Method for improving drought tolerance of macadamia rootstock by overexpressing minac2a gene
By overexpressing the MiNAC2a gene through genetic engineering, the problem of insufficient drought resistance in macadamia nuts has been solved, and new drought-resistant varieties with inherent high efficiency and stability have been bred. These varieties are adapted to arid environments, shorten the breeding cycle, and improve breeding efficiency and trait purity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH ASIAN TROPICAL AGRI SCI RES INST OF GUANGXI
- Filing Date
- 2026-02-04
- Publication Date
- 2026-05-29
Smart Images
Figure CN122104773A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of planting technology, and in particular to a method for overexpressing the MiNAC2a gene to improve the drought resistance of macadamia rootstock. Background Technology
[0002] Macadamia nuts, also known as macadamia nuts, originated in the tropical rainforests of northeastern Australia. They are a nutritious and economically valuable tropical and subtropical crop. Since their introduction to my country in the 20th century, they have been widely cultivated in provinces with suitable climates, such as Guangxi, Yunnan, and Guizhou, gradually forming a considerable planting scale and continuously expanding the industry's scope. While these regions possess suitable temperature and sunlight conditions, problems such as seasonal drought or uneven interannual rainfall are prominent. Macadamia nuts are highly sensitive to water during critical growth stages; drought easily inhibits plant growth and poor fruit development, directly affecting nut yield and quality, becoming one of the important factors restricting further expansion and improving the industry's quality and efficiency. Therefore, systematically conducting research on the drought resistance mechanism of macadamia nuts and tackling drought-resistant technologies, including breeding drought-resistant varieties, optimizing water-saving irrigation models, and improving field management measures, is of significant practical importance and long-term value for enhancing the adaptability of macadamia nuts under drought adversity, achieving stable and high yields, and promoting the scientific layout and sustainable development of the industry.
[0003] Grafting, an ancient agricultural technique, has always carried the important mission of preserving and propagating superior varieties. In traditional practice, the focus has been primarily on the scion—the branch bearing high-quality fruit, vibrant flowers, or unique shapes—hoping to perpetuate its characteristics through grafting. In contrast, the rootstock often plays a neglected "behind-the-scenes" role. However, the rootstock is an indispensable cornerstone of the grafting system. It typically originates from seedlings and possesses comprehensive advantages such as well-developed root systems, strong growth vigor, good environmental adaptability, and high stress resistance. It not only delivers water and nutrients to the scion but also directly affects the survival rate, growth rate, and long-term health of the grafted seedling. Therefore, the selection and cultivation of rootstock are crucial to successful grafting. Especially against the backdrop of intensifying global climate change and increasing drought frequency, cultivating rootstocks with outstanding drought resistance is particularly urgent. These rootstocks, with their strong root water absorption capacity and physiological drought tolerance mechanisms, can effectively mitigate the impact of water stress on the scion, significantly improving the survival rate and growth level of grafted seedlings under drought conditions, thereby ensuring the stability and sustainability of agricultural production. In the future, while continuing to optimize the traits of scions, strengthening research on the resistance, adaptability, and interaction mechanisms between rootstocks and scions will be an important direction for promoting the advancement of grafting technology.
[0004] Currently, the most widely used drought-resistant technologies in agricultural production to cope with drought stress mainly include water-saving irrigation technologies such as drip irrigation, and chemical regulation methods that enhance the plant's own stress resistance by spraying exogenous growth regulators. However, for macadamia trees, which are widely planted in mountainous and hilly areas, these conventional technologies face significant practical challenges. On the one hand, due to their complex site conditions and steep slopes, not only are the construction and maintenance of irrigation systems costly, but the soil's water retention capacity is also poor. In the event of prolonged and severe seasonal droughts, the limited amount of drip irrigation water is often insufficient to meet the trees' needs, resulting in a significant reduction in water use efficiency. On the other hand, mountainous terrain makes mechanical operations difficult. Large-scale and frequent spraying of growth regulators is inconvenient and costly, and its effectiveness is often affected by environmental factors, making it difficult to achieve stable and lasting endogenous drought resistance enhancement. Summary of the Invention
[0005] The purpose of this invention is to provide a method for overexpressing the MiNAC2a gene to improve the drought resistance of macadamia rootstock, thereby solving the technical problem of poor drought resistance in existing macadamia trees.
