Efficient Editing of CSLD2 Orthologue by CRISPR/Cas9 Affects Cell Morphogenesis of Root Hair in Spinach

CRISPR/Cas9 is a valuable tool and has been extensively employed to perform gene editing in plants. However, CRISPR/Cas9 has not been successfully used in spinach, an important leafy vegetable crop. Here, we precisely edited Spo23361 and Spo10340, two cellulose synthase-like D (CSLD) genes involved in root hair formation of spinach hairy roots, using CRISPR/Cas9 system. Four mutation types (i.e., replacement, insertion, deletion, and combined mutations) were observed, among which the deletion accounted for the vast majority (about 64.1%). Mutation rate differed largely among different targets. Seven homozygous/bi-allelic and eight heterozygous/chimeric mutated lines of Spo23361 were obtained from 15 independent transgenic hairy root lines. All of the seven homozygous/bi-allelic lines displayed bulking and short root hairs, which exhibited the characteristics of Arabidopsis csld2 mutants. Thirteen heterozygous/chimeric mutated lines, but no homozygous/bi-allelic lines, of Spo10340 were obtained from 15 independent transgenic hairy root lines, all of which showed similar phenotype of root hair with normal hairy roots. The transcriptomic analysis further revealed that multiple gene expressions for cell wall modulation and membrane trafficking were disturbed, which might result in the inhibition of root hair growth in Spo23361 mutants. Our results indicate that Agrobacterium rhizogenes-mediated transformation using CRISPR/Cas9 is a simple and efficient genome editing tool in spinach. It lays a solid foundation for large-scale genome editing in spinach in future.

An Efficient Root Transformation System for Recalcitrant Vicia sativa

Common vetch (Vicia sativa) is a multi-purpose legume widely used in pasture and crop rotation systems. Vetch seeds have desirable nutritional characteristics and are often used to feed ruminant animals. Although transcriptomes are available for vetch, problems with genetic transformation and plant regeneration hinder functional gene studies in this legume species. Therefore, the aim of this study was to develop a simple, efficient and rapid hairy root transformation system for common vetch to facilitate functional gene analysis. At first, we infected the hypocotyls of 5-day-old in vitro or in vivo, soil-grown seedlings with Rhizobium rhizogenes K599 using a stabbing method and produced transgenic hairy roots after 24 days at 19 and 50% efficiency, respectively. We later improved the hairy root transformation in vitro by infecting different explants (seedling, hypocotyl-epicotyl, and shoot) with R. rhizogenes. We observed hairy root formation at the highest efficiency in shoot and hypocotyl-epicotyl explants with 100 and 93% efficiency, respectively. In both cases, an average of four hairy roots per explant were obtained, and about 73 and 91% of hairy roots from shoot and hypocotyl-epicotyl, respectively, showed stable expression of a co-transformed marker β-glucuronidase (GUS). In summary, we developed a rapid, highly efficient, hairy root transformation method by using R. rhizogenes on vetch explants, which could facilitate functional gene analysis in common vetch.

Development of Hairy Root Cultures for Biomass and Triterpenoid Production in Centella asiatica

Centella asiatica (Apiaceae) is a tropical/subtropical medicinal plant, which contains a variety of triterpenoids, including madecassoside, asiaticoside, madecassic acid, and asiatic acid. In this study, we tested the efficiency of hairy root (HR) induction in C. asiatica from leaf and petiole explants. Leaves and petioles collected from C. asiatica plants were suspended in agro-stock for 30 min and co-cultured with Agrobacterium rhizogenes for 3 days to induce HR formation. The transformation efficiency of leaf and petiole explants was approximately 27% and 12%, respectively. A total of 36 HR lines were identified by PCR-based amplification of rol genes, and eight of these lines were selected for further analysis. Among all eight HR lines, the petiole-derived lines HP4 and HP2 displayed the highest growth index (37.8) and the highest triterpenoids concentration (46.57 mg∙g−1), respectively. Although triterpenoid concentration was >2-fold higher in leaves than in petioles of C. asiatica plants, the accumulation of triterpenoids in petiole-derived HR cultures was 1.4-fold higher than that in leaf-derived HR cultures. Additionally, in both leaf- and petiole-derived HR cultures, terpenoid production was higher in HRs than in adventitious roots. These results demonstrate that the triterpenoid content in the explant does not affect the triterpenoid content in the resultant HRs. The HR culture of C. asiatica could be scaled up to enable the mass production of triterpenoids in bioreactors for the pharmaceutical and cosmetic industries.

