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Abstract [Objectives] This study was conducted to improve the Agrobacterium-mediated genetic transformation system of Begonia wallichiana.
[Methods]With sterilized tube seedling leaves as the recipient material and GFP as the reporter gene， optimization experiments were carried out in terms of infection time and method， co-cultivation time and method， and PCR detection technology.
[Results] The transformation effect was better under the conditions of shaking Agrobacterium liquid， infection time of 1-2 h， and co-cultivation on sterilized filter paper for 2 d. After co-cultivation， the recipient material was first subjected to recovery culture， and then used for Hyg gradient screening， which was conducive to obtaining resistant transformants. The designed specific PCR detection technology could quickly identify false positives in resistant regenerated plants， and the proportion of transgenic plants was 16.7%.
[Conclusions]The research results provide a new technical reference for the genetic transformation of ornamental plants.
Key words Begonia wallichiana L.; Genetic transformation; Agrobacterium mediated transformation; PCR detection
Received： March 23， 2021 Accepted： May 27， 2021
Siyu ZHOU （1996-）， female， P. R. China， master， devoted to research about garden plant biotechnology.
*Corresponding author. E-mail： sudatanjz@163.com.
Begonia wallichiana L. is a perennial herb in Begonia of Begoniaceae. It has independent and strong main stem and luxuriant branches and leaves， and the biomass of a single plant is large. It grows fast and has a short life cycle， and can complete one generation within 4-5 months. B. wallichiana flowers in all four seasons， and has large amount of flowers and fruit， very large number of seeds in a single fruit pod[1]. It has strong regeneration ability， and the leaves can be cultured in vitro for 40-60 d to obtain cluster buds， and can be multiplied in a large quantity[2]. It has good tolerance of shade， and is suitable for indoor planting[3]. B. wallichiana is a potential model plant for ornamental plant genetic transformation and molecular biology research.
In terms of the genetic transformation of B. wallichiana， the existing research shows that the infection effect of the Agrobacterium ‘GV3101’ strain is better. The lotus CHS gene[4] and the lotus petal-related NnTP1 gene and NnTP2 genes have been successively transformed into B. wallichiana by the Agrobacterium-mediated transformation method， in order to verify the transgenic function of ornamental plants[5]， but there are still few studies and reports on factors affecting transformation efficiency and rapid transgene identification technology. To this end， in this study， with sterilized test-tube seedling leaves as the recipient material and gene GFP as the reporter gene， the Agrobacterium-mediated transformation conditions and PCR detection technology of B. wallichiana were explored， aiming to construct an efficient and stable genetic transformation system and provide a new technical reference for the genetic transformation research of ornamental plants. Materials and Methods
Test materials
（1） Plant material： B. wallichiana was cultivated and preserved in Shanghai Chenshan Botanical Garden （121°11′ E， 31°04′ N）. The aseptic seedling leaves were cut into leaf discs with veins of about 1 cm×1 cm as recipient materials for genetic transformation.
（2） Strain and vector： The tested Agrobacterium tumefaciens strain was GV3101 （containing plasmid pMP90）. The engineered Agrobacterium constructed here contained the intermediate expression vector pCAMBIA1301-eGFP， which carried the hygromycin （Hyg） resistant marker gene and GFP gene. The engineered Agrobacterium was gifted by teacher Zhang Dasheng from Shanghai Chenshan Botanical Garden， and was stored at -80 ℃ for later use.
（3） Medium： The leaf regeneration medium was MS+1.5 mg/L 6-BA+0.2 mg/L NAA+30 g/L sucrose， pH 5.8. According to the needs of the experimental design， different concentrations of cephalosporin （Cef）， kanamycin （Kan）， hygromycin （Hyg） and acetosyringone （AS） were added.
Preparation of Agrobacterium infection liquid
The preserved strain was thawed on ice and transferred to LB liquid medium （containing 50 mg/L Kan）， and shaken and cultured at 27.5 ℃， 200 r/min on a shaker for 16 h. The bacterial liquid was centrifuged at 18 ℃， 4 000 r/min for 20 min. An Agrobacterium infection liquid was prepared with LB liquid medium， and the OD600 was adjusted to about 0.5.