[0006] To fundamentally improve the drought resistance of macadamia nuts, it is necessary to move beyond external intervention and delve deeper into the plant's own genetic potential. Using modern, mature genetic engineering techniques, we can precisely introduce drought-related genes (such as key genes related to osmotic regulation, stomatal opening and closing, root development, and antioxidant protection) from other organisms or through artificial modification into the somatic cells of macadamia trees. This approach can genetically strengthen the physiological and molecular mechanisms that respond to water stress, such as promoting root extension into deeper soil layers, reducing water transpiration, and enhancing cellular water retention capacity, thereby cultivating new varieties with intrinsic, efficient, and stable drought resistance. This plant-based genetic engineering improvement strategy provides a highly promising fundamental solution for achieving sustainable and stable macadamia production in arid mountain environments. Furthermore, modern transgenic and gene-editing technologies can be used to enhance the drought resistance of macadamia rootstocks by overexpressing the MiNAC2a gene.
[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0008] A method for improving drought resistance in macadamia rootstock by overexpressing the MiNAC2a gene, the method comprising the following steps:
[0009] Step 1: Obtain sterile seedling cotyledons from macadamia nut seeds;
[0010] Step 2: Induce callus tissue using cotyledons of sterile seedlings;
[0011] Step 3: Based on the obtained MiNAC2a gene sequence, construct an overexpression vector and transform it into Agrobacterium;
[0012] Step 4: Inject Agrobacterium into macadamia nut callus tissue;
[0013] Step 5: Cultivate positive callus tissue to grow adventitious buds and roots;
[0014] Step 6: Extract DNA from seedlings and verify whether they are truly positive plants by detecting the bands.
[0015] Step 7: Harden off the seedlings and transplant them to the field to obtain genetically modified drought-resistant rootstock resources;
[0016] Step 8: Strengthen the root system of the transgenic seedlings and improve the drought resistance of the rootstock.
[0017] Furthermore, the specific process of step 1 is as follows:
[0018] Step 1.1: Seed selection and treatment. First, select healthy, mature Guire No. 1 macadamia seeds. Mature seeds have plump kernels and hard shells.
[0019] Step 1.2: Sun-drying the seeds. After harvesting, expose the seeds to the sun for 48-72 hours.
[0020] Step 1.3: Remove the hard shell from the seeds to facilitate sowing and improve the germination rate;
[0021] Step 1.4: Soaking. Soak the seeds in warm water containing 250 mg / L gibberellin for 24 to 48 hours.
[0022] Step 1.5: Use the kernels for tissue culture. Macadamia kernels can provide moisture and nutrients to the culture medium used for tissue culture and maintain a sterile environment.
[0023] Step 1.6: When sowing, place the treated kernels flat on the surface of the culture medium, keep the substrate moist but avoid water accumulation to prevent the seeds from rotting. After sowing, place in a culture room, away from light, and control the temperature at around 30 degrees Celsius.
[0024] Step 1.7: Germination and Management. Macadamia nuts take 5-10 weeks to germinate. During germination, the culture medium should be kept moist, and the germination status of the seeds should be checked regularly. Once the seedlings have sprouted, they should be placed in a well-lit cultivation room to promote the healthy growth of the cotyledons.
[0025] Further, the specific process of step 2 is as follows: newly sprouted macadamia cotyledons are collected and cultured using starter culture medium. During the culture stage, the culture medium is changed regularly. When the cotyledons are cultured for 50 days, they dedifferentiate to form callus tissue, which is light green to yellowish-brown with a loose structure. In order to facilitate contact with the culture medium and subsequent growth of adventitious buds and roots, the callus tissue is cut into slices for further culture.
[0026] Furthermore, the specific process of step 3 is as follows:
[0027] Step 3.1: The MiNAC2a gene information was obtained by referring to whole genome and transcriptome databases. The macadamia nut transcription factor MiNAC2a gene was cloned, sequenced, and an overexpression vector was constructed.
[0028] Step 3.2: Add macadamia nut callus to a culture flask, add 75% alcohol to cover the callus and soak for 1 minute, then rinse 3 times with sterile water, then add 2.5% sodium hypochlorite to soak the callus for 3 minutes, and rinse 5 times with sterile water.
