Application of BjuPP2C52 gene in resistance breeding of Brassica juncea

Abstract

The application belongs to the technical field of plant molecular breeding, and particularly relates to application of BjuPP2C52 gene in resistance breeding of stem knot mustard. The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2. The overexpression vector of the gene is transformed into stem knot mustard, and a large number of epidermal hairs appear on leaves and stems of the obtained transgenic plants. Plant epidermal hair is a sharp protrusion or exogenous epidermal tissue caused by glandular hair and cortical cell deformation, has defense effects of increasing plant strength, avoiding plants from being eaten by animals of various types, and reducing water evaporation. The research result also provides a molecular genetic improvement target for resistance breeding of Brassica plants including stem knot mustard. Aphids and the spread of the virus disease caused by aphids are one of the main factors affecting the yield of stem knot mustard, and cultivating the transgenic plants of the scheme is beneficial to improving the disease and pest resistance of stem knot mustard, and has a great popularization and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically involving the application of the BjuPP2C52 gene in resistance breeding of stem mustard. Background Technology

[0002] Stem mustard (Brassica juncea var. tumida Tsen et Lee), commonly known as green mustard, is a variety of mustard in the Brassica genus of the Brassicaceae family. It is a vegetable endemic to my country with a cultivation history of nearly 300 years. Fuling, Chongqing, is the largest planting and processing base for stem mustard. Its modified stems are its main economic harvest, serving as the primary raw material for Fuling pickled mustard tuber and also edible fresh. "Fuling Green Mustard" has been recognized as "Chongqing's No. 1 Vegetable Brand," and Fuling District is known as "China's Green and Ecological Green Mustard Town." While stem mustard cultivation can bring considerable economic benefits, aphids and the viral diseases they cause are major factors affecting yield. How to efficiently and safely control these pests is a long-standing technical challenge that those skilled in the art have been working to solve.

[0003] Plant epidermal trichomes are pointed protrusions or exophytic growths of epidermal tissue formed by the deformation of glandular hairs and cortical cells. They are widely distributed on the leaves, fruits, stems, and other organs of many plants. Leaf epidermal trichomes possess various defensive characteristics, which can be classified into three types according to their functions: protective, secretory, and absorptive. They can prevent external mechanical damage and pests and diseases (Kenzo et al., 2008; Balkunde et al., 2010; Zaluki et al., 2012; Hamaoka et al., 2017). Therefore, by promoting the formation and growth of epidermal trichomes in *Strombyx mori* (a type of leaf mustard), the plant's defense capabilities can be enhanced to some extent, reducing the damage caused by pests and diseases.

[0004] Those skilled in the art have conducted research on the genes and mechanisms of action that influence the development of epidermal trichomes in Arabidopsis thaliana. They have found that the epidermal trichome trait in Arabidopsis thaliana is controlled by two pairs of nuclear genes, and the presence of epidermal trichomes is incompletely dominant over the absence of epidermal trichomes, exhibiting an additive effect (Liu et al., 2006). Arabidopsis thaliana has approximately 40-70 epidermal trichome-related genes, among which the major gene GL1 (Glabrous 1) inhibits epidermal trichome formation (Kim et al., 2010). Genes such as GL1, GL3 (Glabrous 3), and TTG1 (Transparent testa glabra 1) are all involved in the transcriptional regulation of plant epidermal trichomes, thus indirectly affecting their morphology and formation (Payne et al., 2000). GL1 is highly expressed during development and participates in regulating the early development of epidermal cell trichomes, GL3 mainly controls the number of epidermal trichomes, and TTG1 participates in their formation. GL1, GL3, and TTG1 encode R2R3-type MYB transcription factors, bHLH transcription factors, and WD40 protein, respectively. These three proteins together form the WD40-bHLH-MYB protein complex, which regulates the expression of downstream genes and ultimately leads to the formation of epidermal hairs (Payne et al., 2000; Zhao et al., 2008; Pesch et al., 2009). However, no genes have yet been identified that can directly regulate the development of epidermal hairs in *Strombyx mori*. The lack of such molecular genetic targets for insect resistance modification presents a significant obstacle to the genetic breeding of *Strombyx mori*. Summary of the Invention

[0005] The present invention aims to provide the application of the BjuPP2C52 gene in the resistance breeding of stem mustard, in order to solve the technical problem of the lack of molecular genetic modification targets for the insect-repellent effect of stem mustard in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Application of the BjuPP2C52 gene in resistance breeding of stem mustard. The nucleotide sequence of the BjuPP2C52 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the protein sequence encoded by the BjuPP2C52 gene is shown in SEQ ID NO.2.

