Agrobacterium-mediated Genetic Transformation Research Articles

Development of sorghum mutants with improved in vitro protein digestibility by CRISPR/Cas9 editing of kafirin genes

Sorghum (Sorghum bicolor (L.) Moench) is a major world crop that is a reliable source of fodder and food grain in arid regions. However, unlike other cereals, sorghum grain has low nutritional value, owing mainly to the resistance of its storage proteins (kafirins) to protease digestion. Changing the composition of kafirins or their primary structure may address this problem. To induce mutations in kafirin-encoding genes that were expected to disturb their accumulation in endosperm cells, we used a genome-editing approach. By Agrobacterium-mediated genetic transformation of immature embryos of cv. Avans, we obtained 14 transgenic plants with genetic constructs for site-directed mutagenesis of the k1C5 and gKAF1 genes encoding 22 kDa α- and 28 kDa γ-kafirins, respectively. Sequencing of 5 regenerants obtained by using k1C5-addressing vector revealed two plants with mutations. T1 progeny of these mutants had higher in vitro digestibility of endosperm proteins (86%–92%), in comparison with the donor Avans (63%–67%). The kernels of these plants had a thick vitreous endosperm. A mutant with increased in vitro protein digestibility and vitreous endosperm, carrying a mutation in the target sequence, was also obtained by use of the gKAF1-addressing vector. Thus, using genome editing technology, we have obtained mutants with improved kafirin digestibility that can be used in sorghum breeding.

Enhancing alfalfa resistance to Spodoptera herbivory by sequestering microRNA396 expression.

Sequestering microRNA396 by overexpression of MIM396 enhanced alfalfa resistance to Spodoptera litura larvae, which may be due to increased lignin content and enhanced low-molecular weight flavonoids and glucosinolates biosynthesis. Alfalfa (Medicago sativa), the most important leguminous forage crop, suffers from the outbreak of defoliator insects, especially Spodoptera litura, resulting in heavy losses in yield and forage quality. Here, we found that the expression of alfalfa microRNA396 (miR396) precursor genes and mature miR396 was significantly up-regulated in wounding treatment that simulates feeding injury by defoliator insects. To verify the function of miR396 in alfalfa resistance to insect, we generated MIM396 transgenic alfalfa plants with significantly down-regulated miR396 expression by Agrobacterium-mediated genetic transformation. The MIM396 transgenic alfalfa plants exhibited improved resistance to Spodoptera litura larvae with increased lignin content but decreased JA accumulation. Most of the miR396 putative target GRF genes were up-regulated in MIM396 transgenic lines, and responded to the wounding treatment. By RNA sequencing analysis, we found that the differentially expressed genes related to insect resistance between WT and MIM396 transgenic plants mainly clustered in biosynthesis pathways in lignin, flavonoids and glucosinolates. In addition to the phenotype of enhanced insect resistance, MIM396 transgenic plants also displayed reduced biomass yield and forage quality. Our results broaden the function of miR396 in alfalfa and provide genetic resources for studying alfalfa insect resistance.

Heterologous expression of HsPstS gene reducing arsenic accumulation and improving As-tolerance in transgenic tobaccos by enhancing CAT activity

Arsenic (As) is a toxic metalloid element that affects plant growth and development. Reducing the uptake of arsenic by plants via genetic engineering strategy can effectively improve the tolerance and safety of economic crops in As-contaminated soil. In this paper, the HsPstS gene coded ABC-type periplasmic phosphate-binding protein (PBP) of Halomonas strain GFAJ-1 was introduced into tobacco K326 by Agrobacterium-mediated genetic transformation to create transgenic tobaccos. Under As stress, NBT and DAB staining of tobacco leaves showed significant accumulation of H2O2 in wild-type and CK plants, and the further determination showed that the H2O2 content in CK plants was higher than that in transgenic plants except for L35S-2 and LREL-4 at 3 d after stress. Generally, the activity of antioxidant enzymes (CAT and POD) in tobaccos increased first and then decreased under As stress, and the CAT activity in most transgenic tobacco plants was significantly higher than that in wild-type and CK plants at 5 d after stress. By contrast, POD activity in CK and wild-type plants was significantly higher than that in transgenic tobaccos except for L35S-2. Additionally, As content determination showed that all transgenic tobacco plants except for CK showed the characteristic of low As-accumulation, especially in transgenic tobaccos L35S-2 and LREL-4, which suggested that the introduction of HsPstS could significantly reduce the As absorption in HsPstS-contained transgenic tobaccos, while there was no significant influence on agronomic traits and photosynthetic characteristics of transgenic tobaccos compared with wild-type ones. Interestingly, the introduction of HsPstS gene also reduced the content of nicotine and nornicotine in transgenic tobacco plants, while there was no significant difference on K content between transgenic and non-transgenic tobaccos. These results above provided ideal parental materials for cultivating tobacco germplasm with the characteristic of low As-accumulation.

