An Agrobacterium-mediated genetic transformation method for macadamia nuts

Abstract

This invention discloses an Agrobacterium-mediated genetic transformation method for macadamia nuts: (1) pre-culturing macadamia nut explants on callus induction medium to obtain pre-cultured explants; (2) transferring a plant expression vector carrying the target gene into Agrobacterium to obtain engineered bacteria, and immersing the explants in an infection solution containing engineered bacteria for infection; (3) transferring to a co-culture medium and culturing in the dark; (4) conducting alternating light and dark culture in a shoot induction medium, screening to obtain positive shoots, transferring them to a shoot elongation medium for culture, and transferring the elongated seedlings to a rooting medium for culture. This invention achieves efficient regeneration of macadamia nuts through in vitro culture of macadamia cotyledons, and establishes the first Agrobacterium-mediated genetic transformation of macadamia nuts by using visual markers and non-selective screening agents; the method of this invention can rapidly and effectively induce macadamia nut cotyledon explants to produce callus with regenerative potential, and use it to obtain transgenic regenerated plants, with simple operation.

Description

Technical Field

[0001] This invention relates to the fields of plant biotechnology and molecular breeding, specifically to an Agrobacterium-mediated genetic transformation method for macadamia nuts. Background Technology

[0002] Macadamia spp., also known as Hawaiian nut, Queensland chestnut, and Australian walnut, is a perennial evergreen fruit tree belonging to the genus Macadamia in the family Proteaceae. Native to the tropical rainforests of eastern Australia, it has been domesticated and widely cultivated as an economic tree species in South Africa, China, Australia, Kenya, and the United States. Macadamia kernels have excellent flavor and are rich in oils, primarily composed of unsaturated fatty acids, making them widely used in the food, cosmetics, and bio-health industries, thus possessing high economic value. However, traditional macadamia breeding is limited by its long juvenile stage, narrow genetic background, and high heterozygosity, resulting in low efficiency in selecting new varieties. While genetic transformation technology provides a pathway for efficient trait improvement and breeding, woody plants have long faced bottlenecks such as a lack of plant regeneration systems, strong genotype dependence, and low transformation efficiency. Currently, a stable and efficient macadamia plant regeneration system and usable genetic transformation technologies are lacking, severely limiting the analysis of its functional genes and the progress of molecular breeding. Summary of the Invention

[0003] To address the technical problems of difficult macadamia plant regeneration and low genetic transformation efficiency in the prior art, this invention provides an Agrobacterium-mediated macadamia genetic transformation method, aiming to obtain an efficient and stable in vitro macadamia plant regeneration and genetic transformation system.

[0004] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0005] An Agrobacterium-mediated genetic transformation method for macadamia nuts includes the following steps:

[0006] (1) Pre-culture: Macadamia nut explants were pre-cultured on callus induction medium for 7-28 days to obtain pre-cultured explants; the callus induction medium contained thiazophenylurea (TDZ) and nonpiperidine (FPX); the concentration of thiazophenylurea (TDZ) in the medium was 15-30 μM, and the concentration of nonpiperidine (FPX) in the medium was 10-20 μM.

[0007] (2) Infection: The plant expression vector carrying the target gene is transferred into Agrobacterium to obtain engineered bacteria. The explants obtained from the pre-culture in step (1) are immersed in the infection solution containing engineered bacteria for infection treatment. The infection solution is a mixture of resuspension and Agrobacterium. The OD600 value of the mixture is 0.5. The resuspension is prepared by 10 mM 2-morpholine ethanesulfonic acid (MES), 10 mM MgSO4 and 100 μM acetylsyl syringone.

[0008] (3) Co-culture: The infected explants from step (2) were transferred to a co-culture medium and co-cultured in the dark;

[0009] (4) Screening and regeneration: The explants obtained after co-culture in step (3) are transferred to the shoot induction medium for alternating light and dark culture to induce the formation of shoot primordia and differentiation of shoots. Positive transgenic regenerated shoots are screened and transferred to the shoot elongation medium for culture. The elongated transgenic regenerated shoots are transferred to the rooting medium for culture to induce rooting and obtain transgenic positive seedlings.

[0010] Preferably, the macadamia nut explants in step (1) are obtained by selecting mature macadamia kernels, disinfecting them, removing the cotyledons, cutting off the embryo and surface tissue, and cutting the cotyledons into pieces; wherein, the size of the explants is 1~2 cm × 1~2 cm × 0.5~1 cm, preferably 1.5 cm × 1.5 cm × 0.5 cm; the mature macadamia kernels are selected from macadamia germplasm HAES900.