Test zoning and treatment method
Infection time and method： The leaf disc explants were infected for 4， 8， and 12 h respectively. Among them， each treatment groups with infection time of 4， and 8 h had two treatment methods： static infection （denoted as A）， 100 r/min shaker infection （denoted as B）. The survival rate of leaves was counted on the 10th， 20th， and 30th d after inoculation.
Co-cultivation time and method： After the leaf disc explants were infected for 1 h， the explants were wiped dry with sterile filter paper and transferred into regeneration medium （containing 200 mg/L AS and 10 g/L glucose） with a layer of sterile filter paper. Cultivation was performed in the dark for 0， 4， and 6 d. Among them， the 4 d co-cultivation treatment area was further divided into two treatment methods： placing a layer of filter paper （denoted as 4-1） between the explants and the medium， and placing the explants directly on the medium （denoted as 4-2）. The survival rates of leaves were counted on the 10th， 20th， and 30th d of inoculation. After the co-cultivation was completed， the explants are transferred to regeneration medium supplemented with 100-300 mg/L Cef for recovery culture. Hyg gradient screening： The above-mentioned explants that were still alive after 1 month of recovery culture were cut into small pieces with a size of about 1 cm3， and then transferred to media supplemented with 10， 30 and 50 mg/L Hyg （containing 300 mg/L Cef） in sequence to perform concentration gradient screening. The survival rate was calculated after 7 d of screening and culture.
PCR identification of resistant regenerated plants
The DNA of Hyg-resistant regenerated plants was extracted by the CTAB method. The PCR reaction system （10 μl） contained Tag Mix 5 μl， ddH2O 3.5 μl， template DNA and upstream and downstream primers each 0.5 μl. The PCR upstream primer of the GFP gene was 5′-CTGGTCGAGC TGGACGGCGACG-3′， and the downstream primer was 5′-CACGAACTCCGCAGGACCATG-3′. The PCR reaction program was started with pre-denaturation at 94 ℃ for 5 min， denaturation at 94 ℃ for 30 s， annealing at 65 ℃ for 30 s and extension at 72 ℃ for 30 s， a total of 34 cycles， and finally completed with 72 ℃ extension for 5 min. The PCR upstream primer designed for the partial sequence of pMP90 plasmid was 5′-TGTTGCTTTCGCTTTTGGCTTGAC-3′， and the downstream primer was 5′-CCACGATGTAAGGCACGAGGT TC-3′. And the annealing temperature was 60 ℃.
Results and Analysis
Effects of the time and method of infection on the survival of explants and plant regeneration
After being infected by Agrobacterium at different times， the survival rate of explants gradually decreased with the culture time， and there were significant differences between different treatment zones at 30 d of culture （Table 1）. Specifically， the survival rate was the lowest with the infection time of 12 h， only 9.54%; and the survival rate and vigor of explants infected for 1 and 2 h were relatively high， and the remaining treatment zones were between the two. Meanwhile， it was found that when the infection time was long， the adventitious bud differentiation rate of the explants gradually slowed down， especially in the 12-h treatment zone where the adventitious buds gradually whiten and died in the later period of culture. Furthermore， the Hyg concentration gradient screening effect was also relatively good， which was manifested as a higher survival rate and more resistant regenerated plants. Therefore， 1-2 h of bacterial infection with shaking was the suitable infection condition for the leaf disc genetic transformation of B. wallichiana.
Lowercase letters in the table indicate significant differences at the level of α 0.05; the same below; The medium composition is MS+1.5 mg/L 6-BA+0.2 mg/L NAA+100 mg/L Cef+30 g/L sucrose+1.4 g/L agar. Effect of co-cultivation time and method on the survival of transformed materials and plant regeneration
The test results showed （Table 2） that with the increase of co-cultivation time， the survival rate showed a trend of increasing first and then decreasing. Among them， the survival rate of co-cultivation for 2 d was the highest， reaching 63.81%; and the survival rate of co-cultivation for 4-6 d was significantly reduced， and the explants gradually turned white from the edge， and died of whitening within a few days. From the perspective of culture method， when co-cultured for 4 d with filter paper， most of the surviving explants grow well， and they could differentiate into adventitious buds in a short period of time （4-1 in Table 2）， while without filter paper， the explanted directed contact the medium， and most of the explants died of whitening （4-2 in Table 2）. Moreover， the Hyg screening effect also showed a similar tendency. It could be seen that appropriate co-cultivation time was beneficial to the survival of explants and the differentiation of adventitious buds， and the co-cultivation time of 2 d was suitable.
