Application of High-Efficiency Regeneration Line Screening Methods for Malus 'Apple' in Genetic Transformation

Abstract

This invention relates to a method for screening highly efficient regenerated lines of *Malus argentea* and its application in genetic transformation. The regenerated line screening method described in this invention can rapidly and effectively screen out *Malus argentea* tissue culture seedlings with high regeneration capacity. Furthermore, overexpression of the MdSBRL gene in *Malus argentea* tissue culture seedlings significantly increases the number of roots, thereby enhancing the application value of *Malus argentea* as a grafting rootstock for apples.

Description

Technical Field

[0001] This invention relates to the fields of crop genetics and breeding and plant genetic engineering, specifically to the application of a highly efficient regenerative line screening and genetic transformation of Malus baccata. Background Technology

[0002] Root systems are fundamental to plant growth and survival, and their health and rooting ability are crucial. They not only absorb water and nutrients, providing essential resources, but also offer robust support, helping plants withstand environmental adversities such as drought and soil infertility. A strong root system also contributes to plant survival and reproduction in adverse conditions, playing a key role in ecosystems and maintaining ecological balance and biodiversity. Therefore, root systems and rooting ability are critical factors for plant adaptation to their environment and sustainable growth and reproduction.

[0003] Malus × Robusta is a species of apple tree belonging to the genus Malus in the family Rosaceae. It is a hybrid of Malus baccata and Malus macrantha. Because apples are self-incompatible and have a highly heterozygous genetic background, apple seedlings are generally propagated through vegetative propagation methods such as layering and grafting. To promote early and high yields and enhance resistance, apple varieties are typically grafted onto rootstocks with robust root systems and strong resistance to adverse conditions, thereby improving their drought tolerance, salt and alkali tolerance, tolerance to poor soil, cold resistance, and disease resistance. Malus × Robusta is an excellent apple rootstock, exhibiting strong resistance to waterlogging and salinity, making it the most suitable apple rootstock for low-lying plains, coastal areas, and inland saline-alkali regions.

[0004] Rooting is of great significance to Malus halliana. It not only improves the plant's reproductive efficiency and ease of cultivation and management, but also enhances its adaptability to adverse conditions such as drought and saline-alkali soil, ensuring the plant's health and disease resistance. At the same time, it provides an important selection basis for genetic improvement, thereby cultivating varieties with greater adaptability and ornamental value.

[0005] The SBRL gene has been shown to promote the growth and development of seedling roots in tomatoes (Omary M et al., 2022), but it has not been reported in Rosaceae plants such as apples. It is necessary to screen for an efficient genetic transformation genotype of apple rootstock Malus baccata and use this germplasm as test material to overexpress the rooting gene and create new Malus baccata rootstock materials. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for screening highly efficient regenerated lines of *Malus argentea* and its application in genetic transformation. This regenerated line screening method can rapidly and effectively screen out *Malus argentea* tissue culture seedlings with high regeneration capacity. Furthermore, overexpression of the MdSBRL gene in *Malus argentea* tissue culture seedlings significantly increases the number of roots, thereby enhancing the application value of *Malus argentea* as a grafting rootstock for apples.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for screening highly efficient regenerated lines of Malus macrantha, characterized in that the method includes the following steps:

[0009] Multiple samples of Malus halliana seedlings were obtained, and tissue culture regeneration data and morphological index data of each sample were obtained.

[0010] Correlation analysis was performed on the tissue culture regeneration data and morphological index data of each sample to determine the evaluation model for the regeneration capacity of tissue culture Malus halliana seedlings.

[0011] The evaluation model for the regeneration capacity of tissue-cultured Malus octagonalis seedlings is as follows:

[0012] Y = -0.522 + 0.734 * basal diameter + 3.752 * germination rate;

[0013] The regeneration ability of tissue-cultured Malus halliana seedlings was evaluated using the above-mentioned evaluation model.

[0014] Based on the above plan,

[0015] The tissue culture regeneration data include: callus formation rate, budding rate, and average number of regenerated buds;

[0016] The morphological data include: plant height, stem diameter, leaf length, leaf width, leaf thickness, number of branches, and number of internodes.

[0017] An application of the MdSBRL gene in promoting plant rooting ability, characterized in that the application increases plant rooting ability by overexpressing the MdSBRL gene in plants.

