Callus Lines Research Articles

Comparative morpho-physiological response of in vitro selected somaclones of wheat (Triticum aestivum L.) and explant donor parent to drought stress

Drought stress is the most prevalent environmental factor limiting wheat productivity globally and demands integration of new plant improvement approaches. Plant tissue culture, being a mutagenic process induces somaclonal variations, which can be manipulated for improving drought tolerance of wheat. An in vitro selection system was employed to regenerate PEG-6000 tolerant callus lines into R1 somaclones. Comparative morpho-physiologcial responses of R1 somaclones and explant donor parent cultivar to osmotic stress were studied at seedling and booting stages by inducing artificial drought stress along with the control of unstressed. Both R1 somaclones and explant donor parent exhibited differential responses to drought stress at both growth stages and the selected somaclones were less affected by drought stress over their donor parent. At seedling stage, the R1 somaclones exhibited significantly higher root length, shoot length, root to shoot length ratio and relative water contents (RWC) under PEG- 6000 induced osmotic stress. Similarly, at booting stage the R1 somaclones accumulated significantly higher proline, protein, sugar and potassium ion (K+) contents and maintained higher membrane stability index (MSI) than the original cv. GA-2002 under drought stress conditions. The results revealed that the R1 somaclones of wheat regenerated from PEG-6000 tolerant calli have better drought tolerance than the explant donor parent cv. GA-2002, suggesting that in vitro immature embryo culture can be employed to produce drought tolerant plants of wheat.

Callus induction and plant regeneration of Spirodela polyrhiza

This study reports efficient protocols of tissue culture of Spirodela polyrhiza, the only species whose whole genome (including nuclear, mitochondrial and chloroplast genome) has been sequenced in Lemnaceae. The callus induction, callus maintenance and plant regeneration have been established. Sixteen kinds of combinations of phytohormones from the orthogonal combinations of four auxins and four cytokinins, and 64 kinds of orthogonal combinations of concentration for each combination of phytohormones were tested for callus induction. Callus were induced with a high efficiency from the root apical meristem on the MS medium with the phytohormone combination of 2,4-D and thidiazuron by a method called “Rhizoids-hovering”. Unlike other reported species in Lemnaceae whose callus has been induced from frond, frond of S. polyrhiza cannot be induced into callus under all tested conditions in this study. The stable callus line which is cultured on the medium of callus maintenance with reduced phytohormone under dark condition has been maintained healthy proliferation for more than three years. The callus of S. polyrhiza can be easily regenerated under a broad range of concentration of cytokinins.

Kinetics and modeling of cell growth for potential anthocyanin induction in cultures of Taraxacum officinale G.H. Weber ex Wiggers (Dandelion) in vitro

Background: Taraxacum officinale G.H. Weber ex Wiggers is a wild plant used in folk medicine to treat several diseases owing to bioactive secondary metabolites present in its tissue. The accumulation of such molecules in plant cells can occur as a response against abiotic stress, but these metabolites are often deposited in low concentrations. For this reason, the use of a biotechnological approach to improve the yields of technologically interesting bioactive compounds such as anthocyanins is a compelling option. This work focuses on investigating the potential of in vitro T. officinale cultures as an anthocyanin source. Results: To demonstrate the suitability of anthocyanin induction and accumulation in calluses under specific conditions, anthocyanin was induced in the T. officinale callus. A specific medium of 5.5% sucrose supplemented with 6-benzylaminopurine /1-naphthaleneacetic acid in a 10:1 ratio was used to produce an anthocyanin yield of 1.23 mg g-1 fw. An in vitro dandelion callus line was established from this experiment. Five mathematical models were then used to objectively and predictably explain the growth of anthocyanin-induced calluses from T. officinale. Of these models, the Richards model offered the most suitable representation of anthocyanin callus growth in a solid medium and permitted the calculation of the corresponding kinetic parameters. Conclusions: The findings demonstrate the potential of an in vitro anthocyanin-induced callus line from T. officinale as an industrial anthocyanin source.

