FT, A Spontaneous Doubled Haploid Recipient of Transient Transformation in Chinese Cabbage

Special Article: Crop Improvement

Ann Agric Crop Sci. 2023; 8(2): 1132.

‘FT’, A Spontaneous Doubled Haploid Recipient of Transient Transformation in Chinese Cabbage

Han Wu²; Kuangye Zhang²; Jiaxu Wang²; Jia Li²; Youhou Duan²; Zhiqiang Liu²; Ying Zhao¹; Yun Zhang¹

¹College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China

²Sorghum Research Institute, Liaoning Academy of Agricultural Sciences, Shenyang 110161, China

*Corresponding author: Yun Zhang College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China. Email: zhangyun511@syau.edu.cn

Received: April 05, 2023 Accepted: May 03, 2023 Published: May 10, 2023

Abstract

Chinese cabbage is relatively recalcitrant to genetic transformation. Microspore is an ideal haploid single cell recipient for genetic m anipulation, whereby homozygous Doubled Haploid (DH) transformants can be obtained to accelerate the breeding process. In this study, Microspore Derived Embryo (MDE) productive material DH ‘FT’ is screened out from 10 candidates; microspore culture parameters were optimized: bud size ranged from 2.4mm to 2.7mm and microspore density 20000~40000/mL shows highest MDE yield. Antibiotic (chemical) sensitivity assay indicates that 100mg/L Carbenicillin or 300mg/L Timentin can be applied to inhibit the overgrowth of Agrobacterium after transformation; microspores are sensitive to cellulase and lysozyme treatments when liquid cultured, kanamycin slight below 5mg/L can be used as selection marker. ‘FT’ microspore and MDE were successfully transfected by Agrobacterium. Transient expression of embryogenic transcription factor LEC1-GFP in nucleus, rather than cytoplasm, was clearly detected by confocal microscopy. ‘FT’ microspores, 14/28d MDEs can be used as transient transfection recipients in Chinese cabbage. This study provides an efficient and spontaneous doubled haploid recipient system in Brassica rapa.

Keyword: Chinese cabbage; DH line; Microspore culture; Transformation; Recipient

Abbreviations: DH: Doubled Haploid; MDE: Microspore Derived Embryo; PPT: Phosphinothricin

Introduction

Immature pollen of plants, which is known as microspore, has haploid genome and simple cell structure. Under stress or exogenous hormone induction, haploid plantlets can regenerate through embryoid pathway from microspores that is called isolated microspore embryogenesis [2-4]. After colchicin treatment or spontaneous genome doubling, homozygous DH (doubled haploid) lines can be obtained to sharply shorten the breeding cycles [5], which also provides a technical platform for single cell genetic operation. Chinese cabbage is recalcitrant to regenerate after Agrobacterium transfection [6]. Some relative successful attempts have been made in Brassica rapa transformation [7-12]. However, the results showed strong genotype dependency, and the transformation efficiency is hardly stable and repeatable, which is the bottleneck to study gene function in Brassica rapa. An efficient recipient system is urgently needed, especially single cell recipient.

Efficient recipient system is the first step of transformation protocol. In the past two decades, the molecular mechanism [13] of totipotency has been well studied [14]. Morphogenic transcription factors play important roles in embryogenesis [15] and can induce somatic embryogenesis when over-expressed [16]. Some discovered morphogenic transcription factors can be applied to enhance regeneration after transformation, such as BBM [17], WUS [18], GRF–GIF [19,20]. The LEC transcription factors [22] were firstly discovered [21] to improve somatic embryogenesis when ectopically expressed [22]. The LEC-GFP fusion protein can be found in the nucleus of embryogenic cells rather than cytoplasm [4], which marks the initiation of microspore embryogenesis. A microspore-based transient transformation system is suitable to understand gene function, especially in embryogenesis phase.

In this study, a Microspore Derived Embryo (MDE) productive variety DH 'FT' were screened out from 10 candidates, the microspore culture parameters were optimized, and antibiotic sensitivity assay was performed. Microspore and MDE of 'FT' were used as recipient of Agrobacterium mediated transformation. Transient expression of LEC1-GFP, an embryo-expressed transcription factor, were successfully detected in nucleus, rather than cytoplasm, by confocal as expected. We offered an efficient spontaneous doubled haploid recipient system of transformation in Brassica rapa.

Materials and Methods

Plant Materials

The following 10 materials: 'Golden Crow', 'Yangchun' 'Chundajiang’,'Leader', 'Hongkang 1’, 'Rich Spring', 'Beijing New 3', 'Multi-resistant3', 'FT', 'Golden Spring' were used as candidates in microspore culture experiment. They were cultured in a greenhouse. 'FT' is Double Haploid line (DH) from hybrid 'Futian 50' in Brassica rapa.