[0018] The nucleotide sequence of the MdSBRL gene is shown in SEQ ID NO.1, which encodes the protein shown in SEQ ID NO.2.

[0019] The screening method for high-efficiency regenerated lines of Malus macrantha described in this invention and its application in genetic transformation have the following beneficial effects:

[0020] This invention provides a method for easily screening large quantities of highly regenerating Malus halliana germplasm in the field, and creates new Malus halliana germplasm with extremely strong rooting ability through transgenic technology. Attached Figure Description

[0021] The present invention includes the following figures:

[0022] Figure 1 Flowchart for sowing Malus halliana. (a) Stratification treatment of Malus halliana seeds; (b) Selection of healthy and plump seeds with hypocotyls grown to 0.2-0.5 cm; (c) Sowing in seedling substrate blocks in a cold greenhouse; (d) Seedling management.

[0023] Figure 2 To establish a sterile system: (a) morphological parameters of 100 seedlings were measured; (b) stem segments were aseptically treated; (c) explants were grown on MS medium; (d) a sterile system for 100 seedlings was established.

[0024] Figure 3 The growth of BL-57 was shown in different subculture media. Medium A: MS + 0.2 mg / L 6-BA + 0.2 mg / L IAA + 30 g / L sucrose + 7.5 g / L agar; Medium B: MS + 0.3 mg / L 6-BA + 0.2 mg / L IAA + 0.1 mg / L GA3 + 30 g / L sucrose + 7.5 g / L agar; Medium C: MS + 2 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar; Medium D: MS + 0.6 mg / L 6-BA + 0.15 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar; Medium E: MS + 1 mg / L 6-BA + 0.5 mg / L IAA + 2 mg / L GA3 + 30 g / L sucrose + 7.5 g / L agar.

[0025] Figure 4 The germination of BL-57 in different germination media is shown. Leaves A, B, C, D, and E are placed with the upper surface facing up; leaves A', B', C', D', and E' are placed with the lower surface facing up. A and A' contain 2 mg / L TDZ + 0.4 mg / L NAA; B and B' contain 2 mg / L TDZ + 0.5 mg / L NAA; C and C' contain 2 mg / L TDZ + 0.4 mg / L NAA + 0.2 mg / L IAA + 2 mg / L 6-BA; D and D' contain 2 mg / L TDZ + 1.0 mg / L NAA + 5 mg / L 6-BA; E and E' contain 0.8 mg / L IAA + 6 mg / L 6-BA.

[0026] Figure 5The following describes the budding results for different types of leaf damage. A is achieved by making three horizontal cuts on the leaf; B is achieved by cutting the leaf into small squares with scissors; C is achieved by punching holes with a hole punch.

[0027] Figure 6 A gel image for DNA level identification of Malus halliana.

[0028] Figure 7 To compare the rooting of the control line and the transgenic line expressing MdSBRL in Malus baccata. Detailed Implementation

[0029] To further explain the purpose and technical solution of this invention, the invention will be described in further detail below with reference to the accompanying drawings and embodiments. The embodiments given are only for illustrating the invention and are not intended to limit the scope of the invention. Experimental procedures in the following embodiments, unless otherwise specified, were performed under conventional conditions known to those skilled in the art.

[0030] Example 1: A screening method for a highly efficient regenerated Malus baccata apple strain:

[0031] (1) Sowing of Malus halliana seeds: In early March, wash about 10,000 Malus halliana seeds. After washing, wrap the seeds in damp gauze and seal them in a sealed bag. Store them in a refrigerator at 4℃. Wash the seeds and gauze every week and observe the germination. When the germination rate is above 50%, sow 3,000 plump seeds that have germinated and whose hypocotyls have grown to 0.2cm-0.5cm into the seedling substrate blocks in the cold greenhouse, cover with a thin layer of soil, and water thoroughly. Observe the emergence and replace any seeds that fail to emerge in time.

[0032] (2) Seedling management: Ensure sufficient water during the seedling growth stage (April-August). Apply compound fertilizer during the vigorous growth period (June) to meet the growth needs. Spray high-efficiency cyhalothrin in time to prevent pests (June-October). Loosen the soil and remove weeds frequently to ensure soil oxygen content (April-October).

