Arbuscular mycorrhizal fungal diversity in high-altitude hypersaline Andean wetlands studied by 454-sequencing and morphological approaches

Withania somnifera, also known as Indian ginseng is known to contain valuable bioactive compounds, called withanolides that structurally resemble ginsenosides of Panax ginseng. These compounds provide the basis of pharmacological relevance in traditional systems of medicine. In the present study, 150 hairy root lines of W. somnifera were induced of which nine fast growing lines were analysed for their growth and withanolide content. Hairy root line W9 was selected due to its high specific growth rate (0.196 ± 0.005 d^?1) and high withanolide content. The response to different concentrations of elicitors (methyl jasmonate and P. indica cell homogenate) and various exposure durations was assessed in the W9 hairy root line. The withanolide content as well as the pattern of gene expression from MVA, MEP and sterol pathway, was evaluated using qPCR. Though gene expression and withanolide content were found to be elevated in almost all MeJ and CHP treatments, the exposure of hairy roots to 15 μM MeJ for 4 h gave the maximum withanolide yield. The results suggest that the elicitation potential of methyl jasmonate was higher than that of P. indica cell homogenate for increasing withanolide levels in hairy roots of W. somnifera.     

An efficient <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation protocol of <Emphasis Type="Italic">Withania somnifera</Emphasis>

This is the first report on Agrobacterium rhizogenes-mediated transformation of Withania somnifera for expression of a foreign gene in hairy roots. We transformed leaf and shoot tip explants using binary vector having gusA as a reporter gene and nptII as a selectable marker gene. To improve the transformation efficiency, acetosyringone (AS) was added in three stages, Agrobacterium liquid culture, Agrobacterium infection and co-culture of explants with Agrobacterium. The addition of 75 μM AS to Agrobacterium liquid culture was found to be optimum for induction of vir genes. Moreover, the gusA gene expression in hairy roots was found to be best when the leaves and shoot tips were sonicated for 10 and 20s, respectively. Based on transformation efficiency, the Agrobacterium infection for 60 and 120 min was found to be suitable for leaves and shoot tips, respectively. Amongst the various culture media tested, MS basal medium was found to be best in hairy roots. The transformation efficiency of the improved protocol was recorded 66.5 and 59.5?% in the case of leaf and shoot tip explants, respectively. When compared with other protocols the transformation efficiency of this improved protocol was found to be 2.5 fold higher for leaves and 3.7 fold more for shoot tips. Southern blot analyses confirmed 1–2 copies of the gusA transgene in the lines W1-W4, while 1–4 transgene copies were detected in the line W5 generated by the improved protocol. Thus, we have established a robust and efficient A. rhizogenes mediated expression of transgene (s) in hairy roots of W. somnifera.     

Microbial secondary metabolites ameliorate growth, <Emphasis Type="Italic">in planta</Emphasis> contents and lignification in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal

In the present investigation, metabolites of Streptomyces sp. MTN14 and Trichoderma harzianum ThU significantly enhanced biomass yield (3.58 and 3.48 fold respectively) in comparison to the control plants. The secondary metabolites treatments also showed significant augmentation (0.75–2.25 fold) in withanolide A, a plant secondary metabolite. Lignin deposition, total phenolic and flavonoid content in W. somnifera were maximally induced in treatment having T. harzianum metabolites. Also, Trichoderma and Streptomyces metabolites were found much better in invoking in planta contents and antioxidants compared with their live culture treatments. Therefore, identification of new molecular effectors from metabolites of efficient microbes may be used as biopesticide and biofertilizer for commercial production of W. somnifera globally.     

