Construction method of dragon fruit genetic transformation system based on non-tissue culture technology

Abstract

The invention discloses a construction method of a dragon fruit genetic transformation system based on a non-tissue culture technology, and relates to the technical fields of plant genetic engineering, a plant genetic transformation technology, agricultural biology and the like. The method specifically comprises the following steps: treating pitaya seeds, and culturing to obtain explants; constructing a recombinant vector containing the pitaya gene HpCML18, transferring the recombinant vector into K599 agrobacterium rhizogenes, and preparing a resuspension for dark induction; and treating the explant for soaking and dip dyeing, and carrying out co-cultivation induction to generate transgenic hairy roots. The method does not need to depend on tissue culture, effectively overcomes the defects of tedious operation, high cost, strong genotype dependence and the like of traditional tissue culture transformation, fills the technical blank of a pitaya non-tissue culture technology, and provides a comprehensive and effective solution for solving the bottleneck of a pitaya genetic transformation technology; the method has great significance in promoting the dragon fruit functional genome research and molecular breeding process.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, plant genetic transformation technology, agricultural biology, and specifically relates to a construction method of a genetic transformation system of pitaya based on non-tissue culture technology. BACKGROUND

[0002] Pitaya (Hylocereus spp.) is also known as red dragon fruit, dragon ball fruit, and fairy honey fruit, and is a perennial climbing succulent plant in the Cactaceae family. The fruit is rich in nutrients such as sugar, betalains, vitamins, and amino acids. Regular consumption can lower blood pressure, lower blood lipids, moisten the lungs, detoxify, nourish the skin, and improve eyesight, and is very beneficial to human health, having important edible value and health benefits. However, the current pitaya variety resources are insufficient, and the industry is facing the severe challenge of lagging behind in breeding technology.

[0003] The key approach for plant genetic engineers to create new pitaya germplasm is current genetic transformation, which mainly relies on Agrobacterium-mediated stereoscopic regeneration technology. This technology involves introducing foreign genes into plant explants, and obtaining transgenic plants through dedifferentiation and redifferentiation processes. However, there are systematic defects in its application to pitaya. First, a high-efficiency and stable in vitro regeneration system has not been established, resulting in low transformation efficiency and poor reproducibility. Second, the tissue culture operation is tedious and requires strict aseptic conditions, making it susceptible to microbial contamination. Third, there is a strong genotype dependence, and the regenerated plants have poor adaptability. Finally, traditional identification methods require expensive equipment or complex substrates, and have low screening efficiency.

[0004] Non-tissue culture genetic technology can bypass the tissue culture regeneration process, simplifying the process and reducing costs. It has been successfully applied to species such as licorice, Sansevieria trifasciata, Kalanchoe blossfeldiana, and even woody plants such as walnut, but it is still in the blank stage for pitaya. There is a lack of systematic research on its core key parameters. Furthermore, this technology is highly species-specific and lacks universality. There is also a lack of experimental systems for studying the function of pitaya root genes, which hinders the rapid verification of candidate genes related to stress resistance and quality. This severely restricts the development of pitaya gene function research and molecular breeding.

[0005] In summary, the systematic defects of tissue culture technology and the species adaptation blank of non-tissue culture technology severely restrict the development of pitaya gene function research and molecular breeding. Therefore, there is an urgent need to establish a high-efficiency non-tissue culture genetic transformation system suitable for pitaya, which has important strategic value for promoting the sustainable development of the pitaya industry. SUMMARY

[0006] The main purpose of the present application is to provide a construction method of a genetic transformation system of pitaya based on non-tissue culture technology, so as to solve the problems of the existing genetic transformation technology of pitaya tissue culture, such as complicated operation, easy pollution, strong genotype dependence, low transformation efficiency and other defects, and the technical bottleneck problem that the non-tissue culture genetic transformation technology is blank in the field of pitaya, and to provide key technical support for gene function research and breeding technology innovation of pitaya and other succulent stem plants, and to provide an efficient molecular breeding method for promoting the sustainable development of pitaya related industry.

[0007] Based on the first main aspect of the present application, a construction method of a genetic transformation system of pitaya based on non-tissue culture technology is provided, comprising the following steps:

[0008] The pitaya seeds are stored at 4 DEG C after treatment, and are transplanted to a first artificial climate incubator for culture after germination by gibberellin soaking to obtain explants; the explants include any one or more of 30-day-old pitaya seedlings and 70-day-old succulent stems;

[0009] A specific plasmid is used as a carrier to construct a recombinant plant expression vector of pitaya gene HpCML18, K599 agrobacterium is transformed by freeze-thaw method, and single colonies are screened;

[0010] An agrobacterium resuspension solution is prepared from the single colonies and the explants are immersed respectively after dark induction;

[0011] The immersed explants are inserted into a mixed substrate, and are cultured in the dark first and then are transferred to a second artificial climate incubator for culture to induce the generation of pitaya transgenic red hairy roots.

