Withania somnifera (L.) Dunal: Enhanced production of withanolides and phenolic acids from hairy root culture after application of elicitors.

Withania somnifera (L.) Dunal is an important indigenous medicinal plant with extensive pharmaceutical potential. The root is the main source of major bioactive compounds of this plant species including withanolides, withanine, phenolic acids, etc. Hairy root culture (HRC) is a crucial method for low-cost production of active compounds on a large scale. Four different Agrobacterium rhizogenes strains have been used for the hairy root induction. Maximum transformation efficiency (87.34 ± 2.13%) was achieved with A4 bacterial strain-mediated transformed culture. The genetic transformation was confirmed by using specific primers of seven different genes. Seven HR (Hairy root) lines were selected after screening 29 HR lines based on their fast growth rate and high accumulation of withanolides and phenolic acids content. Two biotic and three abiotic elicitors were applied to the elite root line to trigger more accumulation of withanolides and phenolic acids. While all the elicitors effectively increased withanolides and phenolic acids production, among the five different elicitors, salicylic acid (4.14 mg l-1) induced 11.49 -fold increase in withanolides (89.07 ± 2.75 mg g-1 DW) and 5.34- fold increase in phenolic acids (83.69 ± 3.11 mg g- 1 DW) after 5 days of elicitation compared to the non-elicited culture (7.75 ± 0.63 mg g-1 DW of withanolides and 15.66 ± 0.92 mg g-1 DW of phenolic acids). These results suggest that elicitors can tremendously increase the biosynthesis of active compounds in this system; thus, the HRC of W. somnifera is cost-effective and can be efficiently used for the industrial production of withanolides and phenolic acids.

Revealing the phosphate-solubilizing characteristics and mechanisms of the plant growth-promoting bacterium Agrobacterium deltaense C1.

AIMS: This study explores the phosphate (Pi)-solubilizing characteristics and mechanisms of a novel phosphate-solubilizing bacterium, Agrobacterium deltaense C1 (C1 hereafter). METHODS AND RESULTS: The growth-promoting effects of C1 were investigated by gnotobiotic experiments, and the Pi-solubilizing mechanism was revealed by extracellular metabolomics, liquid chromatography analysis, and reverse transcription quantitative polymerase chain reaction. Results showed that C1 significantly increased Arabidopsis biomass and total phosphorus (P) content under P deficiency. Under Ca3(PO4)2 condition, the presence of C1 resulted in a significant and negative correlation between available P content and medium pH changes, implying that Pi dissolution occurs through acid release. Metabolomics revealed C1's ability to release 99 organic acids, with gluconic acid (GA), citric acid, and α-ketoglutaric acid contributing 64.86%, 9.58%, and 0.94%, respectively, to Pi solubilization. These acids were significantly induced by P deficiency. Moreover, C1's Pi solubilization may remain significant even in the presence of available P, as evidenced by substantial pH reduction and high gcd gene expression. Additionally, C1 produced over 10 plant growth-promoting substances. CONCLUSIONS: C1 dissolves Pi primarily by releasing GA, which enhances plant growth under P deficiency. Notably, its Pi solubilization effect is not significantly limited by available Pi.

Adsorption of Selenium and Lanthanum by Agrobacterium-mediated Hairy Roots of Hybanthus Enneaspermus (L.) F. Muell: A Greener Phytoremediation Strategy.

In this study, phytoremediation potential of toxic metals like selenium (Se) and lanthanum (La) by transformed hairy roots was investigated and compared with plantlets under in-vitro conditions. Agrobacterium rhizogenes A4RS could induce hairy roots with higher biomass in 5-7 days of infection on in-vitro leaves of Hybanthus enneaspermus. The ICP-OES data indicated that the hairy roots were able to accumulate both selenium and lanthanum efficiently compared to plantlets. The hairy roots and plantlets show optimum absorption at 50 ppm under both individual and combined metal supply. The metal accumulation performances increased by 13.6% (La) and 10.9% (Se) in hairy roots with combined metal supply (La and Se) compared to individual supply (La or Se) conditions. The Se accumulated more than La, but the La accumulation percentage was found to increase substantially under combined metal supply conditions, shows the suitability and potential of hairy roots for phytoremediation of La and Se.

