Efficient Transformation System Research Articles

Tissue culture and Agrobacterium-mediated genetic transformation of the oil crop sunflower

Sunflower is one of the four major oil crops in the world. ‘Zaoaidatou’ (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.

An efficient genetic transformation system mediated by Rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion.

Genetic transformation is a critical tool for gene editing and genetic improvement of plants. Although many model plants and crops can be genetically manipulated, genetic transformation systems for fruit trees are either lacking or perform poorly. We used Rhizobium rhizogenes to transfer the target gene into the hairy roots of Malus domestica and Actinidia chinensis. Transgenic roots were generated within 3 weeks, with a transgenic efficiency of 78.8%. Root to shoot conversion of transgenic hairy roots was achieved within 11 weeks, with a regeneration efficiency of 3.3%. Finally, the regulatory genes involved in stem cell activity were used to improve shoot regeneration efficiency. MdWOX5 exhibited the most significant effects, as it led to an improved regeneration efficiency of 20.6% and a reduced regeneration time of 9 weeks. Phenotypes of the overexpression of RUBY system mediated red roots and overexpression of MdRGF5 mediated longer root hairs were observed within 3 weeks, suggesting that the method can be used to quickly screen genes that influence root phenotype scores through root performance, such as root colour, root hair, and lateral root. Obtaining whole plants of the RUBY system and MdRGF5 overexpression lines highlights the convenience of this technology for studying gene functions in whole plants. Overall, we developed an optimized method to improve the transformation efficiency and stability of transformants in fruit trees.

Establishment of an Efficient Sugarcane Transformation System via Herbicide-Resistant CP4-EPSPS Gene Selection

Sugarcane (Saccharum spp.), a major cash crop that is an important source of sugar and bioethanol, is strongly influenced by the impacts of biotic and abiotic stresses. The intricate polyploid and aneuploid genome of sugarcane has shown various limits for conventional breeding strategies. Nonetheless, biotechnological engineering currently offers the best chance of introducing commercially significant agronomic features. In this study, an efficient Agrobacterium-mediated transformation system that uses the herbicide-resistant CP4-EPSPS gene as a selection marker was developed. Notably, all of the plants that were identified by PCR as transformants showed significant herbicide resistance. Additionally, this transformation protocol also highlighted: (i) the high yield of transgenic lines from calli (each gram of calli generated six transgenic lines); (ii) improved selection; and (iii) a higher transformation efficiency. This protocol provides a reliable tool for a routine procedure for the generation of resilient sugarcane plants.

A rapid and efficient Agrobacterium-mediated transient transformation system in grape berries.

Transient transformation is extremely useful for rapid in vivo assessment of gene function, especially for fruit-related genes. Grape berry, while an important fruit crop, is recalcitrant to transient transformation, due to the high turgor pressure in its mesocarp cells that limits the ability of Agrobacterium to penetrate into the tissue. It is urgent to establish a simple transient transformation system for rapid analysis of gene function. In this study, different injection methods, grape genotypes, and developmental stages were tested in order to develop a rapid and efficient Agrobacterium-mediated transient transformation methodology for grape berries. Two injection methods, namely punch injection and direct injection, were evaluated using the β-glucuronidase (GUS) gene and by x-gluc tissue staining and 4-methylumbelliferyl-β-D-glucuronide fluorescence analysis. The results indicated that there were no significant differences on transformation effects between the two methods, but the latter was more suitable because of its simplicity and convenience. Six grape cultivars ('Hanxiangmi', 'Moldova', 'Zijixin', 'Jumeigui', 'Shine-Muscat', and 'A17') were tested for transient transformation. 'Hanxiangmi', 'Moldova', and 'Zijixin' grape berries were not suitable for agroinfiltration due to frequently fruit cracking, browning, and formation of scar skin. The fruit integrity rates of 'Jumeigui', 'Shine-Muscat', and 'A17' berries were all above 80%, and GUS activity was detected in the berries of the three cultivars 3-14days after injection with the Agrobacterium culture, while higher GUS activities were observed in the 'Jumeigui' berries. The levels of GUS activity in injected berries at 7-8weeks after full blooming (WAFB) were more than twice at 6 WAFB. In subsequent assays, the over-expression of MYB transcription factor VvMYB44 via transient transformation accelerated the anthocyanin accumulation and fruit coloring through raising the expression levels of VvLAR1, VvUFGT, VvLDOX, VvANS, and VvDFR, which verified the effectiveness of this transformation system. These experiments finally identified the reliable grape cultivars and suitable operational approach for transient transformation and further indicated that this Agrobacterium-mediated transient transformation system was efficient and suitable for the elucidation of gene function in grape berries.

