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Choosing the proper starting material is crucial to the entire regeneration[30]. In this study, Chrysanthemum cultivar 'Yuhualuoying' obtained from the Chrysanthemum Germplasm Resource Preserving Centre, Nanjing Agricultural University, China was chosen as the wild-type. The aseptic seedlings for genetic transformation were obtained depended on the space and technology provided by the Ornamental Horticulture Genetics and Breeding Laboratory of Nanjing Agricultural University. Young shoots were surface sterilized by initial immersion in 70% ethanol for 30 s, then in 30% H2O2 for 10 min, and finally being rinsed three times in sterile distilled water[31]. They were then incubated for 3 weeks on solidified Murashige and Skoog (MS) medium[32]. In order to obtain the best results, we took the tissue culture seedlings with the seedling age about 30 d as materials for subsequent experiments and operations (Fig. 5a).
Figure 5.
Tissue culture seedlings from explants sterilized and stem material preparation. (a) Stem explants were taken from plants growing on land, young shoots were surface sterilized and tissue seedlings were cultured in a sterile environment. Note: steam internode explants with a seedling age of 30−40 d were used. (b) Remove the steam tips and steam leaves and cut the explants into 0.5 cm sections containing undifferentiated axillary buds. (c) Pre-culture the explants on MS basal medium containing 2.0 mg/L 6-BA and 0.1 mg/L NAA.
Recipes for modified MS medium in each step are listed in Table 1. MS basal medium was used to provide nutrients for culture seedlings, 6-benzylaminopurine (6-BA) and Napthalene acetic acid (NAA) are two hormones for dedifferentiation and differentiation of explants[33]. While Carbenicillin (Carb) was used for inhibiting the outbreak of Agrobacterium, and hygromycin (Hyg) was used as a selectable marker for transformation[10]. Throughout the whole cultivation process, the temperature was maintained at 25 °C. Except the co-cultivation stage, during which the materials were put under dark conditions at all times, there would be 16 h of illumination and 8 h in darkness set by hand. The plant hormones and chemical reagents used in this research were provided by Sigma, and the various enzymes were provided by Takara and Invitrogen.
Table 1. Several culture mediums used during the training process.
Medium name Code Medium composition Pre-culture and co-culture medium MS1 MS + 6-BA 2.0 mg/L + NAA 0.1 mg/L Decarboxylation medium MS2 MS + 6-BA 2.0 mg/L + NAA 0.1 mg/L + Carb 350 mg/L Select medium MS3 MS + 6-BA 2.0 mg/L + NAA 0.1 mg/L + Carb 350 mg/L Select medium MS4 MS + 6-BA 2.0 mg/L + NAA 0.1 mg/L + Carb 80 mg/L + Hyg 8 mg/L rooting medium MS5 MS + Hyg 9 mg/L MS: Murashige and Skoog basal medium (Potassium Nitrate 1,900 mg/L, Ammonium Nitrate 1,650 mg/L, Potassium Phosphate Monobasic 170 mg/L, Magnesium Sulfate 370 mg/L, Calcium Chloride 440 mg/L, Potassium Iodide 0.83 mg//L, Boric Acid 6.2 mg/L, Manganese Sulfate 22.3 mg/L, Zinc Sulfate 8.6 mg/L, Sodium Molybdate 0.25 mg/L, Cupric Sulfate 0.025 mg/L, Cobalt Chloride·6H2O 0.025 mg/L, Ferrous Sulfate 27.8 mg/L, Myo-Inositol 100 mg/L, Glycine 2 mg/L, Thiamine Hydrochloride 0.1 mg/L, Pyridoxine Hydrochloride 0.5 mg/L, Nicotinic Acid 0.5 mg/L, Sucrose 30 g/L, Sugar 6.5−7 g/L, PH = 5.8, adjusted with NaOH); 6-BA: 6-benzylaminopurine; Carb: Carbenicillin; NAA: Napthaleneacetic acid; Hyg: hygromycin. CmLEC1 gene isolation and vector construction
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In order to achieve the CmLEC1 open reading frame (ORF), bioinformatics analysis was conducted according to the full length sequence of CL4474.Contig1 in 'Yuhualuoying' transcriptome database and NCBI[34]. The CmLEC1 fragments were obtained by polymerase chain reaction (PCR) with primers designed (Table 2) and RNA extracted from the embryo. Finally, we got the full length sequence of the gene, which is 1002 bp, and the ORF is 660 bp that can encode a protein of 219 amino acids. Specific primers were designed to import Sal I and Not I restriction sites, and link the CmLEC1 to the vector pENTR1A (Table 2). The amplicons were gelling purified and linearized by endonuclease PvuI. Then LR recombination reaction was carried out to subclone the amplicons into pMDC32 which contains the CaMV35S promoter and the nos terminator. The schematic diagram of the vector is shown in Fig. 2. Constructs were transformed into Agrobacterium tumefaciens strain EHA105 using a freeze–thaw method[35].
