Agrobacterium Rhizogenes Research Articles

Response surface methodology-based optimization of hairy roots cultures for in vitro AM production

Arbuscular mycorrhizal fungi can do wonders in promoting the crop growth as well maintaining soil health. In vitro root organ culture technology is an exciting avenue for AM biofertilizer production. This study aims to optimize the key influencing factors for enhancing the hairy root production used for in vitro AMF culturing using response surface methodology, a statistical and mathematical tool used for designing optimization studies. Study uses Rhizobium rhizogenes MTCC 2364 for transformation in carrot explant. Factors considered for optimization are concentration of gelling agent (phytagel), carbon source (sucrose) and pH of the Modified Strullu and Romand medium (MSR). Design Expert software uses second order polynomial regression equation for predicting the outcome of each experiment. Totally, 17 experiments were run following Box-Behnken design and average hairy root length and average side branch emergence were taken as response. ANOVA analysis reveals that the concentration of phytagel and sucrose had a strong influence on root length, while the phytagel and pH of the medium had a strong effect on side branch emergence. Overall, taking into account the both responses, concentration of phytagel had a significant impact on hairy root production. The maximum average hairy root length obtained was 1.12 cm and number of side branches emerged were 6.78 per day. Based on these results, the optimal parameters were MSR medium with 3gL-1 phytagel, 11gL-1 sucrose and a pH of 4 for boosting hairy root development. This study is a cost-effective approach and minimizes the time taken for establishing the hairy root technology.

Further enhancement of cold tolerance in rice seedlings by Piriformospora indica collaborating with plant growth-promoting bacteria: Evidence from the antioxidant defense, osmoregulation, photosynthesis, and related genes

As the global population continues to grow, so does the demand for food. However, cold stress due to global climate change greatly threatens the safe production of rice. Piriformospora indica plays a pivotal role as a functional microbiota in improving cold tolerance in rice. However, the addition of P. indica alone has a limited effect. In this study, an attempt was made to select plant growth-promoting bacteria (Agrobacterium rhizogenes and Bacillus subtilis) as helper bacteria to help P. indica function better and further enhance rice cold tolerance. Under cold stress, the co-addition of the three beneficial microorganisms significantly increased the biomass, photosynthetic performance indicators, osmoregulatory substance contents, and antioxidant enzyme activities of rice. P. indica and helper bacteria significantly reduced the cold stress-induced increases in malondialdehyde content and electrolyte leakage rate in rice leaves and triggered a substantial upregulation of cold-tolerant genes in rice. This suggests that P. indica and helper bacteria can synergistically improve the cold resistance of rice. The findings of the research offer a conceptual foundation and practical assistance for enhancing rice's resilience to cold temperatures.

Variability in growth and biosynthetic activity of Calendula officinalis hairy roots.

Calendula officinalis is a widespread medicinal plant with a sufficiently well-studied chemical composition. Secondary metabolites synthesized by C.officinalis plants have pharmacological value for treating numerous diseases, and various types of aseptic in vitro cultures can be used as a source of these compounds. From this perspective, hairy roots attract considerable attention for the production of bioactive chemicals, including flavonoids with antioxidant activity. This paper shows the possibility of C.officinalis hairy roots obtaining with 100% frequency by Agrobacterium rhizogenes genetic transformation. Hairy root lines differed in growth rate and flavonoid content. In particular, flavonoids were accumulated in the amount of up to 6.68 ± 0.28 mg/g of wet weight. Methyl jasmonate in the concentration of 10 µM inhibited root growth to a small extent but stimulated the synthesis of flavonoids. The antioxidant activity and the reducing power increased in the roots grown in the medium with methyl jasmonate. The strong correlation of antioxidant activity and reducing power with flavonoid content was detected. The influence of extraction conditions on the content of flavonoids in the extracts and their bioactivity was determined. The potent reducing activity of extracts from hairy roots allowed the production of silver nanoparticles, which was confirmed by transmission electron microscopy.

Modeling Of the Structure of the tRolC Protein of Nicotiana tabacum and Its Functional Relation to Other Proteins

The trolC gene is a homologue of the rolC oncogene of Agrobacterium rhizogenes and most likely got into Nicotiana tabacum genome during horizontal gene transfer. This gene is quite conserved and is expressed in young organs, but its functions, like the agrobacterium rolC gene, remain unknown. The putative structure of the tRolC protein of Nicotiana tabacum, predicted using the I-TASSER service, was analyzed in the study. The presence of amino acid residues in the tRolC protein of the active center and binding site, as well as a protein-binding and ARTT loop, similar in structure to protein 6b of Agrobacterium vitis, indicates that they perform similar biological functions. Based on the data, a hypothesis was put forward about the presence of ADP-ribosyltransferase activity in the tRolC protein, which allows it to influence the growth and stress tolerance, as well as the biosynthesis of secondary metabolites by influencing the regulation of gene expression through interaction with transcription factors, RNA interference, or the manifestation of histone-like activity.

