Genetic transformation system construction of local rice variety

Abstract

The invention discloses construction of a genetic transformation system of local rice varieties, and belongs to the technical field of agricultural biology. According to the method, the problem of low genetic transformation efficiency of local rice varieties in the prior art is solved by optimizing culture conditions such as an induction culture medium, a subculture culture medium, infection conditions and a differentiation and rooting culture medium. The method specifically comprises the following steps: respectively adopting different hormone combinations to induce calluses for japonica rice and indica rice, adding maltose and Vc to inhibit callus browning, optimizing agrobacterium infection time and AS concentration, and adopting a specific differential culture medium and a rooting culture medium to promote regeneration. Experimental results show that by means of the system, the positive transformation rate of japonica rice can reach 43.24%-52.38%, the positive transformation rate of indica rice can reach 40.00% or above, and a stable technical basis is provided for efficient genetic transformation of local rice varieties.

Description

Technical Field

[0001] This invention application relates to the field of agricultural biotechnology, specifically to the construction of genetic transformation systems for local rice varieties. Background Technology

[0002] Rice genetic transformation technology is the core method of molecular breeding. Current technology generally involves inducing callus tissue from seeds, then differentiating and culturing it to form regenerated plants, and finally introducing and integrating target genes through the Agrobacterium-mediated system.

[0003] Different rice varieties require different conditions in the dedifferentiation and differentiation stages of culture. Existing studies often focus on a single step (such as optimizing infection conditions only), neglecting the synergistic optimization of callus quality (such as resistance to browning), differentiation efficiency (such as KT concentration), and antibacterial measures (such as antibiotic selection).

[0004] Secondly, the synergy between the regeneration system and Agrobacterium infection is insufficient. For example, browning of callus in indica rice leads to decreased cell activity, making it difficult to tolerate the stress of Agrobacterium infection. Furthermore, the weak response of cytokinins (such as KT) during the differentiation stage results in low shoot regeneration efficiency, preventing transgenic cells from developing into complete plants and causing a break in the transformation chain. These factors contribute to the low efficiency of the genetic transformation system in local rice varieties.

[0005] Therefore, it is urgent to build a regeneration system that is collaborative throughout the entire process, and to overcome the genotype limitations of indica rice by targeted improvement of callus quality, differentiation efficiency and low-toxicity antibacterial scheme, so as to provide a stable receptor basis for the efficient transformation of Agrobacterium. Summary of the Invention

[0006] To address the problems mentioned in the background art, this invention provides a genetic transformation system for local rice varieties to solve the aforementioned problems.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The genetic transformation system for local rice varieties was constructed. The components of the induction medium used to form primary callus from rice seeds were as follows: N6 medium with macroelements (Zhu Zhiqing formula, containing KNO3 2.8 g / L, (NH4)2SO4 0.463 g / L, etc.).

[0009] Gamborg B5 medium contains trace elements (including MnSO4·4H2O 10 mg / L, ZnSO4·7H2O 2 mg / L, etc.).

[0010] Gamborg B5 organic ingredients (containing inositol 100 mg / L, thiamine·HCl 10 mg / L, etc.);

[0011] Fe-EDTA chelated iron (containing FeSO4·7H2O 27.8 mg / L, Na2EDTA·2H2O 37.3 mg / L);

[0012] L-Glutamic acid: 0.2 g / L; L-proline: 0.1 g / L; Acid-hydrolyzed casein: 0.1 g / L; Sucrose: 30 g / L; Agar: 8 g / L; Add different concentrations of 1-4 mg 2,4-D and 0-2.5 mg NAA to the above-mentioned basic components of the induction medium;

[0013] The pH of the induction medium was 5.8±0.1; the induction culture temperature was 28±2℃, the humidity was 60%~70%, and the culture was carried out in the dark.

[0014] As preferred, japonica rice varieties were treated with a hormone combination containing 2 mg / L 2,4-D + 1 mg / L NAA, and indica rice varieties were treated with a hormone combination containing 3 mg / L 2,4-D + 1 mg / L NAA. Embryogenic callus was induced by dark culture for 15 days.

[0015] As a preferred method, rice seeds were placed in an induction medium and cultured for 15 days to obtain primary callus tissue, which was then transferred to a new induction medium for subculture for 15 days. The induction medium for subculture was supplemented with 20 g / L maltose and 40 mg / L Vc.

[0016] Preferably, the callus tissue induced for 30 days was placed in the infection solution for 30 minutes and then air-dried on sterile filter paper. The infection solution was prepared by Agrobacterium tumefaciens bacterial suspension and infection buffer at an OD600 of 0.6-0.8. The infection buffer contained the following components: AA macroelements + Gamborg B5 microelements + Gamborg B5 organic components + iron salts; MES buffer: 3.9 g / L; hydrolyzed casein (CH): 0.5 g / L.

[0017] As a preferred option, the callus tissue induced for 30 days was placed in the infection solution for 30 minutes, then dried on sterile filter paper, transferred to the symbiotic culture medium, and co-cultured at 28°C for 3 days.

[0018] The symbiotic culture medium consists of the following components: N6 macroelements + B5 microelements + B5 organic compounds + iron salts + inositol 2g / L + MES 3.9g / L + CH 0.5g / L + 30g / L sucrose + 8g / L agar (pH=5.5).

[0019] As a preferred option, the callus tissue is transferred to a recovery medium after being cultured on a symbiotic medium for further culture.

[0020] The basic components of the recovery medium include: N6 macroelements; B5 microelements; B5 organics; iron salts; 0.2 g / L Glu; 0.1 g / L L-Pro; 0.1 g / L CH; 2.0 mg / L 2,4-D; 30 g / L sucrose; 8 g / L agar; the pH of the recovery medium is adjusted to 5.8 ± 0.1.

[0021] Different concentrations of 0-500 mg / L cefotaxime and 0-500 mg / L termethin were added to the basic components of the above-mentioned recovery culture medium.

[0022] As a preferred option, the differentiation medium used for callus differentiation comprises: N6 macroelements + B5 microelements + B5 organic compounds + iron salts + 0.2 g / L Glu + 0.1 g / L L-Pro + 0.1 g / L CH + 30 g / L sucrose + 8 g / L agar; kinetin KT (1-3 mg / L), α-naphthaleneacetic acid (1-2 mg / L), and 6-benzylaminopurine (1-3 mg / L) are added to the above basic components of the differentiation medium.

[0023] As a preferred option, the differentiation medium used for japonica rice varieties is supplemented with 3 mg / L KT, 1 mg / L NAA and 2 mg / L 6-BA; the differentiation medium used for indica rice varieties is supplemented with 3 mg / L KT, 2 mg / L NAA and 3 mg / L 6-BA.

[0024] Preferably, 200 μM acetylsuccinone (AS) is added to the infection solution, and the infection time is set according to genotype differences: 10 min for japonica rice and 5 min for indica rice.

[0025] Preferably, the rooting medium used in the callus rooting process is 1 / 2 MS + 0.2 mg / L NAA (MA treatment), the components of which include: 1 / 2 MS basal salt (containing iron salt and trace elements); 0.2 mg / L α-naphthaleneacetic acid (NAA); 30 g / L sucrose + 8 g / L agar (pH 5.8±0.1).