[0012] As a further preferred scheme, in the foregoing method, the pitaya seed treatment specifically includes: removing the mucus on the surface of the pitaya seeds by rubbing with gauze, and then drying in the air;

[0013] The sealed storage operation is to seal the dried pitaya seeds and silica gel desiccant together in a centrifuge tube.

[0014] As a further preferred scheme, in the foregoing method, the concentration of gibberellin is 100 mg / L, and the soaking time is 12-16 h.

[0015] As a further preferred scheme, in the foregoing method, the culture conditions of the first artificial climate incubator are temperature 25 DEG C, relative humidity 70%, and light intensity 14000 Lx.

[0016] As a further preferred scheme, in the foregoing method, the specific plasmid is pCAMBIA1301-RUBY-35S-EGFP plasmid;

[0017] The recombinant plant expression vector is pCAMBIA1300-HpCML18-RUBY-35S.

[0018] As a further preferred solution, in the foregoing method, the freeze-thaw method comprises the following steps:

[0019] The K599 Agrobacterium tumefaciens competent cells are thawed on ice, the recombinant plant expression vector is added and mixed, and then sequentially subjected to ice bath standing for 30 min, liquid nitrogen quick freezing for 5 min, 37°C water bath for 5 min, and ice bath for 2 min.

[0020] 800 μL of antibiotic-free Luria-Bertani liquid medium is added at room temperature, and the mixture is cultured on a 28°C shaking table for 4 h, 200 μL of the K599 Agrobacterium tumefaciens liquid containing the recombinant plant expression vector is taken and uniformly coated on a Luria-Bertani agar plate; the Luria-Bertani agar plate contains 50 μg / mL kanamycin sulfate and 50 μg / mL streptomycin.

[0021] The Luria-Bertani agar plate is inverted and cultured in a 28°C incubator for 2-3 days to obtain the single colony.

[0022] As a further preferred solution, in the foregoing method, the preparation of the Agrobacterium resuspension solution specifically comprises the following steps:

[0023] The single colony is inoculated in LB liquid medium and cultured at 28°C for 12-16 h to obtain a turbid bacterial liquid.

[0024] The bacterial cells are collected by centrifugation at 4°C and 8000 rpm for 5 min, the bacterial cells are resuspended using a nutrient solution, and the concentration of the Agrobacterium resuspension solution is adjusted to obtain the Agrobacterium resuspension solution.

[0025] As a further preferred solution, in the foregoing method, the components of the nutrient solution for resuspending the bacterial cells are 0.5 mM MES (2-morpholinoethanesulfonic acid) buffer, 1.5 mM MgCl2 (magnesium chloride), 100 μM AS (acetyl-syringone), and 50 g / L sucrose.

[0026] As a further preferred solution, in the foregoing method, the OD 600 of the Agrobacterium resuspension solution is 1.0.

[0027] As a further preferred solution, in the foregoing method, the soaking time is 30 min, and the component ratio of the mixed substrate is: nutrient soil: vermiculite = 3:1.

[0028] The dark culture time is 2 d, and the culture conditions of the second artificial climate chamber are temperature 25°C, relative humidity 70%, and light intensity 20000 Lx.

[0029] Approximately 35 days after the end of infection, red transgenic hairy roots produced by the expression of the RUBY reporter gene can be observed at the wound site of the explant.

[0030] Compared with existing technologies, this invention, through systematic research, has for the first time determined the core technical parameters such as the optimal explant type, efficient infection window period, and co-culture conditions suitable for dragon fruit, successfully filling the long-standing technical gap in the field of non-tissue culture technology and providing the first operable standardized scheme for dragon fruit gene function research.

[0031] Secondly, this invention addresses the highly species-specific nature of non-tissue culture techniques by specifically optimizing the unique tissue structure, wound response mechanism, and Agrobacterium sensitivity of dragon fruit, resulting in a proprietary technology system adapted to the physiological characteristics of dragon fruit. Furthermore, this invention bypasses the complex three-dimensional regeneration process by directly inducing transgenic hairy roots through inoculation of living explants. This overcomes the shortcomings of tissue culture techniques, such as cumbersome operations, demanding sterile environments, susceptibility to contamination, and strong genotype dependence, significantly improving the accessibility and application value of the technology.