Retraction Note: Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants.

Retraction Note: Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants.

Molecular and cytogenetic characterization of Osteospermum fruticosum lines harboring wild type pRi rol genes.

Transgenic lines engineered through wild type Rhizobium rhizogenes display an altered phenotype known as the Ri phenotype. This phenotype includes a more compact plant habit, which has proved useful to obtain more compact varieties that require less chemical growth regulation. Here, we develop a method for the molecular and cytogenetic characterization of Cape daisy (Osteospermum fruticosum Norl.) Ri lines in order to predict segregation of pRi T-DNA genes. Analysis of copy number variation (CNV) by means of digital PCR indicated large variation in the copy number of the inserted root oncogenic loci (rol) genes, ranging from 1 to more than 15 copies. In addition, up to 9 copies of the auxin biosynthesis genes (aux) were present in a single Ri line. Visualization of pRiA4 and pRi1724 rol and aux insertion in 4 Ri lines was performed through Fluorescence In Situ Hybridization. The number of rol integrated loci varied from 1 to 3 loci. In contrast, the different TR-gene copies were confined to a single locus which consistently co-localized with a TL locus, this was demonstrated for the first time. Based on CNV and FISH a single Ri line, harboring 7 pRi1724 rol gene copies dispersed over 3 integration loci, was selected for breeding. Copy number segregation in R1 progeny of 2, 3, 4 and 5 pRi1724 copies was confirmed, indicating that the evaluation of the breeding value of first generation Ri lines is possible through CNV and FISH.

Superior osmotic stress tolerance in oilseed rape transformed with wild-type Rhizobium rhizogenes

Key messageNatural transformation with R. rhizogenes enhances osmotic stress tolerance in oilseed rape through increasing osmoregulation capacity, enhancing maintenance of hydraulic integrity and total antioxidant capacity.Transformation of plants using wild strains of agrobacteria is termed natural transformation and is not covered by GMO legislation in, e.g., European Union and Japan. In this study, offspring lines of Rhizobium rhizogenes naturally transformed oilseed rape (Brassica napus), i.e., A11 and B3 (termed root-inducing (Ri) lines), were investigated for osmotic stress resilience. Under polyethylene glycol 6000 (PEG) 10% (w/v)-induced osmotic stress, the Ri lines, particularly A11, had less severe leaf wilting, higher stomatal conductance (8.2 times more than WT), and a stable leaf transpiration rate (about 2.9 mmol m−2 s−1). Although the leaf relative water content and leaf water potential responded similarly to PEG treatment between the Ri lines and WT, a significant reduction of the turgid weight to dry weight ratio in A11 and B3 indicated a greater capacity of osmoregulation in the Ri lines. Moreover, the upregulation of plasma membrane intrinsic proteins genes (PIPs) in roots and downregulation of these genes in leaves of the Ri lines implied a better maintenance of hydraulic integrity in relation to the WT. Furthermore, the Ri lines had greater total antioxidant capacity (TAC) than the WT under PEG stress. Collectively, the enhanced tolerance of the Ri lines to PEG-induced osmotic stress could be attributed to the greater osmoregulation capacity, better maintenance of hydraulic integrity, and greater TAC than the WT. In addition, Ri-genes (particularly rolA and rolD) play roles in response to osmotic stress in Ri oilseed rape. This study reveals the potential of R. rhizogenes transformation for application in plant drought resilience.

Phytochemical Analysis, Biological Activities, and Docking of Phenolics from Shoot Cultures of Hypericum perforatum L. Transformed by Agrobacterium rhizogenes.