Hairy root culture optimization and resveratrol production from <Emphasis Type="Italic">Vitis vinifera</Emphasis> subsp. <Emphasis Type="Italic">sylvesteris</Emphasis>

Resveratrol is a polyphenolic compound produced in very low levels in grapes. To achieve high yield of resveratrol in wild grape, three Agrobacterium rhizogenes strains, Ar318, ArA4 and LBA9402, were used to induce hairy roots following infection of internodes, nodes or petioles of in vitro grown Vitis vinifera subsp. sylvesteris accessions W2 and W16, and cultivar Rasha. The effects of inoculation time, age of explants, bacterial concentration and co-cultivation times were examined on the efficiency of the production of hairy roots. Strains Ar318, ArA4 and LBA9402 all induced hairy roots in the tested genotypes, but the efficiency of ArA4 strain was higher than the other strains. The highest hairy root production was with using internodes as explants. The transformation of hairy roots lines was confirmed by PCR detection of rolB gene. Half Murashige and Skoog (MS) medium was better for biomass production compared with MS medium. HPLC analysis of resveratrol production in the hairy root cultures showed that all the genotypes produced higher amounts of resveratrol than control roots. The highest amount of resveratrol was produced from W16 internode cultures, which was 31-fold higher than that of control root. Furthermore, TLC analysis showed that treatments of hairy roots with sodium acetate and jasmonate elevated resveratrol levels both in hairy root tissue and excreted into the half MS medium. These results demonstrate that endogenous and exogenous factors can affect resveratrol production in hairy root culture of grape, and this strategy could be used to increase low resveratrol production in grapes.     

Growth kinetics and withanolide production in novel transformed roots of <Emphasis Type="Italic">Withania somnifera</Emphasis> and measurement of their antioxidant potential using chemiluminescence

Markedly increased withanolide content was found in transformed roots (TR) of Withania somnifera germplasm grown in low mineral minimal media and withanolides showed high antioxidant potential when analysed using acidic potassium permanganate chemiluminescence. Transformation frequency of explants infected with Agrobacterium rhizogenes strain A4 varied between the three germplasms tested with the highest observed as 75?±?0.9. Transformed root production was explant specific with leaves being the most productive among the different explants used. Withanolides, namely withaferin A, withanolide A, withanolide B and 12-deoxywithastramonolide were detected in TR cultures and differences in their content were found between germplasms. The highest concentrations of secondary metabolites were found in 4-week-old cultures and concentrations declined by the 8th and 12th week of culture. In 4-week-old cultures, the biomass of TR cultures was 4.5 fold higher than their respective non-transformed roots (NTR). Withaferin A was found in TR at levels that were 28–34 times higher than that found in NTR. A rapid method for the determination of the antioxidant potential of W. somnifera TR extracts was developed using post-column acidic potassium permanganate chemiluminescence (APPC) detection. The APPC chromatographic peaks for extract constituents showed strong alignment with those found for ultraviolet absorbance detection. The methods developed in this study for TR culture establishment and the use of a fast and sensitive way for the qualitative and quantitative determination of the antioxidant activity of their metabolites provides a new platform that will have use for similar studies in other species.     

Pyrethrin accumulation in elicited hairy root cultures of <Emphasis Type="Italic">Chrysanthemum cinerariaefolium</Emphasis>

The flowers of Pyrethrum (Chrysanthemum cinerariaefolium) are known to contain Pyrethrins that are naturally occurring potential insecticide. Hairy roots were induced from leaves of C. cinerariaefolium using Agrobacterium rhizogenes strain A4. The root clones were characterized in to four groups i.e. thick, unbranched (D2 and D5), thin, highly branched (D3), thick, branched (B2) and thick, highly branched (D1, D6). Six established hairy root clones showed the presence of pyrethrin and were selected for elicitation studies. Growth kinetics studies revealed highest growth index in hairy root clone D1 (592.0) followed by D6 and D3 on dry weight basis after 40 days of culture. The maximum pyrethrin content was found in the clone D3 (7.2 mg/g dw) which is comparable to the flowers obtained from the variety “Avadh”. Hairy root clone D2 (5.2 mg/g dw) and D6 (1.3 mg/g dw) contained pyrethrin but in less amount as compared to clone D3. The PCR analysis showed the presence of rol B and rol C genes in all the six hairy root clones while rol A was detected only in D2 clone. The methanolic extract of D3 clone showed antifungal activities against phytopathogenic fungal strains which were found maximum against Curvuleria andropogonis followed by Colletotrichum acutatum and Rhizoctonia solani. Hairy root clones D2, D3 and D6 were elicited with culture filtrate of endophytic fungus (Fusarium oxysporum) and bacteria (Bacillus subtilis). The culture filtrate (4.0?%v/v) of both the fungal and bacterial origin was found to be effective in enhancing the pyrethrin content in all the tested hairy root clones. Clone D3 showed maximum pyrethrin content on elicitation with F. oxysporum (9.7 mg/g dw) and B. subtilis (9.7 mg/g dw) culture filtrate, which is 32?% higher than the non elicited D3 hairy roots (7.2 mg/g dw). F. oxysporum also enhanced the hairy root growth resulting into the higher biomass yield of D3 (50?%) and D2 (76?%) in comparison to control non elicited hairy root clones of D3 and D2, respectively leading to higher pyrethrin yield.     

