SciCombinator

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Concept: Plant reproduction

191

Cliff sides are extreme habitats, often sheltering a rich and unique flora. One example is the dioecious herb Borderea chouardii (Dioscoreaceae), which is a Tertiary, tropical relict, occurring only on two adjacent vertical cliffs in the world. We studied its reproductive biology, which in some aspects is extreme, especially the unusual double mutualistic role of ants as both pollinators and dispersers. We made a 2-year pollination census and four years of seed-dispersal experiments, recording flower visitors and dispersal rates. Fruit and seed set, self-sowing of seeds, seedling recruitment, and fate of seedlings from seeds sowed by different agents were scored over a period of 17 years. The ants Lasius grandis and L. cinereus were the main pollinators, whereas another ant Pheidole pallidula dispersed seeds. Thus ants functioned as double mutualists. Two thirds of all new seedlings came from self-sown seeds, and 1/3 was dispersed by ants, which gathered the seeds with their oil-rich elaiosome. Gravity played a minor role to dispersal. Both ant dispersal and self-sowing resulted in the same survival rate of seedlings. A double mutualism is a risky reproductive strategy, but B. chouardii buffers that by an unusual long-term demographic stability (some individuals exceed 300 years in lifespan) and its presence in a climatically very stable habitat, inaccessible to large herbivores. Such a combination of traits and habitat properties may explain the persistence of this relict species.

Concepts: Reproduction, Plant, Symbiosis, Seed, Plant morphology, Plant reproduction, Sexual reproduction, Mutualism

170

BACKGROUND: Plant grafting techniques have deepened our understanding of the signals facilitating communication between the root and shoot, as well as between shoot and reproductive organs. Transmissible signalling molecules can include hormones, peptides, proteins and metabolites: some of which travel long distances to communicate stress, nutrient status, disease and developmental events. While hypocotyl micrografting techniques have been successfully established for Arabidopsis to explore root to shoot communications, inflorescence grafting in Arabidopsis has not been exploited to the same extent. Two different strategies (horizontal and wedge-style inflorescence grafting) have been developed to explore long distance signalling between the shoot and reproductive organs. We developed a robust wedge-cleft grafting method, with success rates greater than 87%, by developing better tissue contact between the stems from the inflorescence scion and rootstock. We describe how to perform a successful inflorescence stem graft that allows for reproducible translocation experiments into the physiological, developmental and molecular aspects of long distance signalling events that promote reproduction. RESULTS: Wedge grafts of the Arabidopsis inflorescence stem were supported with silicone tubing and further sealed with parafilm to maintain the vascular flow of nutrients to the shoot and reproductive tissues. Nearly all (87%) grafted plants formed a strong union between the scion and rootstock. The success of grafting was scored using an inflorescence growth assay based upon the growth of primary stem. Repeated pruning produced new cauline tissues, healthy flowers and reproductive siliques, which indicates a healthy flow of nutrients from the rootstock. Removal of the silicone tubing showed a tightly fused wedge graft junction with callus proliferation. Histological staining of sections through the graft junction demonstrated the differentiation of newly formed vascular connections, parenchyma tissue and lignin accumulation, supporting the presumed success of the graft union between two sections of the primary inflorescence stem. CONCLUSIONS: We describe a simple and reliable method for grafting sections of an Arabidopsis inflorescence stem. This step-by-step protocol facilitates laboratories without grafting experience to further explore the molecular and chemical signalling which coordinates communications between the shoot and reproductive tissues.

Concepts: Reproductive system, Grafting, Plant stem, Plant reproduction, Rose, Rootstock, Tree shaping, Tomato grafting

169

Vitis vinifera scions are commonly grafted onto rootstocks of other grape species to influence scion vigour and provide resistance to soil-borne pests and abiotic stress; however, the mechanisms by which rootstocks affect scion physiology remain unknown. This study characterized the hydraulic physiology of Vitis rootstocks that vary in vigour classification by investigating aquaporin (VvPIP) gene expression, fine-root hydraulic conductivity (Lp®), % aquaporin contribution to Lp®, scion transpiration, and the size of root systems. Expression of several VvPIP genes was consistently greater in higher-vigour rootstocks under favourable growing conditions in a variety of media and in root tips compared to mature fine roots. Similar to VvPIP expression patterns, fine-root Lp® and % aquaporin contribution to Lp® determined under both osmotic (Lp®(Osm)) and hydrostatic (Lp®(Hyd)) pressure gradients were consistently greater in high-vigour rootstocks. Interestingly, the % aquaporin contribution was nearly identical for Lp®(Osm) and Lp®(Hyd) even though a hydrostatic gradient would induce a predominant flow across the apoplastic pathway. In common scion greenhouse experiments, leaf area-specific transpiration (E) and total leaf area increased with rootstock vigour and were positively correlated with fine-root Lp®. These results suggest that increased canopy water demands for scion grafted onto high-vigour rootstocks are matched by adjustments in root-system hydraulic conductivity through the combination of fine-root Lp® and increased root surface area.