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] 1. A groundbreaking, fully collaborative genetic transformation system for local rice varieties has been established: addressing the problem of low callus induction rate due to genotype dependence in existing technologies, the induction rate has been significantly improved by using hormone combinations specific to japonica and indica rice, and the quality of callus tissue has been significantly improved.

[0028] 2. To address the problem of callus browning in indica rice, an anti-browning combination of 20 g / L maltose and 40 mg / L vitamin C was used, which significantly reduced the browning rate and solved the problem of decreased cell activity during Agrobacterium infection.

[0029] 3. Improvements and adjustments were made to the culture media used in the regeneration system; the bottlenecks of insufficient synergy between the regeneration system and Agrobacterium infection and the fragmentation of technical links in traditional technologies were overcome, providing a systematic solution for the efficient genetic transformation of local rice varieties. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The effects of different concentrations of 2,4-D combined with NAA on callus induction in rice;

[0032] Figure 2 The effect of different carbon source concentrations on browning of Hui 128 callus;

[0033] Figure 3 The effects of different concentrations of vitamin C and PVP on browning of callus tissue from Hui 128;

[0034] Figure 4 The effects of different concentrations of antibacterial agents on the contamination rate and differentiation of rice callus;

[0035] Figure 5 This diagram illustrates the process of callus differentiation into seedlings in four rice varieties.

[0036] Figure 6 The effects of different combinations of AS concentration and infection time on rice callus resistance;

[0037] Figure 7 The effect of different AS concentrations and infection times on GUS staining of rice callus.

[0038] Figure 8 The effects of different rooting media on the root growth of rice regenerated seedlings;

[0039] Figure 9 Images showing PCR positive detection and GUS staining identification of transgenic seedlings;

[0040] Figure 10 A diagram showing the effect of different rooting media on rice root growth;

[0041] Figure 11 This is a reference diagram for grading the browning of callus tissue. Detailed Implementation

[0042] To make the purpose, features, and advantages of this application more apparent and understandable, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0043] Example

[0044] 1. Materials and Methods

[0045] 1.1 Selection of test materials

[0046] Two japonica rice varieties (Xinjing 293 and Wandao 68) and two indica rice varieties (Hui 128 and E33) were used as transformation recipient materials, all bred and provided by Anhui Youxin Agricultural Technology Co., Ltd. All four varieties have relatively tall plant heights and fewer tillers, and are widely planted in the Jianghuai region of Anhui Province. They exhibit stable yields, strong resistance to adverse conditions, and short growth periods, making them suitable for crop rotation in the main grain-producing areas of the transitional zone between northern and southern Anhui.

[0047] 1.2 Construction of callus regeneration system

[0048] 1.2.1 Acquisition of primary callus

[0049] The pretreatment steps for rice seeds are as follows:

[0050] Step 1: Select healthy, plump, mature rice seeds without glumes, and wash them three times with sterile water. The glumes have many folds, making them prone to harboring microorganisms (such as fungal spores). During disinfection, pathogenic bacteria may remain, leading to tissue culture contamination. Removing the glumes allows disinfectants (such as NaClO) to directly contact the seed surface, improving sterilization efficiency. Furthermore, after glumes are removed, the embryo can more easily absorb growth regulators, increasing callus induction rate.

[0051] Step 2: After cleaning, immerse in 75% ethanol for 3 min for disinfection, and then elute with 3% NaClO solution for 40 min (shaking at 180 rpm).

[0052] Step 3: Finally, wash 5 times with sterile water and air dry on sterile filter paper.

[0053] Primary callus formation was induced from rice seeds on an induction medium.

[0054] After sterilization, the seeds were placed in induction medium and cultured for 15 days to obtain primary callus tissue. Then, they were transferred to a new induction medium and subcultured for 15 days (the composition of the induction medium used in both cultures was the same). The size, fresh weight, dry weight and induction rate of the primary callus tissue were investigated.

[0055] The basic components of the induction medium are as follows:

[0056] 1. Basic salt solution:

[0057] N6 medium macro-elements (Zhu Zhiqing formula, containing KNO3 2.8 g / L, (NH4)2SO4 0.463 g / L, etc.);

[0058] Gamborg B5 medium contains trace elements (including MnSO4·4H2O 10 mg / L, ZnSO4·7H2O 2 mg / L, etc.).

[0059] Gamborg B5 organic ingredients (containing inositol 100 mg / L, thiamine·HCl 10 mg / L, etc.);

[0060] Fe-EDTA chelated iron (containing FeSO4·7H2O 27.8 mg / L, Na2EDTA·2H2O 37.3 mg / L);

[0061] 2. Additives:

[0062] L-Glutamic acid: 0.2 g / L;

[0063] L-proline: 0.1 g / L;

[0064] Acid-hydrolyzed casein: 0.1 g / L;

[0065] Sucrose: 30 g / L;

[0066] Agar: 8 g / L;

[0067] 3. pH adjustment:

[0068] Before sterilization, its pH is adjusted to 5.8±0.1.

[0069] The tissue culture room conditions were: temperature 28±2℃, humidity 60%~70%, and dark culture.

[0070] Different concentrations of 2,4-D (1-4 mg) and NAA (0-2.5 mg) were added to the basic components of the above-mentioned induction medium; some of the treatment combinations are shown in Table 1; where 2,4-D is the abbreviation for 2,4-dichlorophenoxyacetic acid, and NAA is the abbreviation for α-naphthaleneacetic acid, both of which belong to the class of auxin plant growth regulators; and the effects of different concentrations of 2,4-D and NAA on primary callus formation were investigated.

[0071] Table 1. Treatment combinations of different concentrations of 2,4-D and NAA

[0072]

[0073] 1.2.2 Browning treatment of callus tissue

[0074] To prevent callus browning, different carbon sources (sucrose and maltose) were used during subculture to reduce the callus browning rate, and the optimal carbon source type and concentration were screened (Table 2). Then, under the optimal carbon source type and concentration, different concentrations of the anti-browning agents vitamin C (Vc) and polyvinylpyrrolidone (PVP) were added, and the callus browning rate and browning index, as well as callus size, fresh weight, and dry weight, were investigated to evaluate the callus's anti-browning ability.

[0075] The degree of browning is calculated as the proportion of the area of ​​browning in each callus to the total area of ​​the callus, and is divided into 5 grades: Grade 0 is basically no browning, with a proportion of <10%; Grade 1 has a browning proportion of 10-35%; Grade 2 has a browning proportion of 36-65%; Grade 3 has a browning proportion of 66-95%; and Grade 4 has a browning proportion of 96-100%.

[0076] Table 2 Different anti-browning treatments

[0077]

[0078] 1.2.3 Pre-infection and recovery culture of callus tissue

[0079] Culture blank Agrobacterium (without transformation plasmid, EHA105, Sangon Biotech, China), collect the bacterial culture by centrifugation, and then dilute the bacterial culture with infection buffer to prepare infection solution (OD600=0.6-0.8).

[0080] Infection buffer: AA macro-elements + B5 micro-elements + B5 organic + iron salts + MES 3.9g / L + CH 0.5g / L (pH=5.5).