[0033] (3) Morphological index measurement: 100 seedlings of different ecotypes (e.g., Malus baccata) were selected during the growth stage of Malus baccata. Figure 1 As shown, growth index data were measured. The indexes included plant height, basal stem diameter, leaf length, leaf width, leaf thickness, number of branches, and number of internodes.

[0034] (4) Establishment of a sterile system: Leaves and stem segments of seedlings growing in a cold greenhouse were used as explants. They were rinsed with tap water for 1 hour, then disinfected with 75% alcohol for 45 seconds, rinsed three times with sterile water, disinfected with 2% sodium hypochlorite for 8 minutes, rinsed three times with sterile water, and then the water was absorbed with sterile filter paper. The leaf margins were removed with sterile scissors, leaving the central veins. The leaves were then laid flat on the regeneration medium, or stem segments were inserted into the medium as explants (e.g., ...). Figure 2 (As shown), 54 explants survived.

[0035] (5) Comparison of regeneration capacity: The leaves of 54 tissue culture seedlings of different ecotypes were placed on the bud induction medium to calculate the callus formation rate, budding rate and average number of regenerated buds.

[0036] (6) Data Analysis: Correlation analysis and principal component analysis were performed on the morphological data (plant height, stem diameter, leaf length, leaf width, leaf thickness, number of branches, number of internodes) measured in the field and the tissue culture regeneration data (callus formation rate, budding rate, average number of regenerated buds) using SPSS software. See Table 1 for details.

[0037] Table 1. Determination of morphological and regeneration indicators among different Malus 'Aglaonema' seedlings.

[0038]

[0039] Table 1 shows the statistical analysis of data from 54 Malus 'Arcticum' seedlings of different ecotypes that successfully established an aseptic system. The morphological indicators and regeneration abilities of the Malus 'Arcticum' seedlings varied among the different ecotypes. Among them, line number 57 had a callus formation rate of 100%, a budding rate of 87%, and the highest average number of regenerated buds (15). The results indicate that line number 57 has the strongest regeneration ability, and further optimization of the regeneration system for this material is possible in the next step.

[0040] Principal component analysis (PCA) can transform numerous test indicators into a smaller number of comprehensive indicators with minimal loss of information, effectively condensing data and simplifying indicators. PCA was performed on standardized data of 10 individual indicators using SPSS software to evaluate the growth capacity of Malus halliana seedlings, establishing a comprehensive evaluation model for the growth capacity of Malus halliana seedlings.

[0041] As shown in Table 2, the main components are divided into four principal components, with contribution rates of 28.970%, 17.006%, 12.432%, and 11.044%, respectively. The cumulative contribution rate reaches 69.451%.

[0042] Table 2 Principal Component Analysis of Regeneration Capacity and Morphological Indicators

[0043]

[0044] As shown in Table 3, correlation analysis revealed a significant correlation between the average number of regenerated buds and basal diameter (P<0.05), and a highly significant correlation with the budding rate (P<0.01), with no significant correlations with other indicators. Plant height showed a highly significant correlation with basal diameter and number of branches (P<0.01), and a significant correlation with the budding rate (P<0.05). Basal diameter showed a highly significant correlation with the number of branches and budding rate (P<0.01). Leaf length showed a highly significant correlation with leaf width (P<0.01). The above results indicate that the factors significantly correlated with the average number of regenerated buds are basal diameter and budding rate, which may be the main factors playing a role in evaluating the regeneration capacity of different ecotypes of Malus halliana.

[0045] Table 3 Correlation Analysis between Regeneration Capacity and Morphological Indicators

[0046]

[0047] As shown in Table 4, the evaluation model equation for the regeneration capacity of Malus halliana can be derived as follows:

[0048] Y = -0.522 + 0.734 * base diameter + 3.752 * germination rate.

[0049] Table 4. Evaluation Model Coefficients for the Regeneration Capacity of Malus 'Octagonus'

[0050]

[0051] Substituting the data of 54 ecotypes of Malus halliana into the above-mentioned evaluation model equation for the regeneration capacity of Malus halliana, the regeneration capacity rating score of Malus halliana was obtained. Further K-Means cluster analysis was used to preliminarily divide the evaluation values ​​of the regeneration capacity of the 54 different ecotypes of Malus halliana into three levels, with classification standard points of 4.118 and 5.388.

