Reduced generation time of apple seedlings to within a year by means of a plant virus vector: a new plant‐breeding technique with no transmission of genetic modification to the next generation

Fruit trees have a long juvenile phase. For example, the juvenile phase of apple (Malus × domestica) generally lasts for 5–12 years and is a serious constraint for genetic analysis and for creating new apple cultivars through cross‐breeding. If modification of the genes involved in the transition from the juvenile phase to the adult phase can enable apple to complete its life cycle within 1 year, as seen in herbaceous plants, a significant enhancement in apple breeding will be realized. Here, we report a novel technology that simultaneously promotes expression of Arabidopsis FLOWERING LOCUS T gene (AtFT) and silencing of apple TERMINAL FLOWER 1 gene (MdTFL1‐1) using an Apple latent spherical virus (ALSV) vector (ALSV‐AtFT/MdTFL1) to accelerate flowering time and life cycle in apple seedlings. When apple cotyledons were inoculated with ALSV‐AtFT/MdTFL1 immediately after germination, more than 90% of infected seedlings started flowering within 1.5–3 months, and almost all early‐flowering seedlings continuously produced flower buds on the lateral and axillary shoots. Cross‐pollination between early‐flowering apple plants produced fruits with seeds, indicating that ALSV‐AtFT/MdTFL1 inoculation successfully reduced the time required for completion of the apple life cycle to 1 year or less. Apple latent spherical virus was not transmitted via seeds to successive progenies in most cases, and thus, this method will serve as a new breeding technique that does not pass genetic modification to the next generation.     

Expression of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> induces precocious flowering in soybean irrespective of maturity group and stem growth habit

The flowering integrator gene FLOWERING LOCUS T (FT) in Arabidopsis thaliana is conserved between diverse plant species and is thought to be the flowering signal ‘‘florigen’’, a universal long-distance mobile signal. In soybean, two FT homologs having a function to induce flowering in Arabidopsis have been identified. In this study, we showed that the expression of FT from Arabidopsis by the Apple latent spherical virus (ALSV) vector promoted precocious flowering and terminated vegetative growth in a wide range of genotypes of soybean, without using a short-day treatment. Four determinate and two indeterminate cultivars, infected with ALSV expressing FT (FT-ALSV), set flower buds on shoot apices and terminated vegetative growth at the fourth- to seventh-node stages under long-day conditions. In contrast, non-infected, healthy plants did not set flower buds on shoot apices at the same stage under the same conditions. After flowering, soybean cultivars infected with FT-ALSV, belonging to different maturity groups and stem growth habits, matured and produced seeds. The results suggest that the basic flowering pathway controlled by FT in A. thaliana might also be conserved in soybean. A system for precocious flowering and shortening of generation time using FT-ALSV would be a useful and novel technology for efficient soybean breeding.     

Virus-induced gene silencing in soybean seeds and the emergence stage of soybean plants with Apple latent spherical virus vectors

Virus-induced gene silencing (VIGS) has great potential as a reverse-genetics tool in plant genomics. In this study, we examined the potential of VIGS in soybean seeds and the emergence stage of soybean plants using Apple latent spherical virus (ALSV) vectors. Inoculation of an ALSV vector (soyPDS-ALSV) carrying a fragment of the soybean phytoene desaturase (soyPDS) gene into soybean seedlings resulted in a highly uniform photo-bleached phenotype, typical of PDS inhibition, on the upper leaves throughout plant growth. The photo-bleached phenotype was also found on all immature pods, all seed coats, and about 50% embryos of seeds on soybean plants infected with soyPDS-ALSV. Infection with an ALSV vector (soyIFS2-ALSV) having a fragment of soybean isoflavone synthase 2 (soyIFS2) gene also led to a reduction of the levels of both soyIFS2- and soyIFS1- mRNAs and an isoflavone content in the cotyledons of about 36% mature seeds of infected soybean plants. Furthermore, VIGS of soyPDS was induced in the next generation plants by the seed transmission of soyPDS-ALSV. Thus ALSV vectors will be useful for studying gene functions in the reproductive stages and early growth stages, such as emergence and cotyledon stages, in addition to the vegetative stages of soybean plants.     