Salt stress induced soybean <Emphasis Type="Italic">GmIFS1</Emphasis> expression and isoflavone accumulation and salt tolerance in transgenic soybean cotyledon hairy roots and tobacco

Effects of isoflavones on plant salt tolerance were investigated in soybean (Glycine max L. Merr. cultivar N23674) and tobacco (Nicotiana tabacum L.). Leaf area, fresh weight, net photosynthetic rate (Pn), and transpiration rate (Tr) of soybean N23674 plants treated with 80 mM NaCl were significantly reduced, while a gene (GmIFS1) encoding for 2-hydroxyisoflavone synthase was highly induced, and isoflavone contents significantly increased in leaves and seeds. To test the impact of isoflavones to salt tolerance, transgenic soybean cotyledon hairy roots expressing GmIFS1 (hrGmIFS1) were produced. Salt stress slightly increased isoflavone content in hairy roots of the transgenic control harboring the empty vector but substantially reduced the maximum root length, root fresh weight, and relative water content (RWC). The isoflavone content in hrGmIFS1 roots, however, was significantly higher, and the above-mentioned root growth parameters decreased much less. The GmIFS1 gene was also transformed into tobacco plants; plant height and leaf fresh weight of transgenic GmIFS1 tobacco plants were much greater than control plants after being treated with 85 mM NaCl. Leaf antioxidant capacity of transgenic tobacco was significantly higher than the control plants. Our results suggest that salt stress-induced GmIFS1 expression increased isoflavone accumulation in soybean and improved salt tolerance in transgenic soybean hairy roots and tobacco plants.     

Morphological and Molecular Analysis of Isolated Cultures of Tobacco Adventitious Roots Obtained by the Methods of Biolistic Bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation

Plant infection with Agrobacterium rhizogenes leads to the development of a hairy root disease notable for the rapid agravitropic growth of roots on hormone-free nutrient media. In order to look into the interaction of A. rhizogenes with plants and assess opportunities of practical application of hairy root culture, new approaches to their production are elaborated. A method of bacterium-free and plasmid-free production of genetically modified roots (hairy roots) by means of biolistic transformation of leaf explants with a DNA fragment (size of 5461 bp) consisting of genes rolA, rolB, rolC, and rolD are proposed. In most cases, such transformation resulted in the emergence of only adventitious roots with transient expression of rol-genes, and the growth of such roots on hormone-free media ceased in 2–3 months in contrast to genuine hairy roots capable of unrestricted growth. Molecular analysis of different systems of target genes’ expression showed an important role of transgene rolC and host gene of cyclin-dependent protein kinase CDKB1-1 in the maintenance of rapid growth of hairy roots in vitro (in isolated cultures).     

Acid phosphatase gene <Emphasis Type="Italic">GmHAD1</Emphasis> linked to low phosphorus tolerance in soybean,through fine mapping

Key message

Map-based cloning identified GmHAD1, a gene which encodes a HAD-like acid phosphatase, associated with soybean tolerance to low phosphorus stress.

