An ex vitro hairy root system from petioles of detached soybean leaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

MAIN CONCLUSION: The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

Methyl jasmonate enhances ursolic, oleanolic and rosmarinic acid production and sucrose induced biomass accumulation, in hairy roots of Lepechinia caulescens.

BACKGROUND: Ursolic (UA), oleanolic (OA) and rosmarinic (RA) acids are bioactive metabolites found in Lepechinia caulescens that have generated interest for their health benefits, which include antimicrobial, antioxidant, antimutagenic, gastroprotective, antidiabetic, antihypertensive and anti-inflammatory properties, among others. To date, very few attempts have been made to evaluate the potential for simultaneous production of these bioactive compounds, using a biotechnological approach. Hairy root cultures offer a biotechnology approach that can be used to study the factors affecting the biosynthesis and the production of UA, OA and RA. In the current study, we established hairy root cultures of L. caulescens and evaluated the effect of sucrose on biomass accumulation, and the effect of different concentrations and times of exposure of methyl jasmonate (MeJA), on the accumulation of UA, OA and RA. METHODS: Leaves from plants of L. caulescens were inoculated with Agrobacterium rhizogenes strain ATCC 15834. PCR of rolB gene confirmed the transgenic nature of hairy roots. Hairy roots were subcultured in semisolid MSB5 medium, supplemented with 15, 30, 45 or 60 g/L sucrose and after 4 weeks, dry weight was determined. The accumulation of UA, OA and RA of wild plants and hairy roots were determined by HPLC. Finally, the hairy roots were treated with 0, 100, 200 and 300 µM of MeJA and the content of bioactive compounds was analyzed, after 24, 48 and 72 h. RESULTS: High frequency transformation (75%) was achieved, using leaf explants from axenic seedlings, infected with A. rhizogenes. The hairy roots showed an enhanced linear biomass accumulation, in response to the increase in sucrose concentration. The hairy root cultures in MSB5 medium, supplemented with 45 g/L sucrose, were capable to synthesizing UA (0.29 ± 0.00 mg/g DW), OA (0.57 ± 0.00 mg/g DW) and RA (41.66 ± 0.31 mg/g DW), about two, seven and three times more, respectively, than in roots from wild plants. Elicitation time and concentration of MeJA resulted in significant enhancement in the production of UA, OA and RA, with treatments elicited for 24 h, with a concentration of 300 µM of MeJA, exhibiting greatest accumulation. CONCLUSION: This is the first report on development of hairy root cultures of L. caulescens. Future studies should aim towards further improving triterpenes and polyphenolic compound production in hairy roots of L. caulescens, for use in the pharmaceutical and biotechnological industry.

First Report of Agrobacterium rhizogenes-induced Hairy Root Formation in Selaginella bryopteris: a Pteridophyte Recalcitrant to Genetic Transformation

Abstract we report A. rhizogenes-induced hairy root formation in S. bryopteris, a medicinally and commercially important plant. A. rhizogenes strain LBA1334 co-cultivated with explants (root, rhizophore, stem portion near the root, and stem with intact fronds) for 24 and 48 h after transformation for induction of hairy roots. The induction of hairy root was observed after 6 days of infection in case of 48 h co-cultivation only. PCR with rolA and virC gene specific primers confirmed the induced hairy roots were due to Ri T-DNA integration and not due to contaminating A. rhizogenes. The root network as explants showed the maximum transformation efficiency. We tested different media like MS, SHFR (Stage Hog Fern Root) and KNOP's during transformation for hairy root induction. The SHFR based media showed good response in transformation as well as propagation. Further, transformation efficiency was enhanced by addition of TDZ (2 mg/L) and Bevistin (0.1%) in SHFR media. The present work would be helpful in hairy roots-based in vitro production of secondary metabolites and on aspect of functional genomics of S. bryopteris.

Functional Analysis of Root microRNAs by a Constitutive Overexpression Approach in a Composite Plant System.

Agrobacterium-mediated transformation is a fast and efficient method for genome modification in plants. In this protocol, we apply this technique for the analysis of root microRNA functionality. The induction of hairy roots constitutively overexpressing a given microRNA precursor allows us, in a simple way, to modify the accumulation of specific mature microRNA and analyze the consequence of this alteration on a phenotype of interest. This method generates ready-to-phenotype "composite plants" with untransformed aerial part and microRNA-overexpressing root system, in about 20 days.

A Cytotoxic and Anti-inflammatory Campesterol Derivative from Genetically Transformed Hairy Roots of Lopezia racemosa Cav. (Onagraceae).

