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An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

In April 2019, hydroponically cultivated cucumber plants with characteristic symptoms of crazy root disease were found in two different commercial production cucumber greenhouses in Poland. Due to excessive and inappropriate root growth, this disease led to a reduction in yield and deterioration of the general conditions of infected plants. Bacteria isolated from the roots were subjected to a morphological evaluation, as well as molecular, biochemical and pathogenicity tests. To identify the bacteria causing the disease, Agrobacterium-like colonies were subjected to PCR with primers complementary to the pathogenicity-related genes located on the crazy root-inducing plasmid (Ri-plasmid): the virD2A + virD2E primers complementary to the virD2 gene and the rolBF + rolBR primers complementary to the rolB gene. The pathogenicity of the isolated strains was studied in sunflowers and cucumbers. Twelve strains positive for the Ri plasmid, as determined by PCR, and pathogenic to sunflowers were identified based on sequence analysis of the 16S rRNA and recA genes. One strain was classified as belonging to the genus Pararhizobium, three to Rhizobium, and eight to Agrobacterium biovar 1, with the highest similarity to genomospecies G3. The results of the analyses suggest that these strains may belong to a new, thus far, undescribed species. To confirm this hypothesis, further phylogenetic studies are required.

Bacteria from the rhizobia group have been previously identified as grass-endophytes in grassland with cropping history of legumes. In this paper we aimed to demonstrate that strains of the Rhizobium genus, isolated from the rhizosphere of a rice Cuban cultivar subjected to intensive monoculture management, behave as endophytes and promote its growth. Eleven bacteria previously isolated from the rhizosphere of rice plants, were screened for their plant growth promoting traits. We found that some of them were able to solubilize inorganic phosphate compounds, produce siderophores and biofilm, have exocellulase and protease activity and were able to inhibit the phytopathogen Pyricularia oryzae growth. The phylogenetic analysis using partial sequences of 16S rRNA gene showed that bacteria isolated from rice plant of cultivar INCA LP-5 belonged to Rhizobium, Agrobacterium and Pseudomonas genera. Two strains analyzed, Rhizobium sp. Rpr2 and Rdp16, increased the height and biomass of rice plants, 70 days post-inoculation in greenhouse conditions. According to the quantification of Rhizobium sp. strain Rpd16 in surface-disinfected tissues of root and sheath, together with the analysis of confocal microscopy images, we can conclude that the Rdp16 strain is a rice endophyte of the cultivar INCA LP-5. This investigation is the first evidence of endophytic rhizobia able to promote the rice growth of a Cuban cultivar subjected to intensive monoculture, without rotation with legume plants and without a previous history of rhizobia inoculation.

Regeneration of in vitro multiple shoots was achieved through organogenesis on MS supplemented with 2.0 mg/l BAP and 0.5 mg/l Kn from cotyledonary leaf explants of two local varieties of eggplant (Solanum melongena L.). Elongation of regenerated shoots was obtained on growth regulator free MS. In vitro root induction from excised regenerated shoots was less effective on MS with or without plant growth regulators. On the other hand regenerated shoots treated with 10 mM IBA for 5 min were found to be effective for ex vitro rooting in sterilized soil. Following sufficient development of roots, the ex vitro rooted plantlets were acclimatized in growth room condition, and were transferred to the field having 100% survival rate. The regeneration system developed was utilized for Agrobacterium-mediated genetic transformation using Agrobacterium tumefaciens strain LBA4404/pBI121 containing GUS and nptII genes. Adequate transformation response was obtained from cotyledonary leaf segments with bacterial suspension having an optical density of 0.50 at 600 nm with 30 min incubation followed by co-cultivation period of 72 hrs in Nayantara (BARI Begun-5) variety of eggplant. Selection of transformed shoots was carried out on MS supplemented with 2.0 mg/l BAP, 0.5 mg/l Kn, 300 mg/l carbenicillin and 100 mg/l kanamycin. Stable integration of GUS and nptII genes in Nayantara were confirmed through PCR analysis using the genomic DNA isolated from transformed shoots. Plant Tissue Cult. & Biotech. 31(1): 97-108, 2021 (June)