[0011] Preferably, the callus induction medium in step (1) comprises MS medium, additives, thiazophenylurea (TDZ) and nonpiperidine (FPX); the concentration of thiazophenylurea (TDZ) in the callus induction medium is 30 μM; the concentration of nonpiperidine (FPX) in the callus induction medium is 15 μM; the pre-culture temperature in step (1) is 22°C, and the pre-culture time is 14-21 days.

[0012] Preferably, the contamination process in step (2) includes vacuum-assisted impregnation and oscillation contamination, i.e., vacuum treatment at -80 kPa for 5 minutes, followed by oscillation contamination at 28 ℃ and 120 rpm for 30 minutes.

[0013] Preferably, the plant expression vector carrying the target gene in step (2) is the pKSE402 vector carrying the reporter gene eGFP and the kanamycin resistance gene NPTII, or the pGNP-PAP2 vector carrying the Arabidopsis anthocyanin-regulated transcription factor gene AtPAP2 and NPTII.

[0014] Preferably, the co-culture medium in step (3) comprises MS medium, thiazophenylurea (TDZ), acetylsyleugenone, and agar; its composition is 4.43 g·L⁻¹. -1 MS medium, 15 µM TDZ, 30 g·L -1 Sucrose, 100 µM acetylsalicylic acid, 6 g·L -1 Agar, pH=5.6; the co-culture temperature is 20-25℃, and the time is 2-4 days.

[0015] Preferably, the bud induction medium in step (4) comprises MS medium, antibacterial agent, additives and thiazophenylurea (TDZ).

[0016] Preferably, the shoot elongation medium in step (4) contains gibberellin (GA3) and termethin, wherein the concentration of gibberellin (GA3) in the shoot elongation medium is 0.10-0.20 µM; and the concentration of termethin in the shoot elongation medium is 300 mg·L⁻¹. -1 The rooting medium contains indolebutyric acid (IBA), and the concentration of indolebutyric acid (IBA) in the shoot elongation medium is 3-7 µM.

[0017] Preferably, the shoot elongation medium comprises MS medium, gibberellin (GA3), and additives, wherein the concentration of gibberellin (GA3) in the shoot elongation medium is 0.14 μM; the rooting medium comprises 1 / 2 MS medium and indolebutyric acid (IBA); wherein the concentration of indolebutyric acid (IBA) in the shoot elongation medium is 5 µM.

[0018] Preferably, the alternating light and dark culture in step (4), the culture conditions in the shoot elongation medium, and the culture conditions in the rooting medium are all alternating light and dark conditions of 16 hours / 8 hours, with the light intensity being approximately 50 μmol·m⁻¹. -2 ·s -1 The temperature is 25 ℃.

[0019] Preferably, the additive is one or more of sucrose, coconut powder, or agar.

[0020] The plant expression vector carries a visual screening marker gene; the visual screening marker gene is a fluorescent protein gene (eGFP) or an anthocyanin synthesis-related regulatory gene (AtPAP2); the screening in step (4) is to directly screen positive buds and plants by visually observing color or fluorescence, that is, during the bud induction and elongation stage, by visually observing or irradiating with a handheld excitation light source, buds and plants with characteristic colors (such as red) or emitting characteristic fluorescence (such as green fluorescence) are directly screened, and non-positive regenerated buds are removed.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention achieves efficient regeneration of macadamia nuts through in vitro culture of cotyledons, uses visual markers without screening agents to screen transgenic regenerated shoots, and establishes Agrobacterium-mediated genetic transformation of macadamia nuts for the first time. Furthermore, the method of this invention can rapidly and effectively induce macadamia nut cotyledon explants to produce callus tissue with regenerative potential, and use it to obtain transgenic regenerated plants. The operation is simple. Attached Figure Description

[0023] Figure 1 It refers to the cotyledon regeneration capacity of six representative macadamia nut genotypes.

[0024] Figure 2 This describes the effects of different treatments on the regeneration of macadamia cotyledon buds.

[0025] Figure 3 The effects of different treatments (FPX, 4-Cl-IAA, and NPA) on the in vitro regeneration efficiency of macadamia cotyledons are shown; the blue bars represent the left Y-axis, and the yellow bars represent the right Y-axis.