Unique chromoplast organisation and carotenoid gene expression in carotenoid-rich carrot callus

Main conclusionThe new model orange callus line, similar to carrot root, was rich in carotenoids due to altered expression of some carotenogenesis-associated genes and possessed unique diversity of chromoplast ultrastructure.Callus induced from carrot root segments cultured in vitro is usually pale yellow (p-y) and poor in carotenoids. A unique, non-engineered callus line of dark orange (d-o) colour was developed in this work. The content of carotenoid pigments in d-o callus was at the same level as in an orange carrot storage root and nine-fold higher than in p-y callus. Carotenoids accumulated mainly in abundant crystalline chromoplasts that are also common in carrot root but not in p-y callus. Using transmission electron microscopy, other types of chromoplasts were also found in d-o callus, including membranous chromoplasts rarely identified in plants and not observed in carrot root until now. At the transcriptional level, most carotenogenesis-associated genes were upregulated in d-o callus in comparison to p-y callus, but their expression was downregulated or unchanged when compared to root tissue. Two pathway steps were critical and could explain the massive carotenoid accumulation in this tissue. The geranylgeranyl diphosphate synthase gene involved in the biosynthesis of carotenoid precursors was highly expressed, while the β-carotene hydroxylase gene involved in β-carotene conversion to downstream xanthophylls was highly repressed. Additionally, paralogues of these genes and phytoene synthase were differentially expressed, indicating their tissue-specific roles in carotenoid biosynthesis and metabolism. The established system may serve as a novel model for elucidating plastid biogenesis that coincides with carotenogenesis.

Rapid discrimination of CMS cybrid lines between Brassica oleracea var. capitata and Raphanus sativus L. using Fourier transform infrared (FT-IR) spectroscopy of genomic DNA

The purpose of this study is to establish the rapid discrimination system of cybrid callus lines by Fourier transform infrared (FT-IR) spectroscopy without genetic fingerprinting analysis. Genomic DNA isolated from two parental lines (Brassica oleracea var. capitata and Raphanus sativus L.) and their cybrid callus lines were analyzed by FT-IR spectroscopy in the spectral region from 4000 to 400 cm−1. Several spectral differences between the two parental lines were detected in the frequency regions of N–H stretching (amide I), C=O stretching vibrations (amide II), and PO2− ionized asymmetric and symmetric stretching. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to discriminate cybrid cell lines from the two parental species at the genomic DNA level. PLS-DA analysis provided more clear discrimination between the two parental lines and their progeny cybrid lines in the score plot. PCA loading values also showed that obvious spectral differences played a significant role in discrimination between the two parental lines and their cybrid lines. These spectral differences might be directly related to subtle changes in the base functional groups and backbone structures of genomic DNA. Considering these results, this technique could provide a research foundation for the FT-IR spectral-based diagnosis, selection, and discrimination of parental lines and their cybrids. Furthermore, this technique could be applied in the hybrid seed industry for rapid screening with high heterogeneity.

Over-expression of 3-hydroxy-3- methylglutaryl-coenzyme A reductase 1 (hmgr1) gene under super-promoter for enhanced latex biosynthesis in rubber tree (Hevea brasiliensis Muell. Arg.)

Natural rubber (cis-1, 4-polyisoprene) is being produced from bark laticifer cells of Hevea brasiliensis and the popular high latex yielding Indian rubber clones are easily prone to onset of tapping panel dryness syndrome (TPD) which is considered as a physiological syndrome affecting latex production either partially or completely. This report describes an efficient protocol for development of transgenic rubber plants by over-expression of 3-hydroxy 3-methylglutaryl Co-enzyme A reductase 1 (hmgr1) gene which is considered as rate limiting factor for latex biosynthesis via Agrobacterium-mediated transformation. The pBIB plasmid vector containing hmgr1 gene cloned under the control of a super-promoter was used for genetic transformation using embryogenic callus. Putatively transgenic cell lines were obtained on selection medium and produced plantlets with 44% regeneration efficiency. Transgene integration was confirmed by PCR amplification of 1.8 kb hmgr1 and 0.6 kb hpt genes from all putatively transformed callus lines as well as transgenic plants. Southern blot analysis showed the stable integration and presence of transgene in the transgenic plants. Over expression of hmgr1 transgene was determined by Northern blot hybridization, semi-quantitative PCR and real-time PCR (qRT-PCR) analysis. Accumulation of hmgr1 mRNA transcripts was more abundant in transgenic plants than control. Increased level of photosynthetic pigments, protein contents and HMGR enzyme activity was also noticed in transgenic plants over control. Interestingly, the latex yield was significantly enhanced in all transgenic plants compared to the control. The qRT-PCR results exhibit that the hmgr1 mRNA transcript levels was 160-fold more abundance in transgenic plants over untransformed control. These results altogether suggest that there is a positive correlation between latex yield and accumulation of mRNA transcripts level as well as HMGR enzyme activity in transgenic rubber plants. It is presumed that there is a possibility for enhanced level of latex biosynthesis in transgenic plants as the level of mRNA transcripts and HMGR enzyme activity is directly correlated with latex yield in rubber tree. Further, the present results clearly suggest that the quantification of HMGR enzyme activity in young seedlings will be highly beneficial for early selection of high latex yielding plants in rubber breeding programs.

