Unlocking variability: inherent variation and developmental traits of garlic plants originated from sexual reproduction

Recent collections of fertile garlic (Allium sativum) accessions from Central Asia allow a detailed study of seedling developments and the evaluation of inherent variations. We hereby provide a comprehensive account of the ontogenesis of a population of garlic seedlings and their vegetative and reproductive traits. A nucleotide binding site profiling marker technology was applied to provide conclusive evidence for the cross-pollination nature of garlic, and to compare the levels of polymorphism between progeny derived from a single mother clone fertilized by several pollinators. The seedlings’ population demonstrates a large variation in vegetative and reproductive characters, including bulbing ability, bulb color and size, clove number, and response to environmental conditions, similar to that of the genepool of vegetatively propagated garlic clones. In addition, a large variation in flowering and seed production ability was recorded. The understanding of garlic physiology, the availability of the large variability unleashed by sexual reproduction, and the possible utilization of sexual hybridization opens the way for genetic studies and breeding work. Haim D. Rabinowitch and Rina Kamenetsky contributed equally to this paper.     

Mineral allocation to reproduction in Sorhum bicolor and Sorghum halepense in relation to parental nutrient supply

Summary This experiment investigated the effect of parental nutrient shortage on the allocation of five nutrients to seeds and rhizomes in Sorghum halepense, a perennial, noxious weed, and to seeds in Sorghum bicolor, an annual, cultivated species. Plants from both species were grown from seeds and supplied with fertilizer at three concentrations. The allocation of biomass and nutrients (N, P, K, Ca and Mg) to reproductive and vegetative parts was determined. Relative biomass allocation to reproduction (either sexual or vegetative) remained constant in S. halepense in spite of large differences in total plant weight. In S. bicolor, however, biomass allocation to sexual reproductive structures decreased significantly with decreasing nutrient supply. Individual seed weight was not modified by parental nutrient supply in S. halepense, but it increased with decreasing nutrient availability in S. bicolor. Important differences in mineral allocation to seeds were found between the two species. While S. bicolor seeds were largely buffered from the differences in parental nutrient status, concentration of nutrients in S. halepense seeds decreased significantly with decreasing supply for all the nutrients analyzed except Ca. However, mineral nutrient concentration in S. halepense rhizomes remained remarkably constant despite differences in the external supply, evincing the priority given to vegetative reproduction at the expense of sexual reproduction. Overall, the pattern of nutrient allocation in S. bicolor seeds under different nutrient supply resembled the pattern observed in S. halepense rhizomes, but it had little resemblance to the pattern of nutrient allocation in S. halepense seeds. The results are discussed in terms of differences and similarities in the reproductive strategy of these two species.     

Sexual and vegetative reproduction in the aboveground part of a dioecious clonal plant, <Emphasis Type="Italic">Dioscorea japonica</Emphasis> (Dioscoreaceae)

In dioecious clonal plants, the reproductive effort required to set seeds will be responsible for the larger investment in sexual reproduction by females. If there will be a trade-off in resource allocation between sexual and clonal reproduction, this differential sexual reproduction will lead to sexual differentiation in the relative amount of clonal reproduction. To test this prediction, we studied differences between the sexes in their phenologies and investments in sexual and vegetative reproduction (clonal reproduction by means of bulbils) with respect to ramet size in a dioecious clonal plant, Dioscorea japonica Thunb. The period of bulbil production overlapped the period during which infructescences developed. Females flowered later, produced heavier inflorescences, and fewer flowers per inflorescence than did males. Regression analysis using the size of the individual plants demonstrated that large females made smaller investments in inflorescences and larger investments in sexual reproduction than did large males. In contrast, females invested fewer resources in vegetative reproduction than did males. However, the total investments in sexual and vegetative reproduction did not differ between the sexes. These results supported our hypothesis on the sexual differentiation in sexual and clonal reproduction.     