[0026] Figure 4 The effects of pre-culture time (A) and infection time (B) on conversion efficiency are represented, respectively.

[0027] Figure 5 Phenotypic characteristics of leaf-like organs obtained from direct organogenesis under bright field (A) and excited field (B), and PCR identification of the target gene eGFP (C).

[0028] Figure 6 This describes the developmental process of AtPAP2 transgenic macadamia nut plants.

[0029] Figure 7 It is the regeneration of wild-type (WT) and AtPAP2-OE transgenic macadamia nut buds.

[0030] Figure 8 This is a qRT-PCR analysis of AtPAP2 transcription levels in wild-type (WT) and transgenic lines (#1–#4).

[0031] Figure 9 This is the ImmunoStrip® kit (for neomycin phosphotransferase II (NPTII), STX 73000 / 0050, Agdia, USA) for detecting NPTII protein expression.

[0032] Figure 10This is a sequencing chromatogram of genomic DNA near the T-DNA insertion site in AtPAP2-OE-#1, showing a clear single peak; the vertical line indicates the junction between the macadamia nut genomic region and the T-DNA insertion site.

[0033] Figure 11 It is the sequence of genomic DNA near the T-DNA insertion site in AtPAP2-OE-#1.

[0034] Figure 12 The sequence of genomic DNA near the T-DNA insertion site in AtPAP2-OE-#1. Detailed Implementation

[0035] The following detailed description, in conjunction with the accompanying drawings, outlines specific embodiments. However, it should be understood that the scope of protection of this invention is not limited to these specific embodiments. Unless otherwise specified, all raw materials and reagents used in the examples are commercially available.

[0036] The macadamia nut varieties HAES333, HAES660, HAES695, HAES800, HAES900, GR1, H2, JW, and OC are commercially available.

[0037] The culture media in the examples are as follows:

[0038] The callus induction medium consisted of MS medium, additives, thiazophenylurea (TDZ), and nonpiperidine (FPX), with a specific composition of 4.43 g·L⁻¹. -1 MS medium, 15 µM TDZ, 15 µM FPX, 30 g·L⁻¹ -1 Sucrose, 10 g·L -1 Coconut powder, 6 g·L -1 Agar, pH=5.8

[0039] The co-culture medium (CCM) consisted of MS medium, thiazophenylurea (TDZ), acetylsyleugenone, and agar, with a specific composition of 4.43 g·L⁻¹. -1 MS medium, 15 µM TDZ, 30 g·L -1 Sucrose, 100 µM acetylsalicylic acid, 6 g·L -1 Agar, pH=5.6.

[0040] The bud induction medium consisted of MS medium, antibacterial agent, additives, and thiazophenylurea (TDZ), with a specific composition of 4.43 g•L. -1 MS, 300 mg·L -1 Antibacterial agent, 15 µM TDZ, 30 g•L -1 Sucrose, 10 g•L -1Coconut powder, 6 g•L -1 Agar, pH=5.8; antibacterial agents are termethin or cefotaxime, used alternately.

[0041] The shoot elongation medium consisted of MS medium, gibberellin (GA3), additives, and termethin, with a specific composition of 4.43 g·L⁻¹. -1 MS medium, 0.14 µM GA3, 30 g·L -1 Sucrose, 5 g·L -1 Coconut powder, 6 g·L -1 Agar, 300 mg·L -1 Termetin, pH=5.8.

[0042] Rooting medium: 2.47 g·L -1 ½ MS medium, 5 µM IBA, 30 g·L -1 Sucrose, 6 g·L -1 Agar, pH=5.8.

[0043] Example 1

[0044] Screening of macadamia nut regeneration potential germplasm and establishment of in vitro regeneration system

[0045] 1.1 Experimental Materials

[0046] The test materials consisted of 27 different genotypes of macadamia (Macadamia spp.), whose fruits were harvested from macadamia planting bases in Guangxi and Yunnan provinces during August and September. Healthy, disease-free, and mature fresh fruits were selected, and the green husk and hard shell were removed to extract the kernels for later use.

[0047] 1.2 Explant preparation and disinfection

[0048] After rinsing the kernels thoroughly with running water, immerse them in commercial 84 disinfectant (approximately 5% effective chlorine content) in a clean bench for 1 hour, shaking and agitating them every 15-20 minutes. After disinfection, discard the disinfectant solution and rinse the kernels 3-4 times with sterile water to thoroughly remove any residual disinfectant. Use a sterile scalpel to remove the seed coat and embryo from the kernel surface, and cut the cotyledons into pieces approximately 1.5 cm × 1.5 cm × 0.5 cm in size to serve as explants.