Abstract

Phosphorus (P) deficiency in soils is a major limiting factor for crop growth worldwide. Plants may adapt to low phosphorus (LP) conditions via changes to root morphology, including the number, length, orientation, and branching of the principal root classes. To elucidate the genetic mechanisms for LP tolerance in soybean, quantitative trait loci (QTL) related to root morphology responses to LP were identified via hydroponic experiments. In total, we identified 14 major loci associated with these traits in a RIL population. The log-likelihood scores ranged from 2.81 to 7.43, explaining 4.23–13.98% of phenotypic variance. A major locus on chromosome 08, named qP8-2, was co-localized with an important P efficiency QTL (qPE8), containing phosphatase genes GmACP1 and GmACP2. Another major locus on chromosome 10 named qP10-2 explained 4.80–13.98% of the total phenotypic variance in root morphology. The qP10-2 contains GmHAD1, a gene which encodes an acid phosphatase. In the transgenic soybean hairy roots, GmHAD1 overexpression increased P efficiency by 8.4–16.5% relative to the control. Transgenic Arabidopsis plants had higher biomass than wild-type plants across both short- and long-term P reduction. These results suggest that GmHAD1, an acid phosphatase gene, improved the utilization of organic phosphate by soybean and Arabidopsis plants.

     

Characterisation of a novel quantitative trait locus, <Emphasis Type="Italic">GN4</Emphasis>-<Emphasis Type="Italic">1</Emphasis>, for grain number and yield in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Key message

A novel QTL for grain number, GN4-1, was identified and fine-mapped to an ~ 190-kb region on the long arm of rice chromosome 4.

Abstract

Rice grain yield is primarily determined by three components: number of panicles per plant, grain number per panicle and grain weight. Among these traits, grain number per panicle is the major contributor to grain yield formation and is a crucial trait for yield improvement. In this study, we identified a major quantitative trait locus (QTL) responsible for rice grain number on chromosome 4, designated GN4-1 (a QTL for Grain Number on chromosome 4), using advanced segregating populations derived from the crosses between an elite indica cultivar ‘Zhonghui 8006’ (ZH8006) and a japonica rice ‘Wuyunjing 8’ (WYJ8). GN4-1 was delimited to an ~ 190-kb region on chromosome 4. The genetic effect of GN4-1 was estimated using a pair of near-isogenic lines. The GN4-1 gene from WYJ8 promoted accumulation of cytokinins in the inflorescence and increased grain number per panicle by ~ 17%. More importantly, introduction of the WYJ8 GN4-1 gene into ZH8006 increased grain yield by ~ 14.3 and ~ 11.5% in the experimental plots in 2014 and 2015, respectively. In addition, GN4-1 promoted thickening of the culm and may enhance resistance to lodging. These results demonstrate that GN4-1 is a potentially valuable gene for improvement of yield and lodging resistance in rice breeding.

     

Genetics and fine mapping of a yellow-green leaf gene (<Emphasis Type="Italic">ygl</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis> L.)

Cabbage (Brassica oleracea var. capitata L.) is one of the most popular cultivated vegetables worldwide. Cabbage has rich phenotypic diversity, including plant height, head shape, head color, leaf shape and leaf color. Leaf color plays an important role in cabbage growth and development. At present, there are few reports on fine mapping of leaf color mutants in B. oleracea. In this study, a naturally occurring yellow-green leaf cabbage mutant (YL-1), derived from the self-pollinated progenies of the hybrid ‘Hosom’, was used for inheritance analysis and gene mapping. Segregation populations including F₂ and BC₁ were generated from the cross of two inbred lines, YL-1 and 01–20. Genetic analysis with the F₂ and BC₁ populations demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene, ygl-1. Insertion–deletion (InDel) markers, designed based on the parental re-sequencing data, were used for the preliminary mapping with BSA (bulked segregant analysis) method. A genetic map constructed with 15 InDels indicated that ygl-1 was located on chromosome C01. The ygl-1 gene is flanked by InDel markers ID2 and M8, with genetic distances of 0.4 cM and 0.35 cM, respectively. The interval distance between two markers is 167 kb. Thus, it enables us to locate the ygl-1 gene for the first time in B. oleracea. This study lays the foundation for candidate gene prediction and ygl-1gene cloning.     