Genetic manipulation of bermudagrass photosynthetic biosynthesis using Agrobacterium-mediated transformation.

Bermudagrass is one of the most extensively used warm-season grasses. It is widely used in landscaping, stadium construction and soil remediation due to its excellent regeneration, trampling and stress tolerances. However, studies on its regulatory mechanism and variety improvement by genetic engineering are still at a standstill, owing to its genetic variability and intrinsic limits linked with some resistance to Agrobacterium infection. In this study, we established a higher efficient Agrobacterium-mediated transformation via screening for vital embryogenic callus and improving infection efficiency. The superior callus was light yellow, hard granular and compact, determined with a differentiation rate of more than 95%. The optimized infestation courses by gentle shaking, vacuuming and sonicating were used. The infested calluses were co-cultured for 3 days, followed by desiccation treatments for 1 day to get higher infection efficiency. Then the CdHEMA1 gene, essential for chlorophyll biosynthesis, was cloned and transferred into bermudagrass to validate the aforementioned optimization procedures integrally. Molecular-level analyses indicated that the CdHEMA1 gene had successfully integrated and was greatly increased in transgenic seedlings. Results of the photosynthetic capacity assessment showed that CdHEMA1 overexpression may considerably enhance the contents of photosynthetic pigments, OJIP curve and reaction center density (RC/CSo) to absorb (ABS/CSo, ABS/CSM) and capture (TRo/CSo) more light energy, hence improve the performance indices PIABS and PICS compared to the wild type. The successful completion of this project would provide a solid platform for further gene function study and molecular breeding of bermudagrass.

Generation of Stable Catharanthus roseus Hairy Root Lines with Agrobacterium rhizogenes.

Agrobacterium rhizogenes is the bacterial agent that causes hairy root disease in dicots and is purposefully engineered for the development of transgenic hairy root cultures. Due to their genetic and metabolic stability, hairy root cultures offer advantages as a tissue culture system for investigating the function of transgenes and as a production platform for specialized metabolites or proteins. The process for generating hairy root cultures involves first infecting the explant with A. rhizogenes, excising and eliminating A. rhizogenes from the emerging hairy roots, selecting for transgenic hairy roots on plates containing the selective agent, confirming genomic integration of transgenes by PCR, and finally adapting the hairy roots in liquid media. Here we provide a detailed protocol for developing and maintaining transgenic hairy root cultures of our medicinal plant of interest, Catharanthus roseus.

Biotechnology for propagation and secondary metabolite production in Bacopa monnieri.