The establishment of cell suspension culture of sabah snake grass (Clinacanthus nutans (Burm.F.) Lindau)

Clinacanthus nutans (Burm.F.) Lindau is an herbaceous plant that has long been used for traditional medicinal purposes in Asia. It has recently gained popularity as an alternative treatment for cancer. The aim of this study was to establish cell suspension cultures of C. nutans and to identify targeted bioactive compounds in the cultures. Young leaf explants were cultured on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin to identify a suitable medium for callus induction and proliferation. Proliferated, friable calluses were cultured in different combinations of plant growth regulators (2,4-D, naphthaleneacetic acid [NAA], picloram, kinetin, and 6-benzylaminopurine) in liquid medium to establish cell suspension cultures. Three cell lines of suspension culture, callus, and intact plant parts were subjected to ethyl acetate extraction followed by thin layer chromatography for identification of selected bioactive compounds. Medium supplemented with 0.25 mg L−1 2,4-D and 0.75 mg L−1 kinetin was found to be optimal for callus induction, whereas supplementation with 0.50 mg L−1 2,4-D was efficient for callus proliferation. Liquid medium supplemented with 0.25 mg L−1 2,4-D and 0.50 mg L−1 NAA produced the highest growth index (2.52). Quercetin, catechin, and luteolin were present together in the callus and cell suspension cultures of C. nutans, but all three compounds were detected separately in young leaves, mature leaves, and stems. This study is the first to report the establishment of cell suspension culture of C. nutans with both cell and callus cultures producing quercetin, catechin, and luteolin.

Inducing Ni sensitivity in the Ni hyperaccumulator plant Alyssum inflatum Nyárády (Brassicaceae) by transforming with CAX1, a vacuolar membrane calcium transporter

AbstractThe importance of calcium in nickel tolerance was studied in the nickel hyperaccumulator plant Alyssum inflatum by gene transformation of CAX1, a vacuolar membrane transporter that reduces cytosolic calcium. CAX1 from Arabidopsis thaliana with a CaMV35S promoter accompanying a kanamycin resistance gene was transferred into A. inflatum using Agrobacterium tumefaciens. Transformed calli were sub‐cultured three times on kanamycin‐rich media and transformation was confirmed by PCR using a specific primer for CAX1. At least 10 callus lines were used as a pool of transformed material. Both transformed and untransformed calli were treated with varying concentrations of either calcium (1–15 mM) or nickel (0–500 µM) to compare their responses to those ions. Increased external calcium generally led to increased callus biomass, however, the increase was greater for untransformed callus. Further, increased external calcium led to increased callus calcium concentrations. Transformed callus was less nickel tolerant than untransformed callus: under increasing nickel concentrations callus relative growth rate was significantly less for transformed callus. Transformed callus also contained significantly less nickel than untransformed callus when exposed to the highest external nickel concentration (200 µM). We suggest that transformation with CAX1 decreased cytosolic calcium and resulted in decreased nickel tolerance. This in turn suggests that, at low cytosolic calcium concentrations, other nickel tolerance mechanisms (e.g., complexation and vacuolar sequestration) are insufficient for nickel tolerance. We propose that high cytosolic calcium is an important mechanism that results in nickel tolerance by nickel hyperaccumulator plants.

Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit.

SummaryKiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired‐sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired‐sgRNA cloning, our strategy only requires the synthesis of two gRNA‐containing primers which largely reduces the cost. We further compared efficiencies of paired‐sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA‐sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10‐fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired‐sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418‐resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.

