetic variation in regenerated litchi plants, with a non-uniform mutation spectrum, indicating that this mutagen has good mutagenic efficiency and mutation diversity in litchi. These results provide important genetic resources and a theoretical basis for subsequent screening of superior mutants, discovery of functional genes, and molecular breeding of litchi.

[0088] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, contents, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some basic features can be applied within the scope of the following appended claims.

Claims

1. A method for producing litchi mutants, characterized in that, The process includes the following steps: 7 days of mutagenesis of litchi embryogenic callus was performed using a liquid containing 20 mg / L bleomycin; after recovery culture, the obtained mutagenesis of embryogenic callus was differentiated and matured to obtain mature embryos; the mature embryos were germinated and cultured in germination medium until regenerated plants were obtained, and the obtained regenerated plants are litchi mutants or candidates. The germination medium is a solid medium obtained by adding any one of ornithine, spermine, and spermidine to WPM medium as the basic medium, wherein the concentration of ornithine, spermine, or spermidine is 2-8 mg / L.

2. The method according to claim 1, characterized in that, The liquid containing 20 mg / L bleomycin is a suspension medium containing 20 mg / L bleomycin; the suspension medium containing 20 mg / L bleomycin is obtained by adding bleomycin to a suspension medium to make the bleomycin concentration 20 mg / L; the suspension medium is obtained by adding 2,4-D, inositol, coconut milk and sucrose to MS liquid medium to make the 2,4-D concentration 1 mg / L, the inositol concentration 150 mg / L, the coconut milk concentration 50 mL / L, and the sucrose concentration 20 g / L.

3. The method according to claim 2, characterized in that, The litchi is the litchi variety Fei Zi Xiao, and the germination medium is a solid medium obtained by adding ornithine, sucrose and agar to WPM liquid medium, so that the concentration of ornithine is 8 mg / L, the concentration of sucrose is 30 g / L and the concentration of agar is 7 g / L.

4. The method according to claim 2, characterized in that, The litchi is the litchi variety Shangshuhuai, and the germination medium is a solid medium obtained by adding spermine, sucrose and agar to WPM liquid medium, so that the concentration of spermine is 8 mg / L, the concentration of sucrose is 30 g / L and the concentration of agar is 7 g / L.

5. The method according to claim 2, characterized in that, The litchi is the litchi variety Lingnan 15. The germination medium is a solid medium obtained by adding spermidine, sucrose and agar to WPM liquid medium, so that the concentration of spermidine is 2 mg / L, the concentration of sucrose is 30 g / L and the concentration of agar is 7 g / L.

6. The method according to any one of claims 1-5, characterized in that, The recovery culture is a step of culturing the induced embryogenic callus in a proliferation medium; the proliferation medium is a solid medium obtained by adding 2,4-D, sucrose and gellan gum to MS liquid medium, so that the concentration of 2,4-D is 1 mg / L, the concentration of sucrose is 30 g / L, and the concentration of gellan gum is 3.5 g / L. The differentiation is the step of differentiating the embryogenic callus after recovery culture into a somatic embryo differentiation medium to obtain a somatic embryo; the somatic embryo differentiation medium is a solid medium obtained by adding NAA, KT, inositol, LH, coconut milk, sucrose and agar to MS liquid medium, so that the concentration of NAA is 0.1 mg / L, the concentration of KT is 5 mg / L, the concentration of inositol is 100 mg / L, the concentration of LH is 0.4 g / L, the concentration of coconut milk is 100 mL / L, the concentration of sucrose is 60 g / L and the concentration of agar is 10 g / L; The maturation process involves culturing the somatic embryos in maturation medium C19 until mature somatic embryos are obtained. The maturation medium C19 is a solid culture medium obtained by adding IAA, ABA, sucrose and agar to MS liquid medium, such that the concentration of IAA is 0.5 mg / L, the concentration of ABA is 1 mg / L, the concentration of sucrose is 60 g / L, and the concentration of agar is 10 g / L.

7. The method according to claim 6, characterized in that, The embryogenic callus is an embryogenic callus obtained by proliferation culture in the proliferation medium; the method further includes a step of first suspending the proliferating culture in the suspension medium before mutagenesis.

8. The method according to claim 7, characterized in that, The mutagenesis, proliferation culture, suspension culture, recovery culture, differentiation culture, and maturation culture are all carried out under dark conditions at 23℃-27℃; the germination culture is carried out under normal light conditions at 23℃-27℃, wherein the normal light is 15-17 hours / day and the light intensity is 2000-5000 Lux.

9. The application of the method according to any one of claims 1-8 in litchi breeding.

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