[0032] Meanwhile, this invention introduces the RUBY reporter gene system, constructs an efficient and convenient transgenic identification system, realizes visual identification of transgenic events, eliminates the need for special equipment or reagents such as fluorescence microscopes and chemical substrates, and can be directly performed in vivo and non-destructively under natural light, shortening the time of several hours of traditional methods to instantaneous completion, significantly improving screening efficiency and ease of operation.

[0033] Furthermore, this invention innovatively constructs a dedicated experimental platform for dragon fruit root research. Addressing the current lack of research tools for dragon fruit roots in existing technologies, the transgenic hairy roots obtained are developed into the first dedicated system for studying the biological traits of dragon fruit roots. This provides innovative technical support for key agronomic traits such as root stress resistance mechanisms and nutrient absorption, filling a gap in research in this field.

[0034] Finally, this invention fundamentally avoids the common problem of microbial contamination in tissue culture, maintains stable genetic transformation efficiency, and supports large-scale, high-throughput operations. A single experiment can process hundreds of explants simultaneously, providing a reliable technical guarantee for the rapid creation of new germplasm.

[0035] In summary, this invention provides a comprehensive and effective solution to the bottleneck of dragon fruit genetic transformation technology through a series of technological innovations, and is of great significance to advancing the research on dragon fruit functional genomics and molecular breeding. Attached Figure Description

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only represent some embodiments of the present application, and for those skilled in the art, other drawings can be obtained according to these drawings without any creative effort.

[0037] Figure 1 An implementation flowchart of a method for constructing a genetic transformation system of pitaya based on non-tissue culture technology is shown in an embodiment of the present application.

[0038] Figure 2 The growth of the hairy roots induced by non-tissue culture of the 70d seedling age pitaya fleshy stems is shown in an embodiment of the present application.

[0039] Figure 3 The growth of the hairy roots induced by non-tissue culture of the 30d seedling age pitaya seedlings is shown in an embodiment of the present application. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present application will be described in detail in the following, so as to more clearly understand the purposes, characteristics and advantages of the present application. It should be understood that the following embodiments are not a limitation on the scope of the present application, but only to illustrate the essential spirit of the technical solutions of the present application.

[0041] In the following description, for the purpose of illustrating various disclosed embodiments, specific details are set forth in order to provide a thorough understanding of various disclosed embodiments. However, persons of ordinary skill in the relevant art will recognize that embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures associated with the present application can not be shown or described in order to avoid unnecessarily obscuring the description of embodiments.

[0042] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. In addition, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0043] Main raw and auxiliary materials and experimental apparatus involved in the present application:

[0044] Raw materials: purple pitaya seeds, pCAMBIA1301-RUBY-35S-EGFP plasmid, HpCML18 gene of pitaya, K599 rhizobium agrobacterium competence.

[0045] Reagents: gibberellin (GA3), silica gel desiccant, double-distilled water (ddH2O), Kpnl restriction enzyme, Pstl restriction enzyme, kanamycin sulfate, streptomycin, MES (2-morpholinoethanesulfonic acid), MgCl2(magnesium chloride), AS (acetyl-syringone), sucrose.

[0046] Culture medium and substrate:

[0047] Luria-Bertani liquid medium (10 g / L peptone + 10 g / L sodium chloride + 5 g / L yeast extract),

[0048] Luria-Bertani agar plate (Luria-Bertani liquid medium + 15 g / L agar powder),

[0049] Mixed substrate (nutrient soil: vermiculite = 3:1).

[0050] Experimental apparatus: centrifuge tube, culture dish, seedling pot, artificial climate chamber, constant temperature incubator, pipette, clean bench, shaker, centrifuge, cutting tool.

[0051] Example 1

[0052] Combination Figure 1 As shown in the figure, the method for constructing the genetic transformation system of Hylocereus based on non-tissue culture technology is implemented according to the following steps:

[0053] S110. Experimental material preparation

[0054] The seeds of the Hylocereus variety "Zihonglong" were used as experimental materials. First, the seeds were washed with gauze to remove the mucilage on the surface of the seeds, and then the seeds were dried in a well-ventilated place.

[0055] The dried clean seeds were sealed in 50 mL centrifuge tubes with an appropriate amount of silica gel desiccant, and stored in a 4°C refrigerator for 2.