[0049] 1.3 Culture medium preparation

[0050] The composition of the culture medium used in this embodiment is shown in Table 1. All culture media were autoclaved at 121°C for 20 min.

[0051] Table 1: Culture Medium Composition Table

[0052]

[0053] 1.4 In vitro regeneration culture

[0054] The explants prepared in step 1.2 were inoculated onto callus induction medium (CIM) and cultured in the dark at 25°C for 4 weeks to induce callus formation. After 4 weeks, the explants that produced callus were transferred to shoot induction medium (SIM) and then placed in a light incubator with a 16-hour light / 8-hour dark cycle and a light intensity of approximately 50 μmol•m. -2 •s -1 At a temperature of 25 ℃, shoot differentiation was induced, with the culture medium replaced with fresh medium every 20 days. After approximately 4 weeks, the number of shoot primordia formed on the surface of each explant was counted and recorded. Explants that had differentiated shoot primordia were then transferred to shoot elongation medium (SEM) and cultured under the same alternating light and dark conditions, subculturing every 20 days until shoot elongation formed seedlings with distinct nodes. The average number of elongated shoots on each explant was counted. The elongated seedlings were then cut and transferred to rooting medium (RIM) for root induction, under the same culture conditions as above.

[0055] 1.5 Results Analysis

[0056] Experimental results are as follows Figure 1 (#0547, F3, HAES800, HAES900, GR1, and OC are representative) and as shown in Table 2, cotyledonary explants from all 27 germplasms were able to induce callus tissue on CIM, with induction rates ranging from 79.09% to 95.33%, showing no significant difference (P < 0.05). However, after transformation into SIM, the number of induced shoot primordia among different germplasms showed significant differences (P < 0.05). Germplasm #0057 had the highest number of induced shoot primordia (approximately 10 per explant), while germplasm OV had the lowest (approximately 1.65 per explant). During the shoot elongation stage, germplasm #0547 had the highest average number of shoots, reaching 7.99, while germplasm OV remained the lowest, with only 1.27 shoots. In summary, germplasm #0057, #0547, HAES800, HAES900, HAES695, and F3 exhibited high regeneration potential, making them ideal recipient materials for genetic transformation. These results confirm the necessity of screening for genotypes with high regeneration capacity.

[0057] Table 2. Statistical analysis of cotyledon regeneration efficiency in subculture of different macadamia nut genotypes

[0058]

[0059]

[0060]

[0061]

[0062] In Table 1, the germplasm starting with "#" comes from the Yunnan Tropical Crops Research Institute; "E9" and "F3" come from the South Subtropical Crops Research Institute of Guangxi Academy of Agricultural Sciences in Longzhou City, Guangxi; the remaining germplasm was provided by Guangxi Xiaguo Planting Co., Ltd. in Fusui County, Guangxi.

[0063] Example 2

[0064] The promoting effect of regeneration inducer FPX on in vitro regeneration of macadamia nuts

[0065] 2.1 Experimental Materials and Methods

[0066] Mature cotyledons of HAES900, a high regeneration potential germplasm screened in Example 1, were used as explants. The explant preparation method was the same as 1.2 in Example 1.

[0067] Different callus induction media: Based on the basal medium (containing 15 μM TDZ), different concentrations of FPX (15, 30, 50 μM), 4-Cl-IAA (4-chloroindole-3-acetic acid) (10, 15 μM), and NPA (naphthylazine) (10 μM) were added to create treatment groups. The medium containing only 15 μM TDZ served as the control group.

[0068] Explants were first inoculated into callus induction medium (CIM) for each of the above treatment groups and cultured in the dark for 10 days. After 10 days, the explants were transferred to conventional CIM / SIM medium containing 15 μM TDZ but without the corresponding regulator, and cultured in the dark to complete callus induction. Subsequently, they were transferred to light for shoot induction and differentiation. The remaining culture steps were the same as in Example 1. The callus induction rate, the number of induced shoots, and the rooting status were recorded.