[0081] The callus tissue induced for 30 days was placed in the infection solution for 30 minutes, then dried on sterile filter paper, transferred to the symbiotic culture medium, and co-cultured at 28°C for 3 days.

[0082] The basic components of the symbiotic culture medium are as follows:

[0083] 1. Basic salt solution:

[0084] N6 medium macro-elements (Zhu Zhiqing formula, containing KNO3 2.8 g / L, (NH4)2SO4 0.463 g / L, etc.);

[0085] Gamborg B5 medium contains trace elements (including MnSO4·4H2O 10 mg / L, ZnSO4·7H2O 2 mg / L, etc.).

[0086] Gamborg B5 organic ingredients (containing inositol 100 mg / L, thiamine·HCl 10 mg / L, etc.);

[0087] Fe-EDTA chelated iron (containing FeSO4·7H2O 27.8 mg / L, Na2EDTA·2H2O 37.3 mg / L);

[0088] 2. Additives:

[0089] Inositol: 2g / L;

[0090] MES: 3.9g / L;

[0091] Hydrolyzed casein: 0.5 g / L;

[0092] Sucrose: 30 g / L;

[0093] Agar: 8 g / L;

[0094] 3. pH adjustment:

[0095] Before sterilization, its pH is adjusted to 5.8±0.1.

[0096] The tissue culture room conditions were: temperature 28±2℃, humidity 60%~70%, and dark culture.

[0097] Then, wash the callus tissue 6-7 times with sterile water (containing 400 mg / L Cef) and dry it on sterile filter paper for 30 minutes.

[0098] Finally, the callus tissue was transferred to recovery culture medium. It was then placed in a tissue culture room for further recovery culture for 7 days, during which the contamination rate and initial differentiation rate were calculated.

[0099] The basic components of the recovery culture medium are:

[0100] 1. Basic salt solution:

[0101] N6 medium macro-elements (Zhu Zhiqing formula, containing KNO3 2.8 g / L, (NH4)2SO4 0.463 g / L, etc.);

[0102] Gamborg B5 medium contains trace elements (including MnSO4·4H2O 10 mg / L, ZnSO4·7H2O 2 mg / L, etc.).

[0103] Gamborg B5 organic ingredients (containing inositol 100 mg / L, thiamine·HCl 10 mg / L, etc.);

[0104] Fe-EDTA chelated iron (containing FeSO4·7H2O 27.8 mg / L, Na2EDTA·2H2O 37.3 mg / L);

[0105] 2. Additives:

[0106] L-Glutamic acid: 0.2 g / L;

[0107] L-proline: 0.1 g / L;

[0108] Hydrolyzed casein: 0.1 g / L;

[0109] 2,4-D (2,4-dichlorophenoxyacetic acid): 2.0 mg / L;

[0110] Sucrose: 30 g / L;

[0111] Agar: 8 g / L;

[0112] 3. pH adjustment:

[0113] Before sterilization, its pH is adjusted to 5.8±0.1.

[0114] Different concentrations of cefotaxime (0-500 mg / L) and termethin (0-500 mg / L) were added to the basic components of the above-mentioned recovery culture medium; some of the treatment combinations are shown in Table 3. Tissue culture room conditions: temperature 28±2℃, humidity 60%~70%, photoperiod 16 h light / 8 h dark, light intensity 1000~2500 lux.

[0115] Table 3. Treatment combinations of different antibacterial agents and concentrations

[0116]

[0117] 1.2.4 Callus Differentiation and Culture

[0118] After 45 days of recovery culture, the callus tissue was transferred to differentiation medium and continued to grow for 30 days. The differentiation rate of the callus tissue was then investigated.

[0119] The basic components of the differentiation culture medium are:

[0120] 1. Basic salt solution:

[0121] N6 medium macro-elements (Zhu Zhiqing formula, containing KNO3 2.8 g / L, (NH4)2SO4 0.463 g / L, etc.);

[0122] Gamborg B5 medium contains trace elements (including MnSO4·4H2O 10 mg / L, ZnSO4·7H2O 2 mg / L, etc.).

[0123] Gamborg B5 organic ingredients (containing inositol 100 mg / L, thiamine·HCl 10 mg / L, etc.);

[0124] Fe-EDTA chelated iron (containing FeSO4·7H2O 27.8 mg / L, Na2EDTA·2H2O 37.3 mg / L);

[0125] 2. Additives:

[0126] L-Glutamic acid: 0.2 g / L;

[0127] L-proline: 0.1 g / L;

[0128] Hydrolyzed casein: 0.1 g / L;

[0129] Sucrose: 30 g / L;

[0130] Agar: 8 g / L;

[0131] 3. pH adjustment:

[0132] Before sterilization, its pH is adjusted to 5.8±0.1.

[0133] Add kinetin KT (1-3 mg / L), α-naphthaleneacetic acid (1-2 mg / L), and 6-benzylaminopurine (1-3 mg / L) to the basic components of the differentiation medium as described above; some of the treatment combinations are shown in Table 4.

[0134] The conditions in the tissue culture room during the callus differentiation and culture process were as follows: temperature 28±2℃, humidity 60%~70%, photocycle 16 h light / 8 h dark, and light intensity 1000~2500 lux.

[0135] Table 4 Different PGRs treatment combinations

[0136]

[0137] 1.3 Construction of the genetic transformation system

[0138] 1.3.1 Preparation of the Infecting Engineering Solution

[0139] The expression vector pCAMBIA2301-GUS (CaMV35S promoter) was provided by our laboratory. The plasmid was extracted and transformed into Agrobacterium EHA105. The Agrobacterium-derived target plasmid was streaked on LB solid medium (5 g / L yeast extract + 10 g / L tryptone + 10 g / L NaCl + 15 g / L agar (pH=7); also containing 50 mg / L Kan+ and 100 mg / L Rif+) for 3 days at 28°C. Single colonies were picked and placed in 50 mL of LB liquid medium (containing the same antibiotics, 50 mg / L Kan+ and 100 mg / L Rif+) and cultured with shaking at 28°C for 24 h. The bacterial culture was collected by centrifugation and then diluted with infection buffer to an OD600 value of 0.6-0.8 for later use.

[0140] 1.3.2 Callus infection and recovery culture

[0141] Using an optimized callus regeneration system, callus tissue induced and cultured for 30 days was immersed in Agrobacterium infection medium (containing pCAMBIA2301-Gus plasmid), and subjected to shaking infection for different time periods (28°C, 180 rpm). After co-culturing for 3 days, the tissue was washed with sterile water and transferred to recovery medium. Fresh recovery medium was added every 15 days to induce callus growth. The callus resistance rate was calculated after 45 days. Different concentrations of AS were added to the Agrobacterium infection medium, and different infection time periods were set (Table 5).

[0142] Table 5 Treatment combinations with different AS concentrations and infection times

[0143]

[0144] Select pale yellow, firm, and uncontaminated callus tissue, and use a small portion for GUS staining. Immerse the callus tissue in GUS staining solution at 37°C for 12 hours, then decolorize the tissue by shaking with 30%, 40%, 50%, 60%, and 70% ethanol for 1 hour (180 rpm) respectively, and observe and photograph under a microscope. Refer to the instruction manual for the preparation method of GUS working solution. The remaining selected callus tissue was transferred to differentiation medium (containing 300 mg / L TMT) and cultured in a tissue culture room for 15 days to promote the growth of shoot clusters. The differentiation rate was recorded.