Bacopa monnieri (L.) Wettst. or water hyssop commonly known as "Brahmi" is a small, creeping, succulent herb from the Plantaginaceae family. It is popularly employed in Ayurvedic medicine as a nerve tonic to improve memory and cognition. Of late, this plant has been reported extensively for its pharmacologically active phyto-constituents. The main phytochemicals are brahmine, alkaloids, herpestine, and saponins. The saponins include bacoside A, bacoside B, and betulic acid. Investigation into the pharmacological effect of this plant has thrived lately, encouraging its neuroprotective and memory supporting capacity among others. Besides, it possesses many other therapeutic activities like antimicrobial, antioxidant, anti-inflammatory, gastroprotective properties, etc. Because of its multipurpose therapeutic potential, it is overexploited owing to the prioritization of natural remedies over conventional ones, which compels us to conserve them. B. monnieri is confronting the danger of extinction from its natural habitat as it is a major cultivated medico-botanical and seed propagation is restricted due to less seed availability and viability. The ever-increasing demand for the plant can be dealt with mass propagation through plant tissue culture strategy. Micropropagation utilizing axillary meristems as well as de novo organogenesis have been widely investigated in this plant which has also been explored for its conservation and production of different types of secondary metabolites. Diverse in vitro methods such as organogenesis, cell suspension, and callus cultures have been accounted for with the aim of production and/or enhancement of bacosides. Direct shoot-organogenesis was initiated in excised leaf and internodal explants without any exogenous plant growth regulator(s) (PGRs), and the induction rate was improved when exogenous cytokinins and other supplements were used. Moreover, biotechnological toolkits like Agrobacterium-mediated transformation and the use of mutagens have been reported. Besides, the molecular marker-based studies demonstrated the clonal fidelity among the natural and in vitro generated plantlets also elucidating the inherent diversity among the natural populations. Agrobacterium-mediated transformation system was mostly employed to optimize bacoside biosynthesis and heterologous expression of other genes. The present review aims at depicting the recent research outcomes of in vitro studies performed on B. monnieri which include root and shoot organogenesis, callus induction, somatic embryogenesis, production of secondary metabolites by in vitro propagation, acclimatization of the in vitro raised plantlets, genetic transformation, and molecular marker-based studies of clonal fidelity. KEY POINTS: • Critical and up to date records on in vitro propagation of Bacopa monnieri • In vitro propagation and elicitation of secondary metabolites from B. monnieri • Molecular markers and transgenic studies in B. monnieri.

Selenium-oxidizing Agrobacterium sp. T3F4 steadily colonizes in soil promoting selenium uptake by pak choi (Brassica campestris).

Selenium (Se) deficiency in soil is linked to its low content in edible crops, resulting in adverse impacts on the health of 15% of the global population. The crop mainly absorbs oxidized selenate and selenite from soil, then converts them into organic Se. However, the role of Se-oxidizing bacteria in soil Se oxidation, Se bioavailability and Se absorption into plants remains unclear. The strain Agrobacterium sp. T3F4, isolated from seleniferous soil, was able to oxidize elemental Se into selenite under pure culture conditions. The green fluorescent protein (gfp)-gene-marked strain (T3F4-GFP) and elemental Se or selenite (5 mg·kg-1) were added to pak choi (Brassica campestris ssp. chinensis) pot cultures. Observation of the fluorescence and viable counting indicated that GFP-expressing bacterial cells steadily colonized the soil in the pots and the leaves of the pak choi, reaching up to 6.6 × 106 and 2.0 × 105 CFU g-1 at 21 days post cultivation, respectively. Moreover, the total Se content (mostly organic Se) was significantly increased in the pak choi under T3F4 inoculated pot culture, with elemental Se(0) being oxidized into Se(IV), and soil Se(IV) being dissolved before being absorbed by the crop. After strain T3F4 was inoculated, no significant differences in microbial diversity were observed in the soils and roots, whereas the abundance of Rhizobium spp. was significantly increased. To our knowledge, this is the first time that Se-oxidizing Agrobacterium sp. T3F4 has been found to steadily colonize soil and plant tissues, and that its addition to soil increases the absorption of Se in plants. This study provides a potential strategy for Se biofortification.

Application of Hairy Root Culture for Bioactive Compounds Production in Medicinal Plants.

Medicinal plants are rich sources of natural bioactive compounds used to treat many diseases. With the development of the health industry, the market demands for Chinese medicine have been rapidly increasing in recent years. However, over-utilization of herbal plants would cause serious ecological problems. Therefore, an effective approach should be developed to produce the pharmaceutically important natural drugs. Hairy root culture induced by Agrobacterium rhizogenes has been considered to be an effective tool to produce secondary metabolites that are originally biosynthesized in the roots or even in the aerial organs of mature plants. This review aims to summarize current progress on medicinal plant hairy root culture for bioactive compounds production. It presents the stimulating effects of various biotic and abiotic elicitors on the accumulation of secondary metabolites. Synergetic effects by combination of different elicitors or with other strategies are also included. Besides, the transgenic system has promising prospects to increase bioactive compounds content by introducing their biosynthetic or regulatory genes into medicinal plant hairy root. It offers great potential to further increase secondary metabolites yield by the integration of manipulating pathway genes with elicitors and other strategies. Then advances on two valuable pharmaceuticals production in the hairy root cultures are illustrated in detail. Finally, successful production of bioactive compounds by hairy root culture in bioreactors are introduced.