The rolB plant oncogene affects multiple signaling protein modules related to hormone signaling and plant defense

The rolB plant oncogene of Agrobacterium rhizogenes perturbs many biochemical processes in transformed plant cells, thereby causing their neoplastic reprogramming. The oncogene renders the cells more tolerant to environmental stresses and herbicides and inhibits ROS elevation and programmed cell death. In the present work, we performed a proteomic analysis of Arabidopsis thaliana rolB-expressing callus line AtB-2, which represents a line with moderate expression of the oncogene. Our results show that under these conditions rolB greatly perturbs the expression of some chaperone-type proteins such as heat-shock proteins and cyclophilins. Heat-shock proteins of the DnaK subfamily were overexpressed in rolB-transformed calli, whereas the abundance of cyclophilins, members of the closely related single-domain cyclophilin family was decreased. Real-time PCR analysis of corresponding genes confirmed the reliability of proteomics data because gene expression correlated well with the expression of proteins. Bioinformatics analysis indicates that rolB can potentially affect several levels of signaling protein modules, including effector-triggered immunity (via the RPM1-RPS2 signaling module), the miRNA processing machinery, auxin and cytokinin signaling, the calcium signaling system and secondary metabolism.

NUTRITIONAL COMPOSITION OF DIOSCOREA HISPIDA FROM DIFFERENT LOCATIONS AROUND LEUSER ECOSYSTEM AREA

NUTRITIONAL COMPOSITION OF DIOSCOREA HISPIDA FROM DIFFERENT LOCATIONS AROUND LEUSER ECOSYSTEM AREA

Overexpression of EcGSH1 induces glutathione production and alters somatic embryogenesis and plant development in Hevea brasiliensis

Oxidative stress occurring during in-vitro culture is detrimental for clonal propagation by somatic embryogenesis in particular in Hevea brasiliensis. In previous work, overexpression of the gene encoding the cytosolic reactive oxygen species detoxification enzyme HbCuZnSOD led to reduce somatic embryo regeneration in Hevea. In this study, the role of antioxidant was tested by overexpression of the E.coli GSH1 gene involved in glutathione biosynthesis in rubber embryogenic callus lines. Transgenic lines were successfully established and some plants were regenerated. Overexpression of EcGSH1 gene led to glutathione over-accumulation, and affected dramatically the somatic embryogenesis process and plant development. Upon a water deficit treatment, these plants displayed the greatest drop in photosynthetic nitrogen use efficiency, higher proline content, and higher glutathione reductase activity. Changes induced in transgenic lines overexpressing HbCuZnSOD and EcGSH1 were discussed as well as possible applications on plant material propagation, overcoming loss of natural rubber production through Tapping Panel Dryness, and tropical soil remediation.

Strategies for Fast Multiplication and Conservation of Forest Trees by Somatic Embryogenesis and Cryopreservation: a Case Study with Cypress (Cupressus sempervirens L.)

Common cypress (Cupressus sempervirens L.) is one of the most widespread species in the Mediterranean area. It has been traditionally cultivated for its ornamental value, becoming a typical feature of urban and rural landscapes, and high timber quality. In the last 30 years, cypress has been subjected to important breeding programmes, aimed to select clones tolerant to the widespread canker, caused by the pathogenic fungus Seiridium cardinale, leading to various patented varieties today available on the market, as well as for genotypes producing null or low amount of allergenic pollen. Somatic embryogenesis is a suitable in vitro regeneration method for fast cloning of conifer trees, and the cryopreservation of embryogenic callus is a significant tool for the safe long-term conservation of valuable cell lines. Recently, a complete protocol for the production of cypress plants from somatic embryogenesis was developed for the patented clone ‘Mediterraneo’. Here, the coupling of somatic embryogenesis and cryopreservation may offer a superior tool to propagate and maintain selected genotypes of cypress by overcoming repetitive subculturing of selected embryogenic callus lines. For the above, this study aimed to compare different cryopreservation techniques (PVS2-based vitrification and slow cooling) with the ‘Mediterraneo’ embryogenic callus line. Best results were obtained after the optimization of a slow cooling procedure, based on the 30-min treatment of embryogenic masses with a cryoprotective solution containing 180 g l-1 sucrose and 7.5% DMSO, followed by the reduction of the temperature at a rate of -1 °C min-1 up to -40 °C and the subsequent immersion in liquid nitrogen (“two-step freezing”).

