Establishment of highly efficient Agrobacterium rhizogenes-mediated transformation for Stevia Rebaudiana Bertoni explants

Leaves and internodes from Stevia rebaudiana Bertoni plants growing in different conditions were used for transformation with two strains of Agrobacterium rhizogenes: ATCC 15384 and LBA 9402. Hairy roots formation was observed and the percentage of the transformed explants depended on the type of explant, time of inoculation and inoculum concentration.Inoculation of explants from ex vitro and in vitro plants with LBA9402 strain led to higher efficiency of transformation than inoculation with ATCC 15384 strain. Growth rate of hairy roots in liquid culture was assessed under light and dark conditions. It was found that the growth of hairy roots decreased significantly under light conditions. Transformation of hairy roots growing in different culture conditions was confirmed at the molecular level using PCR method with primers constructed against rolB and rolC genes from A rhizogenes. Growth rate of hairy roots in liquid culture was assessed under light and dark conditions. It was found that the growth of hairy roots decre ased significantly under light conditions . Transformation of hairy roots growing in different culture conditions was confirmed at the molecular level using PCR method with primers constructed against rolB and rolC genes from A . rhizogene

Influence of anti- and prooxidants on rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured in vitro

The enrichment with antioxidants (glutathione or ascorbate) or prooxidants (alloxan, methylviologen, hydrogen peroxide) of root inducing medium significantly decreased rhizogenesis frequency (alloxan, hydrogen peroxide) or inhibited roots regeneration (ascorbate, methylviologen) during the in vitro culture of Mesembryanthemum crystallinum L. hypocotyls. The adventitious roots morphology, root hairs length and density, was also influenced. Changes in the rhizogenesis course were related to the differences in hydrogen peroxide concentration during following days of culture between explants exhibiting morphogenic potential and those without the ability to form adventitious roots. In explants with morphogenic potential, rhizogenesis induction was always accompanied by a high level of hydrogen peroxide followed by the decrease in H_{2}O_{2} content in following days. In contrast, in the explants without regeneration potential, the level of hydrogen peroxide was increasing during the culture period. Activity patterns of superoxide dismutase (SOD) and guaiacol peroxidase (POX) in the following days of culture were similar in the explants exhibiting regeneration potential cultured on different media. Total activity of SOD decreased during initial days of culture and then increased due to the activation of additional SOD isoform described as MnSODII. The activity of POX was low during the rhizogenesis induction, and then increased during following days of culture; the increase was correlated with the decrease in hydrogen peroxide content. In the explants without the ability to regenerate roots, the total activity of SOD was low throughout the whole culture period, whereas the POX activity was significantly higher than in hypocotyls with regeneration potential. It might be concluded that the increase in hydrogen peroxide during initial stages of rhizogenesis and the induction of MnSODII are prerequisites for adventitious roots formation from hypocotyls of M. crystallinum, independently in the presence of anti- or prooxidant in the culture medium

The localization of NADPH oxidase and reactive oxygen species in in vitro-cultured Mesembryanthemum crystallinum L. hypocotyls discloses their differing roles in rhizogenesis

This work demonstrated how reactive oxygen species (ROS) are involved in the regulation of rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured on a medium containing 1-naphthaleneacetic acid (NAA). The increase of NADPH oxidase activity was correlated with an increase of hydrogen peroxide (H2O2) content and induction of mitotic activity in vascular cylinder cells, leading to root formation from cultured hypocotyls. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, inhibited H2O2 production and blocked rhizogenesis. Ultrastructural studies revealed differences in H2O2 localization between the vascular cylinder cells and cortex parenchyma cells of cultured explants. We suggest that NADPH oxidase is responsible for H2O2 level regulation in vascular cylinder cells, while peroxidase (POD) participates in H2O2 level regulation in cortex cells. Blue formazan (NBT) precipitates indicating superoxide radical (O2 •−) accumulation were localized within the vascular cylinder cells during the early stages of rhizogenesis and at the tip of root primordia, as well as in the distal and middle parts of newly formed organs. 3,3′-diaminobenzidine (DAB) staining of H2O2 was more intense in vascular bundle cells and in cortex cells. In newly formed roots, H2O2 was localized in vascular tissue. Adding DPI to the medium led to a decrease in the intensity of NBT and DAB staining in cultured explants. Accumulation of O2 •− was then limited to epidermis cells, while H2O2 was accumulated only in vascular tissue. These results indicate that O2 •− is engaged in processes of rhizogenesis induction involving division of competent cells, while H2O2 is engaged in developmental processes mainly involving cell growth