Agrobacterium salinitolerans DG3-1 is an endophytic bacterium isolated from cotton root tissue. Our previous work has shown that it can inhibit the growth of Fusarium and Verticillium wilt pathogens as well as increase the chlorophyll content of cotton leaves. Here, we reported the complete genome sequence of strain DG3-1, which was analyzed by sequence reads generated from Nanopore PromethION and Illumina NovaSeq PE150 platforms. This genome sequence could be used to clarify the possible mechanism of DG3-1 at the gene level. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Sugarcane (Saccharum officinarum L.) is the most important food and energy crop worldwide. In the present study, an efficient Agrobacterium mediated transformation and regeneration system for sugarcane cultivar SPF-234 was established. Agrobacterium tumefaciens strains EHA101and LBA4404 using vector pIG121 Hm, having GUS, HPTII and NPTII genes were used. Polymerase chain reaction (PCR) and histochemical assays confirmed the GUS gene expression. A 620 bp fragment from GUS positive plants was amplified. The GUS expressing putative transformants were 35% of the total plants formed under 30 minute immersion time and 72 hr of incubation period. The co-cultivation media having 60 µM acetosyringone produced 66% GUS expressing plants for LBA4404 and 58% for EHA101. The maximum average number of directly produced shoot (59.5%) from leaf explant was in M6 media having 1.00 mg/l 6-Benzylaminopurine (BAP) and 2.5 mg/l Naphthaleneacetic acid (NAA). A significant decrease (17%) was observed when auxin (NAA) concentration was increased to 4.0 mg/l. The best response of shoot elongation was observed in SE4 media having equal concentration (2.00 mg/l) of both kinetin and BAP. Increased concentrations of kinetin significantly decreased shoot elongation of the subject cultivar. Agrobacterium strain LBA4404 performed better for genetic transformation of the said sugarcane cultivar.This quick and less expensive transformation and direct regeneration system could be exploited for sugarcane on commercial scale in general, and for this elite cultivar in particular.

“Hairy” roots obtained through genetic transformation of plants by Agrobacterium rhizogenes, a soil phytopathogen, are valuable producers of important secondary metabolites possessing medicinal properties as well as a useful model system for studying plant responses to impacts of unfriendly environmental conditions. This study compares a postponed response of Althaea officinalis L. “hairy” roots to the impacts of short-term cold- and high-temperature stress factors. The results obtained by the study have shown that “hairy” roots from different A. officinalis lines (individual transformational events) are characterized by different sensitivity to short-term temperature stress impacts, regardless of the transformation vectors or the presence of the human interferon(ifn)-α2b gene. High temperature caused a significant level of growth inhibition in roots of all lines, except those with the highest flavonoid content under the control conditions. On the other hand, a short-term cultivation of “hairy” roots at a low temperature did not cause growth suppression. In parallel with growth inhibition caused by a temperature increase, the activation of flavonoid synthesis, which was probably a response of plants to high temperature as a stress factor, was observed. The study has shown a strong (R2 = 0.78) linear dependence between the antioxidant activity of extracts from “hairy” roots and their flavonoid content. Thus, it is obvious that flavonoids participate in the process of response and adaptation of roots to impacts of high-temperature stress.

