EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine

Key message

Embryogenic suspension cultures are suitable for EMS mutagenesis in grapevine, and HRM prescreening of EMS-treated somatic embryo clusters allows rapid detection of point mutations before plant regeneration.

Abstract

Somatic embryogenesis is an excellent system for induced mutagenesis and clonal propagation in woody plants. Our work was focused on establishing a procedure for inducing ethyl methanesulfonate (EMS) mutagenesis in grapevine. Embryogenic cell aggregates (ECAs) growing in liquid medium were treated with increasing concentrations of EMS. We found that EMS dramatically affects the viability of ECAs at concentrations above 20 mM (25.5 ± 2.9 % survival), whereas concentrations above 10 mM affect embryogenic potential (22.1 ± 1.7 % of ECAs gave rise to embryos). Embryo masses generated from EMS-treated embryogenic cell aggregates were prescreened by quantitative PCR-High Resolution Melting (qPCR-HRM) to detect single nucleotide polymorphisms (SNPs) in a 1,000-bp VvNCED1-encoding DNA fragment, which served as the target gene. Detected mutations were verified in regenerated plants by PCR and sequencing. qPCR-HRM analysis of the difference plots for the fluorescence signals allowed detection of a mutation in a sample from an embryogenic aggregate treated with 10 mM EMS. To confirm the nature of the mutation, embryos from this aggregate were recovered and germinated, and leaves were collected for PCR and sequencing analysis. The alignment of sequences from regenerated plants with the wild-type sequence revealed a transitional mutation (G/C to A/T) in the 1,000-bp VvNCED1-encoding region. To our knowledge, this is the first time that EMS mutagenesis has been performed using an embryogenic cell suspension of grapevine.     

Somatic embryogenesis from stamen filaments in grapevine (Vitis vinifera L. cv. Mencía): changes in ploidy level and nuclear DNA content

Somatic embryogenesis was induced from stamen filaments and an embryogenic suspension culture was established in the grapevine cultivar Mencía using thidiazuron and 2,4-dichlorophenoxyacetic acid. Four combinations of each growth regulator were assessed for somatic embryo induction in a basal medium containing Nitsch and Nitsch salts and Murashige and Skoog vitamins, and an embryogenic suspension was established in liquid medium containing 1 μM 2,4-dichlorophenoxyacetic acid plus 4.5 μM thidiazuron. By using thidiazuron instead of benzyladenine, induction rates were improved over those previously reported for this cultivar and were relatively high compared with previous results in other cultivars. Three combinations of indole-3-acetic acid and benzyladenine and two inoculum levels were tested in a differentiation medium containing activated charcoal. The size of the inoculum affected the developmental stage of the somatic embryos, whereas the type of growth regulator did not. Both the germination and plant conversion rates were high (87.8% and 88.2%, respectively). An analysis of plant ploidy levels by flow cytometry revealed that 5.6% of the somatic embryo-derived plants were tetraploid. The mean nuclear DNA content of the diploid somatic embryo-derived plants was, on average, 6.7% lower than that of diploid field-grown plants, indicating that this protocol produces low levels of somaclonal variation. The results obtained here indicate that such variations in grapevine can occur both through changes in the ploidy level and by loss of genetic material during somatic embryogenesis.     

Kanamycin selection in temporary immersion bioreactors allows visual selection of transgenic citrus shoots

In mature citrus transformation, the nptII gene is most commonly used for selection and it is confounded by the high number of non-transformed, escaped shoots that develop on semi-solid kanamycin selection medium, even at high concentrations. Selection in liquid medium with kanamycin in temporary immersion bioreactors might provide a better means of distinguishing between transformed and non-transformed shoots. A dose-response curve was constructed for wild-type Carrizo rootstock in liquid medium to evaluate the effects of kanamycin concentration on the number and the length of microshoots. Kanamycin at 200 mg/l was chosen as the optimal concentration for selection of transgenic mature citrus shoots in bioreactors. At this dose, most non-transgenic microshoots turned yellow and their lengths and numbers were significantly reduced in comparison to the no kanamycin controls. Selection of transgenic shoots in bioreactors was tested after Agrobacterium transformations of mature Carrizo and Valencia using three different binary vectors containing nptII as the selectable marker. Shoots developed on semi-solid medium and were transferred to temporary immersion bioreactors containing liquid MS medium with 200 mg/l kanamycin. After two weeks of culture in bioreactors, 21 dark green shoots were visually selected on the basis of color from a total of 6882 microshoots, and 17 of them (81%) were confirmed as transgenic with either the GUS histochemical assay, GFP fluorescence or PCR. Yellow shoots (5675) to be discarded from pTLAB21 and pCAMBIA2301 transformations were also tested for GUS or GFP expression and only one (0.01%) was positive. Kanamycin selection of mature transgenic shoots in temporary immersion bioreactors permitted transgenics to be visually distinguished on the basis of color, and reduced labor and consumable costs for PCR screening, particularly when reporter genes were not used.     

Biolistic transformation of Carrizo citrange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> Osb. × <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.)

Key message

The development of transgenic citrus plants by the biolistic method.

Abstract

A protocol for the biolistic transformation of epicotyl explants and transgenic shoot regeneration of immature citrange rootstock, cv. Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.) and plant regeneration is described. Immature epicotyl explants were bombarded with a vector containing the nptII selectable marker and the gfp reporter. The number of independent, stably transformed tissues/total number of explants, recorded by monitoring GFP fluorescence 4 weeks after bombardment was substantial at 18.4 %, and some fluorescing tissues regenerated into shoots. Fluorescing GFP, putative transgenic shoots were micro-grafted onto immature Carrizo rootstocks in vitro, confirmed by PCR amplification of nptII and gfp coding regions, followed by secondary grafting onto older rootstocks grown in soil. Southern blot analysis indicated that all the fluorescing shoots were transgenic. Multiple and single copies of nptII integrations were confirmed in five regenerated transgenic lines. There is potential to develop a higher throughput biolistics transformation system by optimizing the tissue culture medium to improve shoot regeneration and narrowing the window for plant sampling. This system will be appropriate for transformation with minimal cassettes.