Modern Trends in the In Vitro Production and Use of Callus, Suspension Cells and Root Cultures of Medicinal Plants.

This paper studies modern methods of producing and using callus, suspension cells and root cultures of medicinal plants in vitro. A new solution for natural product production is the use of an alternative source of renewable, environmentally friendly raw materials: callus, suspension and root cultures of higher plants in vitro. The possibility of using hairy root cultures as producers of various biologically active substances is studied. It is proven that the application of the genetic engineering achievements that combine in vitro tissue culture and molecular biology methods was groundbreaking in terms of the intensification of the extraction process of compounds significant for the medical industry. It is established that of all the callus processing methods, suspension and root cultures in vitro, the Agrobacterium method is the most widely used in practice. The use of agrobacteria has advantages over the biolistic method since it increases the proportion of stable transformation events, can deliver large DNA segments and does not require special ballistic devices. As a result of the research, the most effective strains of agrobacteria are identified.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2-3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Characterization and improvement of curdlan produced by a high-yield mutant of Agrobacterium sp. ATCC 31749 based on whole-genome analysis.

Curdlan is a bacterial, water-insoluble, linear homopolysaccharide that has been widely used in the food industry. In this study, genome information of strain CGMCC 11546, a UV-induced high-yield mutant of the model curdlan-producing strain Agrobacterium sp. ATCC 31749, was used to investigate the molecular mechanism of curdlan biosynthesis. The maximum curdlan yield of 47.97 ± 0.57 g/L was obtained from strain CGMCC 11546 by using optimal media containing 60 g/L sucrose, 6 g/L yeast, 2 g/L KH2PO4, 0.4 g/L MgSO4·7H2O, 2 g/L CaCO3, 0.1 g/L FeSO4·7H2O, 0.04 g/L MnSO4, and 0.02 g/L ZnCl2 at 30 °C and 280 rpm after 96 h of fermentation. The gel strength of curdlan was improved by 41 % by knocking out the ß-1,3-glucanase genes exoK and exsH of strain CGMCC 11546. Furthermore, the application of curdlan from the ΔexoK-exsH strain in noodles significantly improved the eating quality of both raw and cooked noodles.

Establishment of hairy root cultures of Salvia bulleyana Diels for production of polyphenolic compounds.

This study was to obtain stable transformed roots of Salvia bulleyana using A. rhizogenes strain A4 and then evaluate their phytochemical profile and selected the most productive clone. Our results indicated that the type of explant and medium used for bacterium and explant incubation had an influence on the frequency of hairy root formation. The best response was obtained on leaves infected with bacteria cultivated on YMB medium supplemented with acetosyringone. Of the four selected transformed root clones, after five-week cultivation in Woody Plant (WP) medium, the highest growth indexes were demonstrated for line C1: i.e. 13 for fresh and 15 for dry weight (81.4 and 8.2 g/l fresh and dry weight, respectively). The qualitative analysis of hydromethanolic extracts of hairy roots of S. bulleyana using UPLC-PDA-ESI-MS/MS method showed the presence of 10 polyphenolic compounds including predominant rosmarinic acid (RA), its derivatives (hexoside and methyl rosmarinate), caffeic acid, its derivatives and several salvianolic acids: K, E and F. Their production varied among the four root clones studied; the highest RA (39.6 mg/g dry weight) and total polyphenol (48.9 mg/g dry weight) level were found in the roots of C4 clone. These values were significantly higher than those of the roots of plants grown for several years under field conditions. The transformation of the obtained root cultures was confirmed by polymerase chain reaction using aux1, aux2, rolB, rolC and rolD primers.

Generation of transgene-free PDS mutants in potato by Agrobacterium-mediated transformation.