Siyu ZHOU et al. Study on the Genetic Transformation Conditions of Begonia wallichiana L. with Leaf Disc Method and Corresponding Identification Techniques
Analysis of Hyg gradient screening effect
In this study， it was found that after Agrobacterium infection and co-cultivation， when directly transferred to Hyg screening culture， the growth of explants was relatively slow， and it was difficult for them to differentiate to adventitious buds. However， the transfer of explants to the Hyg-free recovery medium was beneficial to the rapid induction of callus formation （Fig. A-B）. After 1 month of culture， the surviving explants had completely differentiated to adventitious buds and were in a rapid proliferation stage （Fig. C）， and transferring to Hyg gradient screening at this time could achieve a good effect. With the increase of Hyg concentration， most of the explants （adventitious buds） grew poorly and gradually whitened and died （Fig. D-F）.
PCR identification of resistant regenerated plants
Samples to be tested were randomly selected from the resistant regenerated plants obtained after Hyg gradient screening， and specific PCR identification was performed with the GFP gene as the target gene. As a result， amplified bands were detected in some samples （Fig. lanes 4-9）， and not detected in the rest of the samples （Fig. lanes 10-15）， that is， the detection rate of the GFP gene introduced into the resistant regenerated plants was 50%. Secondly， primers were designed for specific PCR based on the partial sequence of the plasmid pMP90 carried by Agrobacterium GV3101. The results showed that in the above-mentioned treatment groups with positive GFP gene identification， only two samples were not detected with PCR amplified bands （Fig. 3， Lanes 4 and 9）， indicating that samples No. 4 and No. 9 were free of Agrobacterium contamination and had indeed successfully transformed with the GFP gene， with a transformation efficiency of 16.7%， while the possibility of Agrobacterium contamination could not be ruled out for the remaining samples.
Conclusions and Discussion
During plant genetic transformation， cold treatment， pressurization， vacuum and ultrasound are usually used to increase the success rate of infection[6-7]. It was found in this study that under the two set of infection methods， the overall transformation effect of 1-2 h of infection with shaking was better. However， when the infection time was 1 h， the survival rate of bacterial infection with shaking was higher， and the result was the opposite when the infection time was longer. It was speculated that the reason was that part of the poorly growing leaf discs were broken during the long-term bacterial infection with shaking， and the remaining intact leaf discs had relatively high growth vigor after inoculation.
Most plants are usually screened for antibiotic resistance directly after co-cultivation[8-10]. However， it was found in this study that most of the explants only stayed at the callus stage and could not differentiate into adventitious buds when the Hyg screening culture was directly carried out， while carrying out Hyg gradient screening after bacteriostatic recovery for a period of time was beneficial to restore the vitality of the explants. Furthermore， when cultured to 2 weeks， most of the explants were induced to form callus and adventitious buds， and some began to differentiate and proliferate in large numbers， which might be mixed with more false positives. Therefore， it is necessary to transfer to screening culture in time before large number of adventitious buds are formed， and use a higher intensity （Hyg 30 mg/L or more） screening pressure to reduce the number of screening times and false positive plants.