[0145] 1.3.3 Rooting Culture of Regenerated Seedlings

[0146] Regenerated seedlings with uniform growth and good condition were selected from the differentiation medium and inoculated into the rooting medium. After 15 days, the plant height and stem diameter of the regenerated seedlings were measured. Root system indicators such as total root length, average root diameter, root surface area, and root volume were analyzed using a root system analysis system and auxiliary equipment scanner.

[0147] Rooting medium preparation: N (N6 medium), NA (N6 medium + 0.2 mg / L NAA), M (1 / 2 MS medium) and MA (1 / 2 MS medium + 0.2 mg / L NAA).

[0148] 1.4 Positive detection of transgenic materials

[0149] 1.4.1 Tissue staining of transgenic seedlings

[0150] Leaves and root tips of 120-day-old seedlings were immersed in GUS staining solution at 37°C for 12 hours to decolorize. The decolorized leaves or root tips were then observed under a microscope.

[0151] 1.4.2 PCR positive detection

[0152] After 120 days of growth, individual transgenic seedlings were tagged, and leaves of T0 generation transgenic rice were collected, quick-frozen in liquid nitrogen, ground, and DNA samples were extracted using the CTAB method. Then, PCR was used to identify the transformation rate of each individual plant.

[0153] The detection primers GUS-PTF1 / R1 are 5'-CACTGAAGCGGGAAGGGACT-3' and 3'-CGATACCGTAAAGCACGAGGAA-5', respectively.

[0154] PCR system: 7.5 μL of 2×PCR Taq Mix (PC106, Wuhan Sanying Biotechnology Co., Ltd., China); 2.5 μL of forward and reverse primers (2 μmol); 2 μL of DNA template; ddH2O to a final volume of 15 μL. PCR amplification program: pre-denaturation 94℃, 5 min; 35 cycles (denaturation 94℃, 30 s; annealing 52℃, 30 s; extension 72℃, 4 min), final extension 72℃, 5 min; storage at 4℃. Agarose gel electrophoresis.

[0155] 1.5 Data Analysis

[0156] One-way ANOVA was performed on the obtained data using SPSS 18.0 software. Duncan's method was used for multiple comparisons to assess significance. The optimal tissue culture formulation was evaluated using the D-value calculated based on membership functions. The data were processed and plotted using Graphpad Prism 8 and R language. The formulas for calculating callus-related indicators are as follows:

[0157] Induction rate (%) = Number of induced callus tissues / Number of cultured mature seeds.

[0158] Browning rate (%) = Number of browned callus / Number of subcultured callus

[0159] Browning index (%) = (number of calluses per browning level × number of calluses at each browning level) / (number of calluses produced × highest browning level)

[0160] Contamination rate (%) = Number of contaminated callus / Number of callus infected with Agrobacterium

[0161] Initial differentiation rate (%) = Number of callus tissues showing signs of differentiation / Total number of callus tissues

[0162] Differentiation rate (%) = Number of shoot-forming callus / Total number of callus

[0163] Resistance rate (%) = Number of resistant callus / Number of callus infected with Agrobacterium

[0164] GUS staining rate (%) = Number of successfully stained callus tissues with GUS / Number of callus tissues infected with Agrobacterium

[0165] Positive rate (%) = Number of PCR-positive plants / Number of T0 plants

[0166] 2 Results and Analysis

[0167] 2.1 Effects of different concentrations of 2,4-D combined with NAA on callus formation in rice

[0168] Different concentrations of 2,4-D combined with NAA were used to induce callus formation in mature rice embryos. The results showed that after 7 days of culture on the induction medium, a large number of callus tissues appeared, and after 21 days, compact, light yellow embryogenic callus tissues were successfully obtained.

[0169] Experimental results are as follows Figure 1 As shown, where Figure 1 In the table, (A) represents callus induced from mature seeds of four varieties; (B) represents the callus induction rate from rice seeds; (C) represents the size of the callus; (D) represents the fresh weight of the callus; and (E) represents the dry weight of the callus. Lowercase letters indicate significant differences between different treatments of the same variety (P<0.05). Data are expressed as mean ± standard error (n=15).

[0170] When 2,4-D was used alone for induction (A1-A4), the average induction rate for each variety ranged from 53.17% to 57.44%. When combined with NAA (A5-A20), the average induction rate for each variety increased to 57.35%-63.48%, indicating that the combination of 2,4-D and NAA can promote callus formation. Callus tissue measurements showed that under 2,4-D treatment alone, the average callus size for each variety ranged from 10.72 to 10.97 mm², with a fresh weight of 32.70-40.35 mg and a dry weight of 5.94 to 7.36 mg. After the combination of 2,4-D and NAA, the average size of callus tissue for each variety increased to 21.99-27.29 mm², the fresh weight reached 34.84-72.93 mg, and the dry weight reached 5.98-9.95 mg. This indicates that the combined treatment increased the size of the callus tissue, resulting in an increase in both fresh and dry weight, and the callus tissue became denser and yellower.

[0171] Among them, under the A9 treatment, the induction rates of Xinjing 293 and Wandao 68 reached as high as 79.34% and 83.89%, respectively, with callus sizes of 27.40 mm² and 38.53 mm², fresh weights of 53 mg and 81.5 mg, and dry weights of 12.52 mg and 23.68 mg, respectively. Under the A13 treatment, the induction rates of Xiandao Hui 128 and E33 reached as high as 75.87% and 86.61%, respectively, with callus sizes of 40.28 mm² and 43.53 mm², fresh weights of 59.4 mg and 229 mg, and dry weights of 9.68 mg and 12.6 mg, respectively, all higher than other treatments. This indicates that there are differences in the optimal response concentrations of 2,4-D and NAA between japonica and indica rice.

[0172] Based on the comprehensive evaluation of membership functions, the treatments that had the best effect on callus formation of japonica rice Xinjing 293 and Wandao 68 were A9, A7, A8, A10 and A6, respectively, while the treatments that had the best effect on indica rice Hui 128 and E33 were A13, A17, A3, A9 and A6, respectively.

[0173] 2.2 Effects of different additives on browning of rice callus

[0174] During the callus subculture process, it was found that the browning rate of Indica rice Hui 128 was significantly higher than that of other rice varieties, reaching 59.55%. Therefore, using Indica rice Hui 128 as the research material, different concentrations of carbon source were set to inhibit the browning of callus tissue.

[0175] Experimental results and data analysis are as follows Figure 2 As shown, where Figure 2The values ​​in the table represent: (A) callus browning phenotype; (B) browning rate; (C) browning index; and (D), (E), and (F) callus size, fresh weight, and dry weight, respectively. An asterisk indicates significant differences in the same indicator between different treatments, with * and ** representing P < 0.05 and P < 0.01, respectively. (n=15)

[0176] Compared to the control (S3), the browning rate of callus tissue was significantly reduced after treatments S1 and S4, while the browning rate was extremely significantly reduced after treatment S5. The browning index of S1, S4, and S5 treatments was extremely significantly lower than that of the control, with the lowest browning index of 29.66% observed in treatment S4. The fresh weight of callus tissue after treatment S1 was significantly lower than that of the control, while there were no significant differences in callus size and dry weight.

