María Victoria González

Detection of somaclonal variants in somatic embryogenesis-regenerated plants of Vitis vinifera by flow cytometry and microsatellite markers

Flow cytometry and microsatellite analyses were used to evaluate the trueness-to-type of somatic embryogenesis-regenerated plants from six important Spanish grapevine (Vitis vinifera L.) cultivars. Tetraploid plants were regenerated through somatic embryogenesis from all of the cultivars tested with the exception of ‘Merenzao’. In addition, an octoploid plant was obtained in the cv. ‘Albariño’, and two mixoploids in ‘Torrontés’. The most probable origin of these ploidy variations is somaclonal variation. The cv. ‘Brancellao’ presented significantly more polyploids (28.57%) than any other cultivar, but it must be noted that 50% of the adult field-grown ‘Brancellao’ mother plants analysed were mixoploid. Hence, it is probable that these polyploids originated either from somaclonal variation or by separation of genotypically different cell layers through somatic embryogenesis. Microsatellite analysis of somatic embryogenesis-regenerated plants showed true-to-type varietal genotypes for all plants except six ‘Torrontés’ plants, which showed a mutant allele (231) instead of the normal one (237) at the locus VVMD5. There was not a clear relationship between the occurrence of the observed mutant regenerated plants and the callus induction media composition, the developmental stage of the inflorescences, the type of explant used for starting the cultures or the type of germination (precocious in differentiation medium or normal in germination medium) in any of the cultivars tested, except ‘Torrontés’. The mutant plants described herein have been transplanted to soil for future evaluation of putative phenotypic traits of interest. These mutants can be useful both for breeding programs and for functional genomic approaches aimed at increasing knowledge of the biology of grapevine.

Suboptimal temperature favors reserve formation in biennial carrot (Daucus carota) plants.

In response to suboptimal temperatures, temperate annual plants often increase root:shoot ratios, build-up carbohydrates and display typical morphological and anatomical changes. We know less about the responses of biennials such as carrot. As a model plant, carrot has the additional feature of two functionally and morphologically distinct root parts: the taproot, which stores carbohydrate and other compounds, and the fibrous root system involved in acquisition of water and nutrients. Here, we analyze the effects of temperature (12 vs 25°C) on growth, carbohydrate accumulation and whole-plant morphology in two carrot cultivars. Our working hypothesis is that suboptimal temperature favors active formation of reserve structures, rather than passive accumulation of storage carbohydrates. In comparison with plants grown at 25°C, plants grown at 12°C had: (1) higher fibrous root:shoot ratio (13%) , (2) thicker (10-15%) and smaller (up to two- to three-fold) leaves, (3) lower leaf cuticular permeance (two- to four-fold), (4) higher taproot:shoot ratio (two-fold), (5) higher phloem:xylem ratios in taproot (two- to six-fold), (6) unchanged percentage dry matter content (%DMC) in leaves, petioles or fibrous roots and (7) higher %DMC in taproot (20%). However, %DMC of individual taproot tissues (phloem and xylem) was unaffected by temperatures and was consistently higher in the phloem (up to 30%). Therefore, the higher %DMC of whole taproots at 12°C was attributed solely to the increased development of phloem tissue. Carrot, therefore, shares many of the most conspicuous elements of temperate plant responses to low temperatures. Consistently with our hypothesis, however, carrots grown at suboptimal temperature promoted reserve structures, rather than the increase in carbohydrate concentration typical of most temperate annual species and woody perennials.

Identification and sequence characterisation of molecular markers polymorphic between male kiwifruit (Actinidia chinensis var. deliciosa (A. Chev.) A. Chev.) accessions exhibiting different flowering time

Fruit set in kiwifruit is strongly dependent on pollination, which is limited by the lack of efficient male pollen donors, among other factors. We searched for molecular markers that could be polymorphic in relation to flowering time in order to classify male kiwifruit plants to discard those that are not likely to perform as efficient pollen donors. Random amplified polymorphic DNA (RAPD) and modified amplified fragment length polymorphism (AFLP) markers were generated using 41 male kiwifruit plants in two flowering groups, early- and late-flowering males (with respect to the female cultivar ‘Hayward’). One RAPD and nine modified-AFLP markers polymorphic between male plants exhibiting different flowering time were identified, sequenced and analysed in databases. Unweighted pair group method with arithmetic average (UPGMA) clustering and multidimensional scaling showed that these markers could be used to classify the male plants into flowering groups. Analysis of molecular variance (AMOVA) agreed with this classification, showing that most of the genetic variation is found between flowering groups. Sequence analysis based on a database search revealed that the polymorphism PolM contains a 7-nucleotide long element involved in the repression of the phytochrome A gene, that Pol4 is a partial sequence of a phytochrome B gene, and that sequences Pol3, Pol5, Pol7, and Pol9 show high identity with ESTs from kiwifruit buds treated with hydrogen cyanamide. Clustering analysis supported the previous classification of males into flowering groups, making it feasible to predict male plants’ flowering times with respect to the cultivar ‘Hayward’ based upon these molecular markers.