[0056] S120. Seedling cultivation

[0057] Before sowing, the Hylocereus seeds were soaked in a solution of gibberellin (GA3) with a concentration of 100 mg / L for 12-16 h to promote germination.

[0058] Subsequently, the treated seeds were transferred to a culture dish lined with sterile filter paper, and ddH2O was used to keep the filter paper moist to create a suitable germination environment.

[0059] After the seeds germinated for 7 days, the seedlings were transplanted into seedling pots containing nutrient soil and cultured in an artificial climate chamber.

[0060] The cultivation conditions were set as follows: temperature 25℃, relative humidity 70%, and light intensity 14000 Lx.

[0061] By controlling the cultivation time, dragon fruit seedlings aged 30 days and fleshy stems aged 70 days were obtained respectively.

[0062] S130. Construction of plant expression vectors and Agrobacterium-mediated transformation

[0063] Using pCAMBIA1301-RUBY-35S-EGFP plasmid as the initial vector, a plant expression vector for the dragon fruit gene HpCML18 was constructed.

[0064] The pCAMBIA1301-RUBY-35S-EGFP vector was double-digested with two restriction endonucleases, Kpn I and Pst I, to remove the EGFP fragment. The HpCML18 gene of dragon fruit was then cloned into this site, successfully constructing the recombinant plant expression vector pCAMBIA1300-HpCML18-RUBY-35S.

[0065] Subsequently, using the freeze-thaw method, 30 µL of competent cells of K599 Agrobacterium rhizogenes stored at -80℃ were thawed on ice and 5 µL of pCAMBIA1300-HpCML18-RUBY-35S recombinant plasmid was added.

[0066] After mixing with a pipette, the mixture was placed in an ice bath for 30 min, then flash-frozen in liquid nitrogen for 5 min, then in a 37°C water bath for 5 min, and finally in an ice bath for 2 min.

[0067] After the ice bath, add 800 µL of antibiotic-free Luria-Bertani liquid medium (10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract) at room temperature, and incubate at 28°C with shaking for 4 h.

[0068] In a clean bench, 200 µL of K599 bacterial suspension was evenly spread onto Luria-Bertani agar plates (Luria-Bertani liquid medium + 15 g / L agar powder) containing 50 μg / mL kanamycin sulfate and 50 μg / mL streptomycin.

[0069] After coating, invert the plate and incubate it in a 28°C incubator for 2-3 days until a single colony is obtained.

[0070] S140. Preparation of Agrobacterium resuspension

[0071] K599 Agrobacterium rhizogenes containing the recombinant plasmid pCAMBIA1300-HpCML18-RUBY-35S was used to prepare a resuspension.

[0072] First, single colony was picked and inoculated into LB liquid medium, and incubated at 28°C for 12-16 h to get turbid bacterial suspension.

[0073] Then, the bacterial suspension was collected by centrifugation at 4°C, 8000 rpm for 5 min.

[0074] The bacterial suspension was resuspended in the nutrient solution with the following components: 0.5 mM MES (2-morpholinoethanesulfonic acid) buffer, 1.5 mM MgCl2(magnesium chloride), 100 µM AS (acetyl-syringone), and 50 g / L sucrose, and the bacterial suspension was adjusted to OD 600 =1.0.

[0075] The adjusted bacterial suspension was placed in a 28°C incubator in the dark for 3 h and then used to infiltrate pitaya explants.

[0076] S150. Explant treatment and genetic transformation

[0077] The present application uses two types of explants for transformation:

[0078] (1) Pitaya 70-day-old seedling succulent stems:

[0079] The seedlings with stem length of 4-5 cm were removed from the cotyledons and both ends of the stem section, and the middle succulent stem part was cut into 1.0-1.5 cm long segments to create a wound surface.

[0080] (2) Pitaya 30-day-old seedlings:

[0081] The intact seedlings were directly used, and a mechanical wound was created on the hypocotyl cotyledon part.

[0082] The two types of treated explants were soaked in the prepared bacterial resuspension (OD 600 =1.0) for 30 min.

[0083] After the infection was completed, the excess bacterial solution on the surface was absorbed with sterile filter paper, and then the explants were inserted into a mixed substrate with a ratio of nutrient soil to vermiculite of 3:1.

[0084] S160. Co-culture and transgenic root induction

[0085] The inoculated material was first cultured in the dark at room temperature for 2 days to complete the co-culture of Agrobacterium and explants.