The genetically transformed hairy root line LRT 7.31 obtained by infecting leaf explants of Lopezia racemosa Cav with the Agrobacterium rhizogenes strain ATCC15834/pTDT, was evaluated to identify the anti-inflammatory and cytotoxic compounds reported previously for the wild plant. After several subcultures of the LRT 7.31 line, the bio-guided fractionation of the dichloromethane-methanol (1:1) extract obtained from dry biomass afforded a fraction that showed important in vivo anti-inflammatory, and in vitro cytotoxic activities. Chemical separation of the active fraction allowed us to identify the triterpenes ursolic (1) and oleanolic (2) acids, and (23R)-2α,3ß,23,28-tetrahydroxy-14,15-dehydrocampesterol (3) as the anti-inflammatory principles of the active fraction. A new molecule 3 was characterized by spectroscopic analysis of its tetraacetate derivative 3a. This compound was not described in previous reports of callus cultures, in vitro germinated seedlings and wild plant extracts of whole L. racemosa plants. The anti-inflammatory and cytotoxic activities displayed by the fraction are associated to the presence of compounds 1-3. The present study reports the obtaining of the transformed hairy roots, the bioguided isolation of the new molecule 3, and its structure characterization.

Establishment, Culture, and Scale-up of Brugmansia candida Hairy Roots for the Production of Tropane Alkaloids.

Brugmansia candida (syn. Datura candida) is a South American native plant that produces tropane alkaloids. Hyoscyamine, 6ß-hydroxyhyoscyamine (anisodamine), and scopolamine are the most important ones due to their anticholinergic activity. These bioactive compounds have been historically and widely applied in medicine and their demand is continuous. Their chemical synthesis is costly and complex, and thereby, these alkaloids are industrially produced from natural producer plants. The production of these secondary metabolites by plant in vitro cultures such as hairy roots presents certain advantages over the natural source and chemical synthesis. It is well known that hairy roots produced by Agrobacterium rhizogenes infection are fast-growing cultures, genetically stable and able to grow in hormone-free media. Additionally, recent progress achieved in the scaling up of hairy root cultures makes this technology an attractive tool for industrial processes. This chapter is focused on the methods for the induction and establishment of B. candida hairy roots. In addition, the scaling up of hairy root cultures in bioreactors and tropane alkaloid analysis is discussed.

AtTCTP2 mRNA and protein movement correlates with formation of adventitious roots in tobacco.

The Translationally Controlled Tumor Proteins, or TCTP, is a superfamily of exclusively eukaryotic proteins essential in the regulation of proliferation and general growth. However, it is clear that these are multifunctional proteins given (1) the pleiotropic effects of its mutations, and (2), the multiple processes in which this protein is involved. TCTP function in general is conserved, since Arabidopsis AtTCTP1 can rescue a Drosophila mutant, and vice versa. It has become clear, however, that these proteins may have "taxon-specific" functions. In the case of plants, mRNA and/or proteins have been found in the phloem translocation stream of different species, suggesting a role in long-distance signaling. We have found that a second Arabidopsis TCTP gene, AtTCTP2, codes for a protein that moves long-distance through a graft union in tobacco. Interestingly, the mRNA is also transported long-distance. Both mRNA and protein move long-distance; interestingly, the movement, while more efficient from source to sink tissues, also occurs in the opposite direction. The protein reaches the nuclei of parenchyma cells and adventitious roots. Furthermore, it is clear that the long-distance delivery of AtTCTP2 protein and mRNA is required for the induction of adventitious roots. A model is presented that accounts for these observations.

AtTCTP2, an Arabidopsis thaliana homolog of Translationally Controlled Tumor Protein, enhances in vitro plant regeneration.

The Translationally Controlled Tumor Protein (TCTP) is a central regulator of cell proliferation and differentiation in animals, and probably also in plants. Arabidopsis harbors two TCTP genes, AtTCTP1 (At3g16640), which is an important mitotic regulator, and AtTCTP2 (At3g05540), which is considered a pseudogene. Nevertheless, we have obtained evidence suggesting that this gene is functional. Indeed, a T-DNA insertion mutant, SALK_045146, displays a lethal phenotype during early rosette stage. Also, both the AtTCTP2 promoter and structural gene are functional, and heterozygous plants show delayed development. AtTCTP1 cannot compensate for the loss of AtTCTP2, since the accumulation levels of the AtTCTP1 transcript are even higher in heterozygous plants than in wild-type plants. Leaf explants transformed with Agrobacterium rhizogenes harboring AtTCTP2, but not AtTCTP1, led to whole plant regeneration with a high frequency. Insertion of a sequence present in AtTCTP1 but absent in AtTCTP2 demonstrates that it suppresses the capacity for plant regeneration; also, this phenomenon is enhanced by the presence of TCTP (AtTCTP1 or 2) in the nuclei of root cells. This confirms that AtTCTP2 is not a pseudogene and suggests the involvement of certain TCTP isoforms in vegetative reproduction in some plant species.