[0177] Although the browning rate was significantly reduced under S5 treatment, the callus quality was not significantly improved. Using S5 treatment as the carbon source substrate, two different concentrations (Vc and PVP) of anti-browning agents were selected to improve the callus quality.

[0178] Experimental results and data analysis are as follows Figure 3 As shown, where Figure 3 (A) shows the browning of callus tissue under different concentrations of anti-browning agent treatment; (B), (C), and (D) show the size, fresh weight, and dry weight of callus tissue under different concentrations of anti-browning agent treatment, respectively; (E) and (F) show the browning rate and browning index of callus tissue under different concentrations of anti-browning agent treatment; asterisks indicate significant differences between different treatments, * indicates P < 0.05, and ** indicates P < 0.01. (n = 15).

[0179] Under vitamin C treatment, the browning rate was significantly lower than the control. Under PVP treatment, P1 and P2 treatments were significantly lower than the control, and P3 treatment was significantly lower than the control. Regarding callus size, V1, V2, and P1 treatments were significantly higher than the control, while V4 treatment was significantly higher. Callus fresh weight was significantly higher under V2 treatment than the control, while there was no significant difference among different PVP treatments. Callus dry weight also showed no significant difference among treatments. Browning index calculations showed that both vitamin C and PVP treatments significantly reduced the browning index, with V2 and P2 treatments showing the best effects, at 2.08% and 7.14%, respectively. The top five treatments in terms of overall anti-browning effect were: V2 > V1 > V4 > P1 > V5.

[0180] In summary, S5 treatment (20 g / L maltose) can be used as the best carbon source substrate for anti-browning treatment of Hui 128 callus, while vitamin C is the most ideal anti-browning agent, with an optimal concentration of 40 mg / L.

[0181] When assessing the browning status of callus tissue, you can refer to... Figure 11 The browning grade of callus tissue, such as Figure 11 The degree of browning shown is based on the proportion of the area of ​​browning to the area of ​​each individual callus, and is divided into 5 levels: Level 0 is basically no browning, with a proportion of <10%; Level 1 has a browning proportion of 10-35%; Level 2 has a browning proportion of 36-65%; Level 3 has a browning proportion of 66-95%; and Level 4 has a browning proportion of 96-100%.

[0182] 2.3 Effects of different antibacterial agents and concentrations on callus contamination rate and differentiation

[0183] Agrobacterium-infected callus was placed in recovery culture media containing cefotaxime (Cef) and termethin (Tmt) respectively, and the contamination status of all four varieties was significantly improved under M3 and M4 treatments.

[0184] The effects of different types of antibacterial agents on callus contamination rate and differentiation in rice, such as Figure 4 As shown, where Figure 4 In the diagram, (A) shows the experimental results of different types of antibacterial agents inhibiting callus contamination; (B) and (C) show the callus contamination rate and initial differentiation rate under different antibacterial agent treatments, respectively. Lowercase letters indicate significant differences (P<0.05) between different treatments for the same variety. (n=15)

[0185] As shown in the figure, under M3 treatment, although there was some contamination and the callus tissue turned brown, new callus tissue grew, indicating that this condition had a certain promoting effect on callus formation. Under M4 treatment, due to the high concentration of antibacterial agent, the callus tissue appeared white, was non-embryonic, and lacked differentiation potential.

[0186] Contamination rate statistics show that the contamination rate decreases with increasing treatment concentration. Specifically, compared to treatment M1, treatment M2 did not significantly improve the callus contamination rate of Xinjing 293 and Wandao 68, while treatments M3 and M4 showed a sharp decrease in contamination rate. This indicates that increasing the concentration of Tmt can effectively reduce the contamination rate of Xinjing 293 and Wandao 68. In Hui 128 and E33, compared to treatment M1, treatment M2 significantly reduced the callus contamination rate. Treatments M3 and M4 showed a significantly higher reduction in Hui 128 compared to M2, indicating that increasing the concentration of Cef or Tmt can effectively inhibit contamination in Hui 128 and E33.

[0187] Differentiation rates showed that Xinjing 293, Wandao 68, Hui 128, and E33 exhibited the highest differentiation rates under M3 treatment, at 18.87%, 8.51%, 21.06%, and 11.93%, respectively. Based on these results, for these four varieties, it is recommended to use a recovery medium containing 300 mg / L tmt (M3) to inhibit callus contamination and promote callus differentiation.

[0188] 2.4 Effects of different PGRs combinations on callus differentiation rate in rice

[0189] Besides the formation of embryogenic callus, efficient regeneration capacity is also an important factor affecting the conversion efficiency of rice.

[0190] The process of differentiation into seedlings of four types of rice on differentiation culture medium is as follows: Figure 5 As shown; where Figure 5 In section A, callus tissue formed bud primordia after being cultured in differentiation medium for 15 days. At this stage, the callus tissue is in a state of hypersensitivity to PGRs, and different combinations of PGRs can effectively promote bud formation. Figure 5 B in the figure represents the new shoots that grew after being cultured on differentiation medium for 30 days, which are visible to the naked eye. Figure 5 C in the table represents these shoots that were subsequently transferred to rooting medium and cultured further to produce regenerated seedlings. The statistical analysis of callus differentiation rate is shown in Table 6.

[0191] Table 6: Effects of different concentrations and PGRs combinations on callus differentiation rate in rice

[0192]

[0193] In Table 6, lowercase letters indicate significant differences (P < 0.05) between different treatments of the same variety. Data are expressed as mean ± standard error (n = 15). Analysis of the data in the table shows that:

[0194] The differentiation rate of Xinjing 293 ranged from 5.56% to 32.50%, with a coefficient of variation of 48.51% under different treatments. Treatment G7 showed the highest differentiation rate, while treatment G4 showed a relatively lower rate, with a significant difference between the two. The differentiation rate of Wandao 68 ranged from 10.00% to 29.17%, with a coefficient of variation of 34.45% under different treatments. Treatment G8 significantly improved the differentiation rate, while treatment G6 showed a relatively poor improvement. The differentiation rate of Hui 128 varied considerably, ranging from 5.00% to 72.50%, with a coefficient of variation reaching 80.02%. Treatment G9 showed the highest differentiation rate, while treatment G4 showed a relatively lower rate, with a significant difference between the two, reflecting the high sensitivity of Hui 128 to different PGRs combinations. The differentiation rate of E33 also fluctuated significantly, ranging from 12.50% to 60.00%, with a coefficient of variation of 65.26%. The differentiation rate was higher under the G7 treatment, while it was lower under the G2 and G4 treatments.

[0195] Comprehensive analysis showed that Hui 128 and E33 performed relatively well in callus differentiation, especially Hui 128, which had a high differentiation rate under the G9 treatment, indicating its positive response to high concentrations of kinetin (KT) and 6-benzylaminopurine (6-BA). For the four rice varieties, the optimal PGRs combination for callus differentiation was the G7, G8, and G9 treatments, all of which had a high KT content of 3 mg / L, demonstrating that high KT concentrations effectively promote callus shoot formation.