Sahendian savory (Satureja sahendica Bornm) is a medicinal plant species which produces valuable secondary metabolites including thymol, p-cymene and γ-terpinene. In this study, optimization of hairy roots culture in S. sahendica using Agrobacterium rhizogenes was efficiently established. High frequency of genetic transformation was obtained using node and internode explants and three bacterial strains (ATCC 15834, A4, and LBA 9402). However, leaf explants showed no susceptibility to induce hairy roots with the A. rhizogenes strains. The node explants and LBA 9402 strain were the best combination for root induction. Infection time of 20 min caused greater transformation occurrence compared to 10 and 30 min, respectively. Node explants infected with LBA 9402 were found to produce more transformants upon co-cultivation for 2 days. The maximum induction of hairy roots was obtained from bacterial infection at a density of OD600 = 0.8. Polymerase chain reaction by gene-specific primers from rol B gene confirmed the positive transformation events. S. sahendica efficiently provided and produced a large number of hairy roots which could be used for the production of secondary metabolites or other molecular and biotechnological applications. Production of thymol an important secondary metabolite of S. sahendica from hairy root cultures was confirmed by TLC and GC analysis. Elicitor-treated hairy root cultures with methyl jasmonate and ammonium nitrate produced more amount of thymol compared to untreated roots. These results demonstrate that secondary metabolite production such as thymol in hairy root culture of Sahendian savory could be used to increase low secondary metabolite production in this valuable medicinal plant.

Species of the genus Aspergillus have a variety of effects on humans and have been considered industrial cell factories due to their prominent ability for manufacturing several products such as heterologous proteins, secondary metabolites, and organic acids. Scientists are trying to improve fungal strains and re-design metabolic processes through advanced genetic manipulation techniques and gene delivery systems to enhance their industrial efficiency and utility. In this review, we describe the current status of the genetic manipulation techniques and transformation methods for species of the genus Aspergillus. The host strains, selective markers, and experimental materials required for the genetic manipulation and fungal transformation are described in detail. Furthermore, the advantages and disadvantages of these techniques are described.

Application of plant hormones is an effective strategy to further improve the accumulation of microalgae biomass. In this study, the IAA (indole-3-acetic acid) production capacity of symbiotic bacteria isolated from the culture system of Ochromonas and Chlorella was detected and compared, with the bacterial community structure revealed. The results demonstrated that Algoriphagus, Porphyrobacter, Roseococcus, and Rhizobium were the dominant bacterial genera both in Ochromonas and Chlorella system, distributed in Proteobacteria and Bacteroidetes. Sixteen of 40 strains classified into 15 genera isolated from the two genera of microalgae could produce IAA, with Agrobacterium and Rhizobium as the main representatives with highest IAA production level (22.53–34.18 mg/L in 30 h). And through determination of changes of chlorophyll content in the co-culture system of microalgae and the supernatant of IAA high-yielding bacteria suspension from medium with or without tryptophan (synthetic precursor of IAA), Rhizobium (C-3) and Agrobacterium (O-16, C-2) were proved to promote the growth of Ochromonas and Chlorella through secreting IAA. The biomass yield of Ochromonas and Chlorella could be respectively increased by 78.98% and 72.73% through the co-culture with Agrobacterium (C-2), by 68.15% and 69.93% for Rhizobium (C-3) and by 80.89% and 65.73% for Agrobacterium (O-16). Moreover, compared with the application of exogenous IAA to improve the microalgae biomass, it is more efficient and economical to construct algae bacteria symbiosis system using IAA secretory bacteria commonly existed in microalgae culture system. In addition, the growth promoting effect of IAA secretory bacteria on microalgae was different among the species of algae.