A protocol for Agrobacterium-mediated genetic transformation of Lens culinaris Medik (lentil)

A reliable protocol for Agrobacterium-mediated genetic transformation of Lens culinaris Medik (lentil) was developed. Using cultivar Laird, the protocol yielded rooted shoots from an average of 6.8 independent events per hundred seeds. The protocol utilized longitudinal slices of embryo axes from imbibed mature seed as a starting explant and a plasmid containing a β-glucuronidase:neomycin phosphotransferase (gus:nptII) fusion gene in Agrobacterium strain EHA105. A series of four media, each with appropriate levels of kanamycin selection were identified and other factors tested included the optical density of the Agrobacterium suspension, and type and concentration of plant growth regulators. The expression of the gus reporter gene was visualized through histochemical staining, and further molecular analysis through PCR, qPCR, ddPCR and Southern hybridization confirmed transformation and provided copy number. The inserted genes were inherited into the T1 generation and chimaeras were not identified. The time from co-cultivation to the planting of rooted shoots ranged from 4 to 7 months, as transgenic clusters continue to produce additional clonal shoots.

ApGA20ox1, a key gibberellin biosynthesis gene, regulates somatic embryogenesis and plant height in Agapanthus praecox

Agapanthus praecox is cultivated worldwide as an ornamental plant, and a dwarf ideotype has become more popular due to its superior lodging resistance and its suitability for use in cut flowers. The main bioactive gibberellin (GA) controlling scape height in A. praecox is GA4. A GA biosynthesis gene, ApGA20ox1, controls GA4 biosynthesis in this species. Here, we report the development of an A. praecox dwarf phenotype, which was generated by targeted suppression of the biosynthetic pathway for GAs. We utilized Agrobacterium-mediated genetic transformation using embryogenic callus with RNA interference (RNAi) to selectively reduce the expression of the ApGA20ox1 gene. Interestingly, suppression of ApGA20ox1 resulted in poor somatic embryogenesis potential in A. praecox, which was accompanied by a higher accumulation of hydrogen peroxide and poorer catalase activity. Subsequently, the RNAi plantlets showed lower GA levels, leading to inhibition of cell longitudinal size in leaves, which reduced plant height. Furthermore, the dwarf (RNAi) plantlets accumulated higher levels of saccharides and plant hormones such as indole acetic acid (IAA) and abscisic acid (ABA) than the wild type accumulated. However, the levels of endogenous hormones such as GAs, brassinosteroids (BRs), cytokinins (CTKs), which respond to cell proliferation and cell expansion, were significantly decreased. Isobaric tags for relative and absolute quantification (iTRAQ) proteomics revealed that differentially accumulated proteins (DAPs) were enriched differently between control and RNAi plantlets in the categories of cell wall, hormone metabolism, carbohydrate metabolism and stress response. This study provides new insights into the role of GAs in the process of somatic embryogenesis and plant height control in A. praecox.

Synthetic apomixis with normal hybrid rice seed production

Synthetic apomixis with normal hybrid rice seed production

In vitro propagation and Agrobacterium-mediated genetic transformation of caraway (Carum carvi L.)

In vitro propagation and Agrobacterium-mediated genetic transformation of caraway (Carum carvi L.)

Agrobacterium-mediated Genetic Transformation of Cotton and Regeneration via Somatic Embryogenesis.

Cotton is a significant industrial crop, playing an essential role in the global economy that suffers several setbacks due to biotic and abiotic adversities. Despite such problems, biotechnological advances in cotton are limited because of genetic transformation and regeneration limitations. Here, we present a detailed protocol optimized based on previously published papers, along with our modifications. These involve changes in Agrobacterium concentration, co-cultivation time and temperature, hormones used for regeneration, media manipulation for embryogenic callus production, and efficient rescue of deformed embryos. Further, this protocol has been used in genetic studies on biotic and abiotic stress in cotton. This protocol assures a reproducible stable transgenic cotton development procedure via somatic embryogenesis that can be used by researchers worldwide. This protocol was validated in: Nat Biotechnol (2016), DOI: 10.1038/nbt.3665.