Hypericum perforatum transformed shoot lines (TSL) regenerated from corresponding hairy roots and non-transformed shoots (NTS) were comparatively evaluated for their phenolic compound contents and in vitro inhibitory capacity against target enzymes (monoamine oxidase-A, cholinesterases, tyrosinase, α-amylase, α-glucosidase, lipase, and cholesterol esterase). Molecular docking was conducted to assess the contribution of dominant phenolic compounds to the enzyme-inhibitory properties of TSL samples. The TSL extracts represent a rich source of chlorogenic acid, epicatechin and procyanidins, quercetin aglycone and glycosides, anthocyanins, naphthodianthrones, acyl-phloroglucinols, and xanthones. Concerning in vitro bioactivity assays, TSL displayed significantly higher acetylcholinesterase, tyrosinase, α-amylase, pancreatic lipase, and cholesterol esterase inhibitory properties compared to NTS, implying their neuroprotective, antidiabetic, and antiobesity potential. The docking data revealed that pseudohypericin, hyperforin, cadensin G, epicatechin, and chlorogenic acid are superior inhibitors of selected enzymes, exhibiting the lowest binding energy of ligand-receptor complexes. Present data indicate that H. perforatum transformed shoots might be recognized as an excellent biotechnological system for producing phenolic compounds with multiple health benefits.

Elicitor-enhanced steroidal sapogenin accumulation in hairy root cultures of Trigonella foenum-graecum

In current work, we studied hairy root induction in Trigonella foenum graecum, which is an important medicinal plant, and examined the impact of different elicitors on some phytochemical characteristics and metabolites production in hairy root cultures. Accordingly, some factors such as five strain types of Agrobacterium rhizogenes (1724, 15834, A4, A13 and MSU) and three different explants, namely leaf, cotyledon and hypocotyl were studied. The results showed that different A. rhizogenes strains exhibited different infection efficiency. MSU and 15834 had highest efficiency of hairy root induction than other strains. Also, hairy root induction frequency in leaf explants was higher than in other explants. Salicylic acid (SA), nitric oxide (NO), CaCl2 and penconazole (PEN) were used in elicitation process. Hairy roots were treated with SA (0.1 and 0.5 mM), NO (10 and 50 µM), CaCl2 (5 and 10 mM) and PEN (5 and 10 mg/L). Applied elicitors enhanced antioxidant enzymes activities and reduced oxidative stress markers; this observation might be ascribed to regulation of the oxidative status of the elicited cells. Significant increase of antioxidant metabolites (total phenol, flavonoid and anthocyanin) in PEN-treated hairy roots was associated to phenylalanine ammonia lyase activity, indicating an up-regulation of phenylpropanoid/flavonoid metabolism. PEN and CaCl2 treatment enhanced steroidal sapogenin in hairy root cultures. These results suggested that use of elicitors can enhance the production of secondary metabolites in transformed hairy roots. Among the elicitors applied, CaCl2 and PEN were the most effective in increasing secondary metabolite production in transformed hairy roots of T. foenum graecum.

Tapping the potential of Calotropis procera hairy roots for cardiac glycosides production and their identification using UHPLC/QTOF-MS.

The present work deals with the establishment of hairy root cultures from different explants of C. procera using Agrobacterium rhizogenes strain A4. A high transformation frequency (95%) was obtained from leaves followed by cotyledons (81.6%) and hypocotyls (38.3%). Genetic transformation of hairy roots was confirmed through PCR by amplifying a 400 bp fragment of the rolB gene. Hairy roots were highly branched, possessed plagiotropic and rapid growth on hormone-free ½ B5 medium. Ten cardiac glycosides, including calotropagenin, calotoxin, frugoside, coroglaucigenin, calotropin, calactin, uzarigenin, asclepin, uscharidin, and uscharin, based on their specific masses and fragmentation properties were identified in ethanolic extracts of hairy roots by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry UHPLC/QTOF-MS. This protocol could be used as a powerful tool for large-scale in vitro production of highly valued cardiac glycosides and for further transcriptomics or metabolomics studies.

Enhanced root system architecture in oilseed rape transformed with Rhizobium rhizogenes

Transformation of plants using wild strains of agrobacteria is termed natural transformation and is not covered by GMO legislation in e.g. European Union and Japan. In the current study, offspring lines (A11 and B3) of Rhizobium rhizogenes naturally transformed oilseed rape (Brassica napus) were randomly selected to characterize the morphological traits, and analyze the implications of such morphological changes on plant drought resilience. It was found that the introduction of Ri-genes altered the biomass partitioning to above- and under-ground parts of oilseed rape plants. Compared to the wild type (WT), the A11 and B3 lines exhibited 1.2–4.0 folds lower leaf and stem dry weight, leaf area and plant height, but had 1.3–5.8 folds greater root dry weight, root length and root surface area, resulting in a significantly enhanced root: shoot dry mass ratio and root surface area: leaf area ratio. In addition, the introduction of Ri-genes conferred reduced stomatal pore aperture and increased stomatal density in the B3 line, and increased leaf thickness in A11 line, which could benefit plant drought resilience. Finally, the modulations in morphological traits as a consequence of transformation with Ri-genes are discussed concerning resilience in water-limited conditions. These findings reveal the potential of natural transformation with R. rhizogenes for drought-targeted breeding in crops.