PCR is the most direct and effective method for the detection of transgenic plants， and the dual PCR technology has been widely used in rapid detection[11]. In this study， an experimental method similar to double PCR was used. Specifically， specific primers based on the GFP gene and the pMP90 sequence of Agrobacterium’s plasmid were designed respectively to perform PCR detection on resistant regenerated plants. For the materials tested positive with the former （GFP gene） and negative with the latter， the false positives caused by Agrobacterium contamination could be ruled out， which helped to improve the efficiency and reliability of the identification of transgenic plants. References
[1] TIAN DK， ZHANG DS， CHEN Q， et al. Method for in vitro regeneration of Begonia wallichiana[P]. Shanghai： CN106386491B， 2019-04-09. （in Chinese）
[2] CHEN Q， ZHANG DS， TIAN DK， et al. Establishment of tissue culture regeneration system of Begonia wallichiana[J]. Acta Horticulturae Sinica， 2016， 43（S1）： 2676. （in Chinese）
[3] ZHAO B， FU DF， XIANG YC， et al. Effects of light intensity on the growth of Begonia cucullata and B. wallichiana[J]. Acta Agriculturae Shanghai， 2016， 32（6）： 128-133. （in Chinese）
[4] YANG MJ， ZHANG DS， CHEN Q， et al. Genetic transformation of Begonia wallichiana Lehm.[J]. Molecular Plant Breeding， 2018， 16（14）： 4632-4637. （in Chinese）
[5] CHEN Q. Study on the mechanism of lotus NnTP1 and NnTP2 genes regulating the flower formation of "Qianbanlian"[D]. Fuzhou： Fujian Agriculture and Forestry University， 2017. （in Chinese）
[6] LIU YR， CEN HF， YAN JP， et al. Inside out： high-efficiency plant regeneration and Agrobacterium-mediated transformation of upland and lowland switchgrass cultivars[J].Plant Cell Reports，2015，34（7）：1099-1108.
[7] MARTINA BERANOVá， et al. Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax （Linum usitatissimum L.）[J]. Plant Cell， Tissue and Organ Culture， 2008， 94（3）： 253-259.
[8] FAN JP， LIU DY， ZHANG JH， et al. Establishment of genetic transformation system of Asarina procumbens ‘Victoria Falls’[J]. Journal of Northeast Agricultural University， 202 52（2）： 10-16. （in Chinese）
[9] SUN ZL， LI X， ZHOU W， et al. Agrobacterium-mediated genetic transformation of Chinese chestnut （Castanea mollissima Blume）[J]. Plant Cell， Tissue and Organ Culture， 2020， 140（5）： 95-103.
[10] YAN R， WANG Z， REN Y， et al. Establishment of efficient genetic transformation systems and application of CRISPR/ Cas9 genome editing technology in Lilium pumilum DC. Fisch. and Lilium longiflorum white heaven[J]. Int. J. Mol. Sci. 2019， 20（12）： 2920-2937.
[11] GUAN ZY， LI HM， HE MW， et al. Application of multiplex PCR technology in rapid detection[J]. Shandong Chemical Industry， 202 50（3）： 85-88. （in Chinese）
[Methods]With sterilized tube seedling leaves as the recipient material and GFP as the reporter gene， optimization experiments were carried out in terms of infection time and method， co-cultivation time and method， and PCR detection technology.
[Results] The transformation effect was better under the conditions of shaking Agrobacterium liquid， infection time of 1-2 h， and co-cultivation on sterilized filter paper for 2 d. After co-cultivation， the recipient material was first subjected to recovery culture， and then used for Hyg gradient screening， which was conducive to obtaining resistant transformants. The designed specific PCR detection technology could quickly identify false positives in resistant regenerated plants， and the proportion of transgenic plants was 16.7%.
[Conclusions]The research results provide a new technical reference for the genetic transformation of ornamental plants.
Key words Begonia wallichiana L.; Genetic transformation; Agrobacterium mediated transformation; PCR detection
Received： March 23， 2021 Accepted： May 27， 2021
Siyu ZHOU （1996-）， female， P. R. China， master， devoted to research about garden plant biotechnology.
*Corresponding author. E-mail： sudatanjz@163.com.
Begonia wallichiana L. is a perennial herb in Begonia of Begoniaceae. It has independent and strong main stem and luxuriant branches and leaves， and the biomass of a single plant is large. It grows fast and has a short life cycle， and can complete one generation within 4-5 months. B. wallichiana flowers in all four seasons， and has large amount of flowers and fruit， very large number of seeds in a single fruit pod[1]. It has strong regeneration ability， and the leaves can be cultured in vitro for 40-60 d to obtain cluster buds， and can be multiplied in a large quantity[2]. It has good tolerance of shade， and is suitable for indoor planting[3]. B. wallichiana is a potential model plant for ornamental plant genetic transformation and molecular biology research.