High-quality genome assembly and genetic transformation system of Lasiodiplodia theobromae strain LTTK16-3, a fungal pathogen of Chinese hickory.

Lasiodiplodia theobromae, as one of the causative agents associated with Chinese hickory trunk cankers, has caused huge economic losses to the Chinese hickory industry. Although the biological characteristics of this pathogen and the occurrence pattern of this disease have been well studied, few studies have addressed the related mechanisms due to the poor molecular and genetic study basis of this fungus. In this study, we sequenced and assembled L. theobromae strain LTTK16-3, isolated from a Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Phylogenetic analysis and comparative genomics analysis presented crucial cues in the prediction of LTTK16-3, which shared similar regulatory mechanisms of transcription, DNA replication, and DNA damage response with the other four Chinese hickory trunk canker-associated Botryosphaeria strains including, Botryosphaeria dothidea, Botryosphaeria fabicerciana, Botryosphaeria qingyuanensis, and Botryosphaeria corticis. Moreover, it contained 18 strain-specific protein clusters (not conserved in the other L. theobromae strains, AM2As and CITRA15), with potential roles in specific host-pathogen interactions during the Chinese hickory infection. Additionally, an efficient system for L. theobromae protoplast preparation and polyethylene glycol (PEG) -mediated genetic transformation was firstly established as the foundation for its future mechanisms study. Collectively, the high-quality genome data and the efficient transformation system of L. theobromae here set up the possibility of targeted molecular improvements for Chinese hickory canker control.IMPORTANCEFungi with disparate genomic features are physiologically diverse, possessing species-specific survival strategies and environmental adaptation mechanisms. The high-quality genome data and related molecular genetic studies are the basis for revealing the mechanisms behind the physiological traits that are responsible for their environmental fitness. In this study, we sequenced and assembled the LTTK16-3 strain, the genome of Lasiodiplodia theobromae first obtained from a diseased Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Further phylogenetic analysis and comparative genomics analysis provide crucial cues in the prediction of the proteins with potential roles in specific host-pathogen interactions during the Chinese hickory infection. An efficient PEG-mediated genetic transformation system of L. theobromae was established as the foundation for the future mechanisms exploration. The above genetic information and tools set up valuable clues to study L. theobromae pathogenesis and assist in Chinese hickory canker control.

Establishment of an Efficient Genetic Transformation System in Sanghuangporus baumii.

(1) Background: Sanghuangporus baumii, a valuable medicinal fungus, has limited studies on its gene function due to the lack of a genetic transformation system. (2) Methods: This study aimed to establish an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for S. baumii. This study involved cloning the promoter (glyceraldehyde-3-phosphate dehydrogenase, gpd) of S. baumii, reconstructing the transformation vector, optimizing the treatment of receptor tissues, and inventing a new method for screening positive transformants. (3) Results: The established ATMT system involved replacing the CaMV35S promoter of pCAMBIA-1301 with the gpd promoter of S. baumii to construct the pCAMBIA-SH-gpd transformation vector. The vectors were then transferred to A. tumefaciens (EHA105) for infection. This study found that the transformation efficiency was higher in the infection using pCAMBIA-SH-gpd vectors than using pCAMBIA-1301 vectors. The mycelia of S. baumii were homogenized for 20 s and collected as the genetic transformation receptor. After 20 min of co-culture and 48 h of incubation in 15 mL PDL medium at 25 °C, new colonies grew. (4) Conclusions: These colonies were transferred to PDA medium (hygromycin 4 μg/mL, cefotaxime 300 μg/mL), and the transformation efficiency was determined to be 33.7% using PCR.