BACKGROUND: Gene editing using the CRISPR/Cas9 system has become a routinely applied method in several plant species. The most convenient gene delivery system is Agrobacterium-mediated gene transfer with antibiotic selection and stable genomic integration of transgenes, including Cas9. For elimination of transgenes in the segregating progeny, selfing is applied in many plant species. This approach, however, cannot be widely employed in potato because most of the commercial potato cultivars are self-incompatible. RESULTS: In this study, the efficiency of a transient Cas9 expression system with positive/negative selection based on codA-nptII fusion was tested. The PHYTOENE DESATURASE (PDS) gene involved in carotenoid biosynthesis was targeted. A new vector designated PROGED::gPDS carrying only the right border of T-DNA was constructed. Using only the positive selection function of PROGED::gPDS and the restriction enzyme site loss method in PCR of genomic DNA after digestion with the appropriate restriction enzyme, it was demonstrated that the new vector is as efficient in gene editing as a traditional binary vector with right- and left-border sequences. Nevertheless, 2 weeks of positive selection followed by negative selection did not result in the isolation of PDS mutants. In contrast, we found that with 3-day positive selection, PDS mutants appear in the regenerating population with a minimum frequency of 2-10%. Interestingly, while large deletions (> 100 bp) were generated by continuous positive selection, the 3-day selection resulted in deletions and substitutions of only a few bp. Two albinos and three chimaeras with white and green leaf areas were found among the PDS mutants, while all the other PDS mutant plants were green. Based on DNA sequence analysis some of the green plants were also chimaeras. Upon vegetative propagation from stem segments in vitro, the phenotype of the plants obtained even by positive selection did not change, suggesting that the expression of Cas9 and gPDS is silenced or that the DNA repair system is highly active during the vegetative growth phase in potato. CONCLUSIONS: Gene-edited plants can be obtained from potatoes by Agrobacterium-mediated transformation with 3-day antibiotic selection with a frequency high enough to identify the mutants in the regenerating plant population using PCR.

Agrobacterium rhizogenes-mediated transformation of grain (Amaranthus hypochondriacus) and leafy (A. hybridus) amaranths.

KEY MESSAGE: Transgenic A. hypochondriacus and A. hybridus roots were generated. Further, a distinct plant regeneration program via somatic embryos produced from hairy roots was established. Work was implemented to develop an optimized protocol for root genetic transformation of the three grain amaranth species and A. hybridus, their presumed ancestor. Transformation efficiency was species-specific, being higher in A. hypochondriacus and followed by A. hybridus. Amaranthus cruentus and A. caudatus remained recalcitrant. A reliable and efficient Agrobacteruim rhizogenes-mediated transformation of these species was established using cotyledon explants infected with the previously untested BVG strain. Optimal OD600 bacterial cell densities were 0.4 and 0.8 for A. hypochondriacus and A. hybridus, respectively. Hairy roots of both amaranth species were validated by the amplification of appropriate marker genes and, when pertinent, by monitoring green fluorescent protein emission or ß-glucuronidase activity. Embryogenic calli were generated from A. hypochondriacus rhizoclones. Subsequent somatic embryo maturation and germination required the activation of cytokinin signaling, osmotic stress, red light, and calcium incorporation. A crucial step to ensure the differentiation of germinating somatic embryos into plantlets was their individualization and subcultivation in 5/5 media containing 5% sucrose, 5 g/L gelrite, and 0.2 mg/L 2-isopentenyladenine (2iP) previously acidified to pH 4.0 with phosphoric acid, followed by their transfer to 5/5 + 2iP media supplemented with 100 mg/L CaCl2. These steps were strictly red light dependent. This process represents a viable protocol for plant regeneration via somatic embryo germination from grain amaranth transgenic hairy roots. Its capacity to overcome the recalcitrance to genetic transformation characteristic of grain amaranth has the potential to significantly advance the knowledge of several unresolved biological aspects of grain amaranths.