In terms of the genetic transformation of B. wallichiana， the existing research shows that the infection effect of the Agrobacterium ‘GV3101’ strain is better. The lotus CHS gene[4] and the lotus petal-related NnTP1 gene and NnTP2 genes have been successively transformed into B. wallichiana by the Agrobacterium-mediated transformation method， in order to verify the transgenic function of ornamental plants[5]， but there are still few studies and reports on factors affecting transformation efficiency and rapid transgene identification technology. To this end， in this study， with sterilized test-tube seedling leaves as the recipient material and gene GFP as the reporter gene， the Agrobacterium-mediated transformation conditions and PCR detection technology of B. wallichiana were explored， aiming to construct an efficient and stable genetic transformation system and provide a new technical reference for the genetic transformation research of ornamental plants. Materials and Methods
Test materials
（1） Plant material： B. wallichiana was cultivated and preserved in Shanghai Chenshan Botanical Garden （121°11′ E， 31°04′ N）. The aseptic seedling leaves were cut into leaf discs with veins of about 1 cm×1 cm as recipient materials for genetic transformation.
（2） Strain and vector： The tested Agrobacterium tumefaciens strain was GV3101 （containing plasmid pMP90）. The engineered Agrobacterium constructed here contained the intermediate expression vector pCAMBIA1301-eGFP， which carried the hygromycin （Hyg） resistant marker gene and GFP gene. The engineered Agrobacterium was gifted by teacher Zhang Dasheng from Shanghai Chenshan Botanical Garden， and was stored at -80 ℃ for later use.
（3） Medium： The leaf regeneration medium was MS+1.5 mg/L 6-BA+0.2 mg/L NAA+30 g/L sucrose， pH 5.8. According to the needs of the experimental design， different concentrations of cephalosporin （Cef）， kanamycin （Kan）， hygromycin （Hyg） and acetosyringone （AS） were added.
Preparation of Agrobacterium infection liquid
The preserved strain was thawed on ice and transferred to LB liquid medium （containing 50 mg/L Kan）， and shaken and cultured at 27.5 ℃， 200 r/min on a shaker for 16 h. The bacterial liquid was centrifuged at 18 ℃， 4 000 r/min for 20 min. An Agrobacterium infection liquid was prepared with LB liquid medium， and the OD600 was adjusted to about 0.5.
Test zoning and treatment method
Infection time and method： The leaf disc explants were infected for 4， 8， and 12 h respectively. Among them， each treatment groups with infection time of 4， and 8 h had two treatment methods： static infection （denoted as A）， 100 r/min shaker infection （denoted as B）. The survival rate of leaves was counted on the 10th， 20th， and 30th d after inoculation.
Co-cultivation time and method： After the leaf disc explants were infected for 1 h， the explants were wiped dry with sterile filter paper and transferred into regeneration medium （containing 200 mg/L AS and 10 g/L glucose） with a layer of sterile filter paper. Cultivation was performed in the dark for 0， 4， and 6 d. Among them， the 4 d co-cultivation treatment area was further divided into two treatment methods： placing a layer of filter paper （denoted as 4-1） between the explants and the medium， and placing the explants directly on the medium （denoted as 4-2）. The survival rates of leaves were counted on the 10th， 20th， and 30th d of inoculation. After the co-cultivation was completed， the explants are transferred to regeneration medium supplemented with 100-300 mg/L Cef for recovery culture. Hyg gradient screening： The above-mentioned explants that were still alive after 1 month of recovery culture were cut into small pieces with a size of about 1 cm3， and then transferred to media supplemented with 10， 30 and 50 mg/L Hyg （containing 300 mg/L Cef） in sequence to perform concentration gradient screening. The survival rate was calculated after 7 d of screening and culture.
PCR identification of resistant regenerated plants
The DNA of Hyg-resistant regenerated plants was extracted by the CTAB method. The PCR reaction system （10 μl） contained Tag Mix 5 μl， ddH2O 3.5 μl， template DNA and upstream and downstream primers each 0.5 μl. The PCR upstream primer of the GFP gene was 5′-CTGGTCGAGC TGGACGGCGACG-3′， and the downstream primer was 5′-CACGAACTCCGCAGGACCATG-3′. The PCR reaction program was started with pre-denaturation at 94 ℃ for 5 min， denaturation at 94 ℃ for 30 s， annealing at 65 ℃ for 30 s and extension at 72 ℃ for 30 s， a total of 34 cycles， and finally completed with 72 ℃ extension for 5 min. The PCR upstream primer designed for the partial sequence of pMP90 plasmid was 5′-TGTTGCTTTCGCTTTTGGCTTGAC-3′， and the downstream primer was 5′-CCACGATGTAAGGCACGAGGT TC-3′. And the annealing temperature was 60 ℃.