Selection of the optimal concentration of indole-3-butyric acid (IBA) in the nutrient medium for effective micropropagation of Populus × berolinensis

The study describes the features of the effective micropropagation of Populus berolinensis using indolyl-3-butyric acid (IBA). Internode sections without axillary buds are the most frequently used type of explant for Agrobacterium-mediated genetic transformation of woody plants. The application of growth regulators, such as indolyl-3-butyric acid (IBA), allows to increase the length of the internodes and thereby solve the problem of the availability of a sufficient amount of material for genetic transformation. Our results showed that 0.15 mg L-1 of IBA in the nutrient medium is the most suitable concentration for the effective micropropagation of Berlin poplar. Shoots rooted or regenerated from axillary buds in case of replanting of the rooted stump at medium supplemented with IBA in mentioned concentration, have expanded internodes in comparison with the control group of plants and may be successfully used as a good source of internode sections in Agrobacterium-mediated plant transformation.

Ectopic expression of a grapevine alkaline α-galactosidase seed imbibition protein VvSIP enhanced salinity tolerance in transgenic tobacco plants.

Alpha-galactosidase seed imbibition protein (VvSIP) isolated from Vitis vinifera is up-regulated upon salt stress and mediates osmotic stress responses in a tolerant grapevine cultivar. So far, little is known about the putative role of this stress-responsive gene. In the present study, VvSIP function was investigated in model tobacco plants via Agrobacterium-mediated genetic transformation. Our results showed that overexpression of VvSIP exhibited increased tolerance to salinity at germination and late vegetative stage in transgenic Nicotiana benthamiana compared to the nontransgenic plants based on the measurement of the germination rate and biomass production. High salt concentrations of 200 and 400mM NaCl in greenhouse-grown pot assay resulted in better relative water content, higher leaf osmotic potential, and leaf water potential in transgenic lines when compared to the wild-type (WT) plants. These physiological changes attributed to efficient osmotic adjustment improved plant performance and tolerance to salinity compared to the WT. Moreover, the VvSIP-expressing lines SIP1 and SIP2 showed elevated amounts of chlorophyll with lower malondialdehyde content indicating a reduced lipid peroxidation required to maintain membrane stability. When subjected to high salinity conditions, the transgenic tobacco VvSIP exhibited higher soluble sugar content, which may suggest an enhancement of the carbohydrate metabolism. Our findings indicate that the VvSIP is involved in plant salt tolerance by functioning as a positive regulator of osmotic adjustment and sugar metabolism, both of which are responsible for stress mitigation. Such a candidate gene is highly suitable to alleviate environmental stresses and thus could be a promising candidate for crop improvement.

Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation.

Paeonia lactiflora Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue culture techniques and genetic transformation systems has become a master obstacle for P. lactiflora research. The purpose of the present study focuses on obtaining an efficient and stable genetic transformation method using callus as the receptor and exploring an efficient protocol for callus induction and proliferation associated with P. lactiflora. Callus induction and proliferation were performed using MS medium with various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (6-BA) and thidiazuron (TDZ). The sensitivity of callus to kanamycin and cefotaxime was determined. Several parameters such as Agrobacterium cell density, infection time and co-culture duration were studied to optimize transformation efficiency. Agrobacterium strains EHA105 and pBI121 binary vector harboring the β-glucuronidase (GUS) gene were used for transformation. Expression of the GUS reporter gene was detected by GUS assay, polymerase chain reaction (PCR) and Quantitative Real-time PCR (RT-qPCR). The MS medium containing 1.0 mg·L-1 NAA, 0.5 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was optimal for callus induction and MS medium containing 0.5 mg·L-1 NAA, 1.0 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was the best for callus proliferation. The concentrations of kanamycin and cefotaxime used for screening positive callus were 125 mg·L-1 and 200 mg·L-1, respectively. Among various combinations analyzed, the best transformation result was obtained via the 25 min of infection of Agrobacterium at 0.6 OD600 and 3 d of co-culture. Overall, this study provided technical support and theoretical guidance for improving the callus induction and proliferation efficiency and the study of gene function in P. lactiflora.

Agrobacterium-mediated Genetic Transformation of Cassava.