Development of Stephania tetrandra S. MOORE hairy root culture process for tetrandrine production

Tetrandrine, a bioactive active compound mainly found in the roots of Stephania tetrandra, exhibits various pharmacological properties. In vitro hairy root (HR) culture may serve as a promising solution for the extraction of tetrandrine, overcoming the limitations of natural cultivation. The present study describes the consistent production of tetrandrine from S. tetrandra hairy roots induced by different strains of Agrobacterium rhizogenes. Cultivation in woody plant medium (WPM) resulted in the highest HR biomass (0.056 g/petri-dish) and tetrandrine content (7.28 mg/L) as compared to other media. The maximum HR biomass (6.95 g dw/L) and tetrandrine production (68.69 mg/L) were obtained in the fifth week of cultivation. The presence of ammonium nitrate (800 mg/L), calcium nitrate (1156 mg/L), sucrose (20 g/L) and casein (2 g/L) enhanced the tetrandrine production. Moreover, the fed-batch cultivation demonstrated that the NH4NO3 (1200 mg/L) was an important growth limiting factor that yielded the highest tetrandrine amount (119.59 mg/L). The cultivation of hairy roots in a mist trickling bioreactor for eight weeks was less (26.24 mg/L) than in the flask. Despite a lower tetrandrine yield observed in bioreactors compared to flask cultures, refining the growth medium and fine-tuning bioreactor operations hold promise for boosting tetrandrine yield.

Efficient production and enhanced accumulation of Valerenic acid in Valeriana officinalis: Early identification of high-performing hairy root clones and Pioneering use of hydrogen peroxide as an elicitor

Valeriana officinalis (valerian) roots and rhizomes possess a long history of medicinal use due to their sedative, antiepileptic, and anticonvulsant properties. Valerenic acid, a bioactive sesquiterpene with therapeutic potential, is present in limited quantities within these tissues. This study explores the application of hairy root cultures for enhanced valerenic acid production. Hairy root induction was attempted on valerian leaves and petioles using three Rhizobium rhizogenes strains (ATCC15834, A4, and MSU440) across three culture media (Murashige and Skoog (MS), Gamborg's B5 (B5), and Schenk and Hildebrandt (SH)). A non-destructive imaging system and periodic analyses were employed to identify superior hairy root clones exhibiting increased branching frequency. Leaf explants co-cultured with R. rhizogenes strain ATCC15834 yielded the most promising clones, characterized by the highest dry weight (1.03 mg) and valerenic acid content (0.384 mg/g dry weight) when grown in a half-strength SH liquid medium. Following strain and media optimization, the impact of 50 mM hydrogen peroxide as an elicitor on valerenic acid production was investigated. This treatment resulted in a significant 1.76-fold increase in valerenic acid accumulation compared to the control group at the first day post-treatment. This approach presents a valuable strategy for the early identification of high-yielding hairy root lines. Moreover, the utilization of hydrogen peroxide, a safe and cost-effective elicitor, offers a rapid method for enhancing valerenic acid production in the selected superior clone. This study establishes a promising platform for the sustainable production of valuable plant compounds within both research and industrial settings.

Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute (Corchorus capsularis)

Jute (Corchorus capsularis L.) is the second most important natural plant fiber source after cotton. However, developing an efficient gene editing system for jute remains a challenge. In this study, the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4, an elite jute variety widely cultivated in China. The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation (BiFC) assays. The CHLOROPLASTOS ALTERADOS 1 (CcCLA1) gene, which is involved in the development of chloroplasts, was targeted for editing at two sites in Meifeng 4. Based on this hairy root transformation, the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and -GhU6.9 promoters, respectively, to assess the efficiency of gene editing. Results indicated the 50.0% (GhU6.7) and 38.5% (GhU6.9) editing events in the target 2 alleles (gRNA2), but no mutation was detected in the target 1 allele (gRNA1) in transgenic-positive hairy roots. CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation. Two CcCLA1 mutants were albinic, with a gene editing efficiency of 5.3%. These findings confirm that the CRISPR/Cas9 system, incorporating promoter GhU6.7, can be used as a gene editing tool for jute.