Development of Betalain Producing Callus Lines from Colored Quinoa Varieties (Chenopodium quinoa Willd)

Betalains are water-soluble plant pigments of hydrophilic nature with promising bioactive potential. Among the scarce edible sources of betalains is the grain crop quinoa (Chenopodium quinoa Willd), with violet, red, and yellow grains being colored by these pigments. In this work, callus cultures have been developed from differently colored plant varieties. Stable callus lines exhibited color and pigment production when maintained on Murashige and Skoog medium supplemented with the plant growth regulators 6-benzylaminopurine (8.88 μM) and 2,4-dichlorophenoxyacetic acid (6.79 μM) with a reduction of the nitrogen source to 5.91 mM. Pigment analysis by HPLC-DAD and ESI-MS/MS fully describes the content of individual pigments in the cell lines and allows the first report on the pigments present in quinoa seedlings. Phyllocactin and vulgaxanthin I are described as novel pigments in the species and show the potential of C. quinoa culture lines in the production of compounds of nutritional value.

The effect of light quality on the pro-/antioxidant balance, activity of photosystem II, and expression of light-dependent genes in Eutrema salsugineum callus cells.

The antioxidant balance, photochemical activity of photosystem II (PSII), and photosynthetic pigment content, as well as the expression of genes involved in the light signalling of callus lines of Eutrema salsugineum plants (earlier Thellungiella salsuginea) under different spectral light compositions were studied. Growth of callus in red light (RL, maximum 660nm), in contrast to blue light (BL, maximum 450nm), resulted in a lower H2O2 content and thiobarbituric acid reactive substances (TBARS). The BL increased the activities of key antioxidant enzymes in comparison with the white light (WL) and RL and demonstrated the minimum level of PSII photochemical activity. The activities of catalase (CAT) and peroxidase (POD) had the highest values in BL, which, along with the increased H2O2 and TBARS content, indicate a higher level of oxidative stress in the cells. The expression levels of the main chloroplast protein genes of PSII (PSBA and PSBD), the NADPH-dependent oxidase gene of the plasma membrane (RbohD), the protochlorophyllide oxidoreductase genes (POR B, C) involved in the biosynthesis of chlorophyll, and the key photoreceptor signalling genes (CIB1, CRY2, PhyB, PhyA, and PIF3) were determined. Possible mechanisms of light quality effects on the physiological parameters of callus cells are discussed.

Green synthesis of silver nanoparticles using transgenic Nicotiana tabacum callus culture expressing silicatein gene from marine sponge Latrunculia oparinae

In the present investigation, transgenic tobacco callus cultures and plants overexpressing the silicatein gene LoSilA1 from marine sponge Latrunculia oparinae were obtained and their bioreduction behaviour for the synthesis of silver nanoparticles (AgNPs) was studied. Synthesized nanoparticles were characterized using UV–visible spectroscopy, Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic flame electron microscopy (AFM) and nanoparticle tracking analysis (NTA). Our measurements showed that the reduction of silver nitrate produced spherical AgNPs with diameters in the range of 12–80 nm. The results of XRD analysis proved the crystal nature of the obtained AgNPs. FTIR analysis indicated that particles are reduced and stabilized in solution by the capping agent, which is likely to be proteins present in the callus extract. Interestingly, the reduction potential of LoSiLA1-transgenic callus line was increased three-fold compared with the empty vector-transformed calli. The synthesized AgNPs were found to exhibit strong antibacterial activity against Escherichia coli and Agrobacterium rhizogenes. The present study reports the first evidence for using genetic engineering for activation of the reduction potential of plant cells for synthesis of biocidal AgNPs.

Novel potato plants with enhanced cadmium resistance and antioxidative defence generated after in vitro cell line selection.

It is of interest to apply plant tissue culture to generate plants resistant to toxic effects of cadmium (Cd) on plant growth. Callus cultures were initiated from leaf explants of micropropagated potato plantlets (Solanum tuberosum L., cv. Iwa) for in vitro selection comprising 18 different Cd treatments varying in Cd exposure timing and duration. Plantlets regenerated from two different lines of Cd-selected calli, L9 and L11, were found to exhibit enhanced resistance to 218 μM Cd compared to control (source plantlets for leaf explants used to initiate callus cultures for Cd resistance). In response to 218 μM Cd, L11 plantlets had lower levels of lipid peroxidation and hydrogen peroxide than control and L9 plantlets. In addition, antioxidative enzyme activities in L11 were generally higher than control. L11 also had a higher level of proline than control.