The storage root crop cassava (Manihot esculenta Crantz) is predicted to remain central to future food and economic security for smallholder farming households and agricultural output in the tropics. Genetic improvement of cassava is required to meet changing farmer and consumer needs, evolving pests and diseases, and challenges presented by climate change. Transgenic and genome editing technologies offer significant potential for introducing desired traits into farmer-preferred varieties and breeding lines, and for studying the biology of this under-investigated crop species. A bottleneck in implementing genetic modification in this species has been access to robust methods for transformation of cassava cultivars and landraces. In this article, we provide a detailed protocol for Agrobacterium-mediated transformation of cassava and regeneration of genetically modified plants. Basic Protocol 1 describes how to establish and micropropagate in vitro cassava plantlets, and Alternate Protocol 1 details how to establish in vitro cultures from field or greenhouse cuttings. Basic Protocol 2 describes all steps necessary for genetic transformation in the model variety 60444, and Alternate Protocol 2 provides details for modifying this method for use with other cultivars. Finally, Basic Protocol 3 describes how to establish plants produced via Basic Protocol 2 and Alternate Protocol 2 in soil in a greenhouse. These methods have proven applications across more than a dozen genotypes and are capable of producing transgenic and gene-edited plants for experimental purposes, for testing under greenhouse and field conditions, and for development of plants suitable for subsequent regulatory approval and product deployment. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment and propagation of in vitro cassava plantlets Alternate Protocol 1: Establishment of in vitro plants from field or greenhouse plants Basic Protocol 2: Genetic transformation of cassava variety 60444 Alternate Protocol 2: Genetic transformation of additional cultivars Basic Protocol 3: Establishment and growth of plants in the greenhouse.

A novel elicitor protein phosphopentomutase from Bacillus velezensis LJ02 enhances tomato resistance to Botrytis cinerea.

The loss of tomatoes caused by Botrytis cinerea (B. cinerea) is one of the crucial issues restricting the tomato yield. This study screened the elicitor protein phosphopentomutase from Bacillus velezensis LJ02 (BvEP) which improves the tomato resistance to B. cinerea. Phosphatemutase was reported to play a crucial role in the nucleoside synthesis of various microorganisms. However, there is no report on improving plant resistance by phosphopentomutase, and the related signaling pathway in the immune response has not been elucidated. High purity recombinant BvEP protein have no direct inhibitory effect on B. cinerea in vitro,and but induce the hypersensitivity response (HR) in Nicotiana tabacum. Tomato leaves overexpressing BvEP were found to be significantly more resistant to B. cinerea by Agrobacterium-mediated genetic transformation. Several defense genes, including WRKY28 and PTI5 of PAMP-triggered immunity (PTI), UDP and UDP1 of effector-triggered immunity (ETI), Hin1 and HSR203J of HR, PR1a of systemic acquired resistance (SAR) and the SAR related gene NPR1 were all up-regulated in transgenic tomato leaves overexpressing BvEP. In addition, it was found that transient overexpression of BvEP reduced the rotting rate and lesion diameter of tomato fruits caused by B. cinerea, and increased the expression of PTI, ETI, SAR-related genes, ROS content, SOD and POD activities in tomato fruits, while there was no significant effect on the weight loss and TSS, TA and Vc contents of tomato fruits. This study provides new insights into innovative breeding of tomato disease resistance and has great significance for loss reduction and income enhancement in the tomato industry.

VyCAS, a calcium sensing receptor from the Chinese wild Vitis yeshanensis, confers drought tolerance in transgenic V. vinifera

The calcium sensing receptor (CAS) plays an important role in plant growth and development and resistance to abiotic stresses. In this study, a VyCAS gene was cloned from the accession 'Yanshan-1′, an extremely drought-tolerant Chinese wild Vitis yeshanensis. To gain insight into the roles of VyCAS, we overexpressed it in the V. vinifera cultivar 'Thompson Seedless' by Agrobacterium-mediated genetic transformation and obtained three transgenic grape lines. Compared with the wild type (WT), the transgenic lines were less damaged by drought, and had lower transpiration rate, stomatal conductance, malondialdehyde (MDA) content, electrolyte leakage, H2O2 and O2− accumulations but higher leaf relative water content, photosynthetic water use efficiency, CAT, POD and SOD activities. Meanwhile, after drought, the transcript levels of some drought-related genes (RD22, DREB2A, NAC72, SRK2A, CBL1, and CYP707A2) in the transgenic lines were much higher than in the WT. Taken together, these results show that VyCAS can improve the drought tolerance of transgenic grapevine. Further experiments confirmed that Arabidopsis with a CAS mutation was more sensitive to drought than the WT. In addition, VyMYB59, a negative regulator of the promoter of VyCAS gene, was screened by the yeast one-hybrid, luciferase and GUS activity assays. This will help us to further explore the drought tolerance mechanism of VyCAS and provide a new thought for the following research.