[0029] Step 3.3: Inoculate the activated Agrobacterium tumefaciens bacterial suspension at a ratio of 1:100 into 100 mL of YEP medium containing rifampicin and kanamycin, and incubate at 28°C with shaking at 200 rpm until OD600 = 0.5. Aliquot the cultured bacterial suspension into 50 mL centrifuge tubes, centrifuge at 3900 rpm for 10 min, and discard the supernatant.
[0030] Step 3.4: Add 50 mL of YEP medium containing only rifampicin to each tube to resuspend the bacterial cells, centrifuge at 3900 rpm for 10 min, discard the supernatant, and repeat this step once;
[0031] Step 3.5: Transfer the two tubes of bacterial culture to the original conical flask, add 100 μL of 1000× acetylsuccinone, 1 mL of 100× cys and 2 mL of 50× Tween-20, and mix well.
[0032] Further, the specific process of step 4 is as follows: The macadamia nut callus tissue is placed in the above-mentioned bacterial suspension and allowed to stand for 4 hours. The conical flask containing the macadamia nut explants is placed in a vacuum pump and vacuumed for 1 minute, repeated 5 times. The conical flask is then placed in a shaker at 28°C and cultured for a total of 10 hours. After culturing, the callus tissue is repeatedly rinsed with sterile water until the Agrobacterium on the surface of the callus tissue is washed away. The macadamia nut callus tissue is then cultured for 7 days. DNA is extracted from the callus tissue for identification. If no DNA is extracted, Agrobacterium is injected again until the callus tissue shows a positive result.
[0033] Further, the specific process of step 5 is as follows: positive callus tissue is cultured on MS as the basic medium to produce adventitious buds. After 30 days of culture, rooting culture is carried out. The adventitious buds are cut open with a scalpel and re-inoculated on rooting medium with 1 / 2 MS as the basic medium. After about 50 days of culture, rooted seedlings are obtained, and the medium is changed to continue the culture.
[0034] Further, the specific process of step 7 is as follows: the obtained macadamia nut positive tissue culture seedlings are hardened off, and a humus:vermiculite ratio of 2:1 is selected as the transplanting substrate. After hardening off in closed bottles for 7 days, the seedlings are hardened off in open bottles for 7 days. After hardening off in open bottles for 7 days, the seedlings are transplanted into a greenhouse for further cultivation. Vermiculite, river sand, peat moss and humus are selected as the cultivation substrate during transplanting to improve the transplant survival rate.
[0035] Furthermore, the specific process of step 8 is as follows: First, by applying nutrients from organic fertilizer and compound fertilizer to the seedlings, the seedlings are pruned and shaped to adjust their form, promote branching, increase the growth rate of the seedlings, make the seedling root system more developed, and better utilize the drought resistance of the rootstock.
[0036] The present invention, by adopting the above-described technical solution, has the following beneficial effects:
[0037] (1) Overcoming reproductive barriers and expanding the gene pool: Traditional hybridization breeding is limited by reproductive barriers between species, making it difficult to utilize the superior drought-resistant genes existing in distantly related species. Transgenic technology can overcome this barrier, precisely introducing key drought-resistant genes (such as MiNAC2a) from bacteria, fungi, and even other drought-resistant plants into macadamia trees. This is equivalent to opening up a nearly unlimited gene pool for crop breeding, enabling it to directly obtain the complex drought-resistant mechanisms honed through long-term evolution in nature. In arid and semi-arid regions with scarce rainfall and high evaporation, cultivating rootstocks with such exogenous strong drought-resistant characteristics is a fundamental way to establish a water-saving agricultural system.
[0038] (2) Achieving precise design and regulation of traits: Gene editing technologies, represented by CRISPR-Cas9 and TALENs, have propelled breeding into a new stage of "molecular design." Researchers can modify the genome at specific sites, much like using a "molecular scalpel": either precisely inserting drought-resistant genes such as MiNAC2a to enable their efficient expression in specific tissues or under adverse conditions; or knocking out certain genes that negatively regulate drought resistance. This precise manipulation of target traits avoids the large number of undesirable trait linkages that accompany superior genes in traditional hybridization breeding, greatly improving breeding efficiency and the purity of target traits.
[0039] (3) Ensuring the stable inheritance and expression of superior traits: Traits introduced or modified through genetic engineering can usually be stably inherited to offspring as single genes or a few major genes. Unlike traditional drought-resistant traits that rely on complex quantitative traits and are easily affected by the environment, the genetic background of drought resistance obtained through transgenic or gene editing is relatively clear, and the probability of maintaining stability in offspring segregation is higher. This ensures that once a superior drought-resistant rootstock variety is successfully cultivated, its core characteristics can be maintained during propagation, providing a reliable germplasm basis for large-scale application.
[0040] (4) Significantly shortens the breeding cycle and accelerates variety iteration: Traditional breeding relies on multiple generations of hybridization, backcrossing, and selection, and it often takes ten years or even longer to cultivate a stable new variety. Transgenic and gene editing technologies can directly target drought resistance, avoiding the lengthy process of gene separation and recombination. This makes it possible to rapidly improve the drought resistance of macadamia rootstocks within a relatively short experimental period, and to respond more quickly to the urgent drought resistance needs brought about by the intensification of climate change. Attached Figure Description
[0041] Figure 1 This is a picture of fresh, appropriately sized macadamia seeds after being sun-dried according to the present invention.
[0042] Figure 2 This is a diagram of macadamia nut seeds after the seed coat has been removed according to the present invention;
[0043] Figure 3 This is a diagram of macadamia nut seed kernel extraction according to the present invention;
[0044] Figure 4 This is a diagram of macadamia nut seeds germinating into seedlings according to the present invention;
[0045] Figure 5 This invention involves constructing an overexpression vector and transforming it into Agrobacterium.
[0046] Figure 6 This is a diagram illustrating the process of injecting Agrobacterium into callus tissue according to the present invention;
[0047] Figure 7 This is a diagram illustrating the continued culture of transgenic callus tissue from this invention.
[0048] Figure 8 This invention uses germination and rooting media to cultivate seedlings, respectively.
[0049] Figure 9 This invention extracts DNA from seedlings and tests whether it is positive.
[0050] Figure 10 This is a diagram showing the transplanting of seedlings into the field for cultivation after hardening off;
[0051] Figure 11This invention describes how to strengthen rootstocks through measures such as topdressing and pruning. Detailed Implementation
[0052] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are merely to provide the reader with a thorough understanding of one or more aspects of the present invention, and these aspects of the invention can be implemented even without these specific details.
[0053] A method for improving drought resistance in macadamia rootstock by overexpressing the MiNAC2a gene, the method comprising the following steps:
[0054] Step 1: Obtain sterile seedlings and cotyledons from macadamia nut seeds, such as Figure 1-2 As shown.
[0055] Seed Selection and Treatment: First, selecting healthy, mature 'Gui Re No. 1' macadamia seeds is crucial. Mature seeds typically have plump kernels and a hard outer shell. Before sowing, the seeds need appropriate treatment to improve germination rate. Sun-drying: After harvesting, sun-dry the seeds for 48-72 hours. Shelling: Remove the hard outer shell of the seeds to facilitate sowing and improve germination rate. Soaking: Soak the seeds in warm water containing 250 mg / L gibberellin (GA3) for a period of time, usually 24 to 48 hours. This helps promote water absorption, thereby accelerating the germination process. Tissue Culture Using Kernels: The culture medium used for macadamia kernel tissue culture only needs to provide moisture and nutrients; a sterile environment must be maintained. Sowing Method: When sowing, place the treated kernels flat on the surface of the culture medium. Keep the substrate moist, but avoid waterlogging to prevent seed rot. After sowing, place in a culture room, away from light, with the ideal temperature controlled around 30 degrees Celsius. Germination and Management: Macadamia nuts have a relatively long germination time, typically requiring 5-10 weeks. During germination, the growing medium needs to be kept moist, and the germination status should be checked regularly. Once the seedlings have sprouted, they should be placed in a well-lit growing room to promote healthy cotyledon growth.
[0056] Step 2: Induce callus tissue using the cotyledons of sterile seedlings, such as Figure 3-4 As shown.
[0057] Newly sprouted macadamia cotyledons were collected and cultured using a starter culture medium (WPM + 6-BA 2.5 mg / L + IBA 1.5 mg / L + 3.0 mg / L ascorbic acid (ASA) + 2.0 mg / L polyvinylpyrrolidone (PVP) + 300 mg / L activated charcoal). Ascorbic acid and PVP were added for antioxidant purposes, and activated charcoal was added to adsorb substances produced by the cotyledons, reducing autotoxicity and contamination rates. During the culture period, the culture medium was changed regularly. After 50 days of culture, the cotyledons dedifferentiated to form callus tissue, which appeared as a loose, light green to yellowish-brown structure, with browning mostly limited to localized areas.
[0058] To facilitate contact with the culture medium and the subsequent growth of adventitious buds and roots, the callus tissue was cut into slices for further culture.
[0059] Step 3: Based on the obtained MiNAC2a gene sequence, construct an overexpression vector and transform it into Agrobacterium, such as... Figure 5-6 As shown.
[0060] Information on the MiNAC2a gene sequence of macadamia nuts, macadamia nut genome annotation
[0061] >GWHPBAUK027332.1Position=GWHBAUK00000011.1, :OriGeneID=Mi03Gene52577, :OriSeqID=Chr6, Homologous gene information: Macadamia integrifolia NAC domain-containing protein 2-like (LOC122083381)
[0062] The nucleotide sequence (882 bases) is shown in SEQ ID NO: 1, and the amino acid sequence (293 amino acids) is shown in SEQ ID NO: 2.
[0063] The MiNAC2a gene information was obtained from whole-genome and transcriptome databases. This study cloned the macadamia nut transcription factor MiNAC2a gene, sequenced it, and constructed an overexpression vector. An appropriate amount of macadamia nut callus was added to a culture flask, and the callus was soaked in 75% ethanol for 1 min, then rinsed three times with sterile water. Next, the callus was soaked in 2.5% sodium hypochlorite for 3 min, and rinsed five times with sterile water. Activated Agrobacterium tumefaciens culture was inoculated at a 1:100 ratio into 100 mL of YEP medium containing rifampicin and kanamycin, and cultured at 28°C with shaking at 200 rpm until OD600 = 0.5. The cultured culture was aliquoted into 50 mL centrifuge tubes, centrifuged at 3900 rpm for 10 min, and the supernatant was discarded. Add 50 mL of YEP medium containing only rifampicin to each tube to resuspend the bacterial cells, centrifuge at 3900 rpm for 10 min, discard the supernatant, and repeat this step once. Then transfer the two tubes of bacterial culture to the original Erlenmeyer flask. Add 100 μL of 1000× acetylsylgenone, 1 mL of 100× cys, and 2 mL of 50× Tween-20, and mix well.
[0064] Step 4: "Injection method" mediates the transfer of Agrobacterium into macadamia nut callus tissue, such as... Figure 6 As shown.
[0065] Macadamia nut callus was placed in the above bacterial suspension and allowed to stand for 4 hours. The conical flask containing the macadamia nut explants was then placed in a vacuum pump and vacuumed for 1 minute, repeated 5 times. The flask was placed in a shaker at 28°C and cultured for 10 hours. Afterward, the callus was repeatedly rinsed with sterile water until the Agrobacterium tumefaciens on the surface was removed. The macadamia nut callus was then cultured for 7 days. DNA was extracted from the callus for identification. If no DNA was extracted, Agrobacterium tumefaciens was injected again until the callus showed a positive result.
[0066] Step 5: Cultivate positive callus tissue to grow adventitious buds and roots, such as... Figure 7-8 As shown.
[0067] Adventitious shoots were generated from positive callus tissue cultured on MS basal medium (MS + 6-BA 2.5 mg / L + KT 0.75 mg / L + activated charcoal 300 mg / L). After 30 days of culture, rooting culture was performed. The adventitious shoots were surgically cut open and re-inoculated onto rooting medium with 1 / 2 MS as the basal medium (6-BA 0.3 mg / L + NAA 1.5 mg / L + IBA 1.5 mg / L + ABA 0.75 mg / L + activated charcoal 300 mg / L). After about 50 days of culture, rooted seedlings were obtained, and the medium was changed for continued culture.
[0068] Step 6: Extract DNA and test for positive plants, such as... Figure 9 As shown.