Spontaneous Regeneration of Plantlets Derived from Hairy Root Cultures of Lopezia racemosa and the Cytotoxic Activity of Their Organic Extracts

A histological analysis was performed with the aim of elucidating the spontaneous regeneration process of the hairy root lines LRT 2.3 and LRT 6.4, derived from Lopezia racemosa leaf explants and genetically transformed with the Agrobacterium rhizogenes strain ATCC15834/pTDT. The analysis showed both lines regenerate via indirect somatic embryogenesis; LRT 6.4 also regenerated by direct organogenesis. The morphogenic characteristics of the regenerated plantlets from both lines showed the typical characteristics, described previously, including a higher number of axillary shoot formation, short internodes, and plagiotropic roots compared with wild-type seedlings. The regeneration process occurred without the addition of plant growth regulators and was linked to the sucrose concentration in the culture medium. Reducing the sucrose concentration from 3% to 2%, 1%, and 0.5% increased the regeneration rate in LRT 6.4; the effect was less pronounced in LRT 2.3. The cytotoxic activity of different organic extracts obtained from roots and shoots were evaluated in the cancer cell lines HeLa (cervical carcinoma), HCT-15 (colon adenocarcinoma), and OVCAR (ovary carcinoma). The hexane and dichloromethane extracts from roots of both lines showed cytotoxic activity against the HeLa cell line. Only the dichloromethane extract from the roots of PLRT 2.3 showed cytotoxic activity against the OVCAR cell line. None of the methanol extracts showed cytotoxic activity, nor the shoot extracts from any solvent.

Hairy Roots

Agrobacterium rhizogenes induces hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection, when cultured in hormone free medium, show high growth rate and genetic stability. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Several elicitation methods can be used to further enhance the production and accumulation of secondary metabolites. Thus, hairy root culture offer promise for high production and productivity of valuable secondary metabolites in many plants. Hairy roots can also produce recombinant proteins from transgenic roots, and thereby hold immense potential for pharmaceutical industry. Hairy root cultures can be used to elucidate the intermediates and key enzymes involved in the biosynthesis of secondary metabolites, and for phytoremediation due to their abundant neoplastic root proliferation property. Various applications of hairy root cultures and potential problems associated with them are discussed in this chapter.

Hypericum Perforatum L. Hairy Root Extracts – Regulation of Glycemic, Metabolic, Serum Enzyme and Lipid Profile in Stz - Induced Diabetic Rats

Apart from currently available therapeutics for the treatment of diabetes mellitus, much attention has been paid to discover phytochemicals from natural resources, mainly due to their low side-effects. Hypericum perforatum hairy root (HR) transformed with Agrobacterium rhizogenes A4 represent prospective experimental system enriched in xanthones, known as potent antidiabetic agents. Thus, the aim of this study was to evaluate HR extracts for their potential antihyperglycemic activity in streptozotocin (STZ)-induced diabetic rats, also compared to the effects of wild-growing Hyperici herba (HH). We conducted an acute-toxicity study, multiple dose study, and 24h blood glucose measurements after a single dose administration of HH and HR (200 mg/kg) in diabetic rats. Furthermore, we examined the effects of 14-days administration of HH and HR extracts on blood glucose levels, metabolic parameters, enzyme, and lipid status in healthy and diabetic rats. Both extracts produced a fall of about 70% in blood glucose level after 24h of administration. Two-week treatment with HH and HR induced a significant decrease (70-72%) in blood glucose levels. Moreover, we found an improvement of the dysregulated metabolic parameters (body weight, food, and water consumption and urine output). Serum enzyme (AST, ALT, and γ-GT) and lipid profile parameters (CHOL, TAG, and HDL) were also improved by both extracts. These findings might provide a new insight for managing diabetic hyperglycemia and dysregulated serum enzyme and lipid profile, using extracts from transgenic roots cultures from H. perforatum.

Polyscias filicifolia (Araliaceae) Hairy Roots with Antigenotoxic and Anti-Photogenotoxic Activity

Hairy root cultures are considered as a valuable source of bioactive phytoconstituents with expanding applicability for their production. In the present study, hairy root cultures of Polyscias filicifolia (Araliaceae), a traditional Southeast Asian medicinal plant, were established. The transformation with Agrobacterium rhizogenes ATCC 15834 allowed to obtain 15 root lines. The K-1 line, demonstrating the highest growth capabilities, was subjected to further investigations. To enhance the biosynthetic potential of hairy roots, methyl jasmonate elicitation approach was applied (MeJA; at different doses and exposure time), with subsequent transfer of elicited roots to control medium. This strategy resulted in chlorogenic acid production up to 1.59 mg/g dry weight. HPLC-PDA-ESI-MS analysis demonstrated variation in extracts composition and allowed to identify different caffeic and ferulic acid derivatives. Next, cytotoxic, antigenotoxic, and anti-photogenotoxic properties of hairy roots extracts were determined. None of the tested extracts were cytotoxic. In addition, they demonstrated significant antigenotoxic activity with the highest protective potential; up to 52% and 49% of inhibition of induction ratio (IR) induced by the 2-aminoanthracene was revealed for extracts derived from hairy roots elicited for 3 days with 50 µM MeJA and roots elicited for 7 days with 100 µM MeJA and then transferred for 30 days to control medium, respectively. These same extracts exhibited the highest anti-photogenotoxic potential, up to 36% of inhibition of chloropromazine-induced genotoxicity.