Optimum reproduction and dispersal strategies of a clonal plant in a metapopulation: a simulation study with Hieracium pilosella

Clonal spread is favoured in many plants at the expense of seed production in order to expand rapidly into open habitats or to occupy space by forming dense patches. However, for the dynamics of a population in a patchy landscape seed dispersal remains important even for clonal plants. We used a spatially explicit individual-based metapopulation model to examine the consequences of two trade-offs in Hieracium pilosella L: first, between vegetative and sexual reproduction, and second, between short and far-distance dispersal of seeds. Our main question was, what are the environmental conditions that cause a mixed strategy of vegetative and sexual reproduction to be optimal. The model was parameterised with field data on local population dynamics of H. pilosella. Patch dynamics were given firstly by disturbance events that opened patches in a matrix of a clonal grass that were colonisable for H. pilosella, and secondly by the gradual disappearance of H. pilosella patches due to the expanding grass. Simulations revealed opposing selection pressures on traits determined by the two trade-offs. Vegetative reproduction is favoured by local dynamics, i.e. the need for maintenance and expansion of established populations, whereas seed production is favoured by the necessity to colonise empty habitats. Similar pressures act on the proportion of seeds dispersed over short and far distances. Optimum reproductive and dispersal strategies depended on habitat quality (determined by seedling establishment probability), the fraction of dispersed seeds, and the fraction of seeds lost on unsuitable ground. Under habitat conditions supporting moderate to low seedling establishment, between 20% and 40% of reproductive effort in H. pilosella should be devoted to sexual reproduction with at least 10% of the seeds dispersed over distances suitable to attain empty patches. We conclude that in a spatially heterogeneous landscape sexual seed production in a clonal plant is advantageous even at the expense of local vegetative growth.     

Effects of intraspecific competition on size variation and reproductive allocation in a clonal plant

Clonal plants grow in diameter rather than height, and therefore competition among genets is likely to be symmetric and to result in smaller variation in size of genets than in non-clonal plants. Moreover, clonal plants can reproduce both sexually and vegetatively. We studied the effects of density on the size of rosettes and of clones, variation in the size of rosettes and of clones, and allocation to sexual and vegetative reproduction in the clonal herb Ranunculus reptans . We grew plants from an artificial population of R. reptans in 32 trays at two densities. After four months, differences in density were still apparent, although clones in the low-density treatment had on average 155% more rosettes and 227% more rooted rosettes than clones in the high-density treatment. The coefficient of variation of these measures of clone size was 15% and 83% higher, respectively, in the low-density treatment. This indicates that intraspecific competition among clones of R. reptans is symmetric and increases the effective population size. Rooted rosettes were larger and varied more in size in the low-density treatment. The relative allocation of the populations to sexual and to vegetative reproduction was 19% and 13% higher, respectively, in the high-density treatment. Moreover, seeds produced in the high-density treatment had a 24% higher mass and a 7% higher germination percentage. This suggests that with increasing density, allocation to sexual reproduction increases more than allocation to vegetative reproduction in R. reptans , which corresponds to the response of some other species with a spreading growth form but not of species with a compact growth form. We conclude that intraspecific competition is an important factor in the life-history evolution of R. reptans because intraspecific competition affects its clonal life-history traits and may affect evolutionary processes such as genetic drift and selection through its effect on the effective population size.     

Application of Esterase Isozymes for Garlic Ecotype Identification

Expression of esterase isozymes from cloves of bolting and non-bolting garlic clones belonging to winter and summer ecotypes were analyzed. The isozyme patterns were obtained by means of vertical block electrophoresis in polyacrylamide gels. Quantitative and qualitative differences between loci of winter and summer garlic ecotype were found. This fact substantiates application of the isoesterases for identification of garlic clones.     

Interspecific hybrids between Medicago sativa L. and annual Medicago containing Alfafa weevil resistance

Non-embryogenic protoplasts of Medicago rugosa and M. scutellata were electro-fused with iodoacetic acid-treated protoplasts of M. sativa (alfalfa). Putative somatic hybrid callus were obtained and some plants regenerated from both combinations. Hybridity of regenerants was confirmed by morphology, molecular means and cytological observations. Parental specific bands were recognized in somatic hybrids by Southern analysis. The somatic hybrids were perennial and their morphology was similar to M. sativa. Cytological observations were carried out on the somatic hybrids, their vegetative clones and self-pollinated offspring. Original somatic hybrids were aneuploids (2n=31–59), but during vegetative proliferation, their chromosome numbers reduced to 32. Those clones of hybrids formed seeds from M. sativa (+) M. rugosa by self-crossing. Chromosomal rearrangements within the parental genomes were observed in vegetative clones of hybrids and their S₁ offspring by Genomic in situ Hybridization (GISH). Some of S₁ offspring from M. sativa (+) M. rugosa showed better spring growth than parental M. sativa and tend to be tolerant to Alfalfa weevil. It was considered that these traits were introduced from the genome transferring M.␣rugosa chromosome to M. sativa. The cell fusion may still have a potential in transferring alien chromosomes in order to increase the genetic variation for crop breeding.     