A Highly Efficient Agrobacterium rhizogenes-Mediated Hairy Root Transformation Method of Idesia polycarpa and the Generation of Transgenic Plants.

Idesia polycarpa is a promising woody oilseed species because of its high oil yield. However, its use is greatly limited due to the lack of varieties with good qualities; additionally, gene function has been less studied in this plant because an efficient transformation method has not been established yet. In this study, we established a rapid and efficient hairy root transformation method by infecting the whole seedling, the rootless seedling, and the leaf petiole with Agrobacterium rhizogenes using different infection methods. Among these transformation methods, a higher transformation efficiency was obtained using the whole seedling, which could reach up to 71.91%. Furthermore, we found that the seedling age significantly affected the transformation efficiency, either using whole or rootless seedlings. Additionally, we found that the transgenic roots could regenerate transgenic shoots. Taken together, our study lays the foundation for future study and for genetically modifying wood traits in the future.

Improvement of sulforaphane production in hairy root cultures of broccoli (Brassica oleracea L. var. italica) by eliciting Myrosinase gene expression and its effect on breast cancer cells

Plant-derived hairy root cultures have the potential to be a significant biotechnological tool for producing valuable secondary metabolite compounds. This study deals with the production of broccoli’s hairy root cultures via the mediation of Agrobacterium rhizogenes and investigates the effects of different concentrations of copper sulfate (CuSO4) and zinc sulfate (ZnSO4) as two elicitors on the production of sulforaphane in broccoli's hairy root. The expression of the Myrosinase (MY) gene, associated with sulforaphane production, was quantified using real-time PCR. Moreover, the cytotoxic effect of different treatments of sulforaphane on the breast cancer cell line, MDA-MB-231, was estimated and the transcription levels of apoptosis-associated genes were also determined. The highest amount of sulforaphane was produced at 4 µM CuSO4 after 16 h and 16 µM ZnSO4 after 8 h of elicitation. Overexpression of the MY gene was found to be associated with increased production of sulforaphane. All sulforaphane treatments induced a dose-dependent reduction in the viability of cancer cells. The sulforaphane extracted from broccoli's hairy roots treated with CuSO4 exerted a higher cytotoxic effect on the MDA-MB-231 breast cancer cell line than the sulforaphane extracted from broccoli's hairy root treated with ZnSO4. All treatments of sulforaphane up-regulated the Apoptosis Regulator (Bax) gene, Proapoptotic Bcl2 Associated X, Caspase-3, Caspase-8, and Caspase-9, while down-regulating the B-cell lymphoma 2 (Bcl-2) gene (antiapoptotic) transcription. The overall results showed an antiapoptotic effect of sulforaphane extract derived from broccoli's hairy roots on MDAMB-231 breast cancer cells, thereby establishing the potential anticancer activity of the sulforaphane produced in broccoli's hairy root cultures.

Development of a rapid and efficient system for CR genes identification based on hairy root transformation in Brassicaceae

Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection. Breeding of clubroot resistant cultivars based on mapping and cloning of resistant genes is commonly regarded as the most cost-effective and efficient way to fight against this disease. The traditional way of R gene functional validation requires stable transformation that is both time- and labor-consuming. In this study, a rapid and efficient hairy-root transgenic protocol mediated by Agrobacterium rhizogenes was developed. The transformation positive rate was over 80% in Brassica napus showed by GUS reporter gene and this transformation only took 1/6 of the time compared with stable transformation. The system was applicable to different B. napus varieties and other cruciferous crops including Brassica rapa and Brassica oleracea. In particular, two known CR genes, CRA3.7.1 and CRA8.2.4 were used respectively, as example to show that the system works well for CR gene study combined with subsequent P. brassicae infection in B. napus. Most importantly, it works both in over-expression that led to disease resistance, as well as in RNAi which led to disease susceptible phenotype. Therefore, this system can be used in batch-wise identification of CR genes, and also offered the possibility of manipulating key genes within the P. brassicae genome that could improve our knowledge on host–pathogen interaction.