[0052] As shown in Table 5, the evaluation criteria for the tissue culture regeneration ability of Malus halliana seedlings are as follows:

[0053] Strong: If the Y value of the evaluation model is ≥5.388, then the tissue culture regeneration ability of the seedling is strong;

[0054] In the middle: if the evaluation model has 4.118 < Y value < 5.388, then the tissue culture regeneration ability of the seedling is moderate;

[0055] Weak: If the evaluation model's Y value is ≤ 4.118, then the tissue culture regeneration ability of the seedling is weak;

[0056] Table 5. Grading of Regeneration Capacity of Different Malus spectabilis species

[0057]

[0058] Example 2 investigates the effects of different hormone combinations on the succession of BL-57.

[0059] Sterile seedlings of Malus halliana BL-57 (strain number 57 in Table 5) with uniform growth vigor were selected, and the top stem segments were taken for subculture. Subculture was carried out on subculture media with five different hormone combinations. After 30 days, the proliferation coefficient was calculated and the status was observed. The formulations of each culture medium are as follows: Medium A: MS + 0.2 mg / L 6-BA + 0.2 mg / L IAA + 30 g / L sucrose + 7.5 g / L agar; Medium B: MS + 0.3 mg / L 6-BA + 0.2 mg / L IAA + 0.1 mg / L GA3 + 30 g / L sucrose + 7.5 g / L agar; Medium C: MS + 2 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar; Medium D: MS + 0.6 mg / L 6-BA + 0.15 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar; Medium E: MS + 1 mg / L 6-BA + 0.5 mg / L IAA + 2 mg / L GA3 + 30 g / L sucrose + 7.5 g / L agar.

[0060] Table 6 shows that the C subculture medium (containing MS + 2 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar) produced the highest proliferation coefficient for Malus halliana BL-57, reaching 5. However, as... Figure 3 It was observed that plants grown on subculture medium C were relatively small, with weak growth and thin, pale green leaves. The purpose of optimizing the subculture medium was to promote vigorous plant growth and thick, dark green leaves. Furthermore, although the proliferation coefficients of subculture media B and D were the same, the leaves of plants grown on subculture medium B were larger and thicker than those on subculture medium D. Therefore, subculture medium B (containing MS + 0.3 mg / L 6-BA + 0.2 mg / L IAA + 0.1 mg / L GA3 + 30 g / L sucrose + 7.5 g / L agar) was chosen as the subculture medium for Malus halliana BL-57.

[0061] Table 6. Growth of Malus 'Oryza sativa' BL-57 under different subculture media.

[0062]

[0063] Example 3 investigates the effects of different hormone combinations and leaf placement methods on bud emergence of BL-57 leaves.

[0064] Select BL-57 tissue culture seedlings of the same seedling stage, cut them into small squares of the same size, and place them in two ways: face up and back up. Place them on germination medium with five different hormone combinations for 30 days of dark culture, and then place them under light for one week. Then count the average number of regenerated buds. The formulations of each culture medium are as follows: A. Germination medium contains MS + 2 mg / L TDZ + 0.4 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar; B. Germination medium contains MS + 2 mg / L TDZ + 0.5 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar; C. Germination medium contains MS + 2 mg / L TDZ + 0.5 mg / L NAA + 0.2 mg / L IAA + 2 mg / L 6-BA + 30 g / L sucrose + 7.5 g / L agar; D. Germination medium contains MS + 2 mg / L TDZ + 1 mg / L NAA + 5 mg / L 6-BA + 30 g / L sucrose + 7.5 g / L agar; E. Germination medium contains MS + 0.8 mg / L IAA + 0.6 mg / L 6-BA + 30 g / L sucrose + 7.5 g / L agar.

[0065] From Table 7 and Figure 4 It is known that the number of buds varies on different budding media, and the way the leaves are placed also affects the number of buds. The average number of buds is highest on budding medium B, and the number of buds is highest when the leaves are placed with the upper surface facing upwards. Therefore, budding medium B with the upper surface facing upwards is selected as the culture method for BL-57 budding.

[0066] Table 7. Effects of different germination media and leaf placement methods on budding of BL-57.