TIME LAPSE ANALYSES OF SEXUAL REPRODUCTION IN CLOSTERIUM EHRENBERGII (CONJUGATOPHYCEAE)1

The process of sexual differentiation was studied using heterothallic clones of Closterium ehrenbergii Meneghini. The first visible sign of sexual reproduction was agglutination of two or more cells in a group and this was followed by gametangiogenic division and conjugation of gametangial cells. Movements of gametangial cells were carefully studied. Gametangial cells occasionally participated again in gametangiogenesis instead of proceeding directly to the formation of conjugation papilla. The whole process of sexual differentiation from vegetative cell to zygospore was considered to be basically similar in both of the two closely related mating groups, A and B, of C. ehrenbergii. Nevertheless, there were some differences between the two groups in patterns of the sexual differentiation. In Group A, vegetative cell division was completely suppressed by mixing the two complementary mating type clones together into the same medium with high light illumination. This suppression was not caused by the nitrogen depletion in the medium, but by the presence of cells of opposite mating type. In Group B, vegetative cell division and sexual reproduction occurred side by side repeatedly for several days.     

Diversity in fertility potential and organo-sulphur compounds among garlics from Central Asia

Extending the collection of garlic (Allium sativum L.) accessions is an important means that is available for broadening the genetic variability of this cultivated plant, with regard to yield, quality, and tolerance to biotic and abiotic traits; it is also an important means for restoring fertility and flowering. In the framework of the EU project Garlic and Health, 120 garlic accessions were collected in Central Asia – the main centre of garlic diversity. Plants were documented and thereafter maintained in field collections in both Israel and The Netherlands. The collection was evaluated for biological and economic traits. Garlic clones vary in most vegetative characteristics (leaf number, bulb size and structure), as well as in floral scape elongation and inflorescence development. A clear distinction was made between incomplete bolting and bolting populations; most of the accessions in the latter populations produced flowers with fertile pollen and receptive stigma. Wide variations were recorded with regard to differentiation of topsets, their size, number and rapidity of development. Furthermore, significant variation in organo-sulphur compounds (alliin, isoalliin, allicin and related dipeptides) was found within garlic collections and between plants grown under differing environmental conditions. Genetic fingerprinting by means of AFLP markers revealed three distinct groups within this collection, differing also in flowering ability and organo-S content.     

The effects of seed weight on growth and competitive ability of Rumex acetosella from two successional old-fields

Summary The effects of seed size on growth, biomass allocation and competitive ability of Rumex acetosella plants grown either individually or in competition were studied in two populations (6 months and 15 years old respectively) sampled from a postcultivation successional gradient. For plants grown individually there were highly significant effects of seed weight on growth after 43 days, with a higher relative growth rate (RGR) observed for plants raised from heavier seeds. However at the end of the experiment, seedlings developed from lighter seeds had a RGR 2 times greater than those from heavier seeds. Final biomass of the two types was not significantly different after 73 days of growth. When plants were grown individually, there were only slight differences between populations, but when grown in monocultures of 4 plants per pot, plants from the old population had higher root and leaf biomass per pot whereas those from the young population had a higher reproductive effort per pot. This suggests that a trade-off between allocation to sexual and vegetative reproduction occurs over successional time. In mixtures of light and heavy seeds, plants from light seeds were shorter, had fewer leaves and lower biomass than plants from heavy seeds, which were also taller and produced more dry matter than plants grown from heavy seeds in monoculture. The significant effects of seed weight and population on biomass parameters persisted unit the end of the experiment. Seedlings from heavy seeds were strong competitors: those from the young population grew better in the presence of neighbors than in monoculture and those from the late successional population suppressed the more the growth of their partners. Seedlings from light seeds were subordinate competitors. These results suggest that seedlings from seeds of different sizes benefit from contrasting ecological conditions and that selection acts on reproductive output along successional gradients.     