[0069] 2.2 Results Analysis

[0070] Experiments showed that the callus induction rate was above 80% in all treatment groups, with no significant difference. However, significant differences were observed among the different treatments during the shoot induction stage:

[0071] a. FPX treatment group: Explants treated with 15 μM and 30 μM FPX, after being transferred to conventional culture medium, showed significantly different morphology of the bud primordia formed on the callus surface (type II) compared to the control group (type I), being more dense and having more bud points. Figure 2 Statistical results showed that the average number of buds in the 15 μM and 30 μM FPX treatment groups reached 7.7 and 14.9, respectively, with the latter significantly higher than the approximately 5-6 in the control group (based on previous experimental data, compared with the HAES900 data in Table 2). Figure 3When the FPX concentration increased to 50 μM, although the callus induction rate slightly increased, bud differentiation was completely inhibited, and the callus continued to proliferate without forming buds. Figure 3 ).

[0072] b. 4-Cl-IAA treatment group: Under 10 μM 4-Cl-IAA treatment, the average number of buds induced was 4.8, which was not significantly higher than that of the control group. However, some explants under this treatment simultaneously induced buds and roots. Figure 3 When the concentration of 4-Cl-IAA was increased to 15 μM, the explants completely lost their ability to differentiate into shoots, and only differentiated into roots. Figure 2 ,3).

[0073] c. NPA treatment group: Adding 10 μM NPA alone had no significant effect on bud induction efficiency, and was comparable to the control group. Figure 3 ).

[0074] These results indicate that FPX is a highly efficient regeneration inducer, which can significantly enhance the shoot regeneration ability of macadamia nuts at appropriate concentrations (especially 30 μM), overcoming the limitations of the traditional TDZ system. Meanwhile, 4-Cl-IAA showed strong root induction potential.

[0075] Example 3

[0076] Establishment of an Agrobacterium-mediated macadamia nut genetic transformation system

[0077] 3.1 Plant expression vectors and Agrobacterium strains

[0078] This embodiment uses two binary vectors: the pKSE402 vector carrying the reporter gene eGFP and the kanamycin resistance gene NPTII, and the pGNP-PAP2 vector carrying the Arabidopsis anthocyanin-regulating transcription factor genes AtPAP2 and NPTII. The vectors were transformed into competent cells of Agrobacterium tumefaciens strain EHA105 using a freeze-thaw method, in a solution containing 50 μg / mL of [amount missing]. -1 Rifampicin 50 μg·mL -1 Positive clones were screened on YEB plates (commercially available YEB solid medium) containing kanamycin and verified by PCR. Single positive clones were picked for subsequent experiments.

[0079] 3.2 Optimization of transformation parameters (taking pKSE402-eGFP as an example)

[0080] Cotyledonary explants from germplasm HAES900 were used as material, sterilized and cut into pieces according to the method in Example 1. Optimization experiments were conducted for the following parameters, with at least three replicates per group and 10 explants per replicate. Transformation efficiency was assessed as the percentage of callus exhibiting green fluorescence under a stereofluorescence microscope within 4 weeks after co-culture.

[0081] Pre-culture time: Explants were pre-cultured on CIM for 0, 7, 14, 21, and 28 days respectively before infection. Infection conditions were uniformly set as follows: OD 600 =0.5, after vacuum (-80 kPa, 5 min) assisted infiltration, inoculate at 28 ℃ with shaking at 120 rpm for 30 min. After co-culturing for 3 days, transfer to a solution containing 300 mg·L⁻¹ -1 Timentin was used for recovery culture and screening in SIM.

[0082] Infection time: Explants pre-cultured for 14 days were inoculated with the same infection solution concentration and under vacuum conditions, and then shaken for 10, 20, 30, and 40 min respectively. Subsequent treatments were the same as above.

[0083] 3.3 Genetic transformation process

[0084] 3.3.1 Preparation of engineered bacteria: EHA105 glycerol bacteria containing the target vector, stored at -80℃, were streaked onto YEB plates containing the corresponding antibiotics for activation and cultured at 28℃ for 2 days. Single colonies were picked and inoculated into 5 mL of YEB liquid medium (containing antibiotics) and cultured overnight at 28℃ and 220 rpm. The culture was then transferred to fresh YEB liquid medium at a 1:50 ratio and cultured until the logarithmic growth phase. The cells were collected by centrifugation at 5000 rpm for 10 min at 4℃ and resuspended in a resuspension buffer (10 mM MES, 10 mM MgSO4, 100 μM acetylsyleugenol, pH=5.6) to OD200. 600 =0.5, let stand at 28 ℃ for 1 hour to activate, and the infection solution is obtained for later use.