Peroxidase production from hairy root cultures of red beet (Beta vulgaris)

The genetically transformed roots of red beet have been shown, for the first time, to produce very high levels of peroxidase (POD; EC 1.11.1.7) accounting for 1.21 x 10(6) Units L-1. Of the ten clones established using different strains of Agrobacterium rhizogenes, one was that from the strain LMG-150, three each from A 2/83, A 20/83 and A4. All the clones showed true integration of T-DNA when tested by PCR and Southern hybridization methods. Each clone differed significantly from the others in growth, hormone dependency and POD production where LMG-150 produced highest biomass (140 g FW L-1) as well as POD (ranging from 8000-9000 U g-1 FW and 1.18 x 10(6) U L-1 with a specific activity of 600 U mg-1 protein) on hormone-free medium, both in shake-flask as well as in bioreactor with a further enhancement to 1.21 x 10(6) U L-1 upon the addition of extra calcium chloride (5 mM). PAGE with active staining showed 4 distinct bands of Rm 0.06, 0.16, 0.25, 0.38 and 0.46 in the biomass and bands at Rm 0.06, 0.16, 0.25 and one extra band of Rm 0.575 in the spent medium where isozymes of Rm 0.38 and 0.46 were totally absent. The pH optima and other properties were grossly comparable with the standard horse-radish POD (HRP) with better thermal stability than HRP and therefore, the present source appears to offer a cheaper and additional alternative for the commercial production of POD.

Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes-transformed tissues.

Transgenic Mexican lime [Citrus aurantifolia (Christm.) Swing] plants were regenerated from tissues transformed by Agrobacterium rhizogenes strain A4, containing the wild-type plasmid pRiA4 and the binary vector pESC4 with nos-npt II and cab-gus genes. Transgenic shoots were generated by two different approaches. The first approach used internodal stem segments cocultured with A. rhizogenes. These were placed onto regeneration medium containing Murashige and Skoog salts and B5 organic compounds supplemented with 8 g ⋅ l-1 agar, 7.5 mg ⋅ l-1 6-benzylaminopurine, 1.0 mg ⋅ l-1 -naphthaleneacetic acid, 300 mg ⋅ l-1 cefotaxime and 80 mg ⋅ l-1 kanamycin as a selective agent, and incubated under continuous light at 25 °C. Under these conditions, 76% of the explants produced shoots directly with no hairy root phase, with a mean of 1.3 shoots per explant, and 88% of these shoots were genetically transformed as determined by ß-glucuronidase (GUS) assays. In the second approach, segments of transformed roots (15 mm long) obtained from internodal stem segments cocultured with A. rhizogenes were cultured on the above regeneration medium under similar conditions. Forty-one percent of these transformed root segments produced adventitious shoots, with a mean of 2.2 shoots per explant and with 90% of shoots transformed. GUS activity was evident in the transformed roots and in all parts of both transformed shoots and regenerated plants. The presence of the npt II and rolB genes in the regenerated plants was confirmed by PCR analysis. The presence of the npt II gene in the regenerated plants was also confirmed by Southern blot. Using these transformation systems, more than 300 Mexican lime transgenic plants were obtained, 60 of which were adapted to growing in soil.

Changes in peroxidase and polypeptide profiles in Nicotiana tabacum L. transformed with Agrobacterium rhizogenes / Mudanças nos perfis de peroxidase e polipeptídeos em Nicotiana tabacum L. transformada com Agrobacterium rhizogenes

Morphological and proteic modifications in Nicotiana tabacum L. transformed by Agrobacterium rhizogenes were evaluated by the comparison of normal and transformant plants regenerated from hairy-roots formed by the strains A4 or IB-642 of A. rhizogenes. Changes in apical dominance were observed in IB-642 transformants, which exhibited an abnormal development of axiliary buds. The electrophoretic analysis indicated an increase in peroxidase activity and the induction of several isozymes of this complex in the transformants. The SDS-PAGE patterns comparison allows to identify several changes, specially, the increase in 31-33 and 54 kD polypeptides in the transformants.. Biochemical analysis suggests the induction of a pathogen or stress like response of the transformants due to the high auxin concentration codified by A. rhizogenes T-DNA incorporated to the plant genome.