[0086] It was then transferred to an artificial climate chamber and cultured at 25°C, 70% humidity, and 20000 Lx light intensity.

[0087] Combination Figure 2 and Figure 3As shown, red transgenic hairy roots generated by RUBY reporter gene expression can be observed at the wound site of explants about 35 days after the end of infection.

[0088] Example 2

[0089] According to the experimental steps in Example 1, during the seedling cultivation in step S120, by controlling the culture time, pitaya seedlings of 25d seedling age and succulent stems of 60d seedling age were obtained, respectively, and the remaining step conditions were unchanged. Red transgenic hairy roots were also obtained.

[0090] 1. Experimental material preparation

[0091] The pitaya variety "Zihonglong" seeds were used as experimental materials. First, the seeds were washed with gauze to remove the mucilage on the surface of the seeds, and then the seeds were dried in a well-ventilated place.

[0092] The dried clean seeds were sealed in a 50 mL centrifuge tube with an appropriate amount of silica gel desiccant, and stored in a 4°C refrigerator for 2 min for standby use.

[0093] 2. Seedling cultivation

[0094] Before sowing, the pitaya seeds were soaked in a gibberellin (GA3) solution with a concentration of 100 mg / L for 12-16 h to promote germination.

[0095] Then, the treated seeds were transferred to a culture dish lined with sterile filter paper, and ddH2O was used to keep the filter paper moist to create a suitable germination environment.

[0096] After the seeds germinated for 7 days, the seedlings were transplanted into a nursery pot containing nutrient soil and placed in an artificial climate chamber for cultivation.

[0097] The culture conditions were set as follows: temperature 25°C, relative humidity 70%, and light intensity 14000 Lx.

[0098] By controlling the culture time, pitaya seedlings of 25d seedling age and succulent stems of 60d seedling age were obtained, respectively.

[0099] 3. Plant expression vector construction and Agrobacterium transformation

[0100] The pCAMBIA1301-RUBY-35S-EGFP plasmid was used as the initial vector to construct the plant expression vector of pitaya gene HpCML18.

[0101] The pCAMBIA1301-RUBY-35S-EGFP vector was double digested with Kpn I and Pst I restriction enzymes to remove the EGFP fragment, and the HpCML18 gene of pitaya was cloned into the site to successfully construct the recombinant plant expression vector pCAMBIA1300-HpCML18-RUBY-35S.

[0102] Subsequently, using the freeze-thaw method, the K599 Agrobacterium tumefaciens prepared in advance was thawed on ice, and 30 μL of the prepared K599 Agrobacterium tumefaciens was added to 5 μL of the pCAMBIA1300-HpCML18-RUBY-35S recombinant plasmid.

[0103] After mixing, the mixture was sequentially placed in an ice bath for 30 min, quickly frozen in liquid nitrogen for 5 min, placed in a 37°C water bath for 5 min, and placed in an ice bath for 2 min.

[0104] After ice bath, 800 μL of Luria-Bertani liquid medium (10 g / L of proteose peptone, 10 g / L of sodium chloride, 5 g / L of yeast powder) without antibiotics was added at room temperature, and the mixture was cultured at 28°C for 4 h.

[0105] In the clean bench, 200 μL of K599 bacterial liquid was taken and uniformly coated on Luria-Bertani agar plates (Luria-Bertani liquid medium + 15 g / L agar powder) containing 50 μg / mL kanamycin sulfate and 50 μg / mL streptomycin.

[0106] After coating, the plates were inverted and cultured in a 28°C incubator for 2-3 days until single colonies were obtained.

[0107] 4. Preparation of Agrobacterium resuspension

[0108] K599 Agrobacterium tumefaciens containing the pCAMBIA1300-HpCML18-RUBY-35S recombinant plasmid was used to prepare the resuspension.

[0109] First, a single colony was inoculated in LB liquid medium and cultured at 28°C for 12-16 h until the bacterial liquid was turbid.

[0110] Subsequently, the bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 5 min.

[0111] The bacterial cells were resuspended in a nutrient solution containing 0.5 mM MES (2-morpholinoethanesulfonic acid) buffer, 1.5 mM MgCl2 (magnesium chloride), 100 μM AS (acetyl-syringone), and 50 g / L sucrose, and the bacterial liquid concentration was adjusted to OD 600 = 1.0.

[0112] The adjusted resuspension liquid was placed in a 28°C constant temperature incubator for 3h of dark induction before being used for dipping pitaya explants.