Effect of long photoperiod on the reproductive and bulbing processes in garlic (Allium sativum L.) genotypes

Garlic (Allium sativum L.) genotypes differ considerably in scape length, flowering ability, and seed production. In bolting clones reproductive processes are facilitated by a combination of specific temperature and photoperiod regimes. However, it is yet unclear whether the phase-specific photoperiod effect on florogenesis is universal for all garlic genotypes. We investigated the effect of ambient day length interruption by 10 or 30 days of long photoperiod in ten garlic accessions belonging to four bio-morphological groups. It was evident that garlic genotypes vary significantly in response to long photoperiod, and that in some clones the treatment enhances florogenesis, flower stalk elongation and bulbing. The competition for resources by the simultaneously developing bulb and inflorescence sinks differentially determines the fate of stalk elongation and flower development in the investigated genotypes. It is concluded that florogenesis in garlic can be promoted by exposure to proper environmental stimuli, during pre-planting storage and sprouting to the later growth stages. The genetic variability acquired from sexual reproduction and vegetatively preserved collections can serve in genetic and physiological studies as well as in breeding programs.     

The reproductive biology of the genusBaldellia (Alismataceae)

The reproductive biology of the genusBaldellia was studied under cultivation and in nature.B. ranunculoides subsp.repens is self-incompatible whereasB. ranunculoides subsp.ranunculoides andB. alpestris are self-compatible. Bud-pollination occurs in theB. alpestris. Self-incompatibility correlates with the production of fewer, larger, more showy flowers, higher pollen production (but reduced pollen grain size), fewer carpels and smaller seeds. Resource allocation to sexual reproduction is inversely correlated with the degree of vegetative growth.B. ranunculoides subsp.repens produces more ramets than subsp.ranunculoides, which reproduces mainly by seeds.B. alpestris is intermediate between the two since it is self-compatible, produces fewer, larger seed than subsp.ranunculoides and is also able to propagate by vegetative means.     

The frequency of marker changes in sugarcane plants regenerated from callus culture

Leaf tissue from five sugarcane clones with distinctive markers was cultured on a medium favoring callus growth. Transferred to a differentiation medium, calli produced over 5000 plants. Plants differentiated from two clones with stem markers exhibited a high rate of remission of the marker, but the marker reappeared in the vegetative progeny of these plants, and remission was, therefore, transient. Plants differentiated from callus from two clones with leaf markers showed a low rate of remission (2 or 3 per thousand) of the marker and the vegetative progeny was stable. A clone with variegated leaves produced plants with the majority having green leaves, some were albino, and some variegated, suggesting that plant differentiation may start with more than one cell. Permanent phenotypic change may result from tissue culture, but the results suggest that such changes are not frequent and may be confounded by temporary alterations or by chimeras formed in the process of differentiation.     

The Relation Between Growth of Gastrodia elata Protocorms and Fungi

Armillaria mellea penetrated protocorms from seed germination and vegetative multiplication corms of Gastrodia elata with rhizomorph. At beginning, they formed a hypha passing road and a hypha flow in the inner cells of cortex, and then, they both penetrated inside of large cells and penetrated outside of cortical cells. Gastrodia elata seeds depended on digesting Mycenct osmundicola etc gain nutrition to germinate at the stage of sexual reproduction, but its corms of vegetative multiplication must be penetrated by Armillaria mellea obtaining nutrition for normal growth at the stage of vegetative propagation.     

No photoperiodoc control of the formation of turions in eight clones of Spirodela polyrhiza

The influence of daily photoperiod (8, 16, 24 h) on eight clones of Spirodela polyrhiza was tested in two different nutrient media. The number of vegetative fronds and resting turions formed after 50 days of cultivation were scored. The specific turion yield (STY; number of turions formed per vegetative frond) was used to evaluate the effectiveness of turion formation of the tested clones. All clones formed turions in both nutrient media. The STY varied substantially between the different clones, ranging from 0.22 +/- 0.03 (clone SC from Cuba) to 3.9 +/- 0.3 (clone 9256 from Finland) in continuous light. The STY increased with increasing duration of the photoperiod. This increase may have been due to the extended period of photosynthesis rather than that of a photoperiodic long-day response. Shorter photoperiods did not stimulate turion formation in any of the clones. S. polyrhiza is a day-neutral plant with respect to turion formation, as noted previously (Appenroth et al. 1990. Annals of Botany 66: 163-168). In accordance with this conclusion, no correlation was detected between the STY and the latitude at which the clones occur naturally. Environmental factors other than shortening of photoperiods seem to be effective in signalling seasonal changes of growth conditions in advance to S. polyrhiza.     

