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Genetic transformation is a powerful tool to study gene function, secondary metabolism pathways, and molecular breeding in crops. Cotton ( Gossypium hirsutum L.) is one of the most important economic crops in the world. Current cotton transformation methods take at least seven to culture and are labor-intensive and limited to some cultivars. In this study, we first time achieved plantlet regeneration of cotton via embryogenesis from transformed hairy roots. We inoculated the cotyledon explants of a commercial cultivar Zhongmian-24 with Agrobacterium rhizogenes strain AR1193, harboring a binary vector pBI-35S::GFP that contained the NPT II (neomycin phosphotransferase) gene and the GFP (green fluorescent protein) gene as a fluorescent marker in the T-DNA region. 82.6% explants produced adventitious roots, of which 53% showed GFP expression after transformation. 82% of transformed hairy roots produced embryonic calli, 12% of which regenerated into stable transformed cotton plants after 7 months of culture. The integration of GFP in the transformed cotton genomes were confirmed by PCR (Polymerase chain reaction) and Southern blot analysis as well as the stable expression of GFP were also detected by semi-quantitative RT-PCR analysis. The resultant transformed plantlets were phenotypically, thus avoiding Ri syndrome. Here we report a stable and reproducible method for A. rhizogenes -mediated transformation of cotton using cotyledon as explants, which provides a useful and reliable platform for gene function analysis of cotton.

Agrobacterium rhizogenes –mediated (ARM) transformation is an efficient and powerful tool to generate transgenic roots to study root-related biology. For loss-of-function studies, transgenic-root-induced indel mutations by CRISPR/Cas9 only with homozygous/biallelic mutagenesis can exhibit mutant phenotype(s) (excluding recessive traits). However, a low frequency of homozygous mutants was produced by a constitutive promoter to drive Cas9 expression. Here, we identified a highly efficient Arabidopsis thaliana gamma- g lutamyl c ysteine s ynthetase promoter, termed AtGCSpro , with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species. AtGCSpro achieved higher homozygous/biallelic mutation efficiency than the most widely used CaMV 35S promoter in driving Cas9 expression in soybean, Lotus japonicus , and tomato roots. Using the p AtGCSpro -Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p 2 × 35Spro -Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the p AtGCSpro -Cas9 system used in ARM transformation, especially its great potential in diploids with multiple-copy genes targeted mutations and polyploid plants with multiplex genome editing. AtGCSpro is conservatively active in various eudicots species, suggesting that AtGCSpro might be applied in a wide range of dicots species.

Background Chickpea ( Cicer arietinum L.), an important legume crop is one of the major source of dietary protein. Developing an efficient and reproducible transformation method is imperative to expedite functional genomics studies in this crop. Here, we present an optimized and detailed procedure for Agrobacterium rhizogenes -mediated root transformation of chickpea. Results Transformation positive roots were obtained on selection medium after two weeks of A. rhizogenes inoculation. Expression of green fluorescent protein further confirmed the success of transformation. We demonstrate that our method adequately transforms chickpea roots at early developmental stage with high efficiency. In addition, root transformation was found to be genotype-independent and the efficacy of our protocol was highest in two (Annigiri and JG-62) of the seven tested chickpea genotypes. Next, we present the functional analysis of chickpea hairy roots by expressing Arabidopsis TRANSPARENT TESTA 2 ( AtTT2 ) gene involved in proanthocyanidins biosynthesis. Overexpression of AtTT2 enhanced the level of proanthocyanidins in hairy roots that led to the decreased colonization of fungal pathogen, Fusarium oxysporum . Furthermore, the induction of transgenic roots does not affect functional studies involving infection of roots by fungal pathogen. Conclusions Transgenic roots expressing genes of interest will be useful in downstream functional characterization using reverse genetics studies. It requires 1 day to perform the root transformation protocol described in this study and the roots expressing transgene can be maintained for 3–4 weeks, providing sufficient time for further functional studies. Overall, the current methodology will greatly facilitate the functional genomics analyses of candidate genes in root-rhizosphere interaction in this recalcitrant but economically important legume crop.

Limonium bicolor is a perennial herbaceous plant belonging to the Plumbaginaceae family. It can be used as a dried flower or in cut flower arrangements and serves as a model recretohalophyte. Its genome sequencing has been recently completed. However, the research on L. bicolor is limited by the absence of a highly efficient genetic transformation system. In this study, we established a highly efficient Agrobacterium rhizogenes-mediated L. bicolor genetic transformation method. The transgenic hairy roots were induced from the hypocotyl of L. bicolor using A. rhizogenes strain K599 harboring pRdGa4Cas9 plasmid (which carries an expression cassette of 35S::DsRed2). The transgenic shoots were regenerated from hairy root segments (~0.1 cm diameter), and induction efficiency was achieved at 100%. The transgenic shoots with 4–5 rosette leaves were directly planted into the soil to induce the transgenic roots. Therefore, transgenic plantlets were produced. The DsRed2 can be used as a reliable reporter gene in screening transgenic plantlets. Furthermore, we also established a CRISPR/Cas9 system in L. bicolor employing the A. rhizogenes-mediated genetic transformation approach. The highly efficient transformation method and CRIPSP/Cas9 system established will provide a valuable tool for functional genomics investigation and trait improvement in L. bicolor.