VyUSPA3, a universal stress protein from the Chinese wild grape Vitis yeshanensis, confers drought tolerance to transgenic V. vinifera.

VyUSPA3 from the Chinese wild grape Vitis yeshanensis interacts with ERF105, PUB24 and NF-YB3, and overexpression of the VyUSPA3 gene in V. vinifera cv. 'Thompson Seedless' confers drought tolerance. Drought is a major abiotic stress factor that seriously affects the growth and yield of grapevine. Although many drought-related genes have been identified in Arabidopsis and other plants, the functions of only a few of their counterparts have been revealed in grape. Here, a universal stress protein (USP) A from the Chinese wild grape Vitis yeshanensis, VyUSPA3, was identified and its function was subsequently characterized by overexpressing or silencing the VyUSPA3 gene in V. vinifera cv. 'Thompson Seedless' via Agrobacterium-mediated genetic transformation. After 21 d of the drought treatment, most leaves of the untransformed (UT) 'Thompson Seedless' lines wilted, yet UT lines were less damaged compared to the RNAi-VyUSPA3 lines, nonetheless, the OE-VyUSPA3 lines were mostly unaffected. Meanwhile, OE-VyUSPA3 lines showed smaller stomatal aperture, more developed roots, higher leaf relative water content, proline content, and antioxidant enzyme activities, as well as lower malondialdehyde, H2O2 and O2•- accumulation than UT lines, but this response pattern was reversed in the RNAi-VyUSPA3 lines. Besides, the transcript levels of four drought-related genes (RD22, RD29B, DREB2A, and NCED1) in OE-VyUSPA3 lines were greater than those in the RNAi-VyUSPA3 and UT lines. In addition, a yeast two-hybrid assay and a bimolecular fluorescence complementation assay confirmed that VyUSPA3 interacted with ERF105, PUB24, and NF-YB3, respectively. This study revealed that VyUSPA3 improved drought tolerance in transgenic grapevines possibly through interaction with the hormone signaling, ubiquitination system, ethylene-responsive element binding factor and nuclear factors.

Improved and reliable plant regeneration and Agrobacterium-mediated genetic transformation in soybean (Glycine max L.)

Improved and reliable plant regeneration and Agrobacterium-mediated genetic transformation in soybean (Glycine max L.)

CRISPR/Cas9-mediated genome editing of RsGL1a and RsGL1b in radish (Raphanus sativus L.).

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool widely used for genome editing in various organisms, including plants. It introduces and facilitates the study of rare genetic mutations in a short time and is a potent tool to assist in plant molecular breeding. Radish (Raphanus sativus L.) is an important Brassicaceae vegetable cultivated and consumed worldwide. However, the application of the CRISPR/Cas9 system is limited by the absence of an efficient transformation system in radish. This study aimed to establish a CRISPR/Cas9 system in radish employing the Agrobacterium-mediated genetic transformation system reported recently. For this purpose, we performed genome editing using the CRISPR/Cas9 system targeting the GLABRA1 (GL1) orthologs, RsGL1a and RsGL1b, that induces leaf trichome formation in radish. A Cas9/single guide RNA (sgRNA) vector with a common sgRNA corresponding to RsGL1a and RsGL1b was transferred. A total of eight T0 plants were analyzed, of which six (editing efficiency 75%) had a mutated RsGL1a, five (62.5%) had a mutated RsGL1b, and five showed mutations in both RsGL1a and RsGL1b. Most mutations in T0 plants were short (<3 bp) deletions or insertions, causing frameshift mutations that might produce non-functional proteins. Chimeric mutations were detected in several T0 generation plants. In the T1 generation, the hairless phenotype was observed only in plants with knockout mutations in both RsGL1a and RsGL1b. The majority of mutant alleles in T0 plants, with the exception of the chimeric mutant plants detected, were stably inherited in the T1 generation. In conclusion, we successfully knocked out RsGL1a and RsGL1b using the CRISPR/Cas9 system and demonstrated that both RsGL1a and RsGL1b independently contribute to the induction of leaf trichome formation in radish. In this study, genome-edited plants without T-DNA, which are useful as breeding material, were obtained. The findings prove the feasibility of genome editing in radish using a CRISPR/Cas9 system that could accelerate its molecular breeding to improve agronomically desirable traits.

Agrobacterium-mediated genetic transformation of the most widely cultivated superior clone Eucalyptus urophylla × E. grandis DH32-29 in Southern China.