[0196] 2.5 Effects of different AS concentrations and infection time combinations on rice variety transformation

[0197] like Figure 6 The effects of different AS concentrations and infection time combinations on rice callus resistance are shown. Figure 6 (A) is a line graph of rice resistant callus rate; (B) is a graph of callus growth status, showing Xinjing 293 and Wandao 68 (W7 treatment), and Hui 128 and E33 (W6 treatment); (C) is a graph of rice callus differentiation. Lowercase letters indicate significant differences (P<0.05) between different AS concentrations and infection times for the same variety. (n=15)

[0198] It can be seen that the callus resistance rate of the four varieties decreased with increasing infection time. Under W1-W5 treatments (100 µM AS), the resistance rate of all four varieties was relatively high at 5 min of infection (W1). Under W6-W10 treatments (200 µM AS), Xinjing 293 and Wandao 68 had relatively high resistance rates at 10 min of infection (W7), while Hui 128 and E33 had the highest resistance rates at 5 min of infection (W6). All varieties had low resistance rates at 40 min of infection (W5 and W10), indicating that prolonged infection time produces a toxic effect and is not conducive to transformation. Observing the callus, under W6 and W7 treatments, the callus was mainly in a yellow and compact state, with only a very small portion showing brown and dead tissue, indicating that the callus recovered and grew well under W6 and W7 treatments, and the small amount of death may be related to differences in rice genotypes. Meanwhile, compared with the indica rice varieties Hui 128 and E33, the callus tissues of japonica rice varieties Xinjing 293 and Wandao 68 exhibit faster growth rates and higher new shoot formation rates during induction and differentiation.

[0199] Table 7 shows the statistical data on the effects of different AS concentrations and infection times on rice callus differentiation.

[0200] Table 7: Effects of different AS concentrations and infection times on rice callus differentiation

[0201]

[0202] In the table, lowercase letters indicate significant differences (P<0.05) between different treatments of the same variety. Data are expressed as mean ± standard error (n=15).

[0203] The data in Table 7 show that different AS concentrations and infection times significantly affected the differentiation rates of various rice varieties. Xinjing 293 and Wan 68 showed the highest differentiation rates under treatment W7, at 41.71% and 31.15%, respectively, significantly higher than other treatments. Hui 128 and E33 showed the highest differentiation rates under treatment W6, at 28.80% and 23.36%, respectively, also significantly higher than other treatments. In contrast, after Agrobacterium infection, the differentiation rates of japonica rice varieties Xinjing 293 and Wan 68 were relatively high, while the differentiation rates of indica rice varieties Hui 128 and E33 were relatively low, indicating that japonica rice is more suitable for transformation.

[0204] After Agrobacterium-mediated transformation of the GUS gene, the transformed callus tissue was screened and cultured for 45 days, and GUS gene staining was performed to preliminarily calculate the positive staining rate.

[0205] Specific experimental data graphs are shown below. Figure 7As shown in the figure, (A) represents the GUS staining rate of callus tissue; (B) represents GUS staining of rice callus tissue. Lowercase letters indicate significant differences (P<0.05) between different AS concentrations and infection times for the same variety.

[0206] Depend on Figure 7 The following conclusions can be drawn: Xinjing 293 and Wandao 68 had the highest GUS staining rates under W7 treatment, at 13.75% and 17.5% respectively, while Hui 128 and E33 had the highest GUS staining rates under W6 treatment, at 60% and 27.5% respectively.

[0207] This is consistent with the callus resistance rate results under different AS concentrations and infection time combinations. Therefore, Xinjing 293 and Wandao 68 had higher conversion rates in W7 treatment, while Hui 128 and E33 had higher conversion rates in W6 treatment.

[0208] 2.6 Effects of different rooting media on root growth of rice regenerated seedlings

[0209] After the callus tissue differentiated and grew into regenerated shoots, it was transferred to a rooting medium. The growth of the regenerated seedlings showed significant differences under four different rooting medium treatments.

[0210] Rice root growth under different rooting media, such as Figure 10 As shown; where Figure 10 (A) shows the root growth status of four rice varieties seedlings on the rooting medium; (B) shows the root status of four rice varieties seedlings under scanning conditions.

[0211] Data analysis charts as follows Figure 8 As shown, where Figure 8 In this context, SD represents stem diameter (mm); PH represents plant height (cm); RFN represents root branch number (No.); TRL represents total root length (cm); RSA represents root surface area (cm²); RAD represents average root diameter (mm); RV represents root volume (cm³); and RTN represents root tip number (No.). Lowercase letters indicate significant differences (P<0.05) between different treatments for the same indicator of the same variety.

[0212] Depend on Figure 8 The data show that under the MA treatment, the regenerated seedlings had larger stem diameters and greater plant heights, indicating better development. Under the M treatment, the stem diameters and plant heights were smaller. There were significant differences in stem diameter between the two treatments for the four varieties, while only Xinjing 293 and Hui 128 showed significant differences in plant height. Under the MA treatment, except for E33, the other three varieties had the highest total root lengths, showing significant differences compared to the other three treatments.

[0213] The root surface area and volume of all four varieties were highest under the MA treatment, showing significant differences compared to the M treatment. E33 had fewer root tips under the MA treatment, while the other varieties had higher root tip numbers, with significant differences compared to the M treatment. Furthermore, compared to the NA treatment, the regenerated seedlings under the MA treatment exhibited relatively higher stem diameter, total root length, surface area, and volume, suggesting that the MA treatment had a better promoting effect on root growth in the regenerated seedlings.

[0214] Membership function evaluation showed that MA > N > M > NA, confirming that the rooting effect was best under MA treatment. In particular, compared to M treatment, MA treatment resulted in robust root growth with more branches, larger root volume, and more wrinkles and villous protuberances, which helps increase the contact area between the roots and the culture medium, promoting water and nutrient absorption. However, there was no significant difference between N and NA treatments. This indicates that adding NAA (NA) to N6 medium (N) has little effect on the root system of rice regenerated seedlings, but adding NAA (MA) to 1 / 2 MS medium (M) significantly promotes root growth.

[0215] 2.7 Positive detection of transgenic seedlings

[0216] After 120 days of growth, the regenerated seedlings of the four varieties showed heading in Japonica rice varieties Xinjing 293 and Wandao 68, and booting in Indica rice varieties Hui 128 and E33. Individual plants of each variety were labeled and PCR tested.

[0217] Test results as follows Figure 9 As shown, where Figure 9 (A) Transgenic seedlings after 120 days of growth, scale bar 10 cm; (B) PCR positive detection, M is DL2000 marker, N is negative control, lanes 1, 2, 3, 4, and 6 are positive plants, lane 5 is a negative single plant, WT is wild-type recipient; (C) GUS staining of leaves and root tips of transgenic plants (left is WT line, right is transgenic line), scale bar 1 mm; (D) Transformation efficiency of four rice varieties.