[0009] Furthermore, the BjuPP2C52 gene was overexpressed in stem mustard plants.

[0010] Furthermore, the application of the BjuPP2C52 gene in resistance breeding of stem mustard includes the following steps performed sequentially:

[0011] S1: Construct an overexpression vector integrating the BjuPP2C52 gene and transform it into Agrobacterium;

[0012] S2: Agrobacterium tumefaciens was used to infect the cotyledons of stem mustard seedlings, and then the T0 generation plants were obtained through induction culture; after screening, the plants were self-crossed for multiple generations to obtain transgenic stem mustard plants with stable traits.

[0013] Furthermore, a large number of epidermal hairs appeared on the leaves and stem segments of the stem mustard transgenic plants.

[0014] Further, in S1, total RNA was extracted from the flower buds of *Strombyx mori* during the budding stage and reverse transcribed into cDNA. The cDNA was then amplified by PCR using upstream and downstream primers as shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain the BjuPP2C52 gene fragment. The BjuPP2C52 gene fragment was integrated into the p1300-GFP vector to obtain the overexpression vector.

[0015] Furthermore, in S1, the expression vector was transformed into Agrobacterium GV3101 competent cells using a liquid nitrogen freeze-thaw method.

[0016] Furthermore, in S2, Agrobacterium was used to infect the cotyledons of stem mustard seedlings, inducing callus formation, adventitious bud formation, and root formation in sequence, ultimately obtaining T0 generation plants; after screening, multiple generations of self-pollination were conducted to obtain transgenic stem mustard plants with stable traits.

[0017] This technical solution also provides the application of the BjuPP2C52 gene in promoting the appearance of a large number of epidermal hairs on the stems and leaves of stem mustard. The nucleotide sequence of the BjuPP2C52 gene is shown in SEQ ID NO.1; the protein sequence is shown in SEQ ID NO.2.

[0018] This technical solution also provides the application of the BjuPP2C52 gene in enhancing the resistance of stem mustard to pests, characterized in that: the BjuPP2C52 gene is overexpressed in stem mustard plants, promoting the appearance of a large number of epidermal hairs on the leaves and stem segments of stem mustard, thereby enhancing the ability of stem mustard to resist pests; the pests include aphids;

[0019] The nucleotide sequence of the BjuPP2C52 gene is shown in SEQ ID NO.1; the protein sequence is shown in SEQ ID NO.2.

[0020] Among them, pest and disease infection includes damage to plants by pests (such as aphids) and attacks on plants by various harmful microorganisms.

[0021] The principle behind this solution is:

[0022] The inventors discovered the BjuPP2C52 gene and, by overexpressing it in *Musa acutissima*, promoted the growth of numerous epidermal hairs on the leaves and stems, enhancing the plant's defense mechanisms, reducing pest and disease damage, and decreasing pesticide use. Epidermal hairs are a common defense mechanism in plants. Plant breeders generally believe that plants with high density of surface hairs experience a significant reduction in pests because the presence of epidermal hairs prevents the plant from being eaten by various animals (such as insects). Simultaneously, the presence of epidermal hairs increases plant strength, preventing mechanical damage. Furthermore, the growth of a certain amount of epidermal hairs on the stems and leaves can prevent water loss through stomata, reducing water waste, and also reduce direct sunlight, preventing excessively high leaf temperatures, which is beneficial for plant growth. This technical solution provides a regulatory gene for the genetic improvement of *Musa acutissima*, offering more selection possibilities for plant genetic engineering, and also providing support for the molecular modification of *Musa acutissima*.