Eucalyptus, as an economically important species for wood and paper industries, remains a challenge to genetic improvement by transgenic technology owing to the deficiency of a highly efficient and stable genetic transformation system, especially in cultivated superior clones. Eucalyptus urophylla × E. grandis clone DH32-29 is most widely planted in southern China, but it is relatively recalcitrant to adventitious bud regeneration, which blocks the establishment of a genetic transformation system. Here, an efficient adventitious bud regeneration and transformation system of Eucalyptus was established using E. urophylla × E. grandis DH32-29 as material. The in vitro leaves from microshoots that were subcultured for 20-25 days were immersed into liquid Woody Plant Medium supplemented with 0.02 mg·L-1 α-naphthaleneacetic acid (NAA) and 0.24 mg·L-1 forchlorfenuron [callus-inducing medium (CIM)]. After 15 days, explants were transferred to a medium containing 0.10 mg·L-1 NAA and 0.50 mg·L-1 6-benzyladenine (shoot-inducing medium, SIM) for adventitious bud induction. The highest regeneration efficiency of adventitious buds was 76.5%. Moreover, an Agrobacterium tumefaciens-mediated genetic transformation system was optimized. The leaves were precultured for 7 days and infected for 30 min with A. tumefaciens strain EHA105 grown to a bacterial density of 0.3 (OD600). After 72 h of cocultivation in the dark, leaves were transferred to CIM supplemented with 100 mg·L-1 cefotaxime (Cef), 100 mg·L-1 timentin, and 15 mg·L-1 kanamycin (Kan) for 15 days to induce calluses. Then, the explants were transferred to SIM supplemented with the same concentration of antibiotics, and the fresh medium was replaced every 15 days until resistant adventitious buds appeared. After inducing roots in root-inducing medium supplemented with 200 mg·L-1 Cef and 75 mg·L-1 Kan, completely transgenic plants were obtained. Using the aforementioned method, the transformation frequency can reach 1.9%. This provides a powerful approach for genetic improvement of E. urophylla × E. grandis DH32-29 and gene function analysis in Eucalyptus.

Establishment of a genetic transformation system for Codonopsis pilosula callus.

Codonopsis pilosula, a traditional Chinese medicinal and edible plant, contains several bioactive components. However, the biosynthetic mechanism is unclear because of the difficulties associated with functional gene analysis. Therefore, it is important to establish an efficient genetic transformation system for gene function analysis. In this study, we established a highly efficient Agrobacterium-mediated callus genetic transformation system for C. pilosula using stems as explants. After being pre-cultured for 3 days, the explants were infected with Agrobacterium tumefaciens strain GV3101 harboring pCAMBIA1381-35S::GUS at an OD600 value of 0.3 for 15 min, followed by co-cultivation on MS induction medium for 1 day and delayed cultivation on medium supplemented with 250 mg l-1 cefotaxime sodium for 12 days. The transformed calli were selected on screening medium supplemented with 250 mg l-1 cefotaxime sodium and 2.0 mg l-1 hygromycin and further confirmed by PCR amplification of the GUS gene and histochemical GUS assay. Based on the optimal protocol, the induction and transformation efficiency of calli reached a maximum of 91.07%. The establishment of a genetic transformation system for C. pilosula calli lays the foundation for the functional analysis of genes related to bioactive components through genetic engineering technology.

Virus-induced gene silencing shows that LATE FLOWERING plays a role in promoting flower development in soybean

Virus-induced gene silencing (VIGS) is a useful tool to investigate the role of genes particularly in hard-to-transform plant species via the Agrobacterium-mediated genetic transformation process. Soybean is one of the most important crops for the food and protein source, but its low transformation efficiency makes it hard to identify the functions of genes of interest. Here, we adopted tobacco ringspot virus (TRSV)-based VIGS systems and examined the role of a LATE FLOWERING (GmLATE) gene in soybean. Because TRSV induces symptoms affecting leaf senescence and development, we screened soybean genotypes and selected a genotype, named Aram, which shows resistance to TRSV symptoms while is susceptible to TRSV-induced gene silencing. The TRSV-based silencing of GmLATE in soybean showed suppressed flower development with decreased expression of genes related to flowering. These results suggest that GmLATE plays a role in promoting flower development in soybean, which is different from its role as a floral repressor in Arabidopsis. Our results show the novel function of GmLATE and demonstrate that TRSV-based VIGS system can be used as a tool to study genes involved in flowering in soybean.