Results and Analysis
Effects of the time and method of infection on the survival of explants and plant regeneration
After being infected by Agrobacterium at different times， the survival rate of explants gradually decreased with the culture time， and there were significant differences between different treatment zones at 30 d of culture （Table 1）. Specifically， the survival rate was the lowest with the infection time of 12 h， only 9.54%; and the survival rate and vigor of explants infected for 1 and 2 h were relatively high， and the remaining treatment zones were between the two. Meanwhile， it was found that when the infection time was long， the adventitious bud differentiation rate of the explants gradually slowed down， especially in the 12-h treatment zone where the adventitious buds gradually whiten and died in the later period of culture. Furthermore， the Hyg concentration gradient screening effect was also relatively good， which was manifested as a higher survival rate and more resistant regenerated plants. Therefore， 1-2 h of bacterial infection with shaking was the suitable infection condition for the leaf disc genetic transformation of B. wallichiana.
Lowercase letters in the table indicate significant differences at the level of α 0.05; the same below; The medium composition is MS+1.5 mg/L 6-BA+0.2 mg/L NAA+100 mg/L Cef+30 g/L sucrose+1.4 g/L agar. Effect of co-cultivation time and method on the survival of transformed materials and plant regeneration
The test results showed （Table 2） that with the increase of co-cultivation time， the survival rate showed a trend of increasing first and then decreasing. Among them， the survival rate of co-cultivation for 2 d was the highest， reaching 63.81%; and the survival rate of co-cultivation for 4-6 d was significantly reduced， and the explants gradually turned white from the edge， and died of whitening within a few days. From the perspective of culture method， when co-cultured for 4 d with filter paper， most of the surviving explants grow well， and they could differentiate into adventitious buds in a short period of time （4-1 in Table 2）， while without filter paper， the explanted directed contact the medium， and most of the explants died of whitening （4-2 in Table 2）. Moreover， the Hyg screening effect also showed a similar tendency. It could be seen that appropriate co-cultivation time was beneficial to the survival of explants and the differentiation of adventitious buds， and the co-cultivation time of 2 d was suitable.
Siyu ZHOU et al. Study on the Genetic Transformation Conditions of Begonia wallichiana L. with Leaf Disc Method and Corresponding Identification Techniques
Analysis of Hyg gradient screening effect
In this study， it was found that after Agrobacterium infection and co-cultivation， when directly transferred to Hyg screening culture， the growth of explants was relatively slow， and it was difficult for them to differentiate to adventitious buds. However， the transfer of explants to the Hyg-free recovery medium was beneficial to the rapid induction of callus formation （Fig. A-B）. After 1 month of culture， the surviving explants had completely differentiated to adventitious buds and were in a rapid proliferation stage （Fig. C）， and transferring to Hyg gradient screening at this time could achieve a good effect. With the increase of Hyg concentration， most of the explants （adventitious buds） grew poorly and gradually whitened and died （Fig. D-F）.
PCR identification of resistant regenerated plants
Samples to be tested were randomly selected from the resistant regenerated plants obtained after Hyg gradient screening， and specific PCR identification was performed with the GFP gene as the target gene. As a result， amplified bands were detected in some samples （Fig. lanes 4-9）， and not detected in the rest of the samples （Fig. lanes 10-15）， that is， the detection rate of the GFP gene introduced into the resistant regenerated plants was 50%. Secondly， primers were designed for specific PCR based on the partial sequence of the plasmid pMP90 carried by Agrobacterium GV3101. The results showed that in the above-mentioned treatment groups with positive GFP gene identification， only two samples were not detected with PCR amplified bands （Fig. 3， Lanes 4 and 9）， indicating that samples No. 4 and No. 9 were free of Agrobacterium contamination and had indeed successfully transformed with the GFP gene， with a transformation efficiency of 16.7%， while the possibility of Agrobacterium contamination could not be ruled out for the remaining samples.