Concepts: Gene, Grafting, Root, Plant reproduction, Grape, Viticulture, Phylloxera, Rootstock

165

Background and AimsIntraspecific reproductive differentiation into sexual and apomictic cytotypes of differing ploidy is a common phenomenon. However, mechanisms enabling the maintenance of both reproductive modes and integrity of cytotypes in sympatry are as yet poorly understood. This study examined the association of sexual and apomictic seed formation with ploidy as well as gene flow towards sexuals within populations of purely polyploid Potentilla puberula.MethodsThe study is based on 22 populations representing various combinations of five polyploid cytotypes (tetraploid-octoploid) from East Tyrol, Austria. Embryo ploidy and the endosperm/embryo ploidy ratio obtained by a flow cytometric seed screen were used to infer reproductive modes of seed formation and to calculate the male and female genomic contributions to the embryo and endosperm. Self-incompatibility (SI) patterns were assessed and a new indirect approach was used to test for the occurrence of intercytotype matings based on the variation in the male genomic contribution to sexually derived embryos on the level of developed seed.Key ResultsTetraploids formed seeds almost exclusively via sexual reproduction, whereas penta- to octoploids were preferentially apomictic. Non-random distribution of reproductive modes within maternal plants further revealed a tendency to separate the sexual from the apomictic mode among individuals. Self-incompatibility of sexuals indicated functionality of the gametophytic SI system despite tetraploidy of the nuclear genome. We found no indication for significant cross-fertilization of tetraploids by the high polyploids.ConclusionsThe study revealed a rare example of intraspecific differentiation into sexual and apomictic cytotypes at the polyploid level. The integrity of the sexual tetraploids was maintained due to reproductive isolation from the apomictic higher polyploids. Functionality of the gametophytic SI system suggested that the tetraploids are functional diploids.

Concepts: Genetics, Reproduction, Genome, Seed, Asexual reproduction, Rosaceae, Plant reproduction, Parthenogenesis

136

The production of tuberous roots is usually reduced by vigorous vegetative growth because of the competition for resource between the vegetative parts and reproductive organs. In this study, we conducted root pruning to examine the vigorous vegetative growth by regulating root growth, subsequently limiting vegetative growth and improving tuber yield. Compared with the control, stem, tuber, and root biomasses were all improved, whereas both flower and leaf biomasses were increased. Tuber biomass was improved by 23.48% to 50.32%, with the largest tuber biomass obtained at root cutting radius 4/5 R. With delayed root cutting time, tuber and root biomasses increased first and then decreased. The largest tuber biomass was obtained at 65 seedling stage. With a delay in root cutting time, the trend line of aboveground, underground, and total biomasses changed gradually. However, whereas underground and total biomasses showed a gradually increasing, aboveground biomass showed a decreasing. The values of stem-leaf and shoot-root ratios under different root cutting were higher than those of the control. With a delay in root cutting time, stem-leaf ratio showed an initial increase and then decreased with largest value being obtained at 80 seedling stage, whereas the largest shoot-root ratio was obtained at 115 seedling stage.

Concepts: Root, Plant morphology, Plant stem, Asexual reproduction, Tuber, Plant reproduction, Rhizome, Vegetative reproduction

30

Potato (Solanum tuberosum L.) originates from the Andes and evolved short-day-dependent tuber formation as a vegetative propagation strategy. Here we describe the identification of a central regulator underlying a major-effect quantitative trait locus for plant maturity and initiation of tuber development. We show that this gene belongs to the family of DOF (DNA-binding with one finger) transcription factors and regulates tuberization and plant life cycle length, by acting as a mediator between the circadian clock and the StSP6A mobile tuberization signal. We also show that natural allelic variants evade post-translational light regulation, allowing cultivation outside the geographical centre of origin of potato. Potato is a member of the Solanaceae family and is one of the world’s most important food crops. This annual plant originates from the Andean regions of South America. Potato develops tubers from underground stems called stolons. Its equatorial origin makes potato essentially short-day dependent for tuberization and potato will not make tubers in the long-day conditions of spring and summer in the northern latitudes. When introduced in temperate zones, wild material will form tubers in the course of the autumnal shortening of day-length. Thus, one of the first selected traits in potato leading to a European potato type is likely to have been long-day acclimation for tuberization. Potato breeders can exploit the naturally occurring variation in tuberization onset and life cycle length, allowing varietal breeding for different latitudes, harvest times and markets.