Endophytes colonizing plant tissue play an essential role in plant growth, development, stress tolerance and plant protection from soil-borne diseases. In this study, we report the diversity of cultivable endophytic bacteria associated with marigold (Calendula officinalis L.) by using 16S rRNA gene analysis and their plant beneficial properties. A total of 42 bacterial isolates were obtained from plant tissues of marigold. They belonged to the genera Pantoea, Enterobacter, Pseudomonas, Achromobacter, Xanthomonas, Rathayibacter, Agrobacterium, Pseudoxanthomonas, and Beijerinckia. Among the bacterial strains, P. kilonensis FRT12, and P. rhizosphaerae FST5 showed moderate or vigorous inhibition against three tested plant pathogenic fungi, F. culmorum, F. solani and R. solani. They also demonstrated the capability to produce hydrolytic enzymes and indole-3-acetic acid (IAA). Five out of 16 isolates significantly stimulated shoot and root growth of marigold in a pot experiment. The present study reveals that more than half of the bacterial isolates associated with marigold (C. officinalis L.) provided antifungal activity against one or more plant pathogenic fungi. Our findings suggest that medicinal plants with antimicrobial activity could be a source for selecting microbes with antagonistic activity against fungal plant pathogens or with plant growth stimulating potential. These isolates might be considered as promising candidates for the improvement of plant health.

Elsinoë ampelina is an ascomycetous fungus that causes grape anthracnose, a potentially devastating disease worldwide. In this study, a dual RNA-seq analysis was used to simultaneously monitor the fungal genes related to pathogenesis and grape genes related to defense during the interaction at 2, 3, 4, and 5 days postinoculation. Consistent with their potential roles in pathogenicity, genes for carbohydrate-active enzymes, secondary metabolite synthesis, pathogen-host interaction, and those encoding secreted proteins are upregulated during infection. Based on Agrobacterium tumefaciens–mediated transient assays in Nicotiana benthamiana, we further showed that eight and nine candidate effectors, respectively, suppressed BAX- and INF1-mediated programmed cell death. The host response was characterized by the induction of multiple defense systems against E. ampelina, including synthesis of phenylpropanoids, stilbenes, and terpenoid biosynthesis, cell-wall modifications, regulation by phytohormones, and expression of defense-related genes. Together, these findings offer new insights into molecular mechanisms underlying the grape–E. ampelina interaction. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Tripogon loliiformis, an Australian native resurrection grass having an abundant gene pool for combating desiccation, can be the putative model system for functional characterization of stress tolerance genes due to its diploid genome and being a monocotyledonous plant and member of the grass family (Poaceae), like many important cereal crops. For developing callus mediated regeneration from mature grains of Tripogon, Murashige and Skoog medium containing growth regulator,2, 4-dichlorophenoxyacetic acid (2,4-D) at 1.0 mgL-1 was the optimum concentration for induction and proliferation of healthy cream calli. Successful regeneration of shoots from callus clumps in MS medium supplemented with 2.0mgL-1 6-benzylaminopurine (BAP) and 0.5mgL-1 α-naphthalene acetic acid (NAA) was obtained from 2 consecutive rounds subculturing of the calli at 3 weeks interval. In addition, rooting needed another 2 rounds within the same media with 2.0mgL-1 BAP but with 0.25mgL-1 NAA. The transient expression of UidA gene at 3 days after Tripogon callus transformation, performed with Agrobacterium tumefaciens strains AGL1 and LBA4404 following rice and Brachypodium distachyon transformation protocols, indicates successful Agrobacterium infection and gene delivery in calli. A stable transformation system for Tripogon loliiformis can be developed near future following the protocols in this study.

Diazotrophic endophytic bacteria contribute to the plant growth using various plant growth promoting traits as well as by inducing the plant defence mechanism upon pathogen attack. Aim of the present study was to isolate true diazotrophic endophytic bacteria from the total diazotrophic bacterial community of wheat and pearl-millet plants. Further, these isolates were evaluated for their potential of plant growth promotion by in vitro and in vivo experiments. We isolated total three diazotrophic endophytic bacteria based on their growth after repetitive subculturing on nitrogen free medium. Molecular characterization of all three isolates showed affiliation to the genera Agrobacterium, Pseudomonas and Brevundimonas. In vitro analysis of plant growth promoting traits showed positive result for indole acetic acid (IAA) production, cellulase and protease activity. The IAA production is found between the ranges of 98 to 113 µg ml-1. All the endophytic bacterial isolates showed the PCR amplification of NifH gene and ammonia production. Diazotrophic endophytic bacterial isolates (PR1, JB3 and JB6) were tested for their capacity to promote the plant growth using pot experiment. All the diazotrophic endophytic bacteria differ in their plant growth promotion ability as analyzed by various plant growth parameter compared to the untreated control plants. In Pot experiment, PR1 and JB6 showed a significant difference in dry weight of root, whereas JB3 and JB6 showed the significant differences in shoot length and total nitrogen content (p≤0.05) compared to the untreated plants.