[0069] DNA was extracted from the seedlings and the bands were detected to verify whether they were truly positive plants.
[0070] Step 7: Harden off the seedlings and transplant them to the field to obtain genetically modified drought-resistant rootstock resources, such as... Figure 10 As shown.
[0071] Positive tissue culture seedlings of macadamia nuts were obtained and hardened off using a 2:1 ratio of humus to vermiculite as the transplanting substrate. After 7 days of closed-bottle hardening, the seedlings were opened and hardened off again for 7 days before being transplanted to a greenhouse for further cultivation. Using vermiculite, river sand, peat moss, and humus as the cultivation substrate during transplanting can improve the survival rate.
[0072] Step 8: Strengthen the root system of the transgenic seedlings and improve the drought resistance of the rootstock, such as... Figure 11 As shown.
[0073] First, apply appropriate amounts of organic fertilizer and compound fertilizer to the seedlings to provide the necessary elements and promote their healthy growth. Second, take appropriate preventative measures to avoid damage from pests and diseases. Finally, prune and shape the seedlings to adjust their form, promote branching, increase their growth rate, and make their root systems more developed, thus better utilizing the drought resistance of the rootstock.
[0074] Taking the overexpression of the MiNAC2a gene as an example, modern molecular breeding technology, with transgenic and gene editing technologies at its core, has demonstrated comprehensive advantages in improving the drought resistance of crops (especially rootstocks, which serve as the foundation for growth), including cross-resource utilization, precision, stability, and high efficiency. This not only provides a revolutionary tool for breeding new macadamia nut varieties adapted to drought environments but also holds the promise of fundamentally enhancing the resilience of orchards under adversity, providing a solid technical guarantee for sustainable agricultural production and food security in arid regions. This technology uses transgenic technology to cross boundaries, overcome reproductive barriers, broaden the gene resource pool, and provide ideas for the precise introduction of key drought-resistant genes from bacteria, fungi, and even other drought-resistant plants into macadamia nut trees.
[0075] Matters not covered in this invention are common knowledge.
[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for improving drought resistance of macadamia rootstock by overexpressing the MiNAC2a gene, characterized in that, The method includes the following steps: Step 1: Obtain sterile seedling cotyledons from macadamia nut seeds; Step 2: Induce callus tissue using cotyledons of sterile seedlings; Step 3: Based on the obtained MiNAC2a gene sequence, construct an overexpression vector and transform it into Agrobacterium; Step 4: Inject Agrobacterium into macadamia nut callus tissue; Step 5: Cultivate positive callus tissue to grow adventitious buds and roots; Step 6: Extract DNA from seedlings and verify whether they are truly positive plants by detecting the bands. Step 7: Harden off the seedlings and transplant them to the field to obtain genetically modified drought-resistant rootstock resources; Step 8: Strengthen the root system of the transgenic seedlings and improve the drought resistance of the rootstock.
2. The method for improving drought resistance of macadamia rootstock by overexpressing the MiNAC2a gene according to claim 1, characterized in that, The specific process of step 1 is as follows: Step 1.1: Seed selection and treatment. First, select healthy, mature Guire No. 1 macadamia seeds. Mature seeds have plump kernels and hard shells. Step 1.2: Sun-drying the seeds. After harvesting, expose the seeds to the sun for 48-72 hours. Step 1.3: Remove the hard shell from the seeds to facilitate sowing and improve the germination rate; Step 1.4: Soaking. Soak the seeds in warm water containing 250 mg / L gibberellin for 24 to 48 hours. Step 1.5: Use the kernels for tissue culture. Macadamia kernels can provide moisture and nutrients to the culture medium used for tissue culture and maintain a sterile environment. Step 1.6: When sowing, place the treated kernels flat on the surface of the culture medium, keep the substrate moist but avoid water accumulation to prevent the seeds from rotting. After sowing, place in a culture room, away from light, and control the temperature at around 30 degrees Celsius. Step 1.7: Germination and Management. Macadamia nuts take 5-10 weeks to germinate. During germination, the culture medium should be kept moist, and the germination status of the seeds should be checked regularly. Once the seedlings have sprouted, they should be placed in a well-lit cultivation room to promote the healthy growth of the cotyledons.