[0023] Protein phosphatases (PPs) participate in the regulation of protein phosphorylation by reversing the action of protein kinases. They comprise two main classes: tyrosine phosphatases and serine / threonine phosphatases. Serine / threonine phosphatases can be further divided into protein phosphatase 1 (PP1) and protein phosphatase 2 (PP2), with PP2 further subdivided into PP2A, PP2B, and PP2C. PP2Cs primarily regulate protein kinase pathways and are negative regulators activated by various environmental stresses and developmental signals. They are classified into different subfamilies based on sequence similarity (Kerk et al., 2002; Liu et al., 2013). PP2C family proteins contain a highly conserved catalytic domain at their C-terminus and a variable domain at their N-terminus, containing sequences for protein interaction, localization, and autoinhibition of phospholipase activity, playing a crucial role in plant signal transduction. Studies have found that PP2Cs A family genes in plants are negative regulators of the ABA response; B family genes mainly regulate stomatal aperture, seed germination, and abscisic acid-induced gene expression. Furthermore, B family genes can bind to MAPK and activate its activity (Umbrasaite et al., 2010). In Arabidopsis, the AtPP2C52 gene belongs to the PP2Cs E family, and AtPP2C52 can interact with AtAGB1 to participate in the regulation of the Arabidopsis GA signal transduction pathway (Tsugama et al. 2012; Umbrasaite et al., 2010). Therefore, in Arabidopsis, the function of the AtPP2C52 gene is not directly related to plant defense mechanisms such as the formation of epidermal hairs. While there have been studies on the PP2C52 gene in Arabidopsis, there are no reports on the function of the BjuPP2C52 gene in *Bruguiera gymnorhiza*, and its specific function remains unclear. Prior to this discovery, the inventors had not anticipated the connection between the PP2C52 gene (BjuPP2C52 gene) in Brassica oleracea and the formation of epidermal hairs. This patented technology fills a gap in existing technology, creating conditions for molecular genetic breeding of Brassica oleracea and other plants, and can enhance the crop's resistance to mechanical damage and insect repellency by overexpressing the BjuPP2C52 gene.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) This invention is the first to clone the full-length sequence of the BjuPP2C52 gene in Brassica oleracea, obtain its encoded protein sequence, and overexpress this gene in Brassica oleracea. Experimental results show that overexpression of this gene can cause leaves and stem segments to grow a large number of epidermal hairs. This invention provides an important target for the molecular genetic improvement of insect resistance in Brassica oleracea, and has important value and significance for the breeding and production of new varieties of Brassica oleracea.

[0026] (2) This invention, through observation of overexpression and wild-type plant phenotypes, found that overexpression of BjuPP2C52 can induce the production of abundant epidermal hairs on leaves and stem segments, providing an effective gene for the genetic engineering of stem mustard and other plants. The method of this invention lays the foundation for the future cultivation of new insect-resistant germplasm of stem mustard. Therefore, this invention has strong application prospects and promotional value. Attached Figure Description

[0027] Figure 1 This is an amplification diagram (M: Marker) of the BjuPP2C52 gene from stem mustard in Example 1 of the present invention.

[0028] Figure 2 The image shows the electrophoresis results of PCR detection of genomic DNA of transgenic stem mustard in Example 2 of this invention (lanes W1-W3 are wild-type controls, M is the marker, and 1-20 are the 20 T0 generation transgenic plants obtained).

[0029] Figure 3 This is a diagram showing the results of BjuPP2C52 mRNA expression detection in transgenic stem mustard in Example 2 of the present invention (WT is the wild-type control, and OE1, OE2 and OE3 are the three BjuPP2C52 overexpressing plants obtained through screening).

[0030] Figure 4 Phenotypic images of leaves and stem segments of transgenic stem mustard and wild-type plants in Example 2 of this invention (WT is the wild-type control, and OE1, OE2 and OE3 are three BjuPP2C52 overexpressing plants obtained through screening).