Table 2. Primers used in CmLEC1gene isolation and vector construction.
Primer Sequence (5'-3') CmLEC1 forward ATGGGTTACAATTGTGATTACTGTGG CmLEC1 reverse TCAGAAACTTGTTGCTTCATTCATGG CmLEC1-SalI forward TTCAGTCGACATGGGTTACAATTGTGATTA CmLEC1-NotI reverse GAGTGCGGCCGCGAGAAACTTGTTGCTTCATTCA Manual vacuum infiltration treatments
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In this study, we took the healthy and thriving tissue culture seedlings with a seedling age about 30−40 d, removed the steam tips and steam leaves from the base of the petiole, and cut the explants into 0.5 cm sections containing undifferentiated axillary buds (Fig. 5b & c). MS basal medium containing 2.0 mg/L 6-BA and 0.1 mg/L NAA was used for pre-culturing the stem internode. One hundred cut stem segments were put on five petri dishes. This operation was repeated three times. Agrobacterium infection solution was used to introduce the CmLEC1 gene. Agrobacterium was activated and cultured overnight with YEB medium (Yeast extract and peptone) containing 50 mg/L kanamycin and 50 mg/L rifampin on an orbital shaker at 250 rpm at 28 °C (Fig. 6a). When OD600 reached 5.0−6.0, select bacteria by centrifugation at 4,000 rpms for 15−20 mins (Fig. 6b). Bacteria were resuspended in liquid MS medium to OD600 1.0 (Fig. 6c). After 3 d of pre-culture, the stem segments were picked from the solid medium and placed in a syringe (30 mL in volume, Fig. 6d). Then, approximately 8−10 mL of the infection solution was drawn in the syringe so that the stem section could be completely immersed in the infection liquid (Fig. 6e). We removed the needle, drained the gas in the syringe, and used the left thumb to block the front opening of the syringe, or burned the orifice impermeable with an alcohol lamp, then gently pinched and sealed (Fig. 6f). Then pulled the syringe plunger and shaked to release the interstitial air, which infiltrated the fluid into the organization simultaneously due to the negative pressure, this was carried out 5−8 times, for ten seconds each time. We used a 10mL tube as an aid to ease operation and hold the syringe, keep the negative pressure state, shake the syringe from time to time. This step was the most important for determining the effect of infection and transformation and 5−10 mins was necessary to ensure almost all steam sections sink into the bottom of the liquid (Fig. 6g). Then filtered off the infection liquid, put the infected stem segments on clean, sterilized filters (Fig. 6h), dried with sterile wind and replaced on new medium.
Figure 6.
Agrobacterium infection solution preparation and manual vacuum infiltration treatment. (a) Agrobacterium was cultured overnight with YEB medium. (b) Bacteria were selected by centrifugation. (c) Bacteria were resuspended in liquid MS medium. (d) Stem segments were picked and placed in a 30 mL syringe. (e) Infection liquid was drawn in the syringe. (f) Through a series of simple operations to create a vacuum environment. (g) Pull the syringe plunger and shake to release the interstitial air, keeping the negative pressure state for 5−10 min, until all steam sections sink to the bottom of the liquid. (h) Dry the infected stem and replace on new medium.
Culture process
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The stem internode explants treated as above were pre-cultured on MS1 (Table 1) medium, containing 2.0 mg/L 6-BA and 0.1 mg/L NAA for 3 d. We then introduced the CmLEC1 by vacuum infiltration manually by the methods provided in this article, dried the infected stem segments and replaced on new MS1 medium, and co-cultivated with Agrobacterium containing the recombinant vector pMDC32-CmLEC1 for 2 or 3 d under dark conditions. When the edge of the explants showed spot-shaped Agrobacterium colonies, they were transferred to the Decarboxylation culture (MS2), in which Carbenicillin was used to inhibit the outbreak of A. tumefaciens (Table 1, 7 d or more is necessary). The infected stem segments were cultured and selected under the stress of hygromycin on MS3 and MS4 medium (Table 1)[36], and each 15 d was subcultured once. In each subculture, the small lateral buds should be separated from the mother and cultured separately, in order to screen continuously and reduce the production of transgenic chimerism. In the screening process, the concentration of the carboxybenzyl penicillin was reduced in sequence according to the gradient. The explants could be cultured on MS4 medium until rooting. If redisdual Agrobacterium was observed this should be remedied in time. The explants could be transferred to the rooting medium MS5 (Table 1) after a month without the outbreak of A. tumefaciens, with the aim of excluding false positives. Rooted plantlets were transplanted into sterile potting mixture and grown in a greenhouse.