[0218] The results showed that ( Figure 9 The GUS gene fragment size was 488 bp, and the band was single and bright, indicating that the four varieties were successfully transformed with the GUS gene, and transgenic lines were obtained. GUS staining was performed on the leaves and root tips of the positive plants from the four varieties after PCR detection. It was found that the mesophyll cells on both sides of the leaf veins and the root tip cells of the transgenic plants stained blue, while the wild-type (WT) lines did not stain, proving that the GUS gene was successfully transformed and stably expressed in rice tissues.

[0219] Under W7 treatment, the positive rates of Xinjing 293 and Wandao 68 reached 43.24% and 52.38%, respectively. Under W6 treatment, the positive rates of Hui 128 and E33 reached 40.00% and 40.74%, respectively. These results indicate that the optimized rice transformation system has high genetic transformation efficiency for the four conventional rice varieties, and the expression of target genes in positive plants is stable, which can provide a basis for the transformation of conventional japonica and indica rice varieties.

[0220] 3. Discussion

[0221] 3.1 Effect of the combination of 2,4-D and NAA on callus induction

[0222] In Agrobacterium-mediated rice transformation, embryogenic callus with cell division potential is a crucial factor determining genetic transformation. Previous studies have shown that 2,4-D is a major growth regulator in rice callus induction and plays an important role in the proliferation of embryogenic callus.

[0223] Typically, a 2 mg / L concentration of 2,4-D is widely used for callus induction in japonica rice (e.g., Nipponbare), achieving an induction rate of 77%. However, using a combination of 2,4-D (3 mg / L) and NAA (2 mg / L) induction medium, the induction rate can be increased to 90%, with better callus growth quality, indicating that the combination of 2,4-D and naphthaleneacetic acid (NAA) can improve callus quality. This study screened different concentrations of 2,4-D and NAA combinations and found that the combination significantly promoted callus formation and growth. However, japonica rice varieties (Xinjing 293 and Wandao 68) responded better to 2 mg / L 2,4-D and 1 mg / L NAA, while indica rice varieties (Hui 128 and E33) showed a better response to the combination of 3 mg / L 2,4-D and 1 mg / L NAA.

[0224] 3.2 Effects of different additives on browning of rice callus

[0225] Browning of callus tissue affects callus quality and may even lead to callus death. In rice transformation, different carbon sources have a significant impact on callus browning.

[0226] In this study, the indica rice variety Hui 128 showed severe browning. After treatment with maltose, the degree of browning was reduced. However, high concentrations of maltose may increase the risk of browning. This may be because excessive carbohydrate supplementation enhances the enzyme activity of callus tissue, leading to an increase in polyphenol oxidase and exacerbating browning.

[0227] Maltose at 20 g / L effectively reduced callus browning but did not improve callus quality. To further explore effective strategies, this study added different concentrations of the anti-browning agents vitamin C and polyphenol oxidase (PVP) to adjust callus quality. Vitamin C inhibited browning by reducing polyphenol oxidase-mediated browning, while PVP prevented browning by chelating metal ions. In comparison, 40 mg / L vitamin C showed higher anti-browning ability and significantly improved callus quality.

[0228] 3.3 Effects of different antibacterial agents and concentrations on callus contamination rate and differentiation

[0229] In Agrobacterium-mediated transformation, cefotaxime (Cef) and termethin (Tmt) are often used to selectively inhibit non-target bacteria and reduce callus contamination. These antibiotics mainly work by inhibiting bacterial cell wall synthesis or interfering with protein synthesis and generally do not affect callus activity.

[0230] This study shows that the contamination rate decreased significantly with increasing concentrations of the antibiotics Cef and Tmt. However, callus differentiation was poor after Cef treatment, while it was relatively better under Tmt treatment. But when the Tmt concentration reached 500 mg / L, callus differentiation was inhibited, indicating that excessively high concentrations of antibiotics have a toxic effect on callus tissue. For these four rice varieties, treatment with a recovery medium containing 300 mg / L Tmt resulted in a lower callus contamination rate and a higher differentiation rate, effectively addressing the problems of callus contamination and differentiation.

[0231] 3.4 Effects of different PGRs combinations on callus differentiation rate in rice

[0232] Research has found that the organ regeneration capacity of callus tissue stems from its unique hormone response characteristics. Callus tissue not only accumulates high concentrations of auxin, but also exhibits hypersensitivity to cytokinin. This dual characteristic enables it to regenerate both roots and shoots. Low concentrations of auxin can induce root differentiation, while high concentrations of cytokinin can activate shoot gene expression and promote shoot development.

[0233] In differentiated somatic cells, auxin and cytokinin inhibit each other, thus lacking the ability to regenerate organs. However, in rice transformation, cytokinin (such as KT and 6-BA) and auxin (such as NAA) play indispensable roles.

[0234] NAA primarily promotes cell division and elongation, and induces shoot formation. Different rice genotypes exhibit varying responses to NAA concentrations.

[0235] In a differentiation medium containing a combination of 2.0 mg / L KT and 0.1 mg / L NAA, the "CBMH" genotype of the rice variety Moroberekan exhibited the highest differentiation rate, while the "JDJ" genotype showed a differentiation rate of only 10%. This study found that KT tends to promote cell division rate and accelerate callus proliferation, while 6-BA is better at promoting the formation and growth of lateral buds.

[0236] Xinjing 293 and E33 showed better lateral bud formation under low NAA concentration (1 mg / L, G7), while Hui 128 was more suitable for higher NAA concentration (2 mg / L, G9). The optimal NAA concentration for Wandao 68 was 1.5 mg / L (G8), which could promote rapid callus differentiation and effectively promote bud formation.

[0237] 3.5 Effects of different AS concentrations and infection time combinations on rice transformation

[0238] Studies have shown that the addition of AS is essential in the transformation of monocotyledonous plants. AS can activate the Vir gene and promote the transfer of T-DNA to plant cells. Appropriate concentrations of AS can prevent plant necrosis and improve the transformation efficiency of rice.

[0239] Meanwhile, an appropriate infection time can ensure instantaneous expression and a high selective differentiation rate in callus tissue. If the infection time is too long, the callus tissue will die due to hypoxia and toxicity. If the infection time is insufficient, the Agrobacterium tumefaciens solution cannot fully contact the callus tissue, thus reducing the callus tissue expression rate.

[0240] Rice callus tissue was soaked in Agrobacterium infection solutions containing different concentrations of AS, and 200 μM AS was screened as the optimal concentration for rice transformation, with a transformation rate of 10%-30%. In fact, as the AS concentration increases, the rice transformation efficiency also increases accordingly. At 200 μM AS, the transformation rate of rice IET-4786 can reach 33.33%.

[0241] This experiment showed that callus quality was better at 200 μM AS. The resistance rate of callus decreased with increasing infection time. Xinjing 293 and Wandao 68 showed the highest conversion rates after 10 min of infection, at 43.24% and 52.38%, respectively, while Hui 128 and E33 showed the best conversion rates after 5 min of infection, at 40.00% and 40.74%, respectively.

[0242] 3.6 Effects of different rooting media on root growth of rice regenerated seedlings

[0243] This study compared the effects of two widely used basal media on rooting. N6 medium, rich in minerals, is primarily used for tissue culture of monocotyledonous plants, particularly for callus induction, seedling regeneration, and protoplast culture in crops such as rice and maize. 1 / 2 MS medium, rich in inorganic salts, helps maintain a stable ion balance, making it more suitable for rapid root growth in plant seedlings. NAA stimulates root primordium formation, playing a crucial role in the rooting of tissue-cultured seedlings, cuttings, and transplanted seedlings.