Characterization of a Foxtail Mosaic Virus Vector for Rust Fungus Avirulence Gene Expression in Wheat

The wheat fungal pathogens Puccinia graminis f. sp. tritici ( Pgt), P. striiformis f. sp. tritici, and P. triticina, causing stem, stripe, and leaf rust, respectively, pose a threat to global wheat production. Genetic resistance in the form of resistance ( R) genes provides the best protection, but rust fungal populations change frequently by mutating avirulence (AVR) effectors matching specific R genes, thereby defeating resistance. Hence, characterization of AVR effectors is needed to understand the evolution of pathogen populations, provide insights for extending the effectiveness of R genes, and yield tools for the identification and isolation of R genes. Functional characterization of Avr genes in rust fungi is challenging in these biotrophic pathogens that lack a reliable and efficient transformation system. Studies indicate that the recently engineered foxtail mosaic virus (FoMV) shows promise as an expression system in cereals. In this study, we utilized two confirmed AVR effectors from Pgt, AVRSr35 and AVRSr50, to assess the applicability of FoMV for investigating rust fungus Avr genes. We showed that vector FoMV PV101 carrying PgtAvrSr35 induced a hypersensitive response (HR) in wheat having the corresponding Sr35 resistance gene. However, when carrying PgtAvrSr50, no HR or even mild viral symptoms were seen in a wheat line having Sr50, as this particular wheat line was not susceptible to FoMV. Several wheat cultivars did not support FoMV replication. The results here show that FoMV PV101 is effective as an expression vector for studying rust fungi AVR effectors, but its applicability relies on the susceptibility of wheat cultivars to FoMV. [Formula: see text] Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Agrobacterium rhizogenes-mediated marker-free transformation and gene editing system revealed that AeCBL3 mediates the formation of calcium oxalate crystal in kiwifruit

The transformation and gene editing of the woody species kiwifruit are difficult and time-consuming. The fast and marker-free genetic modification system for kiwifruit has not been developed yet. Here, we establish a rapid and efficient marker-free transformation and gene editing system mediated by Agrobacterium rhizogenes for kiwifruit. Moreover, a removing-root-tip method was developed to significantly increase the regeneration efficiency of transgenic hairy roots. Through A. rhizogenes-mediated CRISPR/Cas9 gene editing, the editing efficiencies of CEN4 and AeCBL3 achieved 55 and 50%, respectively. And several homozygous knockout lines for both genes were obtained. Our method has been successfully applied in the transformation of two different species of kiwifruit (Actinidia chinensis ‘Hongyang’ and A.eriantha ‘White’). Next, we used the method to study the formation of calcium oxalate (CaOx) crystals in kiwifruit. To date, little is known about how CaOx crystal is formed in plants. Our results indicated that AeCBL3 overexpression enhanced CaOx crystal formation, but its knockout via CRISPR/Cas9 significantly impaired crystal formation in kiwifruit. Together, we developed a fast maker-free transformation and highly efficient CRISPR-Cas9 gene editing system for kiwifruit. Moreover, our work revealed a novel gene mediating CaOx crystal formation and provided a clue to elaborate the underlying mechanisms.Graphical abstract

Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N.alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.

Cloning, Characterization, and Expression Pattern Analysis of the BBM Gene in Tree Peony (Paeonia ostii)

BABY BOOM (BBM) is one of the members of the plant-specific APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor superfamily. It acts as a key regulator of plant cell pluripotency, playing a significant role in promoting somatic embryogenesis. In this study, a BBM gene named PoBBM was screened, cloned, and identified from the third-generation full-length transcriptome data of Paeonia ostii. Its open reading frame was 2136 bp, encoding 711 amino acids. Sequence feature analysis revealed that it possessed two AP2 conserved domains and eight motifs, including bbm-1. The phylogenetic tree indicated that PoBBM clusters with AtBBM in the euANT group of the Arabidopsis AP2 family, which is most closely related to grape VvBBM and may have the same ancestry as grape. Subcellular localization demonstrated that the PoBBM protein was localized in the nucleus. Semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess the PoBBM transcript levels during ten developmental stages of somatic embryos and in five tissue types of peonies. The results indicate that PoBBM was highly expressed in the early stages of peony somatic embryo development. The expression on 0–15 d was the highest and decreased gradually with somatic embryogenesis. The gene is almost not expressed after 40 d since somatic embryo formation. PoBBM was expressed in roots, stems, leaves, seeds, and calli, with the highest levels in seeds, followed by leaves and calli. The PoBBM protein displayed transcriptional self-activation activity, which may facilitate further research on its relationships with other proteins. The above results provide a key gene PoBBM for somatic embryogenesis in peonies, which is significant for advancing the establishment of a stable and efficient regeneration and genetic transformation system for peonies.