[0085] 3.3.2 Pre-culture and infection: The sterilized cotyledon explants were pre-cultured on CIM at 22℃ for 14-21 days. The pre-cultured explants were then placed in the infection solution prepared in step 3.3.1, and subjected to vacuum treatment (-80 kPa, 5 min), followed by inoculation at 120 rpm in a shaker at 28℃ for 20-30 min.

[0086] 3.3.3 Co-culture: Remove the infected explants, blot the surface bacterial solution with sterile filter paper, dry them in a laminar flow hood, and then transfer them to co-culture medium (CCM) and co-culture them at 22 ℃ in the dark for 3 days.

[0087] 3.3.4 Screening and Regeneration: After co-culture, the explants were removed, the surface bacterial culture was blotted dry with sterile filter paper, and after drying, they were transferred to a solution containing 300 mg·L⁻¹ of bacterial culture. -1 In the bud induction medium (SIM) for termethin (or cefotaxime), the light intensity was approximately 50 μmol•m², with a light intensity of 16 hours of light followed by 8 hours of darkness. -2 •s -1 Shoot induction was performed at a temperature of 25 ℃. The culture medium was replaced with fresh medium every 20 days.

[0088] ① For the pKSE402-eGFP transformation group, observe the green fluorescence using a handheld excitation light source or a fluorescence stereomicroscope during the culture process, and record the time and location of the fluorescence appearance.

[0089] ② For the pGNP-PAP2 transformation group, during the culture process, purplish-red buds and callus tissues were directly selected by visual observation and separated from non-transgenic green buds for separate subculture. Figure 6 , 7 ).

[0090] 3.3.5 Rooting and Transplanting: The selected positive buds (red or green fluorescence) were transferred to bud elongation medium (SEM, containing 300 mg·L⁻¹) -1 Temitin promotes elongation.

[0091] The elongated seedlings are then transferred to a rooting medium to induce rooting. Positive regenerated plants are selected during the culture process.

[0092] 3.4 Results Analysis

[0093] The effect of pre-culture time: The results showed ( Figure 4 A) Explants without pre-culture (day 0) showed high contamination rates and difficulty in callus formation. With prolonged pre-culture time, the GFP-positive callus rate gradually increased, peaking at 29.7% and 26.4% at 14 and 21 days of pre-culture, respectively. The positivity rate significantly decreased at 28 days of pre-culture. This indicates the existence of an "optimal transformation age window," where callus tissue pre-cultured for 14-21 days is most receptive to exogenous genes.

[0094] The effect of infection time: The results showed ( Figure 4(B) At 10 min of infection, the GFP positivity rate was low. The positivity rate increased with prolonged infection time, reaching its peak at 30 min (approximately 29.4%). However, at 30 min and beyond, explant browning and Agrobacterium overgrowth intensified, especially at 40 min, resulting in high explant mortality. Considering both transformation efficiency and explant damage, an infection time of 20 min achieved both high transformation efficiency (approximately 20-25%) and good control over contamination and browning.

[0095] Obtaining transgenic plants: Under optimized conditions, green fluorescence was successfully detected in callus tissue and leaves via pKSE402-eGFP transformation. Figure 5 A, B), and the eGFP-specific band was amplified by PCR ( Figure 5 C) confirmed the integration of exogenous genes.

[0096] Obtaining AtPAP2 gene-transformed plants: Five independent transformation experiments were conducted using pGNP-PAP2 (Table 3). From 1112 explants, five regenerated plants with a distinct red phenotype were finally obtained. Figure 6 7). By qRT-PCR ( Figure 8 ), NPTII immunoassay strip test ( Figure 9 Whole genome sequencing and conjugation PCR ( Figure 10 (11, 12) Successful integration and expression of the exogenous gene were confirmed at the RNA, protein, and DNA levels. The average transformation efficiency was 1.46% (Table 3).

[0097] Table 3: Statistics on the genetic transformation efficiency of macadamia nuts with the AtPAP2 gene

[0098]

[0099] In summary, this invention has successfully established a highly efficient and stable in vitro regeneration and genetic transformation system for macadamia nuts by systematically screening high-regeneration-capacity germplasm, optimizing the regeneration culture medium (especially by introducing FPX), and finely controlling key parameters (pre-culture time and infection time) during the Agrobacterium infection process. This system, combined with visual screening markers, significantly improves transformation efficiency and lays a solid foundation for gene function research and molecular breeding of macadamia nuts.