[0244] This study found that when NAA was added to two different basal culture media, the root growth and branching of tissue culture seedlings of four rice varieties showed significant differences. Among them, the MA-type medium (1 / 2 MS medium + 0.2 mg / L NAA) performed exceptionally well, particularly in terms of root length, root surface area, root volume, and number of root tips. This was mainly because 1 / 2 MS contains abundant trace elements (such as iron, manganese, zinc, copper, boron, molybdenum, etc.), and when combined with an appropriate amount of NAA hormone, these components work together to promote rapid root development, well-developed lateral roots, increased root volume, and enhanced root absorption capacity, which helps the regenerated seedlings grow vigorously.

[0245] 4. Conclusion

[0246] In summary, the above studies indicate that callus induction in japonica and indica rice is best achieved with induction media of A9 (2 mg / L 2,4-D + 1 mg / L NAA) and A13 (3 mg / L 2,4-D + 1 mg / L NAA), respectively, exhibiting high induction rates and good growth. When callus browning occurs during subculture, it can be inhibited by adding 20 g / L maltose and 20 mg / L vitamin C to the subculture medium.

[0247] Under M3 treatment (Tmt 300 mg / L), antibiotics effectively inhibited Agrobacterium contamination. Japonica rice was infected with 200 µM AS infection buffer for 10 minutes, and indica rice for 5 minutes. Under these conditions, the callus tissue exhibited the highest resistance rate and GUS staining rate, and the target gene expression was stable. The transformation positivity rates of Xinjing 293 and Wandao 68 were 43.24% and 52.38%, respectively, while the transformation positivity rates of Hui 128 and E33 were 40.00% and 40.74%, respectively. These results provide a theoretical basis for the efficient genetic transformation of conventional rice.

[0248] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. Construction of a genetic transformation system for local rice varieties, characterized by: The components of the induction medium used for the formation of primary callus from rice seeds are as follows: N6 medium macro-elements (Zhu Zhiqing formula, containing KNO3 2.8 g / L, (NH4)2SO4 0.463 g / L, etc.); Gamborg B5 medium contains trace elements (including MnSO4·4H2O 10 mg / L, ZnSO4·7H2O 2 mg / L, etc.). Gamborg B5 organic ingredients (containing inositol 100 mg / L, thiamine·HCl 10 mg / L, etc.); Fe-EDTA chelated iron (containing FeSO4·7H2O 27.8 mg / L, Na2EDTA·2H2O 37.3 mg / L); L-Glutamic acid: 0.2 g / L; L-proline: 0.1 g / L; Acid-hydrolyzed casein: 0.1 g / L; Sucrose: 30 g / L; Agar: 8 g / L; Add different concentrations of 1-4 mg 2,4-D and 0-2.5 mg NAA to the above-mentioned basic components of the induction medium; The pH of the induction medium was 5.8±0.1; the induction culture temperature was 28±2℃, the humidity was 60%~70%, and the culture was carried out in the dark.

2. The construction of the genetic transformation system for local rice varieties according to claim 1, characterized in that: Japonica rice varieties were treated with a hormone combination containing 2 mg / L 2,4-D + 1 mg / L NAA, while indica rice varieties were treated with a hormone combination containing 3 mg / L 2,4-D + 1 mg / L NAA. Embryogenic callus was induced by dark culture for 15 days.

3. The construction of the genetic transformation system for local rice varieties according to claim 1, characterized in that: Rice seeds were cultured in an induction medium for 15 days to obtain primary callus tissue, which was then transferred to a new induction medium for subculture for 15 days. The induction medium for subculture was supplemented with 20 g / L maltose and 40 mg / L vitamin C.

4. The construction of the genetic transformation system for local rice varieties according to claim 3, characterized in that: The callus tissue induced for 30 days was placed in the infection solution for 30 min, and then air-dried on sterile filter paper. The infection solution was prepared by Agrobacterium tumefaciens bacterial suspension and infection buffer at OD600=0.6-0.

8. The infection buffer contained the following components: AA macroelements + Gamborg B5 microelements + Gamborg B5 organic components + iron salt; MES buffer: 3.9 g / L; hydrolyzed casein (CH): 0.5 g / L.

5. The construction of the genetic transformation system for local rice varieties according to claim 4, characterized in that: The callus tissue induced for 30 days was placed in the infection solution for 30 minutes, then dried on sterile filter paper, transferred to the symbiotic culture medium, and co-cultured at 28°C for 3 days. The symbiotic culture medium consists of the following components: N6 macroelements + B5 microelements + B5 organic compounds + iron salts + inositol 2g / L + MES 3.9g / L + CH 0.5g / L + 30g / L sucrose + 8g / L agar (pH=5.5).

6. The construction of a genetic transformation system for local rice varieties according to claim 5, characterized in that: After being cultured on a symbiotic medium, the callus tissue was transferred to a recovery medium for further culture. The basic components of the recovery medium include: N6 macroelements; B5 microelements; B5 organics; iron salts; 0.2 g / L Glu; 0.1 g / L L-Pro; 0.1 g / L CH; 2.0 mg / L 2,4-D; 30 g / L sucrose; 8 g / L agar; the pH of the recovery medium is adjusted to 5.8 ± 0.

1. Different concentrations of 0-500 mg / L cefotaxime and 0-500 mg / L termethin were added to the basic components of the above-mentioned recovery culture medium.

7. The construction of a genetic transformation system for local rice varieties according to claim 1, characterized in that: The differentiation medium used for callus differentiation is composed of the following components: N6 macroelements + B5 microelements + B5 organic compounds + iron salts + 0.2 g / L Glu + 0.1 g / L L-Pro + 0.1 g / L CH + 30 g / L sucrose + 8 g / L agar; kinetin KT (1-3 mg / L), α-naphthaleneacetic acid (1-2 mg / L), and 6-benzylaminopurine (1-3 mg / L) are added to the above basic components of the differentiation medium.

8. The construction of a genetic transformation system for local rice varieties according to claim 7, characterized in that: The differentiation medium used for japonica rice varieties was supplemented with 3 mg / L KT, 1 mg / L NAA and 2 mg / L 6-BA; the differentiation medium used for indica rice varieties was supplemented with 3 mg / L KT, 2 mg / L NAA and 3 mg / L 6-BA.

9. The construction of a genetic transformation system for local rice varieties according to any one of claims 4-6, characterized in that: The infection solution was supplemented with 200 μM acetylsuccinone (AS), and the infection time was set according to genotype differences: 10 min for japonica rice and 5 min for indica rice.

10. The construction of a genetic transformation system for local rice varieties according to claim 1, characterized in that: The rooting medium used in the callus rooting process was 1 / 2 MS + 0.2 mg / L NAA (MA treatment), and its components included: 1 / 2 MS basal salt (containing iron salts and trace elements); 0.2 mg / L α-naphthaleneacetic acid (NAA); 30 g / L sucrose + 8 g / L agar (pH 5.8 ± 0.1).

Images