[0113] 5. Explant treatment and genetic transformation

[0114] The present application employs two types of explants for transformation:

[0115] (1) Pitaya 60d aged succulent stem:

[0116] The seedlings with stem length of 4-5 cm were removed of cotyledons and stem ends, and the middle succulent stem part was cut into 1.0-1.5 cm long segments to create a wound surface.

[0117] (2) Pitaya 25d aged seedlings:

[0118] The intact seedlings were directly used, and a mechanical wound was created at the hypocotyl cotyledon part.

[0119] The two types of treated explants were respectively soaked in the prepared Agrobacterium resuspension liquid (OD 600 = 1.0) for 30 minutes of infection.

[0120] After the infection was completed, the excess bacterial liquid on the surface was absorbed with sterile filter paper, and then the explants were inserted into a mixed medium with a ratio of nutrient soil: vermiculite = 3:1.

[0121] 6. Co-culture and transgenic root induction

[0122] The inoculated material was first cultured at room temperature in the dark for 2 days to complete the co-culture of Agrobacterium and explants.

[0123] It was then transferred to an artificial climate chamber and cultured at 25°C, 70% humidity, and 20000 Lx light intensity.

[0124] About 35 days after the infection was completed, red transgenic hairy roots generated by RUBY reporter gene expression were observed at the wound site of the explants.

[0125] Example 3

[0126] According to the experimental steps in Example 1, in the seedling cultivation step S120, by controlling the culture time, pitaya seedlings of 35d age and succulent stems of 80d age were respectively obtained, and the remaining step conditions were unchanged, and red transgenic hairy roots were also obtained.

[0127] 1. Experimental material preparation

[0128] The pitaya variety "Zihonglong" seeds were used as experimental materials. First, the seeds were washed with gauze to remove the mucilage on the surface, and then the seeds were dried in a well-ventilated place.

[0129] The dried clean seeds were sealed with an appropriate amount of silica gel desiccant in a 50 mL centrifuge tube and stored at 4°C in the refrigerator for 2.

[0130] 2. Seedling cultivation

[0131] Before sowing, the pitaya seeds were soaked in a gibberellin (GA3) solution with a concentration of 100 mg / L for 12-16 h to promote germination.

[0132] Subsequently, the treated seeds were transferred to a culture dish lined with sterile filter paper, and ddH2O was used to keep the filter paper moist to create a suitable germination environment.

[0133] After the seeds germinated for 7 days, the seedlings were transplanted into seedling pots containing nutrient soil and cultured in an artificial climate chamber.

[0134] The culture conditions were set as follows: temperature 25°C, relative humidity 70%, and light intensity 14000 Lx.

[0135] By controlling the culture time, pitaya seedlings with a 30d seedling age and succulent stems with an 80d seedling age were obtained.

[0136] 3. Plant expression vector construction and Agrobacterium transformation

[0137] Using the pCAMBIA1301-RUBY-35S-EGFP plasmid as the initial vector, a plant expression vector for the pitaya gene HpCML18 was constructed.

[0138] The pCAMBIA1301-RUBY-35S-EGFP vector was double-digested with Kpn I and Pst I restriction enzymes to remove the EGFP fragment, and the pitaya HpCML18 gene was cloned into the site to successfully construct the recombinant plant expression vector pCAMBIA1300-HpCML18-RUBY-35S.

[0139] Subsequently, using the freeze-thaw method, 30 µL of the -80°C stored K599 rhizobium agrobacterium competence was thawed on ice and added to 5 µL of the pCAMBIA1300-HpCML18-RUBY-35S recombinant plasmid.

[0140] After mixing with a pipette gun, it was sequentially placed in an ice bath for 30 min, frozen in liquid nitrogen for 5 min, placed in a 37°C water bath for 5 min, and placed in an ice bath for 2 min.

[0141] After the ice bath, 800 μL of Luria-Bertani liquid medium (10 g / L of proteose peptone, 10 g / L of sodium chloride, 5 g / L of yeast extract) without antibiotics was added at room temperature, and the culture was shaken at 28 °C for 4 h. After the shaking, the culture was terminated.

[0142] In the clean bench, 200 μL of K599 bacterial solution containing the recombinant plasmid pCAMBIA1300-HpCML18-RUBY-35S was uniformly coated on Luria-Bertani agar plates (Luria-Bertani liquid medium + 15 g / L agar powder) containing 50 μg / mL kanamycin sulfate and 50 μg / mL streptomycin.

[0143] After coating, the plates were inverted and cultured in a 28 °C incubator for 2-3 days until single colonies were obtained.