[0031] Figure 5 This is a photograph of the transgenic stem mustard plant grown outdoors in Example 2 of the present invention (example plant 1).

[0032] Figure 6 This is a photograph of the transgenic stem mustard plant grown outdoors in Example 2 of the present invention (Example plant 2). Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods and can be completed according to the described recombination technology (see Molecular Cloning, Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; MaXetal, Arobust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicotplants. Molecular Plant. 2015, 8(8):1274-1284.); the materials and reagents used can all be obtained commercially.

[0034] Example 1: Cloning of the full-length gene of stem mustard BjuPP2C52 and construction of overexpression vector

[0035] Using buds of *Strombyx mori* stored at -80℃ as material, total RNA was extracted using the Tiangen Plant Total RNA Extraction Kit (RNAprep pure Plant Kit) according to the instructions. 2 μL of total RNA from the buds of *Strombyx mori* was reverse transcribed into first-strand cDNA according to the instructions of the All-in-One 5×RT MasterMix (G592, abm).

[0036] The reverse transcription program was: 37℃, 15 min; 60℃, 10 min; 95℃, 3 min.

[0037] Using cDNA synthesized by reverse transcription as a template, the coding sequence of BjuPP2C52 was amplified using a high-fidelity enzyme (MegaFi™ Fidelity 2×PCRMasterMix). The PCR amplification system consisted of: 25 μL Primix, 2 μL forward primer (BjuPP2C52-F, 10 μM), 2 μL reverse primer (BjuPP2C52-R, 10 μM), 2 μL template (cDNA), and sterile ddH2O to a final volume of 50 μL.

[0038] The forward and reverse primers are:

[0039] BjuPP2C52-F: 5'-GAGAACACGGGGGACTCTAGAATGGGAGGTTGTGTGTCGAC-3', (SEQ IDNO.3);

[0040] BjuPP2C52-R: 5'-GCCCTTGCTCACCATTCTAGATCAAGTCTTGGACTTTTCTTC-3', (SEQ ID NO. 4).

[0041] The PCR amplification products were obtained and analyzed by agarose gel electrophoresis, such as... Figure 1 As shown, a specific amplification band can be observed at 1kb-2kb under ultraviolet light irradiation. The band was recovered using an agarose gel recovery kit (D1200, Solarbio) and kept for later use.

[0042] The amplified BjuPP2C52 gene fragment was recombinantly ligated with the p1300-GFP vector linearized using XbaI (Thermo Scienfic FastDigest, 91252720) restriction endonuclease using a recombinant ligase (2×Pro Ligation-Free Cloning MasterMix, E086-1, abm). The product was then transformed into *E. coli* DH5α. Two to three positive clones were picked from LB broth containing kanamycin sulfate (50 mg / L) and analyzed by colony PCR. The resulting bacterial culture, containing the target band size, was then sent to BGI Genomics for sequencing. The results showed that the full-length sequence of the *BjuPP2C52* gene is shown in SEQ ID NO.1, containing a 1398 bp open reading frame (including a stop codon). Based on SEQ ID NO.1, the protein sequence of *BjuPP2C52* was obtained using Primerpremier software, as shown in SEQ ID NO.2, containing 466 amino acids (* indicates a stop signal).

[0043] The nucleotide sequence of the BjuPP2C52 gene is shown in SEQ ID NO.1, and the protein sequence is shown in SEQ ID NO.2.

[0044] SEQ ID NO.1 (5'→3'):

[0045]

[0046] SEQ ID NO.2(N→C):