Conclusions and Discussion
During plant genetic transformation， cold treatment， pressurization， vacuum and ultrasound are usually used to increase the success rate of infection[6-7]. It was found in this study that under the two set of infection methods， the overall transformation effect of 1-2 h of infection with shaking was better. However， when the infection time was 1 h， the survival rate of bacterial infection with shaking was higher， and the result was the opposite when the infection time was longer. It was speculated that the reason was that part of the poorly growing leaf discs were broken during the long-term bacterial infection with shaking， and the remaining intact leaf discs had relatively high growth vigor after inoculation.
Most plants are usually screened for antibiotic resistance directly after co-cultivation[8-10]. However， it was found in this study that most of the explants only stayed at the callus stage and could not differentiate into adventitious buds when the Hyg screening culture was directly carried out， while carrying out Hyg gradient screening after bacteriostatic recovery for a period of time was beneficial to restore the vitality of the explants. Furthermore， when cultured to 2 weeks， most of the explants were induced to form callus and adventitious buds， and some began to differentiate and proliferate in large numbers， which might be mixed with more false positives. Therefore， it is necessary to transfer to screening culture in time before large number of adventitious buds are formed， and use a higher intensity （Hyg 30 mg/L or more） screening pressure to reduce the number of screening times and false positive plants.
PCR is the most direct and effective method for the detection of transgenic plants， and the dual PCR technology has been widely used in rapid detection[11]. In this study， an experimental method similar to double PCR was used. Specifically， specific primers based on the GFP gene and the pMP90 sequence of Agrobacterium’s plasmid were designed respectively to perform PCR detection on resistant regenerated plants. For the materials tested positive with the former （GFP gene） and negative with the latter， the false positives caused by Agrobacterium contamination could be ruled out， which helped to improve the efficiency and reliability of the identification of transgenic plants. References
[1] TIAN DK， ZHANG DS， CHEN Q， et al. Method for in vitro regeneration of Begonia wallichiana[P]. Shanghai： CN106386491B， 2019-04-09. （in Chinese）
[2] CHEN Q， ZHANG DS， TIAN DK， et al. Establishment of tissue culture regeneration system of Begonia wallichiana[J]. Acta Horticulturae Sinica， 2016， 43（S1）： 2676. （in Chinese）
[3] ZHAO B， FU DF， XIANG YC， et al. Effects of light intensity on the growth of Begonia cucullata and B. wallichiana[J]. Acta Agriculturae Shanghai， 2016， 32（6）： 128-133. （in Chinese）
[4] YANG MJ， ZHANG DS， CHEN Q， et al. Genetic transformation of Begonia wallichiana Lehm.[J]. Molecular Plant Breeding， 2018， 16（14）： 4632-4637. （in Chinese）
[5] CHEN Q. Study on the mechanism of lotus NnTP1 and NnTP2 genes regulating the flower formation of "Qianbanlian"[D]. Fuzhou： Fujian Agriculture and Forestry University， 2017. （in Chinese）
[6] LIU YR， CEN HF， YAN JP， et al. Inside out： high-efficiency plant regeneration and Agrobacterium-mediated transformation of upland and lowland switchgrass cultivars[J].Plant Cell Reports，2015，34（7）：1099-1108.
[7] MARTINA BERANOVá， et al. Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax （Linum usitatissimum L.）[J]. Plant Cell， Tissue and Organ Culture， 2008， 94（3）： 253-259.
[8] FAN JP， LIU DY， ZHANG JH， et al. Establishment of genetic transformation system of Asarina procumbens ‘Victoria Falls’[J]. Journal of Northeast Agricultural University， 202 52（2）： 10-16. （in Chinese）
[9] SUN ZL， LI X， ZHOU W， et al. Agrobacterium-mediated genetic transformation of Chinese chestnut （Castanea mollissima Blume）[J]. Plant Cell， Tissue and Organ Culture， 2020， 140（5）： 95-103.
[10] YAN R， WANG Z， REN Y， et al. Establishment of efficient genetic transformation systems and application of CRISPR/ Cas9 genome editing technology in Lilium pumilum DC. Fisch. and Lilium longiflorum white heaven[J]. Int. J. Mol. Sci. 2019， 20（12）： 2920-2937.
[11] GUAN ZY， LI HM， HE MW， et al. Application of multiplex PCR technology in rapid detection[J]. Shandong Chemical Industry， 202 50（3）： 85-88. （in Chinese）