This study evaluated the potential of dwarf morning-glory Convolvulus tricolor (Convolvulaceae) plants and their hairy roots induced by Agrobacterium rhizogenes for rhizoextraction of heavy metals ions from the liquid medium under aseptic growth conditions. Both the young C. tricolor plants and the generated hairy root culture efficiently removed Cr6+, Ni2+, and Pb2+ ions from the liquid cultivation medium. As determined by atomic absorption spectroscopy, the hairy roots demonstrated a high level of heavy metal ions accumulation (μg/g dry weight): 3942 ± 210 of chromium, 1529 ± 312 of nickel, and 2613 ± 373 of lead. These data show that the hairy roots of morning glory might be of interest for some phytoremediation strategies due to their high bioaccumulation abilities. The comparison of bioaccumulation potential of established hairy roots and young C. tricolor plants give grounds to suppose that roots of C. tricolor play an active role in the absorption of Cr6+, Ni2+, and Pb2+ from liquid media, whereas the aboveground part rather serves as a storage for the accumulated metal ions.

Hairy root cultures are often used to produce valuable metabolites. They are grown on sucrose-rich medium, which is highly susceptible to contamination. Trapa natans is a unique plant with photosynthesizing roots. It is a promising object to obtain photoautotrophic hairy root culture. Protocols for transformation of this species are yet unknown. We report that hairy roots can be induced in aquarium and in vitro cultures of T. natans by agrobacterium-mediated and biolistic transformation. 64 roots were induced by Agrobacterium rhizogenes strain 15834, two roots were obtained using strain K599. Strain A4 was not effective. Biolistics with either amplicons of rol genes and 1301 pCAMBIA plasmid carrying rol genes resulted in the formation of six roots. All these roots contained chloroplasts. This achievement opens a prospect for genetic transformation of T. natans and use of its green photosynthesizing hairy root cultures in production of bioactive substances and in phytoremediation. Protocols for the Agrobacterium-mediated and biolistic transformation of Trapa natans to induce hairy roots have been developed for the first time. All induced roots contained chloroplasts.

A stable transformation protocol using Agrobacterium tumefaciens strain EHA 52 harbouring the recombinant plasmid pBI121-CycD2 for anther derived embryos of Eriobotrya japonica (Thunb.) Lindl. cv. ...

Background: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plant-derived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. Results: Pinellia ternata leaf agglutinin gene ( pta ), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium -mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP-9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. Conclusions: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloem-specific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

Transgenic walnuts (Juglans regia L.) were produced in the presence of the Agrobacterium strain LBA4404 and the plasmid pBI121. This plasmid contains a BADH gene driven by a CaMV 35S promoter. It f...

Aim. Optimization of conditions for genetic transformation of new perspective winter wheat genotypes. Methods. Agrobacterium-mediated transformation in culture in vitro. Results. The influence of the optical density of cells of the agrobacterial suspension, the concentration of the antibiotic cefotaxime, the duration of coculture on the frequency of obtaining kanamycin-resistant regenerants of new winter wheat genotypes by genetic transformation of callus cultures were investigated using LBA4404 and AGL0 strains. It is shown that depending on the strain the most optimal is the concentration of agrobacteria 0.2-03 OD, duration of coculture for 2-3 days and the use of cefotaxime at a concentration of 250-500 mg/L. Conclusions. The optimal parameters for conducting Agrobacterium-mediated transformation of callus cultures of new perspective winter wheat genotypes were selected. Keywords: Triticum aestivum, Agrobacterium-mediated transformation, callus cultures.