Identification of transgenes
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Transgenic plants were identified by confirming the certain presence of the CmLEC1 gene[25]. Among all the regenerated seedlings obtained by Agrobacterium infection and control plants (WT), genomic DNA was isolated. Some of the putative transformants were analysed by PCR amplification using a set of specific primers (Table 3), designed by the vector and gene[37]. Plasmid DNA was used as positive control (CK1), and clear water as negative control (CK2) to exclude the impact of environmental factors. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining to confirm integration of CmLEC1 in possible transformants[38].
Table 3. Specific primers designed for identification.
Primer Sequence (5'-3') 35S GACGCACAATCCCACTATCC CmLEC1-TESTreverse ACATTCTTGGATGGTTTCTTTT CmLEC1-RT Forward GGCCATGAGCAAACTAGGGT CmLEC1-RT Reverse ACGTTCTCCGCCATCAAACT CmEF1α Forward TTTTGGTATCTGGTCCTGGAG CmEF1α Reverse CCATTCAAGCGACAGACTCA Transcription of transgenes
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Stable expression of the chrysanthemum CmLEC1 in 'Yuhualuoying' was confirmed by qRT-PCR analysis. The relative expression analysis of CmLEC1 was carried out with wild-type chrysanthemum and transgenic chrysanthemum which was the same age and the same part, and there are three replications for each sample. Total RNA was extracted from with TRIzol reagent (TaKaRa, Tokyo, Japan) following the manufacturer's protocol. Single-stranded cDNA was obtained using an M-MLV Reverse Transcription Kit (TaKaRa), and qRT-PCR was implemented on a LightCycler 480 Real-Time PCR System. The gene-specific primers of CmLEC1 were designed (Table 3) by Primer 5[39]. And EF1α (Elongation Factor 1a) was used as a reference gene, which is stably expressed in chrysanthemum (sequences given in Table 3)[40,41]. The qRT-PCR was performed using a 20 μL reaction system. In addition, relative expression levels were calculated by the 2−ΔΔCᴛ method[42].
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In summary, an efficient Agrobacterium-mediated transformation protocol via vacuum infiltration of chrysanthemum stem internode has been successfully established in the present study. This method not only significantly improves chrysanthemum transformation efficiency, but also dramatically shortens the time to obtain the transgenic positive seedling. Thus, the results of this study will be valuable and useful in gene function and molecular breeding of chrysanthemum, and other plants, in the future.
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About this article
Cite this article
Xu S, Wu Z, Zhao J, Zhang F, Zhang X, et al. 2022. High-efficiency Agrobacterium-mediated transformation of chrysanthemum via vacuum infiltration of internode. Ornamental Plant Research 2:1 doi: 10.48130/OPR-2022-0001
High-efficiency Agrobacterium-mediated transformation of chrysanthemum via vacuum infiltration of internode
- Received: 08 November 2021
- Accepted: 20 December 2021
- Published online: 18 January 2022
Abstract: Leaf disc transformation is one of the traditional methods that are now widely used in chrysanthemum with highly economical and ornamental value in world flower production, but it depends on plant genotypes and is time consuming and complicated. In addition, the transformation success rate of this method is low, generally ranging from 0.1% to 6.25%. Therefore, a highly efficient transformation system is needed. In this study, we are the first to establish a high-efficient chrysanthemum Agrobacterium-mediated transformation system via vacuum infiltration. Chrysanthemum stem internode explants were used as research material and CmLEC1 was used as a reporter gene. After approximately 3 months of culture and selection, the positive transgenic plants were obtained. Additionally, the positive probability was about 42%. The transformation efficiency was up to 37.7%, and if the escapes were removed, it was 16%. Furthermore, stable expression of CmLEC1 in transgenic 'Yuhualuoying' was confirmed by qRT-PCR analysis. These results suggest that this genetic transformation system via vacuum infiltration of chrysanthemum stem internode is highly efficient and convenient, and much better than traditional leaf disc transformation, and it will play an important role in chrysanthemum transformation and functional genetics research.
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Key words:
- Agrobacterium /
- Transformation /
- Method /
- Vacuum /
- Chrysanthemum