Rhizobium rhizogenes-Mediated Genetic Transformation of Antidiabetic Plants

Rhizobium rhizogenes induces a disease syndrome in infected plants commonly known as hairy roots (HR) production. These unique organs are considered of transgenic nature since they derived after horizontal gene transfer from the bacterial root-inducing (Ri) plasmid to the plant genome. The HR culture represents a major biotechnological tool facilitating the production of plant-derived compounds exhibiting interesting features for pharmaceutical, cosmetics and food industries. Moreover, HR cultures can be used for studying the biosynthetic pathways of plant-derived molecules or investigating interactions occurring during phytoremediation. Among this wide range of HR applications, it is important to point out the possibility to synthesize some antidiabetic compounds which are of special interest because diabetes is one of the major and frequently occurring debilitating diseases of the human body in the world. The use of available synthetic antidiabetic drugs has several limitations, including drug resistance, side effects, or toxicity. Therefore, the availability of new antidiabetic drugs from a natural source is of big importance. Stevia rebaudiana Bertoni is one of the promising plant species, constituting the source of desired plant molecules. This plant accumulates steviol glycosides (SGs), which are responsible for the sweet flavour of stevia leaves. These secondary metabolites are considered metabolically inert and therefore could be a safe substitute for sugar for diabetic people. In this chapter, we would like to present recent achievements in the range of antidiabetic substances production via genetic transformation of plants using R. rhizogenes strains. In this context, a key role of the so-called rol genes, carried by Ri plasmids, in the biosynthesis of various secondary metabolites is also discussed. Our aim is to bring closer the issue of HR cultures and their big potential in the pharmacognosy of antidiabetic plants

Engagement of light stress and osmotic stress o steviol glycosides biosynthesis and accumulation in hairy roots from Stevia reabudiana Bertoni. Michalec-Warzecha Zaneta, Zastawny Olga Pistelli Laura, Ruffoni Barbara, Dziurka Michal, Libik-Konieczny Marta,

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Steviol glycosides profile in Stevia rebaudiana Bertoni hairy roots cultured under oxidative stress-inducing conditions

The ability to synthesize particular steviol glycosides (SvGls) was studied in Stevia rebaudiana Bertoni hairy roots (HR) grown in the light or in the dark under the influence of different osmotic active compounds. Manipulation of culture conditions led to changes in the morphology and growth rate of HR, as well as to an increase in oxidative stress manifested as an enhancement in endogenous hydrogen peroxide concentration in the cultured samples. The highest level of H2O2 was noted in HR cultured under light or in the medium with the highest osmotic potential. This correlated with the highest increase in the expression level of ent-kaurenoic acid hydroxylase, responsible for the redirection of metabolic route to SvGls biosynthesis pathway. An analysis of transcriptional activity of some UDPglucosyltransferase (UGT85c2, UGT74g1, UGT76g1) revealed that all of them were upregulated due to the manipulation of culture conditions. However, the level of their upregulation depended on the type of stress factor used in our experiment. Analysis of SvGls content revealed that HR grown under all applied conditions were able to synthesize and accumulate several SvGls but their concentration differed between the samples across the different conditions. The level of rebaudioside A concentration exceeded the content of stevioside in HR in all tested conditions. Concomitantly, the presence of some minor SvGls, such as steviolbioside and rebaudioside F, was confirmed only in HR cultured in the lowest osmotic potential of the medium while rebaudioside D was also detected in the samples cultured in the media supplemented with NaCl or PEG.Key Points● Several steviol glycosides are synthesized in hairy roots of S. rebaudiana.● Light or osmotic factors cause enhancement in oxidative stress level in hairy roots.● It correlates with a significant increase in the level of KAH expression.● UGTs expression and steviol glycosides content depends on culture conditions

Engagements of stress factors on steviol glycosides biosynthesis in in vitro cultures of Stevia rebaudiana

Engagements of stress factors on steviol glycosides biosynthesis in in vitro cultures of Stevia rebaudian