Comparison of Profiling of Hairy Root of Two Tibetan Medicinal Plants Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim.

BACKGROUND: Tropane Alkaloids (TAs) are important drugs for curing many diseases in the medical industry. METHODS: To sustainably exploit TA resources in endangered traditional Tibetan herbs, the hairy root (HR) systems of Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim. were compared under the same culture conditions. RESULTS: The results indicated that both the Agrobacterium rhizogenes strains and explants affected the HR induction frequency, MSU440, A4 and LBA9402 strains could induce hairy roots following infection of cotyledon and hypocotyl of A. tanguticus while LBA9402 could not induce HR on either explants of P. tangutica. The efficiency of LBA9402 was higher than A4 and MSU440 on A. tanguticus and A4 was better strain than MSU440 on P. tangutica. The hypocotyl explant was more suitable for P.tangutica and cotyledon explant was better for A.tangutica with a transformation frequency of 33.3% (P. tangutica) and 82.5% (A. tanguticus), respectively. In a flask reactor system, both the growth curves of HR for two species both appeared to be "S" curve; however, the HR of P. tangutica grew more rapidly than that of A. tanguticus, and the latter accumulated more biomass than the former. As the culture volume increased, the HR proliferation coefficient of both the species increased. HPLC analysis results showed that the content of TAs in the HR of P. tangutica was 257.24mg/100g·DW, which was more than that of A. tanguticus HR (251.08mg/100g·DW), and the anisodamine in the Pt- HR was significantly higher than that in At-HR. Moreover, tropane alkaloids in the HR of the two species were all significantly higher than that of the roots of aseptic seedlings. CONCLUSION: Our results suggest that HR of P. tangutica and A. tanguticus both could provide a useful platform for sustainable utilization of two Tibetan medicinal plants in the Qinghai-Tibetan Plateau in the future.

The effect of purine-type cytokinin on the proliferation and production of phenolic compounds in transformed shoots of Dracocephalum forrestii.

Dracocephalum forrestii is a perennial, endemic to China plant with a number of pharmaceutical properties. Transformed shoots of the species spontaneously regenerated from hairy roots induced by Agrobacterium rhizogenes. The transgenic nature of the shoots was confirmed by polymerase chain reaction (PCR). The shoot culture was multiplied on Murashige and Skoog (MS) medium with 0.2 mg/l IAA and 0.2, 0.5, 1.0, 2.0 or 5.0 mg/l purine-type cytokinins (mT, BAR, BPA or BAP). The highest multiplication rate (about thirteen shoot or buds per explant) was obtained on MS medium with 0.2 mg/l mT after four weeks of culture. The phenolic compounds present in the hydromethanolic extracts from the D. forrestii transgenic shoots were characterized using UPLC-PDA-ESI-MS. The shoots were found to biosynthesize three phenolic acids and five flavonoid glycosides. UHPLC analysis of the hydromethanolic extracts found the predominant phenolic acid to be rosmarinic acid, with its highest content observed in shoots cultivated with 5.0 mg/l BPA. In contrast, the greatest production of flavonoid derivatives (especially acacetin derivatives) was observed in the medium supplemented with 2 mg/l BPA.

Beneficial implications of sugar beet proteinase inhibitor BvSTI on plant architecture and salt stress tolerance in Lotus corniculatus L.