[0047] MGGCVSTSSKSTCSSWSNGEKPMPRPYLGIGCCVSKRAKRTFSDHILSLQNLASIPNRIITSGKSRSSCIFTQQGRKGINQDAMIVWEDFMSEDVTFCGVFDGHGPFGHLVARKVR DTLPVKLQSFFHALQSKQNGRFRRSSSKSAVQEAVKEGTDEDKLKGLWGEAFFKSFKAMDKELRSHPNVDCFCSGSTAVTILKQGSNLFMGNIGDSRAILGSKDSNDTMVATQLTVD LKPDLPREAERIKRCKGRVFALQDEPEVSRVWLPFDDAPGLAAMARAFGDFCLKEYGVISIPEFTHRVLTDKDQFIVLASDGIWDVLSNEEVVDIVASSSSRASAARILVNSAAREWK LKYPTSKMDDCAVVCLFLDGKMDSESDYDEQGFSSATNAVESDDGQRSEPCLQRNFTVRSSSDQENETYGNNVNADTEGEDEKTVGDQNWLGLEGVTRVNSLVQLPRFSEEKSKT*.

[0048] The p1300-BjuPP2C52-GFP plasmid was extracted and purified from *E. coli* using a plasmid extraction kit (D6943, Omega). The constructed p1300-BjuPP2C52-GFP overexpression vector was transformed into *Agrobacterium* GV3101 (AC1001, Shanghai Weidi) competent cells using a liquid nitrogen freeze-thaw method. Single colonies were picked and cultured until the culture became turbid. An equal volume of sterile 50% glycerol was then added, and the cells were stored at -80°C for later use.

[0049] Example 2: Agrobacterium-mediated transformation of stem mustard p1300-BjuPP2C52-GFP and screening of transgenic positive plants

[0050] After sterilizing the seeds of *Strombyx mori*, sow them in 1 / 2 MS medium until both cotyledons are fully expanded. Incubate the preserved *Agrobacterium* GV3101 bacterial suspension containing the p1300-BjuPP2C52-GFP plasmid in YEB (50 mg / mL kanamycin + 20 μg / mL rifampin) liquid medium until the suspension becomes turbid, then scale up the culture at a 1:1000 ratio until OD... 600When the OD value is approximately 0.5, the cells are collected by centrifugation at 3000 rpm for 10 min. The collected cells are then resuspended in 30 g / L sucrose solution, and the OD value is adjusted. 600 =0.01. Next, the cotyledons of the sterile seedlings of *Strombyx mori* were cut off with petioles and soaked in a resuspended *Agrobacterium* solution for 10 minutes. After removing the excess solution with sterile filter paper, the cotyledons were laid flat on a co-culture medium (MS + 2 mg / L 6-BA + 0.15 mg / L NAA + 0.8% agar) and cultured in the dark for 2 days. Then, they were cultured in callus induction medium (MS + 2 mg / L 6-BA + 0.15 mg / L NAA + 20 mg / L hygromycin + 500 mg / L termethin + 0.8% agar). After observing tight, green callus tissue, the callus tissue was cut off and cultured in adventitious shoot differentiation medium (MS + 2 mg / L 6-BA + 0.15 mg / L NAA + 20 mg / L hygromycin + 500 mg / L termethin + 0.8% agar). Finally, the resulting adventitious shoots were cultured in rooting medium (MS + 0.2 mg / L NAA + 20 mg / L hygromycin + 500 mg / L termethin + 0.8% agar) until roots developed. After root development, the plants were transferred to nutrient soil for further culture. The resulting plants were the T0 generation transgenic *Strombyx mori* p1300-BjuPP2C52-GFP. PCR and electrophoresis were performed on the T0 generation transgenic plants, and the results are as follows: Figure 2 As shown, wild-type plants W1-W3 did not show any electrophoretic bands, while the successfully transgenic plants showed the electrophoretic bands indicated by the red arrows. The target of electrophoresis was the tag protein GFP, and the primers for detecting the GFP gene were: GFP-F: 5'-ATGGTGAGCAAGGGCGAGGAG-3' (SEQ ID NO.5); GFP-R: 5'-CTTGTACAGCTCGTCCATGC-3' (SEQ ID NO.6).

[0051] After self-pollinating multiple generations of the obtained *Strombyx mori* p1300-BjuPP2C52-GFP T0 generation plants, leaf genomes were extracted and detected using primers for the GFP tag protein. Finally, the expression level of BjuPP2C52 in the initially screened positive plants was detected using qRT-PCR. The primers for detecting the BjuPP2C52 gene were:

[0052] BjuPP2C52-F: 5'-ATGGGAGGTTGTGTGTCGAC-3', (SEQ ID NO. 7);

[0053] BjuPP2C52-R: 5'-CATCAAATACACCACAAAAC-3', (SEQ ID NO. 8).