3. The method for improving drought resistance of macadamia rootstock by overexpressing the MiNAC2a gene according to claim 1, characterized in that: The specific process of step 2 is as follows: newly sprouted macadamia cotyledons are collected and cultured using starter culture medium. During the culture stage, the culture medium is changed regularly. After 50 days of culture, the cotyledons dedifferentiate to form callus tissue, which is light green to yellowish-brown with a loose structure. In order to facilitate contact with the culture medium and subsequent growth of adventitious buds and roots, the callus tissue is cut into slices for further culture.
4. The method for improving drought resistance of macadamia rootstock by overexpressing the MiNAC2a gene according to claim 1, characterized in that, The specific process of step 3 is as follows: Step 3.1: The MiNAC2a gene information was obtained by referring to whole genome and transcriptome databases. The macadamia nut transcription factor MiNAC2a gene was cloned, sequenced, and an overexpression vector was constructed. Step 3.2: Add macadamia nut callus to a culture flask, add 75% alcohol to cover the callus and soak for 1 minute, then rinse 3 times with sterile water, then add 2.5% sodium hypochlorite to soak the callus for 3 minutes, and rinse 5 times with sterile water. Step 3.3: Inoculate the activated Agrobacterium tumefaciens bacterial suspension at a ratio of 1:100 into 100 mL of YEP medium containing rifampicin and kanamycin, and incubate at 28°C with shaking at 200 rpm until OD600 = 0.
5. Aliquot the cultured bacterial suspension into 50 mL centrifuge tubes, centrifuge at 3900 rpm for 10 min, and discard the supernatant. Step 3.4: Add 50 mL of YEP medium containing only rifampicin to each tube to resuspend the bacterial cells, centrifuge at 3900 rpm for 10 min, discard the supernatant, and repeat this step once; Step 3.5: Transfer the two tubes of bacterial culture to the original conical flask, add 100 μL of 1000× acetylsuccinone, 1 mL of 100× cys and 2 mL of 50× Tween-20, and mix well.
5. The method for improving drought resistance of macadamia rootstock by overexpressing the MiNAC2a gene according to claim 1, characterized in that, Step 4 is as follows: Place the macadamia nut callus tissue into the above bacterial suspension and let it stand for 4 hours. Place the conical flask containing the macadamia nut explants into a vacuum pump and evacuate for 1 minute, repeating this process 5 times. Place the conical flask in a shaker at 28°C and incubate for a total of 10 hours. After 10 hours, remove the flask and rinse the callus tissue repeatedly with sterile water until the Agrobacterium on the surface of the callus tissue is washed away. Continue to incubate the macadamia nut callus tissue. After 7 days of incubation, extract the callus tissue for DNA identification. If no DNA is extracted, continue to inject Agrobacterium until the callus tissue shows a positive result.
6. The method for improving drought resistance of macadamia rootstock by overexpressing the MiNAC2a gene according to claim 1, characterized in that, Step 5 is as follows: positive callus tissue is cultured on MS as the basic medium to produce adventitious buds. After 30 days of culture, rooting culture is carried out. The adventitious buds are cut open with a scalpel and re-inoculated on rooting medium with 1 / 2 MS as the basic medium. After about 50 days of culture, rooted seedlings are obtained. The medium is then changed and culture is continued.
7. The method for improving drought resistance of macadamia rootstock by overexpressing the MiNAC2a gene according to claim 1, characterized in that, The specific process of step 7 is as follows: Obtain macadamia nut positive tissue culture seedlings, harden them off, and use a humus:vermiculite ratio of 2:1 as the transplanting substrate. After hardening off in closed bottles for 7 days, open the bottles for hardening off again. After hardening off in open bottles for 7 days, transplant them into a greenhouse for further cultivation. When transplanting, use vermiculite, river sand, peat moss and humus as the cultivation substrate to improve the transplant survival rate.
8. The method for improving drought resistance of macadamia rootstock by overexpressing the MiNAC2a gene according to claim 1, characterized in that, The specific process of step 8 is as follows: First, apply organic fertilizer and compound fertilizer to the seedlings, prune and shape the seedlings, adjust their shape, promote branching, improve the growth rate of the seedlings, make the seedling roots more developed, and better exert the drought resistance of the rootstock.