[0144] 4. Preparation of Agrobacterium resuspension

[0145] K599 rhizobium containing the pCAMBIA1300-HpCML18-RUBY-35S recombinant plasmid was prepared to prepare a resuspension.

[0146] First, a single colony was picked and inoculated in LB liquid medium, and the culture was shaken at 28 °C for 12-16 h until the bacterial solution was turbid.

[0147] Then, the bacterial cells were collected by centrifugation at 4 °C and 8000 rpm for 5 minutes.

[0148] The bacterial cells were resuspended in a nutrient solution containing 0.5 mM MES (2-morpholinoethanesulfonic acid) buffer, 1.5 mM MgCl2 (magnesium chloride), 100 μM AS (acetyl-syringone), and 50 g / L sucrose, and the bacterial solution was adjusted to OD 600 = 1.0.

[0149] The adjusted resuspension was placed in a 28 °C constant temperature incubator and induced in the dark for 3 h before being used to dip the pitaya explants.

[0150] 5. Treatment of explants and genetic transformation

[0151] The present application uses two types of explants for transformation:

[0152] (1) Pitaya 80d aged succulent stems:

[0153] The seedlings with a stem length of 4-5 cm were removed from the cotyledons and both ends of the stem section, and the middle succulent stem part was cut into 1.0-1.5 cm long segments to create a wound surface.

[0154] (2) Pitaya 35d seedlings:

[0155] Directly use the whole seedlings, and make mechanical wounds on the hypocotyl cotyledon part.

[0156] Soak the two kinds of treated explants in the prepared Agrobacterium resuspension solution (OD 600 =1.0) for 30 minutes.

[0157] After the end of the infection, use sterile filter paper to absorb the excess bacterial solution on the surface, and then insert the explants into a mixed substrate with a ratio of nutrient soil to vermiculite of 3:1.

[0158] 6. Co-culture and transgenic root induction

[0159] Incubate the inoculated material at room temperature in the dark for 2 days to complete the co-culture of Agrobacterium and explants.

[0160] Transfer it to an artificial climate chamber and continue to cultivate it at 25°C, 70% humidity, and 20000 Lx light intensity.

[0161] About 35 days after the end of the infection, red transgenic hairy roots produced by the expression of the RUBY reporter gene can be observed at the wound site of the explant.

[0162] Example 4

[0163] According to the experimental steps in Example 1, the RUBY system can use the mCherry red fluorescent protein reporter system during detection, which includes the following steps:

[0164] Use the pCAMBIA1301-mCherry-35S-EGFP plasmid as the initial vector to construct the plant expression vector of the dragon fruit gene HpCML18.

[0165] Use Kpn I and Pst I restriction enzymes to double digest the pCAMBIA1301-mCherry-35S-EGFP vector to remove the EGFP fragment, and clone the dragon fruit HpCML18 gene to this site, successfully constructing the recombinant plant expression vector pCAMBIA1300-HpCML18-mCherry-35S.

[0166] The remaining steps follow the experimental steps in Example 1 to complete Agrobacterium transformation, explant infection, and co-culture and transgenic root induction. About 35 days after the end of the infection, irradiate the wound site of the explant with natural light or a portable green light source, and red fluorescent hairy roots can be observed.

[0167] Example 5

[0168] According to the experimental steps in Example 1, the HpCML18 gene in the original scheme can be optimized to different foreign genes with agronomic value.

[0169] The exogenous gene can be inserted into the vector through Kpn I and Pst I two restriction enzyme cutting sites, Agrobacterium transformation, immersion and induction culture steps are consistent with example 1.

[0170] In one feasible embodiment, the HpCML18 gene is replaced by the drought stress response gene DREB2A, which specifically includes the following steps:

[0171] The pCAMBIA1301-RUBY-35S-EGFP plasmid is used as the initial vector to construct the plant expression vector of the dragon fruit drought stress response gene DREB2A.

[0172] The pCAMBIA1301-RUBY-35S-EGFP vector is double digested by Kpn I and Pst I two restriction enzymes to remove the EGFP fragment, and the dragon fruit drought stress response gene DREB2A gene is cloned into the site to construct the recombinant plant expression vector pCAMBIA1300-DREB2A-RUBY-35S.

[0173] The remaining method steps are consistent with example 1.

[0174] In the above examples, the technical terms, technical principles or technical means related to the technical solutions of the present application are not described in detail in the above content, which are known to those skilled in the art or common means.

