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

Pattern of antioxidant enzyme activities and hydrogen peroxide content during developmental stages of rhizogenesis from hypocotyl explants of Mesembryanthemum crystallinum L.

KEY MESSAGE: H(2)O(2)is necessary to elicit rhizogenic action of auxin. Activities of specific catalase and manganese superoxide dismutase forms mark roots development. ABSTRACT: Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots on medium containing 2,4-dichlorophenoxyacetic acid. Explants became competent to respond to the rhizogenic action of auxin on day 3 of culture, when hydrogen peroxide content in cultured tissue was the highest. l-Ascorbic acid added to the medium at 5 μM lowered the H(2)O(2) level, inhibited rhizogenesis and induced non-regenerative callus, suggesting that certain level of H(2)O(2) is required to promote root initiation. Coincident with the onset of rhizogenic determination, meristemoids formed at the periphery of the hypocotyl stele and the activity of the manganese form of superoxide dismutase, MnSOD-2 was induced. Once induced, MnSOD-2 activity was maintained through the post-determination phase of rooting, involving root growth. MnSOD-2 activity was not found in non-rhizogenic explants maintained in the presence of AA. Analyses of the maximum photochemical efficiency of photosystem II and the oxygen uptake rate revealed that the explants were metabolically arrested during the predetermination stage of rhizogenesis. Respiratory and photosynthetic rates were high during root elongation and maturation. Changes in catalase and peroxidase activities correlated with fluctuations of endogenous H(2)O(2) content throughout rhizogenic culture. Expression of a specific CAT-2 form accompanied the post-determination stage of rooting and a high rate of carbohydrate metabolism during root growth. On the other hand, the occurrence of MnSOD-2 activity did not depend on the metabolic status of explants. The expression of MnSOD-2 activity throughout root development seems to relate it specifically to root metabolism and indicates it as a molecular marker of rhizogenesis in M. crystallinum

Pathways of ROS homeostasis regulation in "Mesembryanthemum crystallinum" L. calli exhibiting differences in rhizogenesis

A comparison of the hydrogen peroxide (H2O2) content, proline and betacyanin concentration and activities of some antioxidant enzymes (catalase, superoxide dismutase, guaiacol and ascorbate peroxidases) was made in Mesembryanthemum crystallinum L. calli differing in rhizogenic potential. Callus was induced from hypocotyls of 10-day-old seedlings on a medium containing 1 mg l−1 2,4-dichlorophenoxyacetic acid and 0.2 mg l−1 kinetin, which was either supplemented with 40 mM NaCl (CIM-NaCl medium) or did not contain any salt (CIM medium). The callus obtained on CIM-NaCl was rhizogenic, whereas the callus induced on the medium without salt was non-rhizogenic throughout the culture. The rhizogenic callus differed from the non-rhizogenic callus in lower betacyanin and H2O2 content, but the rhizogenic callus displayed a higher proline level. The activity of H2O2 scavenging enzymes, such as catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (POD), was markedly higher in the rhizogenic callus than in the non-rhizogenic callus, but the total activity of superoxide dismutase (SOD) was higher in the non-rhizogenic callus than in the rhizogenic callus. Aminotriazole (CAT inhibitor) and diethyldithiocarbamate (SOD inhibitor) were added solely to the CIM and CIM-NaCl media to manipulate the concentration of reactive oxygen species (ROS) in the cultured tissues. Both CAT and SOD inhibitors brought about an increase in H2O2 content in calli cultured on CIM-NaCl and the loss of rhizogenic potential. Conversely, the addition of inhibitors to the medium without salt led to a decrease in H2O2 content. This corresponded with a significant decrease in the endogenous concentration of betacyanins, but did not change the lack of rhizogenic ability

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