Concepts: Genetics, Solanaceae, Potato, Plant stem, Tuber, Plant reproduction, Andes, Rhizome

28

Elements of micropropagation include establishment of shoot tip cultures, proliferation, rooting, and acclimatization of the resulting plantlets. The wide genetic variation in Pyrus makes micropropagation challenging for many genotypes. Initiation of shoots is most successful from forced dormant shoots or from scions grafted onto seedling rootstocks to impose juvenility. Clean shoots are recovered after testing for contaminants at the initiation stage on ½ strength Murashige and Skoog 1962 medium (MS), at pH 6.9 for 1 week or by streaking on nutrient agar. Although pear species and cultivars are cultured on several well-known media, MS is the most commonly used. Our studies showed that multiplication and growth of shoots are best on Pear Medium with higher concentrations of calcium chloride, potassium phosphate, and magnesium sulfate than MS medium and 4.4 μM N(6) benzyladenine. Pear shoots are often recalcitrant to rooting; however, a 5 s dip in 10 mM indole-3-butyric acid or naphthalene acetic acid before planting on basal medium without plant growth regulators is effective for many genotypes. Pear shoots store well at 1-4°C, and can hold for as long as 4 years without reculture. Cryopreservation protocols are available for long-term storage of pear shoot tips. Acclimation of in vitro-rooted or micrografted shoots in a mist bed follows standard procedures.

Concepts: Grafting, Agar, Plant hormone, Salt, Plant reproduction, Shoot, Rootstock, Plant propagation

28

Jojoba (Simmondsia chinensis (Link) Schn.) is a nontraditional crop in arid and semi-arid areas. Vegetative propagation can be achieved by layering, grafting, or rooting semi-hardwood cuttings, but the highest number of possible propagules is limited by the size of the plants and time of the year. Micropropagation is highly recommended strategy for obtaining jojoba elite clones. For culture initiation, single-node explants are cultivated on Murashige and Skoog medium (MS) supplemented with Gamborg’s vitamins (B5), 11.1 μM BA (N(6)-benzyl-adenine), 0.5 μM IBA (indole-3-butyric acid), and 1.4 μM GA(3) (gibberellic acid). Internodal and apical cuttings proliferate on MS medium containing B5 vitamins and 4.4 μM BA. Rooting is achieved on MS medium (half strength mineral salt) amended with B5 vitamins and 14.7 μM IBA during 7 days and transferred to develop in auxin-free rooting medium. Plantlets are acclimatized using a graduated humidity regime on soil: peat: perlite (5:1:1) substrate. This micropagation protocol produces large numbers of uniform plants from selected genotypes of jojoba.

Concepts: Grafting, Seed, Plant reproduction, Vegetative reproduction, Jojoba, Plant propagation, Horticulture and gardening, Perlite

28

Persimmon (Diospyros kaki Thunb.) is a temperate fruit tree species diffused in all continents. The traditional propagation method adopted by the nursery industry is based on budding/grafting scion cultivars on seedlings from D. kaki, Diospyros lotus, and Diospyros virginiana, the most important species used as rootstock, reproduced by seeds since they are not easy to root. Furthermore, most of nonastringent cultivars of persimmon are not compatible with D. lotus, a rootstock largely utilized because of its hardiness and frost resistance. The main in vitro tissue culture techniques, developed for persimmon, deal with direct regeneration (from dormant buds and root tips), and indirect regeneration through callus from dormant buds, apexes, and leaves. The bottlenecks of micropropagation are (1) the recalcitrance of many cultivars to in vitro establishment, (2) the low multiplication ratio of D. kaki compared to other fruit tree species, (3) the very low rooting ability of ex novo microcuttings both from direct and indirect regeneration, (4) the high sensitivity to transplant from in vitro to in vivo conditions. The development of reliable in vitro regeneration procedures is likely to play a key role for production of both clonal rootstocks and self-rooted cultivars. The general protocol for micropropagation of persimmon reported here is based on the establishment of winter dormant buds in vitro, shoot development, multiplication and elongation, and shoot rooting, using cytokinins (BA or zeatin) in a MS media along with an auxinic pretreatment for rooting induction.

Concepts: In vivo, In vitro, Grafting, Persimmon, Plant morphology, Plant reproduction, Horticulture and gardening, Tissue culture

28

Asparagus officinalis is most extensively studied species within the genus Asparagus, which is well known as garden asparagus. This species is dioecious with unisexual flowers, which means that generative propagation gives roughly equal number of male and female plants. Male plants are high yielders and preferred commercially over female plants. Tissue culture techniques could efficiently promote vegetative propagation of male plants and pave the way for efficient plant breeding.This chapter describes an efficient micropropagation protocol for developing rapid growing in vitro Asparagus shoot cultures. The source of explants, inoculation, and shoot proliferation, followed by shoot propagation, rooting, and acclimatization is described. The optimal medium for Asparagus micropropagation described in this chapter is composed of MS macro- and microelements and a combination of auxins and cytokinins. Plant growth regulators NAA, kinetin, and BA were used in various concentrations. Three different media representing the whole micropropagation protocol of Asparagus are described; medium for shoot initiation, medium for shoot multiplication, and medium for root formation. By in vitro propagation of Asparagus, root initiation is difficult, but can be promoted by adding growth retardant ancymidol which also greatly promotes shoot development and suppresses callus formation.

Concepts: Plant, Root, Seed, Plant sexuality, Plant reproduction, Vegetative reproduction, Perennial plant, Asparagus