Mitochondrial Genome of Callus Protoplast Has a Role in Mesophyll Protoplast Regeneration in Citrus: Evidence From Transgenic GFP Somatic Homo-Fusion

Protoplast fusion has great potential in citrus improvement. Although citrus mesophyll protoplasts usually cannot divide and regenerate, symmetric protoplast fusion of embryogenic callus protoplast + mesophyll protoplast sometimes results in the regeneration of mesophyll-parent-type cybrids. It suggested that mitochondrial DNA (mtDNA) from protoplasts of embryogenic callus parent plays an important role in stimulating division and regeneration of mesophyll protoplasts. Herein, somatic fusion was conducted via electrofusion between callus protoplasts isolated from Valencia orange [Citrus sinensis (L.) Osbeck] cell suspension cultures and transgenic GFP-tagged mesophyll protoplasts from the same genotype, i.e. transgenic Valencia orange plants containing the green fluorescent protein (GFP) gene, in an effort to elucidate whether mtDNA of callus line could stimulate the division and regeneration of mesophyll protoplasts from the same genotype. Two embryoids and one plantlet with GFP expression were successfully obtained and subsequent ploidy analysis by flow cytometry indicated that they were all diploids. The regenerated diploid embryoids and plantlet with GFP expression could be considered as ‘cybrids’ with mtDNA from the callus protoplasts of Valencia orange. The result indicated that citrus mesophyll-parent-type cybrid regeneration needed the stimulation of mtDNA from protoplasts of embryogenic callus parent regardless of their origin either from another genotype or the same genotype as the mesophyll parent.

An efficient method for transgenic callus induction from Vitis amurensis petiole.

Transformation is the main platform for genetic improvement and gene function studies in plants. However, the established somatic embryo transformation system for grapevines is time-consuming and has low efficiency, which limits its utilization in functional genomics research. Vitis amurensis is a wild Vitis species with remarkable cold tolerance. The lack of an efficient genetic transformation system for it has significantly hindered the functional identification of cold stress related genes in the species. Herein, an efficient method was established to produce transformed calli of V. amurensis. Segments of petioles from micropropagated plantlets of V. amurensis exhibited better capacity to differentiate calli than leaf-discs and stem segments, and thus was chosen as target tissue for Agrobacterium-mediated transformation. Both neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (eGFP) genes were used for simultaneous selection of transgenic calli based on kanamycin resistance and eGFP fluorescence. Several parameters affecting the transformation efficiency were optimized including the concentration of kanamycin, Agrobacterium stains, bacterial densities, infection treatments and co-cultivation time. The transgenic callus lines were verified by checking the integration of NPTII gene into calli genomes, the expression of eGFP gene and the fluorescence of eGFP. Up to 20% of the petiole segments produced transformed calli after 2 months of cultivation. This efficient transformation system will facilitate the functional analysis of agronomic characteristics and related genes not only in V. amurensis but also in other grapevine species.

Protoplast fusion in the genus Gentiana: genomic composition and genetic stability of somatic hybrids between Gentiana kurroo Royle and G. cruciata L.

Somatic hybridization by protoplast fusion is used in breeding programs to obtain plant material that has inherited valuable traits from two different species, and in order to broaden plant genetic diversity. Somatic hybrids of the genus Gentiana could provide a useful source of new ornamental cultivars and of secondary metabolites. However, in order to evaluate its further usefulness, detailed characterization of the newly created hybrid is essential. Here, genome composition and stability of interspecific gentian somatic hybrids obtained following electrofusion of cell suspension-derived protoplasts of diploid Gentiana kurroo Royle with leaf mesophyll-derived protoplasts of tetraploid G. cruciata L. were characterized using various molecular markers and flow cytometry. AFLP and ISSR analyses revealed that all 21 hybrid plants and 3 lines of hybrid callus were genetically closer to G. cruciata than to G. kurroo. According to the results of chloroplast DNA analysis with the use of CAPS markers, all somatic hybrids inherited chloroplasts from “mesophyll” fusion partner G. cruciata. Flow cytometry revealed that polyploidization occurred, and probably it took place at the early stage of post-fusion culture. In consequence, gradual elimination of nuclear DNA, mixoploidy, and high genetic instability were observed in most hybrid plants and calli during the subsequent 4 years of in vitro culture.