[0067]

[0068] Example 4 investigates the effects of different injury methods on leaf budding of BL-57.

[0069] Tissue culture seedlings of BL-57 with the same seedling stage were selected and treated with three methods: A) making three cuts, B) cutting into small squares, and C) punching holes with a punch. The treated leaves were placed on germination medium containing MS + 2 mg / L TDZ + 0.5 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar, with the leaf surface facing up, and were cultured in the dark for 30 days. After that, they were placed under light for one week, and the average number of regenerated buds was counted.

[0070] As shown in Table 8 and Figure 5 As shown, the budding rate of leaves treated with different damage methods varies, with the method of making three cuts being the most effective.

[0071] Table 8. Effects of different injury methods on BL-57 budding.

[0072]

[0073] Example 5: Genetic transformation of Malus halliana

[0074] (1) Preparation of bacterial culture

[0075] The nucleotide sequence of the MdSBRL gene is shown in SEQ ID NO.1; the amino acid sequence encoded by MdSBRL is shown in SEQ ID NO.2. According to the apple genome structure annotation provided by the Rosaceae Genome website (https: / / www.rosaceae.org / ; GDR), the full-length CDS of MdSBRL is 720 bp. The full-length CDS of MdSBRL was cloned and constructed into the 35S:GFP overexpression vector pRI101-GFP, which was then transformed into Agrobacterium tumefaciens EHA105. Successfully transformed Agrobacterium tumefaciens bacterial culture was obtained. Positive clones were picked and cultured overnight at 28°C with 1 mL of YEP + 50 mg / L Rif + 25 mg / L Kan liquid medium at 180 rpm with gentle shaking. Then, 1 mL of the bacterial culture was added to 50 mL of YEP + 50 mg / L Rif + 25 mg / L Kan liquid medium for large-scale amplification culture. When the bacterial culture reached OD... 600 When the concentration of bacteria is 0.8-1.0, collect bacteria by centrifugation at 6000 rpm for 8 min at room temperature, and then resuspend in MES buffer (10 mM / L MES-KOH (pH 5.2) + 10 mM / L MgCl2 + 200 mM / L AS) to OD. 600 =0.5, let stand for 1 hour, then use for infection.

[0076] (2) Infection and co-cultivation

[0077] The leaves of plant number 57, selected in step (5) of Example 1, were infected with the prepared bacterial solution. The leaves were immersed in the bacterial solution for 8 minutes, the solution was filtered off, and the bacterial solution on the leaf surface was blotted dry with sterile filter paper sterilized at 121°C for 20 minutes. The dried leaves were then placed on a co-culture medium for 3 days of dark incubation. The co-culture medium consisted of MS medium supplemented with 0.5 mg / L NAA, 2.0 mg / L TDZ, 30 g / L sucrose, and 7.5 g / L agar, with a pH of 5.8.

[0078] (3) Delayed cultivation

[0079] After co-culture, the cells were placed in a delayed culture medium for 3 days, followed by 28 days of dark culture and then light culture until budding. The delayed culture medium consisted of MS medium supplemented with 0.5 mg / L NAA, 2.0 mg / L TDZ, 250 mg / L Cef, 30 g / L sucrose, and 7.5 g / L agar, with a pH of 5.8.

[0080] (4) Identification

[0081] The seedlings regenerated from delayed culture were subjected to DNA-level PCR identification to determine whether they were transgenic positive seedlings. For example... Figure 6 As shown, strain #3 was identified as a positive transgenic seedling.

[0082] (5) Comparison of rooting ability

[0083] Transgenic and non-transgenic Malus halliana plants with similar growth stages were selected and placed in 1 / 2 MS medium supplemented with 15 g / L sucrose, 7.5 g / L agar, and pH 5.8 for rooting. Rooting progress was compared after 30 days. Figure 7 As shown, after 30 days, the rooting ability of transgenic line #3 was significantly improved compared with WT.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0085] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. An application of the MdSBRL gene in promoting plant rooting ability, characterized in that, The application increases the rooting ability of plants by overexpressing the MdSBRL gene, which encodes the protein shown in SEQ ID NO.2; the plant is Malus spectabilis.

2. The application according to claim 1, wherein the nucleotide sequence of the MdSBRL gene is shown in SEQ ID NO.1.

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