[0100] Figure 1 The pink bars represent callus induction rate (left y-axis), and the light green bars represent the number of regenerated shoots per explant (right y-axis). Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test. Different letters indicate statistically significant differences at the P < 0.05 level.

[0101] Figure 2 Representative images showing callus formation and differentiation were displayed in culture media containing 15 µM thiamethoxam (TDZ) (left panel), 15 µM TDZ + 30 µM FPX (middle panel), and 15 µM TDZ + 15 µM 4-Cl-IAA (right panel); FPX treatment enhanced shoot regeneration.

[0102] Figure 6 The purple dashed box represents early transgenic callus tissue, and the purple arrows trace the key developmental transition from callus tissue to bud.

[0103] Figure 7 Wild-type (WT-s1, WT-s2) and transgenic (AtPAP2-OE-#1–5) shoots from cotyledonary explants. WT-s1 and WT-s2 show the same control shoots from the same experimental batch as AtPAP2-OE-#1, exhibiting identical performance at both developmental stages.

[0104] Figure 8 qRT-PCR analysis of AtPAP2 transcription levels in wild-type (WT) and transgenic lines (#1–#4). AtPAP2 transcription was successfully detected in all transgenic lines, but not in wild-type. Expression levels were normalized to the internal reference gene (ACTIN1). Data are presented as mean ± standard deviation (n=3).

[0105] Figure 9 As can be seen, specific positive bands were observed in the positive control (transiently transformed tobacco leaves, vector pGNP-AtPAP2) and all transgenic lines, while the negative control (empty-transformed tobacco and wild-type plants) showed negative results, confirming the successful expression of the selectable marker gene NPTII. Note: AtPAP2-OE-#5 was characterized only by the anthocyanin accumulation phenotype and was not included in qRT-PCR ( Figure 5 ) or NPTII protein analysis ( Figure 6 )middle.

[0106] Figure 10 A sequencing chromatogram of genomic DNA near the T-DNA insertion site in AtPAP2-OE-#1 shows a clear single peak. Vertical lines indicate the junctions between the macadamia nut genomic region and the T-DNA insertion site.

[0107] Figure 11 The sequence of genomic DNA near the T-DNA insertion site in AtPAP2-OE-#1 (sequence listing as shown in SEQ ID NO:1, where...) Figure 10 , Figure 12The sequence shown is also part of this (the T-DNA sequence is marked in red, and the macadamia nut genome sequence is shown in blue). The first base indicated by the magenta arrow is marked "1". The underlined portion of the sequence has significant features, described in detail below. Nucleotides 1–59 represent a portion of the neomycin / kanamycin resistance gene (NeoR / KanR) sequence. Nucleotides 116–175 correspond to the cauliflower mosaic virus (CaMV) 35S polymerized adenosine signal sequence. Nucleotides 412–436 are the left boundary (LB) sequence of the T-DNA, showing a 23-base-pair vector backbone DNA fragment located to the right of the LB being transferred into the plant genome. Nucleotides 437–999 belong to the macadamia nut genome.

[0108] Figure 12 The sequence of genomic DNA near the T-DNA insertion site in AtPAP2-OE-#1. The T-DNA sequence is marked in red, and the macadamia nut genome sequence is shown in blue. The first base indicated by the magenta arrow is marked "1". The underlined portion of the sequence has significant characteristics, described in detail below. Nucleotides 1–59 represent a portion of the neomycin / kanamycin resistance gene (NeoR / KanR) sequence. Nucleotides 116–175 correspond to the cauliflower mosaic virus (CaMV) 35S polymerized adenosine signal sequence. Nucleotides 412–436 are the left boundary (LB) sequence of the T-DNA, showing the 23-base-pair vector backbone DNA fragment located to the right of the LB being transferred into the plant genome. Nucleotides 437–999 belong to the macadamia nut genome.

[0109] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. An Agrobacterium-mediated genetic transformation method for macadamia nuts, characterized in that, The operation includes the following steps: (1) Pre-culture: Macadamia nut explants are pre-cultured on callus induction medium for 7-28 days to obtain pre-cultured explants; the callus induction medium contains thiazophenylurea and nonpiperidine; the concentration of thiazophenylurea in the medium is 15-30 μM, and the concentration of nonpiperidine in the medium is 10-20 μM. (2) Infection: The plant expression vector carrying the target gene is transferred into Agrobacterium to obtain engineered bacteria. The explants obtained from the pre-culture in step (1) are immersed in the infection solution containing engineered bacteria for infection treatment. The infection solution is a mixture of resuspension and Agrobacterium. The OD600 value of the mixture is 0.