Genetic modification using Agrobacterium rhizogenes has been an important biotechnological tool for the improvement of secondary metabolite production in plant species over the past four decades. The tropical vine Pentalinon andrieuxii, a native plant from the Yucatan Peninsula, is known to produce the triterpene betulinic acid and the novel tri-nor-sesquiterpene urechitol A and is used in the Mayan traditional medicine to treat leishmaniasis-derived lesions. Agrobacterium rhizogenes strain ATCC 15834 was used to infect leaf and hypocotyl explants of P. andrieuxii to generate 14 transformed plant lines with increased production of the terpenoids betulinic acid and urechitol A. Insertion of the transgenes was confirmed through PCR amplification of the rolA, rolB, and rolC genes. Betulinic acid and urechitol A were detected and quantified using standardized high-performance liquid chromatography and gas chromatography analyses, respectively. A significant increase in the terpenoid content was observed in some of the transgenic plants with up to 11.37 and 3.35 times more betulinic acid and urechitol A, respectively, when compared with the wild-type control. These new transgenic lines are promising tools to further the studies and knowledge about the biosynthesis of terpenoids in P. andrieuxii, especially regarding the biosynthetic origin of the rare urechitols.

Snapdragon (Antirrhinum majus L.) is a popular ornamental and model plant species, and the recently released reference genome could greatly boost its utilization in fundamental research. However, the lack of an efficient genetic transformation system is still a major limiting factor for its full application in genetic and molecular studies. In this study, a simple method for quick regeneration and efficient Agrobacterium-mediated transformation of snapdragon was developed. Cotyledon petiole and hypocotyl explants derived from two-week-old seedlings were cultured on MS media supplemented with 2 mg/L zeatin (ZT), 0.2 mg/L 1-naphthaleneacetic acid (NAA), and 2 mg/L AgNO3, and adventitious shoots were regenerated through organogenesis with an average regeneration of 48.00% and 41.33%, respectively. By contrast, the regeneration frequency was only 22.67% for cotyledon petiole and 25.67% for hypocotyl explants in the absence of AgNO3. Moreover, the application of AgNO3 promoted indirect shoot organogenesis, while direct shoot organogenesis occurred in the absence of AgNO3 from both hypocotyl or cotyledon petiole explants. Agrobacterium-mediated genetic transformation systems were developed with this high-efficient regeneration system. The transformation efficiency has been improved from 0 to 1% through the direct shoot organogenesis to 3 to 4% via the indirect shoot organogenesis. This efficient regeneration and genetic transformation method could be important for future use of snapdragon as a model plant to address some fundamental questions which are hard to be solved by using other model plant species, and to accelerate the breeding process through CRISPR/Cas9 genome editing. Genetic transformation of snapdragon was significantly improved by applying AgNO3 to induce indirect organogenesis, which will substantially facilitate its fundamental research as well as molecular breeding.

The common ice plant, Mesembryanthemum crystallinum L. provides a useful model for the study of environmentally induced photosynthetic conversion and abiotic stresses tolerance. However, a procedure for the production of transgenic ice plant, which is essential for functional genomics, has not been fully established. Here we tested the factors on the transformation of cotyledonary nodes excised from the ice plant seedlings such as thidiazuron (TDZ), NaCl and phytosulfokine (PSK), a peptidyl plant growth factor using Agrobacterium tumefaciens strains EHA101 and EHA105 harboring binary vector plasmids pBI7EGFP and pCAMBIA1302, respectively. The established procedure is as follows: the explants (cotyledonary nodes) were co-cultivated with Agrobacterium for 3 days, and the explants were cultured in the medium with 0.5 mg l−1 kinetin and 100 mg l−1 carbenicillin for 72 h, and they were cultured in the medium with 0.5 mg l−1 kinetin and 100 nM PSKfor 4 weeks. Thidiazuron and NaCl enhanced the production of multiple adventitious shoot formation during regeneration but reduced the transformation efficiency due to the vitrification of adventitious shoots. PSK was effective in the production of healthy adventitious shoots. The transformation frequency at the stage of whole plants was 0.6% and 4.6% per inoculated cotyledonary nodes using the Agrobacterium strain EHA101 (pBI7EGFP) and EHA105 (pCAMBIA1302), respectively.