Modificações morfológicas e proteicas em transformantes de Nicotiana tabacum L. com Agrobacterium rhizogenes foram avaliadas através da comparação de plantas normais e transformantes regeneradas a partir de raízes pilosas formadas pelas linhagens A4 ou IB-642. Mudanças na dominância apical foram observadas em transformantes com IB-642, os quais exibiram um desenvolvimento anormal dos brotos axilares. As análises eletroforéticas mostraram um aumento na atividade de peroxidase e indução de várias isoenzimas deste complexo nos transformantes. A comparação dos perfis de SDS-PAGE permitiu identificar várias modificações, especialmente, o aumento dos polipeptídios de 31-33 e de 54 kD nos transformantes. As análises bioquímicas sugerem a indução de uma resposta do tipo patógeno ou estresse devida a alta concentração de auxina codificada pelo T-DNA de A. rhizogenes incorporado ao genoma.

Changes in peroxidase and polypeptide profiles in Nicotiana tabacum L. transformed with Agrobacterium rhizogenes

Morphological and proteic modifications in Nicotiana tabacum L. transformed by Agrobacterium rhizogenes were evaluated by the comparison of normal and transformant plants regenerated from hairy-roots formed by the strains A4 or IB-642 of A. rhizogenes. Changes in apical dominance were observed in IB-642 transformants, which exhibited an abnormal development of axiliary buds. The electrophoretic analysis indicated an increase in peroxidase activity and the induction of several isozymes of this complex in the transformants. The SDS-PAGE patterns comparison allows to identify several changes, specially, the increase in 31-33 and 54 kD polypeptides in the transformants.. Biochemical analysis suggests the induction of a pathogen or stress like response of the transformants due to the high auxin concentration codified by A. rhizogenes T-DNA incorporated to the plant genome.

Modificações morfológicas e proteicas em transformantes de Nicotiana tabacum L. com Agrobacterium rhizogenes foram avaliadas através da comparação de plantas normais e transformantes regeneradas a partir de raízes pilosas formadas pelas linhagens A4 ou IB-642. Mudanças na dominância apical foram observadas em transformantes com IB-642, os quais exibiram um desenvolvimento anormal dos brotos axilares. As análises eletroforéticas mostraram um aumento na atividade de peroxidase e indução de várias isoenzimas deste complexo nos transformantes. A comparação dos perfis de SDS-PAGE permitiu identificar várias modificações, especialmente, o aumento dos polipeptídios de 31-33 e de 54 kD nos transformantes. As análises bioquímicas sugerem a indução de uma resposta do tipo patógeno ou estresse devida a alta concentração de auxina codificada pelo T-DNA de A. rhizogenes incorporado ao genoma.

Changes in peroxidase and polypeptide profiles in Nicotiana tabacum L. transformed with Agrobacterium rhizogenes

Morphological and proteic modifications in Nicotiana tabacum L. transformed by Agrobacterium rhizogenes were evaluated by the comparison of normal and transformant plants regenerated from hairy-roots formed by the strains A4 or IB-642 of A. rhizogenes. Changes in apical dominance were observed in IB-642 transformants, which exhibited an abnormal development of axiliary buds. The electrophoretic analysis indicated an increase in peroxidase activity and the induction of several isozymes of this complex in the transformants. The SDS-PAGE patterns comparison allows to identify several changes, specially, the increase in 31-33 and 54 kD polypeptides in the transformants.. Biochemical analysis suggests the induction of a pathogen or stress like response of the transformants due to the high auxin concentration codified by A. rhizogenes T-DNA incorporated to the plant genome.

Modificações morfológicas e proteicas em transformantes de Nicotiana tabacum L. com Agrobacterium rhizogenes foram avaliadas através da comparação de plantas normais e transformantes regeneradas a partir de raízes pilosas formadas pelas linhagens A4 ou IB-642. Mudanças na dominância apical foram observadas em transformantes com IB-642, os quais exibiram um desenvolvimento anormal dos brotos axilares. As análises eletroforéticas mostraram um aumento na atividade de peroxidase e indução de várias isoenzimas deste complexo nos transformantes. A comparação dos perfis de SDS-PAGE permitiu identificar várias modificações, especialmente, o aumento dos polipeptídios de 31-33 e de 54 kD nos transformantes. As análises bioquímicas sugerem a indução de uma resposta do tipo patógeno ou estresse devida a alta concentração de auxina codificada pelo T-DNA de A. rhizogenes incorporado ao genoma.