Tracking organelle activities through efficient and stable root genetic transformation system in woody plants.

Due to the protracted transgenic timeline and low efficiency in stable genetic transformation of woody plants, there has been limited exploration of real-time organelle imaging within stable transgenic woody plant cells. Here, we established an efficient in vivo genetic transformation system for woody plants using an Agrobacterium rhizogenes-mediated approach. This system was successfully validated in multiple perennial woody species. Using citrus as a model, we introduced organelle-targeted fluorescent reporters via genetic transformation and investigated their subcellular localization and dynamics using advanced imaging techniques, such as confocal microscopy and live-cell imaging. Moreover, we subjected transgenic MT-GFP-labeled mitochondria in root cells to stress conditions simulating agricultural adversities faced by fruit crops. The stress-induced experiments revealed notable alterations in mitochondrial morphology. Our study contributes novel insights into membrane trafficking processes, protein localization dynamics, and cellular physiology in woody plants, while also providing stable and efficient genetic transformation methods for perennial woody species.

10k-level integrated rice database shows power for exploiting rare variants.

This Highlight features a recent study by Shang Lianguang and Qian Qian's groups, who re-analyzed published resequencing data covering 10,548 accessions of Asian cultivated rice Oryza sativa and wild rice Oryza rufipogon from 98 countries worldwide to generate a super-large rice genomic variation dataset.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation in citrus seeds and its application in gene functional analysis.

Highly efficient genetic transformation technology is beneficial for plant gene functional research and molecular improvement breeding. However, the most commonly used Agrobacterium tumefaciens-mediated genetic transformation technology is time-consuming and recalcitrant for some woody plants such as citrus, hampering the high-throughput functional analysis of citrus genes. Thus, we dedicated to develop a rapid, simple, and highly efficient hairy root transformation system induced by Agrobacterium rhizogenes to analyze citrus gene function. In this report, a rapid, universal, and highly efficient hairy root transformation system in citrus seeds was described. Only 15 days were required for the entire workflow and the system was applicable for various citrus genotypes, with a maximum transformation frequency of 96.1%. After optimization, the transformation frequency of Citrus sinensis, which shows the lowest transformation frequency of 52.3% among four citrus genotypes initially, was increased to 71.4% successfully. To test the applicability of the hairy roots transformation system for gene functional analysis of citrus genes, we evaluated the subcellular localization, gene overexpression and gene editing in transformed hairy roots. Compared with the traditional transient transformation system performed in tobacco leaves, the transgenic citrus hairy roots displayed a more clear and specific subcellular fluorescence localization. Transcript levels of genes were significantly increased in overexpressing transgenic citrus hairy roots as compared with wild-type (WT). Additionally, hairy root transformation system in citrus seeds was successful in obtaining transformants with knocked out targets, indicating that the Agrobacterium rhizogenes-mediated transformation enables the CRISPR/Cas9-mediated gene editing. In summary, we established a highly efficient genetic transformation technology with non-tissue-culture in citrus that can be used for functional analysis such as protein subcellular localization, gene overexpression and gene editing. Since the material used for genetic transformation are roots protruding out of citrus seeds, the process of planting seedlings prior to transformation of conventional tissue culture or non-tissue-culture was eliminated, and the experimental time was greatly reduced. We anticipate that this genetic transformation technology will be a valuable tool for routine research of citrus genes in the future.

Fast, simple, efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However, the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated (ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches (one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings (Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%–51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin. The one-step method outperformed the two-step method; the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily, quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