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.

Oroxylum indicum (Shyonak) is used as a medicinal herb in the ethnomedicinal system of Asia for prevention and treatment of diseases like respiratory disorder, gastric ulcers, diabetes, cancer, rheumatoid problems, etc. Prior studies have linked the pharmacological properties to flavonoids present in seeds, stems, and root bark. The current research aims to develop a long-term stable system of transgenic hairy roots of O. indicum using Agrobacterium rhizogenes strain ATCC 15834. The best medium, explant type, infection time, co-cultivation period, and anti-browning chemicals were optimized during the hairy root induction. The maximum transformation frequency (49.16 ± 2.44%) was documented for leaf explant after 30 days of inoculation on a semi-solid B5 medium with cefotaxime (200 mg/l), at 48-h co-cultivation period followed by a 2-h infection phase. Explant browning also hindered biomass growth. Hence, different anti-oxidants were used to control cell biomass browning. A transformation frequency up to 65.8% was obtained in B5 media with cysteine (3.71 mM). Successful transformation of the hairy root lines was confirmed using PCR amplification using A. rhizogenes virD, aux1, rolA, rolB, and rolC genes specific primers. Subsequently, in vitro production of four flavonoids viz., baicalein, hispidulin, scutellarin, and biochanin-A, were quantified in hairy root culture of O. indicum and compared with roots of two months old greenhouse cultivated plantlets and roots of in vitro regenerated plants. Higher quantities of baicalein, hispidulin, and scutellarin (viz., 11.47 ± 1.5 μg/g DW, 10.45 ± 0.27 μg/g DW, and 24.75 ± 0.85 μg/g DW, respectively) were quantified in the hairy root cultures of O. indicum.

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.

Virulent strains of Agrobacterium rhizogenes (A. rhizogenes) contain Ri plasmids responsible for hairy root development on susceptible plants. Ri plasmid possesses different gene segments. T-DNA region of Ri plasmid pRi2659 in A. rhizogenes strain K599 carries 11 functional genes/coding regions. In this study, we cloned a complete orf3-coding region in T-DNA of plasmid pRi2659, which is 1479 bp in length and encodes 492 amino acid residues. Then, we developed an orf3-containing expression vector named pRI101-AN-orf3 and generated the orf3-transgenic tobacco plants via A. tumefaciens-mediated method. The orf3-transgenic tobacco displayed altered morphological characters including shortened internodes, dwarfisms, more branches, shortened leaf blade length, early flowering, and weak roots. Compared to those of untransformed tobacco, the contents of two endogenous plant hormones, auxins (IAA) and abscisic acid, were decreased whereas the cytokinin content was increased but gibberellic acid content was not consistently obviously changed. Besides, we also transferred orf3 into Arabidopsis thaliana using floral-dip method and obtained orf3-transgenic Arabidopsis homozygous offspring. The orf3-transgenic Arabidopsis displayed morphological characters of dwarfism and more branches similar to those of the orf3-transgenic tobacco. Together, these results indicate that K599 orf3 possesses the functions of altering plant phenotypes and promotes plants to become dwarf and branch.

Hairy roots derived from the infection of a plant by ) bacteria, can be obtained from a wide variety of plants and allow the production of highly diverse molecules. Hairy roots are able to produce and secrete complex active glycoproteins from a large spectrum of organisms. They are also adequate to express plant natural biosynthesis pathways required to produce specialized metabolites and can benefit from the new genetic tools available to facilitate an optimized production of tailor-made molecules. This adaptability has positioned hairy root platforms as major biotechnological tools. Researchers and industries have contributed to their advancement, which represents new alternatives from classical systems to produce complex molecules. Now these expression systems are ready to be used by different industries like pharmaceutical, cosmetics, and food sectors due to the development of fully controlled large-scale bioreactors. This review aims to describe the evolution of hairy root generation and culture methods and to highlight the possibilities offered by hairy roots in terms of feasibility and perspectives.

Significance We communicate the rather remarkable observation that among 291 tested accessions of cultivated sweet potato, all contain one or more transfer DNA (T-DNA) sequences. These sequences, which are shown to be expressed in a cultivated sweet potato clone (“Huachano”) that was analyzed in detail, suggest that an Agrobacterium infection occurred in evolutionary times. One of the T-DNAs is apparently present in all cultivated sweet potato clones, but not in the crop’s closely related wild relatives, suggesting the T-DNA provided a trait or traits that were selected for during domestication. This finding draws attention to the importance of plant–microbe interactions, and given that this crop has been eaten for millennia, it may change the paradigm governing the “unnatural” status of transgenic crops. Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world’s arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions ( Ib T-DNA1 and Ib T-DNA2) are present in the cultivated sweet potato ( Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. Ib T-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase ( iaaM ), indole-3-acetamide hydrolase ( iaaH ), C-protein ( C-prot ), and agrocinopine synthase ( Acs ) genes of Agrobacterium spp. Ib T-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. Ib T-DNA2 contained at least five ORFs with significant homology to the ORF14 , ORF17n , rooting locus ( Rol ) B/RolC , ORF13 , and ORF18/ORF17n genes of A. rhizogenes . Ib T-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops.