Effect of Stress Signals and Ib-rolB/C Overexpression on Secondary Metabolite Biosynthesis in Cell Cultures of Ipomoea batatas

Ipomoea batatas is a vital root crop and a source of caffeoylquinic acid derivatives (CQAs) with potential health-promoting benefits. As a naturally transgenic plant, I. batatas contains cellular T-DNA (cT-DNA) sequence homologs of the Agrobacterium rhizogenes open reading frame (ORF)14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n of unknown function. This study aimed to evaluate the effect of abiotic stresses (temperature, ultraviolet, and light) and chemical elicitors (methyl jasmonate, salicylic acid, and sodium nitroprusside) on the biosynthesis of CQAs and cT-DNA gene expression in I. batatas cell culture as a model system. Among all the applied treatments, ultraviolet irradiation, methyl jasmonate, and salicylic acid caused the maximal accumulation of secondary compounds. We also discovered that I. batatas cT-DNA genes were not expressed in cell culture, and the studied conditions weakly affected their transcriptional levels. However, the Ib-rolB/C gene expressed under the strong 35S CaMV promoter increased the CQAs content by 1.5-1.9-fold. Overall, our results show that cT-DNA-encoded transgenes are not involved in stress- and chemical elicitor-induced CQAs accumulation in cell cultures of I. batatas. Nevertheless, overaccumulation of RolB/RolC transcripts potentiates the secondary metabolism of sweet potatoes through a currently unknown mechanism. Our study provides new insights into the molecular mechanisms linked with CQAs biosynthesis in cell culture of naturally transgenic food crops, i.e., sweet potato.

Response of physiological parameters in Dionaea muscipula J. Ellis teratomas transformed with rolB oncogene.

BACKGROUND: Plant transformation with rol oncogenes derived from wild strains of Rhizobium rhizogenes is a popular biotechnology tool. Transformation effects depend on the type of rol gene, expression level, and the number of gene copies incorporated into the plant's genomic DNA. Although rol oncogenes are known as inducers of plant secondary metabolism, little is known about the physiological response of plants subjected to transformation. RESULTS: In this study, the physiological consequences of rolB oncogene incorporation into the DNA of Dionaea muscipula J. Ellis was evaluated at the level of primary and secondary metabolism. Examination of the teratoma (transformed shoots) cultures of two different clones (K and L) showed two different strategies for dealing with the presence of the rolB gene. Clone K showed an increased ratio of free fatty acids to lipids, superoxide dismutase activity, synthesis of the oxidised form of glutathione, and total pool of glutathione and carotenoids, in comparison to non-transformed plants (control). Clone L was characterised by increased accumulation of malondialdehyde, proline, activity of superoxide dismutase and catalase, total pool of glutathione, ratio of reduced form of glutathione to oxidised form, and accumulation of selected phenolic acids. Moreover, clone L had an enhanced ratio of total triglycerides to lipids and accumulated saccharose, fructose, glucose, and tyrosine. CONCLUSIONS: This study showed that plant transformation with the rolB oncogene derived from R. rhizogenes induces a pleiotropic effect in plant tissue after transformation. Examination of D. muscipula plant in the context of transformation with wild strains of R. rhizogenes can be a new source of knowledge about primary and secondary metabolites in transgenic organisms.

Ethephon-induced changes in antioxidants and phenolic compounds in anthocyanin-producing black carrot hairy root cultures.

Hairy root (HR) cultures are quickly evolving as a fundamental research tool and as a bio-based production system for secondary metabolites. In this study, an efficient protocol for establishment and elicitation of anthocyanin-producing HR cultures from black carrot was established. Taproot and hypocotyl explants of four carrot cultivars were transformed using wild-type Rhizobium rhizogenes. HR growth performance on plates was monitored to identify three fast-growing HR lines, two originating from root explants (lines NB-R and 43-R) and one from a hypocotyl explant (line 43-H). The HR biomass accumulated 25- to 30-fold in liquid media over a 4 week period. Nine anthocyanins and 24 hydroxycinnamic acid derivatives were identified and monitored using UPLC-PDA-TOF during HR growth. Adding ethephon, an ethylene-releasing compound, to the HR culture substantially increased the anthocyanin content by up to 82% in line 43-R and hydroxycinnamic acid concentrations by >20% in line NB-R. Moreover, the activities of superoxide dismutase and glutathione S-transferase increased in the HRs in response to ethephon, which could be related to the functionality and compartmentalization of anthocyanins. These findings present black carrot HR cultures as a platform for the in vitro production of anthocyanins and antioxidants, and provide new insight into the regulation of secondary metabolism in black carrot.

Agrobacterium rhizogenes mediated transformation of Ficus carica L. for the efficient production of secondary metabolites.

BACKGROUND: Ficus carica L., an ancient source of food and medicines, is rich in valuable nutritional and secondary compounds with antioxidant, antimicrobial, and anticancer effects. The present study is the first attempt to examine hairy root (HR) induction of F. carica (Sabz and Siah) by inoculating the 3-week-old shoots and leaves with different strains of Agrobacterium rhizogenes and also to investigate methyl jasmonate (MeJA) elicitation of HRs to produce a fast and high-yield production method for secondary metabolites. RESULTS: The maximum transformation rate (100%) was achieved by inoculating the shoots with Agrobacterium rhizogenes strain A7. Siah HRs elicited with 100 and 200 µmol L-1 MeJA and Sabz HRs with 100 µmol L-1 MeJA showed the highest total phenolic content. The highest flavonoid content was 3.935 mg QE g-1 DW in Siah HRs treated with 200 µmol L-1 MeJA and 2.762 mg QE g-1 DW in Sabz HRs treated with 300 µmol L-1 MeJA. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and ferric reducing antioxidant power (FRAP) value of HRs were affected by MeJA treatments. Methyl jasmonate elicitation also significantly enhanced the content of six phenolic acids (gallic acid, caffeic acid, chlorogenic acid, coumaric acid, rosmarinic acid, and cinnamic acid) and three flavonoids (rutin, quercetin, and apigenin). Thymol, a monoterpene phenol, was the main HR compound detected in gas chromatography mass spectrometry (GC-MS) analysis of the essential oils. CONCLUSION: Induction of HRs and elicitation of F. carica HRs by MeJA resulted in a significant increase in the production of important phenolic compounds and a significant increase in antioxidant capacity. © 2020 Society of Chemical Industry.

Biosynthesis of Functional Silver Nanoparticles Using Callus and Hairy Root Cultures of Aristolochia manshuriensis.

This study delves into the novel utilization of Aristolochia manshuriensis cultured cells for extracellular silver nanoparticles (AgNPs) synthesis without the need for additional substances. The presence of elemental silver has been verified using energy-dispersive X-ray spectroscopy, while distinct surface plasmon resonance peaks were revealed by UV-Vis spectra. Transmission and scanning electron microscopy indicated that the AgNPs, ranging in size from 10 to 40 nm, exhibited a spherical morphology. Fourier-transform infrared analysis validated the abilty of A. manshuriensis extract components to serve as both reducing and capping agents for metal ions. In the context of cytotoxicity on embryonic fibroblast (NIH 3T3) and mouse neuroblastoma (N2A) cells, AgNPs demonstrated varying effects. Specifically, nanoparticles derived from callus cultures exhibited an IC50 of 2.8 µg/mL, effectively inhibiting N2A growth, whereas AgNPs sourced from hairy roots only achieved this only at concentrations of 50 µg/mL and above. Notably, all studied AgNPs' treatment-induced cytotoxicity in fibroblast cells, yielding IC50 values ranging from 7.2 to 36.3 µg/mL. Furthermore, the findings unveiled the efficacy of the synthesized AgNPs against pathogenic microorganisms impacting both plants and animals, including Agrobacterium rhizogenes, A. tumefaciens, Bacillus subtilis, and Escherichia coli. These findings underscore the effectiveness of biotechnological methodologies in offering advanced and enhanced green nanotechnology alternatives for generating nanoparticles with applications in combating cancer and infectious disorders.

Auxin-dependent regulation of growth via rolB-induced modulation of the ROS metabolism in the long-term cultivated pRiA4-transformed Rubiacordifolia L. calli.

Gene transfer from Agrobacterium to plants is the best studied example of horizontal gene transfer (HGT) between prokaryotes and eukaryotes. The rol genes of A. rhizogenes (Rhizobium rhizogenes) provide uncontrolled root growth, or "hairy root" syndrome, the main diagnostic feature. In the present study, we investigated the stable pRiA4-transformed callus culture of Rubia cordifolia L. While untransformed callus cultures need PGRs (plant growth regulators) as an obligatory supplement, pRiA4 calli is able to achieve long-term PGR-free cultivation. For the first time, we described the pRiA4-transformed callus cultures' PGR-dependent ROS status, growth, and specialized metabolism. As we have shown, expression of the rolA and rolB but not the rolC genes is contradictory in a PGR-dependent manner. Moreover, a PGR-free pRiA4 transformed cell line is characterised as more anthraquinone (AQ) productive than an untransformed cell culture. These findings pertain to actual plant biotechnology: it could be the solution to troubles in choosing the best PGR combination for the cultivation of some rare, medicinal, and woody plants; wild-type Ri-plants and tissue cultures may become freed from legal controls on genetically modified organisms in the future. We propose possible PGR-dependent relationships between rolA and rolB as well as ROS signalling targets. The present study highlighted the high importance of the rolA gene in the regulation of combined rol gene effects and the large knowledge gap in rolA action.

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.

The RolB/RolC homolog from sweet potato promotes early flowering and triggers premature leaf senescence in transgenic Arabidopsis thaliana plants.

Expression of the root oncogenic loci (rol) genes from Agrobacterium rhizogenes provokes multiple divergent effects on physiological properties in transgenic plants and cell cultures. Recently, the homolog of the rolB and rolC oncogenes, named Ib-rolB/C, has been identified in the genome of a naturally transgenic food crop, i.e. sweet potato. In this study, we revealed that the Ipomoea batatas genome contains two full-length copies of Ib-rolB/C. The expression level of Ib-rolB/C in leaves of sweet potato showed a clear age-dependent pattern and increased as leaves senesce. Moreover, dark-induced senescence strongly up-regulates transcription of the Ib-rolB/C gene. Though Ib-rolB/C shares homology with its counterparts in A. rhizogenes, this gene was not capable to induce hairy roots or tumors in kalanchoe and tobacco plants. The Ib-rolB/C gene induced early-flowering phenotype, altered leaf morphology, and promoted premature leaf senescence in transgenic Arabidopsis thaliana plants. At the same time, Ib-rolB/C did not affect root morphology and biomass. Our results suggest that Ib-RolB/RolC participates in both age- and dark-triggered leaf senescence programs.

Segregation of rol Genes in Two Generations of Sinningia speciosa Engineered Through Wild Type Rhizobium rhizogenes.

Rhizobium rhizogenes infects and transforms a wide range of plant species. It thereby introduces new genes located on transfer-DNA of the root inducing plasmid (pRi) into the plant genome and one of its abilities is to alter the host root system. Explants from pRi transformed roots from Sinningia speciosa were regenerated to create naturally transgenic Ri lines. The presence of rol and aux genes in the Ri lines was linked with altered growth characteristics: shorter peduncles, wrinkled leaves, delayed flowering and enhanced root growth. The potential of Ri lines for breeding was evaluated through consecutive backcrossing with the original host genotype. The progeny of reciprocal crosses showed non-Mendelian inheritance suggesting partial transmission of the of the aux and rol genes. The typical Ri phenotype observed in the primary Ri line was partially inherited. These results revealed that the Ri phenotype is a complex trait influenced by the genetic background of the Ri line.

Optimization of cell suspension culture of transformed and untransformed lettuce for the enhanced production of secondary metabolites and their pharmaceutical evaluation.

In vitro suspension culture techniques are cost effective for large-scale production of secondary metabolites. In the present study, firstly, suspension cultures of untransformed Lactuca sativa were prepared using different hormonal combinations and were subjected to different pH, temperature and salt concentrations. Maximum biomass was obtained for suspensions supplemented with 1.5 mg/L BAP and 0.1 mg/L NAA, at pH 5.8, temperature 28 °C and 0 mM NaCl concentration. Using these parameters, suspensions were produced for rol ABC- and rol C-transformed lines of L. sativa. All the transgenic lines showed prominent increase in fresh weight (FW) and dry weight (DW) with maximum values for rol ABC2 line producing 169.8 mg/mL FW and 25.3 mg/mL DW. The exudates of transformed and untransformed plants were tested for the antioxidant activity and in vivo assays on rats. Maximum phenolic content (261 µg/mL) and flavonoid content (637.6 µg/mL) were obtained for rol C1 transgenic line. Total antioxidant capacity was found maximum (1451.7 µg/mL) for untransformed lettuce, whereas rol C1 showed maximum total reducing power activity (637.6 µg/mL). In DPPH assay, maximum activity (104.7 µg/mL) was shown by rol ABC3 line. In rats analgesic assay, maximum activity (74.9%) was shown by rol C2. Line rol C1 showed maximum anti-inflammatory activity (69.2%) and maximum antidepressant activity (minimum immobility time of 55 s). Maximum anticoagulant activity was observed for rol ABC2 with maximum clotting time of 130 s. The present study could help in using lettuce suspension culture as platform for the enhanced production of important metabolites.

Activation of anthraquinone biosynthesis in long-cultured callus culture of Rubia cordifolia transformed with the rolA plant oncogene.

The RolA protein belongs to the RolB class of plant T-DNA oncogenes, and shares structural similarity with the papilloma virus E2 DNA-binding domain. It has potentially as an inducer of plant secondary metabolism, although its role in biotechnology has yet to be realised. In this investigation, a Rubia cordifolia callus culture transformed with the rolA plant oncogene for more than 10 years was analysed. Expression of the rolA gene in the callus line was stable during long-term cultivation, and growth parameters were both elevated and stable, exceeding those of the non-transformed control culture. The rolA-transformed calli not only demonstrated remarkably stable growth, but also the ability to increase the yield of anthraquinones (AQs) in long-term cultivation. After ten years of cultivating rolA callus lines, we observed an activation of AQ biosynthesis from 200 mg/l to 874 mg/l. The increase was mainly due to activation of ruberitrinic acid biosynthesis. The expression of key AQ biosynthesis genes was strongly activated in rolA-transgenic calli. We compared the effects of the rolA gene with those of the rolB gene, which was previously considered the most potent inducer of secondary metabolism, and showed that rolA was more productive under conditions of long-term cultivation.

Plant Development and Organogenesis: From Basic Principles to Applied Research.

The way plants grow and develop organs significantly impacts the overall performance and yield of crop plants. The basic knowledge now available in plant development has the potential to help breeders in generating plants with defined architectural features to improve productivity. Plant translational research effort has steadily increased over the last decade, due to the huge increase in the availability of crop genomic resources and Arabidopsis-based sequence annotation systems. However, a consistent gap between fundamental and applied science has yet to be filled. One critical point is often the unreadiness of developmental biologists on one side, to foresee agricultural applications for their discoveries, and of the breeders on the other, to exploit gene function studies to apply candidate gene approaches when advantageous. In this Special Issue, developmental biologists and breeders make a special effort to reconcile research on basic principles of plant development and organogenesis with its applications to crop production and genetic improvement. Fundamental and applied science contributions interwine and chase each other, giving the reader different but complementary perpectives from only apparently distant corners of the same world.

Plant Cellular and Molecular Biotechnology: Following Mariotti's Steps.

This review is dedicated to the memory of Prof. Domenico Mariotti, who significantly contributed to establishing the Italian research community in Agricultural Genetics and carried out the first experiments of Agrobacterium-mediated plant genetic transformation and regeneration in Italy during the 1980s. Following his scientific interests as guiding principles, this review summarizes the recent advances obtained in plant biotechnology and fundamental research aiming to: (i) Exploit in vitro plant cell and tissue cultures to induce genetic variability and to produce useful metabolites; (ii) gain new insights into the biochemical function of Agrobacterium rhizogenes rol genes and their application to metabolite production, fruit tree transformation, and reverse genetics; (iii) improve genetic transformation in legume species, most of them recalcitrant to regeneration; (iv) untangle the potential of KNOTTED1-like homeobox (KNOX) transcription factors in plant morphogenesis as key regulators of hormonal homeostasis; and (v) elucidate the molecular mechanisms of the transition from juvenility to the adult phase in Prunus tree species.

From A. rhizogenes RolD to Plant P5CS: Exploiting Proline to Control Plant Development.

The capability of the soil bacterium Agrobacterium rhizogenes to reprogram plant development and induce adventitious hairy roots relies on the expression of a few root-inducing genes (rol A, B, C and D), which can be transferred from large virulence plasmids into the genome of susceptible plant cells. Contrary to rolA, B and C, which are present in all the virulent strains of A. rhizogenes and control hairy root formation by affecting auxin and cytokinin signalling, rolD appeared non-essential and not associated with plant hormones. Its role remained elusive until it was discovered that it codes for a proline synthesis enzyme. The finding that, in addition to its role in protein synthesis and stress adaptation, proline is also involved in hairy roots induction, disclosed a novel role for this amino acid in plant development. Indeed, from this initial finding, proline was shown to be critically involved in a number of developmental processes, such as floral transition, embryo development, pollen fertility and root elongation. In this review, we present a historical survey on the rol genes focusing on the role of rolD and proline in plant development.

Transformation of Lettuce with rol ABC Genes: Extracts Show Enhanced Antioxidant, Analgesic, Anti-Inflammatory, Antidepressant, and Anticoagulant Activities in Rats.

Lettuce is an edible crop that is well known for dietary and antioxidant benefits. The present study was conducted to investigate the effects of rol ABC genes on antioxidant and medicinal potential of lettuce by Agrobacterium-mediated transformation. Transgene integration and expression was confirmed through PCR and real-time RT-PCR, respectively. The transformed plants showed 91-102 % increase in total phenolic contents and 53-65 % increase in total flavonoid contents compared to untransformed plants. Total antioxidant capacity and total reducing power increased up to 112 and 133 % in transformed plants, respectively. Results of DPPH assay showed maximum 51 % increase, and lipid peroxidation assay exhibited 20 % increase in antioxidant activity of transformed plants compared to controls. Different in vivo assays were carried out in rats. The transgenic plants showed up to 80 % inhibition in both hot plate analgesic assay and carrageenan-induced hind paw edema test, while untransformed plants showed only 45 % inhibition. Antidepressant and anticoagulant potential of transformed plants was also significantly enhanced compared to untransformed plants. Taken together, the present work highlights the use of rol genes to enhance the secondary metabolite production in lettuce and improve its analgesic, anti-inflammatory, antidepressant, and anticoagulatory properties.

Optimizing culture conditions for establishment of hairy root culture of Semecarpus anacardium L.

Semecarpus anacardium L. is a tree species which produces secondary metabolites of medicinal importance. Roots of the plant have been traditionally used in folk medicines. Different strains of Agrobacterium rhizogenes (A4, ATCC15834 and LBA 9402) were used for induction of hairy roots in in vitro grown tissues of the plant. Hairy root initiation was observed after 25-30 days of infection. Optimum transformation frequency of 61% was achieved on leaf explants with ATCC15834 strain. Infection time of 30 min resulted in greater transformation frequency compared to 10 and 20 min, respectively. The hairy roots cultured in growth regulator-free semi-solid woody plant medium differentiated into callus. Whole shoots infected with ATCC 15834 were found to produce more transformants upon co-cultivation for 4 (65%) and 5 (67%) days. Induction of hairy roots in stem explants infected with ATCC 15834 was lower (52%) compared to leaves (62%) after 4 days of co-cultivation. In A4 and LBA9402 strains transformation efficiency was 49 ± 2.8% and 36 ± 5.7% in shoots after 4 days of co-cultivation. Transformation frequency was higher in ATCC15834 strain, irrespective of explants. The hairy roots of S. anacardium elongated slowly upon transfer to half-strength liquid medium. After 3-4 passages in liquid medium slender hairy roots started differentiating which were separated from the original explants. Visible growth of the roots was observed in hormone-free liquid medium after 2-3 months of culturing. Polymerase chain reaction with gene-specific primers from rol A, B and C genes confirms the positive transformation events.

Transformation of Lactuca sativa L. with rol C gene results in increased antioxidant potential and enhanced analgesic, anti-inflammatory and antidepressant activities in vivo.

Lettuce is an important edible crop which possesses various medicinal properties. In this study Lactuca sativa L. (cv Grand Rapids) was transformed by Agrobacterium-mediated transformation with rol C gene. Transgene integration and expression was confirmed through PCR and semiquantitative RT-PCR. The transformed extracts were evaluated for their in vitro antioxidant and in vivo analgesic, anti-inflammatory and antidepressant activities in rats. The transformed plants showed 53-98 % increase in total phenolic and 45-58 % increase in total flavonoid contents compared with untransformed plants. Results of total reducing power and total antioxidant capacity exhibited 90-118 and 61-75 % increase in transformed plants, respectively. In contrast to control, DPPH, lipid peroxidation and DNA protection assay showed up to 37, 20 and 50 % enhancement in transformed plants, respectively. The extracts showed similar but significant enhancement behavior in hot plate analgesic and carrageenan-induced hind paw edema test. The transformed extracts showed 72.1 and 78.5 % increase for analgesic and anti-inflammatory activities, respectively. The transformants of rol C gene exhibited prominent antidepressant activity with 64-73 % increase compared with untransformed plants. In conclusion, the present work suggests that transformation with rol C gene can be used to generate lettuce with enhanced medicinally important properties, such as antioxidant, analgesic, anti-inflammatory and antidepressant potential.

Cell-wall polysaccharide composition and glycanase activity of Silene vulgaris callus transformed with rolB and rolC genes.

The aim of this research is to investigate the effects of the Agrobacterium rhizogenes rol genes on the composition of cell-wall polysaccharides and glycanase activity in the campion callus. The expression of the rolC gene reduces the yield of campion pectin, while the expression of the rolB or rolC gene inhibits the volumetric production of both pectin and intracellular arabinogalactan. The rol genes are involved in regulating the activity of glycanases and esterases, thereby contributing to the modification of polysaccharide structures, their molecular weight (Mw) and the degree of pectin methyl esterification (DE). The increase in pectin arabinose residue appears to be connected to a decrease in intracellular and extracellular α-l-arabinofuranosidase activity in transgenic campion calluses. In transgenic calluses expressing the rolB and rolC genes, the increase in pectin galactose residue is likely due to a decrease in ß-galactosidase activity. The decrease in the Mw of pectin and its d-galacturonic acid content appears to be connected to an increase in extracellular polygalacturonase activity. Finally, the increase in pectinesterase activity causes a decrease in the DE of pectin. Thus, the expression of rolB and rolC genes in campion callus has a considerable effect on pectin's sugar composition, DE and Mw, while it appears to have an insignificant influence on intracellular and extracellular arabinogalactans.

Thermodynamic and structural properties of tuber starches from transgenic potato plants grown in vitro and in vivo.

Potato plants harboring Phytochrome B (PHYB) gene from Arabidopsis thaliana or rol genes from Agrobacterium rhizogenes were used to study the effect of transgene expression on structure and properties of starch in tubers. Thermodynamic characteristics of starch (melting temperature, enthalpy of melting, thickness of crystalline lamellae) were shown to be variable depending on the transgene expression and plant culturing mode: in vitro or in soil. The expression of rolB or rolC genes in in vitro cultured plants evoked opposite effects on starch melting temperature and crystalline lamellae thickness. AtPHYB or rolB expression in the soil-grown potato led to the formation of more defective or more ordered starch structures, respectively, in comparison with starches of the same lines grown in vitro. On the whole, our study revealed genotype-dependent differences between starches extracted from tubers of in vitro or in vivo grown plants.