Establishment of a Transient Transformation Protocol in Cinnamomum camphora

Cinnamomum camphora is an excellent evergreen broad-leaved tree species with strong stress tolerance, but its molecular character revelation as well as ecological and economic value improvement were limited due to the lack of a genetic transformation system. To establish a simple and efficient transient transformation system for uncovering the molecular mechanism of plant tolerating stresses and promoting the selective breeding of good varieties, the infection method, co-cultivation time, infection solution concentration, and growth density of Agrobacterium tumefaciens containing green fluorescent protein (GFP)-based calmodulin protein 3 gene (GCaMP3) were identified by monitoring the fluorescence emitted from GCaMP3 bound to Ca2+. Meanwhile, the transient transformation effects were evaluated via cytoplasmic Ca2+ concentration variations at high temperatures of 35 °C and 40 °C. When C. camphora leaves were infected with A. tumefaciens containing GCaMP3 via injection and soaking, no significant difference was detected in the fluorescence intensity over 48 h, indicating that the two infection methods had the same transient transformation efficiency. By prolonging the co-cultivation time, the fluorescence intensity gradually increased, reached its strongest at the 48th h, and then gradually declined. For the infection solution concentration, an OD600 of 0.7 led to the strongest fluorescence intensity, with an increase of 42.2%, 13.7%, 4.2%, and 14.2%, respectively, compared to that at OD600 of 0.5, 0.6, 0.8, and 0.9. When A. tumefaciens growth density OD600 was 0.5–0.7, the strongest fluorescence intensity was detected after transient transformation. Combining these optimum conditions, GCaMP3 was transferred into C. camphora, which indicated the variations in cytoplasmic Ca2+ concentration at high temperatures, with the fluorescence intensity at 35 °C and 40 °C increasing by 12.6% and 30.6%, respectively, in contrast to that at 28 °C. Therefore, it should be an efficient transient transformation system for C. camphora, with A. tumefaciens growth density OD600 of 0.5–0.7, infection solution concentration OD600 of 0.7, and co-cultivation time of 48 h by using both injection and soak infection methods, which is beneficial for uncovering the Ca2+ signal transduction in the plant tolerating stresses and promoting its molecular biology development and selective breeding of good varieties.

Effective Agrobacterium-mediated genetic transformation of okra (Abelmoschus esculentus L.) and generation of RNAi plants resistant to Begomovirus infecting okra

ABSTRACT Okra is an important vegetable crop of the Malvaceae family and is infected by varying numbers of viruses of the genus Begomovirus. Regardless of the importance of the crop, very little consideration has been given to its genetic improvement. RNA interference (RNAi), a potent biotechnological tool, is known to control Begomovirus in many crops. For the implementation of successful RNAi, there is a need for an efficient genetic transformation system in okra. In the present study, we developed a procedure for Agrobacterium-mediated tissue culture–dependent regeneration of okra plants for the application of RNAi. Eleven transgenic okra RNAi plants were regenerated by utilising hypocotyls as explants. Transformed plants were screened with hygromycin at the regeneration stage and the presence of transgenes (AC1, AC2 & AC4 codes for replication-associated protein, transcriptional activator protein and suppressor of PTGS) in putative transformed plants was confirmed by polymerase chain reaction (PCR). Wild and transgenic lines were challenged with a dimeric Begomovirus clone or viruliferous whiteflies and the level of resistance was estimated with quantitative real-time reverse transcriptase PCR (qRT-PCR) by utilising viral gene–specific primers. The resistant transgenic lines accumulated very low titres of viral gene products according to the qRT-PCR assays compared to the control plants. This is the first report of tissue culture–mediated RNAi-derived resistance in okra against Begomovirus infection.

Virus-Induced Gene Silencing in the Tea Plant (Camellia sinensis).

The recent availability of a number of tea plant genomes has sparked substantial interest in using reverse genetics to explore gene function in tea (Camellia sinensis). However, a hurdle to this is the absence of an efficient transformation system, and virus-induced gene silencing (VIGS), a transient transformation system, could be an optimal choice for validating gene function in the tea plant. In this study, phytoene desaturase (PDS), a carotenoid biosynthesis gene, was used as a reporter to evaluate the VIGS system. The injection sites of the leaves (leaf back, petiole, and stem) for infiltration were tested, and the results showed that petiole injection had the most effective injection, without leading to necrotic lesions that cause the leaves to drop. Tea leaves were inoculated with Agrobacterium harboring a tobacco rattle virus plasmid (pTRV2) containing a CsPDS silencing fragment. The tea leaves exhibited chlorosis symptoms 7-14 days after inoculation, depending on the cultivar. In the chlorosis plants, the coat protein (CP) of tobacco rattle virus (TRV) was detected and coincided with the lower transcription of CsPDS and reduced chlorophyll content compared with the empty vector control, with 81.82% and 54.55% silencing efficiency of 'LTDC' and 'YSX', respectively. These results indicate that the VIGS system with petiole injection could quickly and effectively silence a gene in tea plants.