[0175] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. Those skilled in the art should understand that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a dragon fruit genetic transformation system based on non-tissue culture technology, characterized in that, Includes the following steps: Dragon fruit seeds were treated and stored in a sealed container at 4°C. After germination by soaking in gibberellin, the seeds were transplanted into an artificial climate incubator for the first time to obtain explants. The explants included any one or more of dragon fruit seedlings aged 30 days and fleshy stems aged 70 days. A recombinant plant expression vector for the dragon fruit gene HpCML18 was constructed using a specific plasmid as a vector. The vector was then transformed into K599 Agrobacterium rhizogenes using a freeze-thaw method, and single colonies were screened. Agrobacterium resuspension was prepared using the single colony and then inoculated into the explants after induction in the dark. The infected explants were inserted into a mixed matrix, cultured in the dark, and then transferred to a second artificial climate chamber for culture to induce the production of transgenic red hairy roots of dragon fruit.

2. The method for constructing a dragon fruit genetic transformation system based on non-tissue culture technology according to claim 1, characterized in that, The dragon fruit seed treatment specifically includes: rubbing the dragon fruit seeds with gauze to remove the mucus on the surface of the seeds, followed by ventilation and drying. The sealing and preservation operation involves sealing the dried dragon fruit seeds together with silica gel desiccant in a centrifuge tube.

3. The method for constructing a dragon fruit genetic transformation system based on non-tissue culture technology according to claim 1, characterized in that, The gibberellin concentration was 100 mg / L, and the soaking time was 12-16 h.

4. The method for constructing a dragon fruit genetic transformation system based on non-tissue culture technology according to claim 1, characterized in that, The first artificial climate incubator was incubated under the following conditions: temperature 25°C, relative humidity 70%, and light intensity 14000 Lx.

5. The method for constructing a dragon fruit genetic transformation system based on non-tissue culture technology according to claim 1, characterized in that, The specific plasmid is pCAMBIA1301-RUBY-35S-EGFP plasmid; The recombinant plant expression vector was pCAMBIA1300-HpCML18-RUBY-35S.

6. The method for constructing a dragon fruit genetic transformation system based on non-tissue culture technology according to claim 1, characterized in that, The freeze-thaw method includes the following steps: Thaw K599 Agrobacterium rhizogenes competent cells on ice, add the recombinant plant expression vector and mix well. Then, incubate on ice for 30 min, freeze in liquid nitrogen for 5 min, incubate in water at 37°C for 5 min, and incubate on ice for 2 min. Add 800 µL of antibiotic-free Luria-Bertani liquid medium at room temperature, and culture on a shaker at 28°C for 4 h. Take 200 µL of the K599 Agrobacterium rhizogenes solution and spread it evenly on a Luria-Bertani agar plate. The Luria-Bertani agar plate contains 50 μg / mL kanamycin sulfate and 50 μg / mL streptomycin. The Luria-Bertani agar plates were inverted and incubated in a 28°C incubator for 2-3 days to obtain the single colonies.

7. The method for constructing a dragon fruit genetic transformation system based on non-tissue culture technology according to claim 1, characterized in that, The preparation of Agrobacterium resuspension specifically includes the following steps: Pick a single colony and inoculate it into LB liquid medium, then incubate at 28°C with shaking for 12-16 h until the bacterial culture becomes turbid; The bacterial cells were collected by centrifugation at 4℃ and 8000 rpm for 5 min. The bacterial cells were resuspended in nutrient solution and the concentration of Agrobacterium resuspension was adjusted to obtain the Agrobacterium resuspension.

8. The method for constructing a dragon fruit genetic transformation system based on non-tissue culture technology according to claim 7, characterized in that, The nutrient solution used to resuspend the bacterial cells consisted of 0.5 mM MES (2-morpholinoethanesulfonic acid) buffer, 1.5 mM MgCl2 (magnesium chloride), 100 µM AS (acetylsyleugenol), and 50 g / L sucrose.

9. The method for constructing a dragon fruit genetic transformation system based on non-tissue culture technology according to claim 1 or 7, characterized in that, The Agrobacterium resuspension OD 600 =1.

0.

10. The method for constructing a dragon fruit genetic transformation system based on non-tissue culture technology according to claim 1, characterized in that, The immersion time is 30 minutes, and the composition ratio of the mixed matrix is: nutrient soil: vermiculite = 3:1; The dark culture period is 2 days, and the culture conditions of the second artificial climate chamber are 25°C, 70% relative humidity, and 20,000 Lx light intensity.

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