Food demands of increasing human population dictate intensification of livestock production, however, environmental stresses could jeopardize producers' efforts. Forage legumes suffer from yield losses and poor nutritional status due to salinity increase of agricultural soils. As tools aimed to reduce negative impacts of biotic or abiotic stresses, proteinase inhibitors (PIs) have been promoted for biotechnological improvements. In order to increase tolerance of Lotus corniculatus L. to salt stress, serine PI, BvSTI, was introduced into this legume using Agrobacterium rhizogenes, with final transformation efficiency of 4.57%. PCR, DNA gel-blot, RT-PCR and in-gel protein activity assays confirmed the presence and activity of BvSTI products in transformed lines. Plants from three selected transgenic lines (21, 73 and 109) showed significant alterations in overall phenotypic appearance, corresponding to differences in BvSTI accumulation. Lines 73 and 109 showed up to 7.3-fold higher number of tillers and massive, up to 5.8-fold heavier roots than in nontransformed controls (NTC). Line 21 was phenotypically similar to NTC, accumulated less BvSTI transcripts and did not exhibit an additional band of recombinant trypsin inhibitor as seen in lines 73 and 109. Exposure of the transgenic lines to NaCl revealed different levels of salt stress susceptibility. The NaCl sensitivity index, based on morphological appearance and chlorophyll concentrations showed that lines 73 and 109 were significantly less affected by salinity than NTC or line 21. High level of BvSTI altered morphology and delayed salt stress related senescence, implicating BvSTI gene as a promising tool for salinity tolerance improvement trials in L. corniculatus.

Production of Rare Ginsenosides Rg3 and Rh2 by Endophytic Bacteria from Panax ginseng.

The ginsenosides Rh2 and Rg3 induce tumor cell apoptosis, inhibit tumor cell proliferation, and restrain tumor invasion and metastasis. Despite Rh2 and Rg3 having versatile pharmacological activities, contents of them in natural ginseng are extremely low. To produce ginsenosides Rh2 and Rg3, the saponin-producing capacity of endophytic bacteria isolated from Panax ginseng was investigated. In this work, 81 endophytic bacteria isolates were taken from ginseng roots by tissue separation methods. Among them, strain PDA-2 showed the highest capacity to produce the rare ginsenosides; the concentrations of rare ginsenosides Rg3 and Rh2 reached 62.20 and 18.60 mg/L, respectively. On the basis of phylogenetic analysis, it was found that strain PDA-2 belongs to the genus Agrobacterium and was very close to Agrobacterium rhizogenes.

Transgene stacking in potato using the GAANTRY system.

OBJECTIVE: GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA). We examined the ability of the GAANTRY Agrobacterium rhizogenes ArPORT1 '10-stack' strain to generate transgenic potato plants. RESULTS: The 28.5 kilobase 10-stack T-DNA, was introduced into Lenape potato plants with a 32% transformation efficiency. Molecular and phenotypic characterization confirmed that six of the seven tested independent transgenic lines carried the entire desired construct, demonstrating that the GAANTRY 10-stack strain can be used can be used in a tissue culture-based callus transformation method to efficiently generate transgenic potato plants. Analysis using droplet digital PCR showed that most of the characterized events carry one or two copies of the 10-stack transgenes and that 'backbone' DNA from outside of the T-DNA was absent in the transgenic plants. These results demonstrate that the GAANTRY system efficiently generates high quality transgenic potato plants with a large construct of stacked transgenes.

The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection.

Higher plants exploit posttranscriptional gene silencing as a defense mechanism against virus infection by the RNA degradation system. Plant RNA viruses suppress posttranscriptional gene silencing using their encoded proteins. Three important motifs (F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region) in P0 of Potato leafroll virus (PLRV) were reported to be essential for suppression of RNA silencing activity. In this study, Agrobacterium-mediated transient experiments were carried out to screen the available amino acid substitutions in the F-box-like motif and G139/W140/G141-like motif that abolished the RNA silencing suppression activity of P0, without disturbing the P1 amino acid sequence. Subsequently, four P0 defective mutants derived from a full-length cDNA clone of PLRV (L76F and W87R substitutions in the F-box-like motif, G139RRR substitution in the G139/W140/G141-like motif, and F220R substitution in the C-terminal conserved region) were successfully generated by reverse PCR and used to investigate the impact of these substitutions on PLRV infectivity. The RT-PCR and western blot analysis revealed that these defective mutants affected virus accumulation in inoculated leaves and systemic movement in Nicotiana benthamiana as well as in its natural hosts, potato and black nightshade. These results further demonstrate that the RNA silencing suppressor of PLRV is required for PLRV accumulation and systemic infection.