Isolated Microspore Culture

The microspore culture protocol in this study is modified from Sato et al., (1989). Buds were selected from healthy inflorescence, 30 buds as a group. 70% ethanol and 2% bleach solution were used for surface disinfection. Vibrate from time to time to drive away the bubbles on surface. Rinse with sterile water 3 times for 1 min, 4 min and 10 min, respectively. Add 5mL NLN liquid medium, crush the buds with sterile grinding rod to press out the microspores. The debris was removed by nylon mesh. The yellow precipitates (microspores) were rinsed 3 times with 5mL B5 medium, centrifuged at 100rpm. 20μL microspore culture was used for DAPI staining to investigate the developmental stages. The microspore concentration was determined by blood cell counting plate to meet the gradient 20000, 40000 and 80000 cells/mL in NLN-13 medium. 1.0mL microspore suspension was transferred into a 3cm sterile culture dish, sealed, and heat shock in precis 32°C for 24h. Then the dishes were transferred to 25°C in dark for 20 days. The microspore derived embryos can be found at the edge of dishes by then.

Antibiotic Sensitivity Assay

Concentration gradient of Carbenicillin (0, 50, 100 and 500mg. L-1 ); Timentin (0, 60, 150, and 300mg. L-1 ); Kanamycin (0, 0.5, 5, 25, 50 and 100mg. L-1 ); Phosphinothricin (PPT) (0, 7.5, 15, 30 and 90mg. L-1 ); Cellulase (0, 10, 20, 25, 30, 40, and 50mg. L-1) and lysozyme (0, 10, 20, 25, 30, 40, and 50mg. L-1) were added to the liquid NLN13 medium respectively for microspore antibiotic sensitivity assay. Each concentration in line with 3 replicates (dishes) for statistics. Suitable concentrations were screened out for downstream experiments.

Ploidy Identification of Regenerated Plants

Ploidy of microspore regenerated plants was identified by FACS calibur flow cytometry in this study. 1mL chopping buffer was added into a culture dish, and 1.5cm of regenerated plant leaves were selected and chopped with blade. The supernatant was filtered with nylon mesh and collected into a tube. Centrifugation was performed at 1000rpm for 10 min at room temperature. Discard the supernatant and add 1mL propidium iodide in dark for 15 minutes, filtered. The cell ploidy was detected on FACS calibur flow cytometry with 3 replicates.

Transformation of Microspore and MDE

The microspore density was kept at 40×103 cells·mL-1. 1μL Agrobacterium stock with OD value 1.0 was added into 10mL microspore suspension containing 0.007% cellulase. 1.0mL microspore suspension was pipetted into a sterile 30mm culture dish, 33°C heat shock for 24h. The Agrobacterium was controlled by washing with 0.01% lysozyme solution, and then rinse twice with NLN13 containing timentin 300mg/L. Finally, the cells were cultured in NLN13 medium containing 100mg/L Timentin and corresponding antibiotics at 25°C. Fresh media were changed weekly to keep the embryo developing. The transient expression of fluorescent markers was traced by confocal. 1 or 3 weeks after heat shock, typical globular MDE, or torpedo embryo were used as transfection recipient. After 24h co-culture, the Agrobacterium were washed with 0.01% lysozyme in order to digest the cell wall of bacterium. Then wash twice with NLN13 medium containing Timentin 300mg/L. Finally, the cells were kept in NLN13 medium containing 150mg/L Timentin and corresponding antibiotics at 25°C. Fresh media were changed weekly. The expression of fluorescent markers was continuously tracked by confocal.

Statistics

Statistics analysis was conducted by SPSS 16.0 software. Statistically significant differences among the number of microspore embryo per dish were calculated using Ducan's t-test (*P<0.05). ANOVA analysis of variance was applied in antibiotic sensitivity experiment.

Results

Genotype Dependency of Microspore Embryogenesis in Brassica rapa

Ten Chinese cabbage varieties 'Golden Crow' 'Yangchun' 'Chundajiang' 'Leader' 'Hongkang 1' 'Rich Spring' 'Beijing New 3' 'Multi-resistant3' 'FT' 'Golden Spring' were used as candidates to induce microspore derived embryos. 14d after microspore isolation, the embryos can be found at the edge of dishes, embryogenesis rate was calculated. 'FT' showed the highest microspore embryogenesis rate, which reached 144 embryos per 10000 microspores. On the contrary, the embryogenesis rate of 'chundajiang' and 'duokangxin 3' had only 1 or 2 embryos per 10000 microspores (Table 1). Microspore embryogenesis shows strong genotype dependency in Brassica rapa.