Hairy roots biochemical characteristics of vegetable pea’s morphotype with modified leaf

BACKGROUND: In the literature, data on the successful production of high-protein pea root cultures with mutant alleles tl and af tl and their biochemical characteristics are not presented. AIM: The aim of this study is the biochemical characteristic of the obtained transformed cultures of mutant pea lines with a modified leaf morphotype was. Rol genes clarification of transformed pea mutant lines by wild strains of Agrobacterium rhizogenes and amino acid analysis of the total protein of the obtained roots cultures were objectives of the research. MATERIALS AND METHODS: Total DNA has been isolated from pea mutant root cultures. The studies were carried out on the equipment of the thermal cycler Tertsik of the company DNA-Technology (Russia). Amplicons were detected by electrophoresis in a 2% agarose gel. The gel was visualized and photographed under ultraviolet light (λ = 312 nm). The quantitative and qualitative amino acid composition of root cultures was determined by ion exchange chromatography on the amino acid analyzer AAA-339 (Microtechna, Czech Republic). RESULTS: PCR analysis revealed the absence of agrobacterial contamination in the transformed cultures and their stable growth on liquid and agar hormone-free media for 5 years. PCR assay showed the presence of rol C and D genes in culture with the tltl genotype and four rol genes A, B, C, and D in culture with the afaftltl genotype. A differentiated content of a number of amino acids in the biomass of transformed cultures was found depending on the genotype of the culture and the insertion of rol genes. Seven essential amino acids were identified in both cultures. CONCLUSIONS: Tryptophan turned out to be the limiting essential amino acid for both cultures. In terms of the sum of essential, ketogenic and sulfur-containing amino acids, the culture with the rol genes A, B, C, and D turned out to be the richest and most balanced.

Optimization of factors affecting Agrobacterium-mediated hairy root induction in Vitex negundo L. (Lamiaceae)

Vitex negundo L. is an aromatic, woody, blooming shrub in the Verbenaceae family which can grow into a small tree. Traditionally, V. negundo root has been used to cure diabetes, colic, boils, leprosy, and rheumatism. Keeping the importance of its roots in mind, an attempt has been taken for development of a protocol for efficient hairy root proliferation system. The Agrobacterium rhizogenes strains (MTCC 532 and MTCC 2364) were used for hairy root induction. For A. rhizogenes infection, both in vitro and in vivo leaves as well as internodes were used as explant. In vitro leaves and internodal explants were obtained by the inoculation of matured nodal segments on the optimum medium [MS + 2.0 mg/L N6-Benzylaminopurine (BAP)] with c.a. 91.6% shoot regeneration and an average of 8.1 shoots per explants. In vitro leaf showed best hairy root induction followed by in vitro internode on ½ MS medium augmented with acetosyringone. Highest transformation efficiency was achieved using MTCC 2364 strain, while no transformation was observed in MTCC 532 strain. Different factors affecting transformation including co-cultivation period, infection time and optical density (O.D.) value were standardized. The highest efficacy, 88.8% hairy root induction was observed in in vitro leaves infected by MTCC 2364 for 60 minutes infection time with an O.D. value of 0.29 maintained over a 44-48 hours of co-cultivation period. The prescribed protocol may be used as a reference for development of industrial scale hairy root production for bioactive compound located in root of V. negundo.

Optimization of Rhizobium rhizogenes-mediated transformation, regeneration and characterization of Malus domesticaBorkh. Ri lines

This study explored the establishment and optimization of Ri (root inducing) technology for apple breeding, using the bacterium Rhizobium rhizogenes to obtain Ri lines with compact shoots and stronger root systems. The transformation and shoot regeneration for Malus domestica cultivars was studied in detail. Various R. rhizogenes strains, scion and rootstock genotypes, explant types, wounding methods and explant orientations were tested for hairy root induction. Most of the 16 tested strains, especially those with plasmid type III, induced hairy roots in the rootstock genotype ‘M26’. Although apple genotypes differed in response, in most of them roots were successfully induced using strains ATCC 15834, LMG 63 and LMG 150, with leaf blades outperforming petioles as explants. Wounding by scratching or sonication further improved transformation efficiency, as did placing leaf blades with their abaxial side upward on root induction medium. The majority (94%) of roots formed in one transformation experiment were tested PCR-positive for at least one T-DNA gene. Shoot regeneration experiments investigated salt concentrations, gelling agents, cytokinin types, concentrations, and a resting period on hormone-free medium. Shoot regeneration was highly genotype-dependent varying between 0 and 83%, whereas only minor, non-significant effects were observed for the treatments tested. Copy numbers of T-DNA genes were estimated using digital PCR for the first time in apple Ri lines. In the greenhouse, two Ri lines showed compact shoots and shorter leaves, but no enhanced root system. The improved protocol provides a valuable tool for breeders and scientists to obtain and further use Ri lines.