Dimorphism in trichome production of <Emphasis Type="Italic">Persicaria lapathifolia</Emphasis> var. <Emphasis Type="Italic">lapathifolia</Emphasis> and Its multiple effects on a leaf beetle

Polymorphisms in plants are main factors that determine the diversity of associated animal communities and their population dynamics. Typically, Persicaria lapathifolia var. lapathifolia (Polygonaceae) has no trichomes on leaf surfaces (glabrous type), but a hairy type does sometimes occur. Based on a cultivation experiment, the presence or absence of trichomes is clarified to be under genetic control. To reveal the defensive function of trichomes against herbivores, laboratory experiments were conducted using a major herbivore, Galerucella grisescens (Coleoptera: Chrysomelidae). In both choice and no-choice feeding tests, the glabrous type was significantly more consumed by G. grisescens adults, while the hairy type was not consumed. In the hairy leaf treatment, larval duration tended to become longer, the adult body weight became significantly lower, and adults laid significantly more eggs than in the glabrous leaf treatment. Hairy leaves contained significantly more total phenolics and condensed tannins than glabrous leaves, suggesting that the hairy type allocates more resources for physical and chemical defence. Because no significant differences in leaf consumption were detected in the feeding experiment using powdered host leaves, G. grisescens seems to have adapted to the chemical defences of P. lapathifolia var. lapathifolia. These results clearly indicate that leaf trichomes of P. lapathifolia var. lapathifolia effectively act as a physical defence against G. grisescens.     

Metabolic Engineering of Glycyrrhizin Pathway by Over-Expression of Beta-amyrin 11-Oxidase in Transgenic Roots of <Emphasis Type="Italic">Glycyrrhiza glabra</Emphasis>

Glycyrrhiza glabra is one of the most important and well-known medicinal plants which produces various triterpene saponins such as glycyrrhizin. Beta-amyrin 11-oxidase (CYP88D6) plays a key role in engineering pathway of glycyrrhizin production and converts an intermediated beta-amyrin compound to glycyrrhizin. In this study, pBI121^GUS-9:CYP88D6 construct was transferred to G. glabra using Agrobacterium rhizogene ATCC 15834. The quantitation of transgene was measured in putative transgenic hairy roots using qRT-PCR. The amount of glycyrrhizin production was measured by HPLC in transgenic hairy root lines. Gene expression analysis demonstrated that CYP88D6 was over-expressed only in one of transgenic hairy root lines and was reduced in two others. Beta-amyrin 24-hydroxylase (CYP93E6) was significantly expressed in one of the control hairy root lines. The amount of glycyrrhizin metabolite in over-expressed line was more than or similar to that of control hairy root lines. According to the obtained results, it would be recommended that multi-genes of glycyrrhizin biosynthetic pathway be transferred simultaneously to the hairy root in order to increase glycyrrhizin content.     

Genetic dissection and validation of candidate genes for flag leaf size in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Key message

Two major loci with functional candidate genes were identified and validated affecting flag leaf size, which offer desirable genes to improve leaf architecture and photosynthetic capacity in rice.

Abstract

Leaf size is a major determinant of plant architecture and yield potential in crops. However, the genetic and molecular mechanisms regulating leaf size remain largely elusive. In this study, quantitative trait loci (QTLs) for flag leaf length and flag leaf width in rice were detected with high-density single nucleotide polymorphism genotyping of a chromosomal segment substitution line (CSSL) population, in which each line carries one or a few chromosomal segments from the japonica cultivar Nipponbare in a common background of the indica variety Zhenshan 97. In total, 14 QTLs for flag leaf length and nine QTLs for flag leaf width were identified in the CSSL population. Among them, qFW4-2 for flag leaf width was mapped to a 37-kb interval, with the most likely candidate gene being the previously characterized NAL1. Another major QTL for both flag leaf width and length was delimited by substitution mapping to a small region of 13.5 kb that contains a single gene, Ghd7.1. Mutants of Ghd7.1 generated using CRISPR/CAS9 approach showed reduced leaf size. Allelic variation analyses also validated Ghd7.1 as a functional candidate gene for leaf size, photosynthetic capacity and other yield-related traits. These results provide useful genetic information for the improvement of leaf size and yield in rice breeding programs.