Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

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.

TÉCNICAS DE TRANSFORMAÇÃO E LINHAGENS De Agrobacterium rhizogenes NA PRODUÇÃO DE HAIRY ROOTS DE Ocimum basilicum L.

O manjericão é uma planta medicinal e seus metabólitos especiais são utilizados com fins terapêuticos e industriais. Este trabalho teve por objetivo avaliar técnicas e linhagens de Agrobacterium rhizogenes na produção de raízes transformadas de duas variedades de Ocimum basilicum L., Dark Opal (roxo) e Minete Anão (verde). Verificou-se o efeito da inoculação com linhagens de A. rhizogenes selvagens (A4, 8196, 9402, 2659, 2659 G, 17242, LBA, 15834) ou transformadas (R1601) em plantas inteiras ou explantes (folhas e segmentos de caule) de manjericão, cultivados em casa de vegetação ou in vitro. A inoculação com as linhagens LBA e 8196 possibilitou redução no número de explantes de folha de manjericão-verde oxidados, quando comparado ao controle. Dentre as linhagens selvagens, as que proporcionaram melhor indução na produção de raízes transformadas foram A4, LBA e 8196, respectivamente. Entretanto, a cepa R1601 apresentou a melhor resposta. Raízes foram eficientemente induzidas mediante a inoculação de explantes de manjericão-roxo e manjericão-verde e expressaram o fenótipo típico de raízes em cabeleira (hairy roots). As culturas clonais de manjericão-verde apresentaram rápido crescimento em meio de cultura livre de reguladores de crescimento.Palavras-chave: agrobactéria, cultura de raízes, manjericão, plantas medicinais. TRANSFORMATION TECHNIQUES AND Agrobacterium rhizogenes Strains In The Production Of Hairy Roots Of Ocimum basilicum L. ABSTRACT:Basil is a medicinal plant and its special metabolites are used for therapeutic or industrial purposes. The aim of this work was to evaluate Agrobacterium rhizogenes techniques and strains in the production of hairy roots of two varieties of Ocimum basilicum L., Dark Opal (purple) and Dwarf Racer (green). The effects of inoculation of A. rhizogene (A4, 8196, 9402, 2659, 2659, 2659 G, 17242, LBA, 15834) or (R1601) lines on whole plants or explants (leaves and stem segments) of basil, grown in a greenhouse or in vitro, in the production of hairy roots and calluses made by subcultures. Inoculation with the LBA and 8196 lines allowed a reduction in the number of oxidized basil-green leaf explants when compared to the control. Among the wild strains, those that had best ability to induce hairy root of hairy roots were A4, LBA and 8196, respectively. However, the R1601 laboratory strain had the best response. Roots were efficiently induced by the inoculation of explants basil-purple and basil-green and expressed the typical phenotype of hairy roots. Green-basal clonal cultures showed rapid growth in culture medium free of growth regulators.Keywords: agrobacterim, basil, medicinal plants, root culture. DOI:

Induction of Platycodon grandiflorum hairy roots through the mediation of four Agrobacterium rhizogenes strains

Balloon flower (Platycodon grandiflorum (Jacq.) A. DC.), the only species in Platycodon genus (Campanulaceae), is mainly distributed in East Asia. The rhizomes of P. grandiflorum, a traditional herbal medicine, have been widely used for the treatment of cough, sore throat, asthma, tuberculosis and other diseases. Recently, pharmacological researches identified important biological activities compounds in the rhizomes. Thus, to study and extract valuable compounds, a hairy root induced technique was achieved on P. grandiflorum for stable material with fast growth rates (in hormone-free media) and metabolites production. To achieve this, the “natural genetic tool” Agrobacterium rhizogenes, which can transfer DNA segments into genome of plant, was exploited. The results suggested two (A. rhizogenes ATCC 15834 and C34) of four A. rhizogenes strains could induce hairy roots. RolB and rolC genes, which are responsible for the induction of hairy roots, were inserted into the genome of hairy roots. Leaves had the highest infection frequency of hairy root induction 100 %. The optimization of protocol, including time of immersion and co-culture, had the best results with 10 and 15 mins (10 mins for A. rhizogenes ATCC 15834 and 15 mins for A. rhizogenes C34) and 72 hours, respectively. In the future, this protocol, which was described in this paper, should be useful for studying and isolating valuable compounds from P. grandiflorum hairy root cultures.

Hairy induction from Impatiens balsamina L. using fourteen Agrobacterium rhizogenes strains

Impatiens balsamina L. is a plant commonly grown in Vietnam. It has long been used as traditional medicine. All of the secondary metabolites produced by Impatiens balsamina L. root possessed biological activities. The main advantage of using hairy root cultures is their ability of growing fast in defined basal media without supplementation of phytohormones. The aim of this research is to invest some factors that could affect on the hairy root production such as A. rhizogenes strain, type of tissue, OD concentration, immersion time and co-cultivation time. The results showed that 3 strain A. rhizogenes C02, C18 and C26 isolated from nature in Vietnam could induce the best hairy root formation at the 0.5–1.0 OD concentration bacteria with 5’ of immersion time and 72 hours of co-cultivating. Among them, C02 is the strain that could offer the best result in B5 medium