[0054] The results are as follows Figure 3 As shown, Figure 3 The phenotypic figures of the BjuPP2C52 transgenic and wild-type stem mustard are shown when they have grown to four true leaves. Figure 3 a represents the observation of BjuPP2C52 overexpression and wild-type plant phenotype. Figure 3 b shows the detection of BjuPP2C52 overexpression and wild-type plant mRNA expression levels. Compared with wild-type plants (WT), the target gene BjuPP2C52 was upregulated in all three representative transgenic lines (OE1, OE2, and OE3), with the highest expression level in OE3, indicating that BjuPP2C52 has been successfully introduced into the BjuPP2C52 genome and transcribed and expressed.

[0055] See leaf phenotype diagrams for transgenic and wild-type stem mustard when they reach four true leaves. Figure 4 . Figure 4 a, b, f, and g represent observations of leaf phenotypes in BjuPP2C52 overexpression and wild-type plants; Figure 4 This study observed the phenotypic characteristics of stem segments from BjuPP2C52 overexpression and wild-type plants. The results showed that BjuPP2C52 overexpression plants produced abundant epidermal hairs on both leaves and stem segments. Epidermal hairs are a plant defense mechanism that helps prevent external mechanical damage and pest infestations. By genetically modifying BjuPP2C52 overexpression in *Mustela stenoptera*, more resistant *Mustela stenoptera* plants can be obtained, thereby reducing the use of pesticides and increasing yield, creating conditions for the development of green agriculture.

[0056] The above experimental results were obtained by cultivating stem mustard in a laboratory greenhouse. The inventors also attempted to transfer the stem mustard plants with the BjuPP2C52 gene to outdoor fields for cultivation. The outdoor field-grown plants had a longer vegetative growth period, were relatively robust, and still produced a large number of epidermal hairs on their leaves and stems. Figure 5 and Figure 6 The image shows photos of BjuPP2C52 gene-overexpressing stem mustard plant grown outdoors two months after sowing, revealing numerous epidermal hairs on its stems and leaves. In contrast, ordinary stem mustard plant grown without transgenic modifications does not exhibit epidermal hairs on its leaves and stems.

[0057] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. Overexpression BjuPP2C52 The application of genes in promoting the production of epidermal hairs in the leaves and stem segments of stem mustard is characterized by: BjuPP2C52 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that: BjuPP2C52 The protein sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The application according to claim 1 or 2, characterized in that: The steps are as follows, performed sequentially: S1: Building and integrating BjuPP2C52 Gene overexpression vectors were then transferred into Agrobacterium; S2: The cotyledons of stem mustard seedlings were infected with Agrobacterium and then induced to grow T0 generation plants. After screening and multiple generations of self-pollination, transgenic stem mustard plants with stable traits were obtained.

4. The application according to claim 3, characterized in that: In S1, total RNA was extracted from flower buds during the budding stage of *Strombyx mori* and reverse transcribed into cDNA. The cDNA was then amplified by PCR using the upstream and downstream primers shown in SEQ ID NO. 3 and SEQ ID NO. 4 to obtain... BjuPP2C52 Gene fragments; BjuPP2C52 Gene fragment integration into p1300-GFP On the vector, an overexpression vector was obtained.

5. The application according to claim 4, characterized in that: In S1, the expression vector was transformed into Agrobacterium GV3101 competent cells using a liquid nitrogen freeze-thaw method.

6. The application according to claim 5, characterized in that: In S2, transgenic Agrobacterium was used to infect the cotyledons of stem mustard seedlings, which sequentially induced callus formation, adventitious bud formation, and root formation, ultimately obtaining T0 generation plants; After screening and multiple generations of self-pollination, transgenic stem mustard plants with stable traits were obtained.

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