5. The resuspension is prepared by 10 mM 2-morpholine ethanesulfonic acid, 10 mM MgSO4 and 100 μM acetylsyl syringone. (3) Co-culture: The infected explants from step (2) were transferred to a co-culture medium and co-cultured in the dark; (4) Screening and regeneration: The explants obtained after co-culture in step (3) are transferred to the shoot induction medium for alternating light and dark culture. Positive transgenic regenerated shoots are screened and transferred to the shoot elongation medium for culture. The elongated transgenic regenerated shoots are transferred to the rooting medium for culture and rooting to obtain transgenic positive seedlings.

2. The Agrobacterium-mediated genetic transformation method for macadamia nuts according to claim 1, characterized in that: The macadamia nut explants mentioned in step (1) are obtained by selecting mature macadamia nut kernels, disinfecting them, removing the surface tissues of the embryo and cotyledons, peeling off the cotyledons and cutting them into pieces; the mature macadamia nut kernels are selected from macadamia nut germplasm HAES900.

3. The Agrobacterium-mediated genetic transformation method for macadamia nuts according to claim 1, characterized in that: The callus induction medium in step (1) comprises MS medium, additives, thiazophenylurea and nonpiperidine; the additives are one or more of sucrose, coconut powder or agar; the concentration of thiazophenylurea in the callus induction medium is 30 μM; the concentration of nonpiperidine in the callus induction medium is 15 μM; the pre-culture temperature in step (1) is 22℃, and the pre-culture time is 14-21 days.

4. The Agrobacterium-mediated genetic transformation method for macadamia nuts according to claim 1, characterized in that: The contamination process described in step (2) includes vacuum-assisted impregnation and oscillation contamination, namely, vacuum treatment at -80 kPa for 5 minutes, followed by oscillation contamination at 28°C and 120 rpm for 30 minutes.

5. The Agrobacterium-mediated genetic transformation method for macadamia nuts according to claim 1, characterized in that: The plant expression vector carrying the target gene mentioned in step (2) is either the pKSE402 vector carrying the reporter gene eGFP and the kanamycin resistance gene NPTII, or the pGNP-PAP2 vector carrying the Arabidopsis anthocyanin-regulated transcription factor genes AtPAP2 and NPTII.

6. The Agrobacterium-mediated genetic transformation method for macadamia nuts according to claim 1, characterized in that: The co-culture medium described in step (3) comprises MS medium, thiazophenylurea, acetylsyleugenone, and agar; its composition is 4.43 g·L⁻¹. -1 MS medium, 15 µM TDZ, 30 g·L -1 Sucrose, 100 µM acetylsalicylic acid, 6 g·L -1 Agar, pH=5.6; the co-culture temperature is 20-25℃, and the time is 2-4 days.

7. The Agrobacterium-mediated genetic transformation method for macadamia nuts according to claim 1, characterized in that: The bud induction medium in step (4) comprises MS medium, antibacterial agent, additives and thiazophenylurea; the additives are one or more of sucrose, coconut powder or agar.

8. The Agrobacterium-mediated genetic transformation method for macadamia nuts according to claim 1, characterized in that: The shoot elongation medium in step (4) contains gibberellin and termethin, wherein the concentration of gibberellin in the shoot elongation medium is 0.10-0.20 µM; and the concentration of termethin in the shoot elongation medium is 300 mg·L. -1 The rooting medium contains indolebutyric acid, and the concentration of indolebutyric acid in the shoot elongation medium is 3-7 µM.

9. The Agrobacterium-mediated genetic transformation method for macadamia nuts according to claim 8, characterized in that: The shoot elongation medium comprises MS medium, gibberellin, and additives, wherein the gibberellin concentration in the shoot elongation medium is 0.14 μM; the rooting medium comprises 1 / 2 MS medium and indolebutyric acid; wherein the indolebutyric acid concentration in the shoot elongation medium is 5 µM; the additives are one or more of sucrose, coconut powder, or agar.

10. The Agrobacterium-mediated genetic transformation method for macadamia nuts according to claim 1, characterized in that: In step (4), the alternating light and dark culture conditions, the culture conditions in the shoot elongation medium, and the culture conditions in the rooting medium are all alternating light and dark conditions of 16 hours / 8 hours, with a light intensity of approximately 50 μmol·m⁻¹. -2 ·s -1 The temperature is 25 ℃.

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