Phoebe bournei, a plant species endemic to China, is a precious timber tree and widely used in landscaping. This tree contains numerous secondary metabolites, underscoring its potential economic value. However, studies on this species, including molecular genetic research, remain limited. In this study, both a somatic embryogenesis (SE) technical system and Agrobacterium-mediated genetic transformation were successfully employed in P. bournei for the first time. The SE technical system was constructed using immature embryos as original material. The primary embryo and embryonic callus induction rates were 30.66% and 41.67%, respectively. The highest rate of embryonic callus proliferation was 3.84. The maximum maturity coefficient and germination rate were 53.44/g and 39%, respectively. Agrobacterium-mediated genetic transformation was performed using the SE technical system, and the highest transformation rate was 11.24%. The results presented here are the first to demonstrate an efficient approach to achieve numerous P. bournei plantlets, which serves as the basis for artificial cultivation and resource conservation. Furthermore, the genetic transformation platform constructed in this study will facilitate assessment of gene function and molecular regulation.

The family Liliaceae (Cronquist system) contains various important ornamental plants. We have been examining for about 20 years the establishment of plant regeneration and genetic transformation systems in liliaceous ornamental plants for their biotechnological breeding and elucidation of the molecular mechanisms determining ornamental traits. In this review, studies on in vitro plant regeneration in 7 genera and on Agrobacterium-mediated production of transgenic plants in 4 genera are described. Plant regeneration was achieved via callus cultures in Agapanthus, Hemerocallis, Hosta, Lilium, Muscari and Tricyrtis. Auxins (2,4-dichrolophenoxyacetic acid, α-naphthaleneacetic acid and/or picloram) were effective for inducing regenerable calli. Tulipa species and cultivars were very recalcitrant to callus induction and plant regeneration. Agrobacterium-mediated transformation was examined in Agapanthus, Lilium, Muscari and Tricyrtis, and transgenic plants were obtained in all genera by using regenerable calli as a target material for Agrobacterium inoculation, inoculation and co-cultivation with Agrobacterium in the presence of acetosyringone, and selection of transgenic tissues and plantlets on hygromycin-containing media. Among 4 genera, Tricyrtis has several advantages for transformation studies: higher transformation efficiency, relatively small plant size, ease of cultivation, and taking only 1 year from in vitro regeneration to flowering. We are now investigating the molecular mechanisms for determining plant form, flower color and flower form by using Tricyrtis spp. as liliaceous model plants.

Tall fescue (Festuca arundinacea Schreb.) is a major cool-season perennial grass grown for forage and turf. We have obtained transgenic tall fescue by Agrobacterium-mediated transformation to improve agronomically important traits. In our protocol, we use embryogenic calli derived from not only mature seeds but also shoot tips. Although tall fescue cultivars consist of various genotypes with different genetic variation, we can produce transgenic plants at any time with calli induced from shoot tips of in vitro-maintained responsive genotypes. When the hygromycin phosphotransferase gene is used as a selectable marker, transformants are selected by incubation with 100 mg l-1 hygromycin in both selection and regeneration media. Since tall fescue is an anemophilous species, the cultivation of transgenic plants poses the risk of transgenic pollen flow. Recently, it has been reported that genome-edited plants without the integration of foreign DNA fragments can be produced by an Agrobacterium-mediated transient gene expression system. We hope that our protocol will contribute to production of transgene-free genome-edited tall fescue.