Agrobacterium rhizogenes-Mediated Genetic Transformation and Establishment of CRISPR/Cas9 Genome-Editing Technology in Limonium bicolor

Limonium bicolor is a perennial herbaceous plant belonging to the Plumbaginaceae family. It can be used as a dried flower or in cut flower arrangements and serves as a model recretohalophyte. Its genome sequencing has been recently completed. However, the research on L. bicolor is limited by the absence of a highly efficient genetic transformation system. In this study, we established a highly efficient Agrobacterium rhizogenes-mediated L. bicolor genetic transformation method. The transgenic hairy roots were induced from the hypocotyl of L. bicolor using A. rhizogenes strain K599 harboring pRdGa4Cas9 plasmid (which carries an expression cassette of 35S::DsRed2). The transgenic shoots were regenerated from hairy root segments (~0.1 cm diameter), and induction efficiency was achieved at 100%. The transgenic shoots with 4–5 rosette leaves were directly planted into the soil to induce the transgenic roots. Therefore, transgenic plantlets were produced. The DsRed2 can be used as a reliable reporter gene in screening transgenic plantlets. Furthermore, we also established a CRISPR/Cas9 system in L. bicolor employing the A. rhizogenes-mediated genetic transformation approach. The highly efficient transformation method and CRIPSP/Cas9 system established will provide a valuable tool for functional genomics investigation and trait improvement in L. bicolor.

Cotyledon peeling method for passion fruit protoplasts: a versatile cell system for transient gene expression in passion fruit (Passiflora edulis).

Passion fruit (Passiflora edulis) is a perennial evergreen vine that grows mainly in tropical and subtropical regions due to its nutritional, medicinal and ornamental values. However, the molecular biology study of passion fruit is extremely hindered by the lack of an easy and efficient method for transformation. The protoplast transformation system plays a vital role in plant regeneration, gene function analysis and genome editing. Here, we present a new method ('Cotyledon Peeling Method') for simple and efficient passion fruit protoplast isolation using cotyledon as the source tissue. A high yield (2.3 × 107 protoplasts per gram of fresh tissues) and viability (76%) of protoplasts were obtained upon incubation in the enzyme solution [1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] for 2 hours. In addition, we achieved high transfection efficiency of 83% via the polyethylene glycol (PEG)-mediated transformation with a green fluorescent protein (GFP)-tagged plasmid upon optimization. The crucial factors affecting transformation efficiency were optimized as follows: 3 μg of plasmid DNA, 5 min transfection time, PEG concentration at 40% and protoplast density of 100 × 104 cells/ml. Furthermore, the established protoplast system was successfully applied for subcellular localization analysis of multiple fluorescent organelle markers and protein-protein interaction study. Taken together, we report a simple and efficient passion fruit protoplast isolation and transformation system, and demonstrate its usage in transient gene expression for the first time in passion fruit. The protoplast system would provide essential support for various passion fruit biology studies, including genome editing, gene function analysis and whole plant regeneration.

An Efficient Transformation System for Fast Production of VcCHS Transgenic Blueberry Callus and Its Expressional Analysis.

The Agrobacterium tumefaciens-mediated transformation for blueberries remains less efficient than is desirable. A new leaf callus regeneration and genetic transformation system was investigated in blueberries in this study. The leaf explants of cv. 'Legacy' and 'Northland' were used to establish the stable callus induction system when placed on the woody plant medium (WPM) supplemented with 1.0 mg·L-1 2, 4-D, 0.4 mg·L-1 6-BA for 30 d; then, the callus was sub-cultured in the proliferation medium supplemented with 1.5 mg·L-1 2, 4-D, 0.4 mg·L-1 6-BA in the darkness at 25 °C every 30 days. The co-cultivation of callus with A. tumefaciens was operated on WPM plus 100 μM acetosyringone for 4 days; then, the transferred callus was grown in WPM supplemented with 1.5 mg·L-1 2,4-D, 0.4 mg·L-1 6-BA, 50 mg·L-1 hygromycin, and 200 mg·L-1 cefotaxime. The VcCHS transgenic blueberry callus with both GFP signal and Hyg resistance was obtained from the transformed callus of cv. 'Northland'. The rate of GFP signal detected in the transformed callus was as high as 49.02%, which was consistent with the PCR assay. Collectively, this study provides a highly efficient genetic transformation system in blueberry callus and a powerful approach for the molecular breeding of blueberries.