Dissection of conjugants and mating type plus and minus cells in selfing clones of the isogamous green alga Closterium ehrenbergii

In the sexual reproduction of the green alga Closterium ehrenbergii, two sexually competent cells that are morphologically indistinguishable from the vegetative cells first come close to each other to form a sexually interacting pair. Each then divides into two gametangial cells. Isogamous conjugation occurs between nonsister gametangial cells of the two resulting pairs. With unusual selfing clones derived from a certain cross of heterothallic strains, we dissected apart a pair of gametangial cells that had already been united together by a delicate transparent tube, into which each gametangial cell was going to develop its conjugation papilla. In spite of such a degree of differentiation, when each was cultured in fresh medium, individual gametangial cells could dedifferentiate into vegetative cells and form subclones. By crossing such subclones with standard stable heterothallic mating-type strains, we show that each selfing clone of this alga actually produces both stable mt ⁺ and stable mt ^- cells, in addition to unstable mt ^- cells with selfing potency, during its mitotic vegetative growth. Although the selfing in C. ehrenbergii studied here differs in certain points from true homothallism, the results of the present study provide insight into how homothallism might have evolved from heterothallism.     

An isozyme study of clone diversity and relative importance of sexual and vegetative recruitment in the grass Brachypodium pinnatum

We asked whether differences in abundance and seed prodtiction of Brachypodium pinnatum after 16 yr of four different experimental land use regimes were reflected in differences in vegetative dispersal distance, clone diversity, clone area, and the proportions of sexual and vegetative recruitment. Mean vegetative dispersal distance was 5.5 mm yr'. Electrophoresis of 5 polymorphic isozyme loci of 20 tillers sampled at defined positions in each of twelve 1 × 6 m sampling areas (3 per treatment) revealed considerable clonal diversity. Per sampling area we found on average 9.98 enzyme phenotypes (clones), mean Simpson index was 0.825. and mean Shannon index 0.801. The mean ratio of sexual vs vegetative recruitment was about 1:32000. Despite this low ratio, clonal diversity within the population of" B. pinnatum was higher than reported for other clonal plant populations, possibly because of its high ramet densities. Moan clone area was 5.73 m²-. i.e. mean clone radius was 1.35 m. None of the 10 pairwise correlations between abundance and seed production on the one hand, and number of clones per plot sample, plot Simpson index, plot Shannon index, ratio of vegetative vs sexual recruitment, and clone area on the other, was significant. Mean clone radius was 245 times larger than the mean distance of yearly vegetative dispersal which suggests old ages and low turnover rates of clones. The time scale of the inert response of clonal diversity of B. pinnatum to changes in land use appears to largely exceed the experimental period of 16 yr.     

Vegetative regeneration on sexual organs inPhycomyces blakesleeanus

Phycomyces blakesleeanus produced an abundance of sexual organs when two mating types met on solid medium, but only about 14.7% of the sexual organs developed to the final stages. On the sexual organs showing arrested development, vegetative hyphae or dwarf sporangiophores (microphores) often regenerated. This vegetative regeneration was accelerated when the paired and looped progametangia were isolated from mycelia, when the counterparts of the progametangial cells constructing the loop were surgically incised, and whenPhycomyces was mated at high temperature (25–27°C). A leaky-carotenogenic mutant, whose sexual reaction was imperfect and arrested at an intermediate stage even when mated with the wild type, also regenerated hyphae with a high frequency on these arrested intermediate organs. The vegetative regeneration seems to result from interruption of a cell-to-cell recognition system between cells of different mating type, which is believed to be essential for the mating process of this fungus in addition to the pheromonal actions.