Development of a transgenic early flowering pear (Pyrus communis L.) genotype by RNAi silencing of PcTFL1-1 and PcTFL1-2

Trees require a long maturation period, known as juvenile phase, before they can reproduce, complicating their genetic improvement as compared to annual plants. 'Spadona', one of the most important European pear (Pyrus communis L.) cultivars grown in Israel, has a very long juvenile period, up to 14?years, making breeding programs extremely slow. Progress in understanding the molecular basis of the transition to flowering has revealed genes that accelerate reproductive development when ectopically expressed in transgenic plants. A transgenic line of 'Spadona', named Early Flowering-Spadona (EF-Spa), was produced using a MdTFL1 RNAi cassette targeting the native pear genes PcTFL1-1 and PcTFL1-2. The transgenic line had three T-DNA insertions, one assigned to chromosome 2 and two to chromosome 14 PcTFL1-1 and PcTFL1-2 were completely silenced, and EF-Spa displayed an early flowering phenotype: flowers developed already in tissue culture and on most rooted plants 1-8?months after transfer to the greenhouse. EF-Spa developed solitary flowers from apical or lateral buds, reducing vegetative growth vigor. Pollination of EF-Spa trees generated normal-shaped fruits with viable F1 seeds. The greenhouse-grown transgenic F1 seedlings formed shoots and produced flowers 1-33?months after germination. Sequence analyses, of the non-transgenic F1 seedlings, demonstrated that this approach can be used to recover seedlings that have no trace of the T-DNA. Thus, the early flowering transgenic line EF-Spa obtained by PcTFL1 silencing provides an interesting tool to accelerate pear breeding.     

Seed germination and seedling allogamy in Rosmarinus officinalis: the costs of inbreeding

• Self‐pollination by geitonogamy is likely in self‐compatible plants that simultaneously expose a large number of flowers to pollinators. However, progeny of these plants is often highly allogamous. Although mechanisms to increase cross‐pollination have been identified and studied, their relative importance has rarely been addressed simultaneously in plant populations.
• We used Rosmarinus officinalis to explore factors that influence the probability of self‐fertilisation due to geitonogamy or that purge its consequences, focusing on their effects on seed germination and allogamy rate. We experimentally tested the effect of geitonogamy on the proportion of filled seeds and how it influences germination rate. During two field seasons, we studied how life history and flowering traits of individuals influence seed germination and allogamy rates of their progeny in wild populations at the extremes of the altitudinal range. The traits considered were plant size, population density, duration of the flowering season, number of open flowers, flowering synchrony among individuals within populations and proportion of male‐sterile flowers.
• We found that most seeds obtained experimentally from self‐pollination were apparently healthy but empty, and that the proportion of filled seeds drove the differences in germination rate between self‐ and cross‐pollination experiments. Plants from wild populations consistently had low germination rate and high rate of allogamy, as determined with microsatellites. Germination rate related positively to the length of the flowering season, flowering synchrony and the ratio of male‐sterile flowers, whereas the rate of allogamous seedlings was positively related only to the ratio of male‐sterile flowers.
• Rosemary plants purge most of the inbreeding caused by its pollination system by aborting the seeds. This study showed that the rates of seed germination and allogamy of the seedlings depend on a complex combination of factors that vary in space and time. Male sterility of flowers, length of the flowering season and flowering synchrony of individuals within populations all favour high rates of cross‐pollination, therefore increasing germination and allogamy rates. Flowering traits appear to be highly plastic and respond to local and seasonal conditions.

     

Seed predation limits post-fire recruitment in the waratah (<Emphasis Type="Italic">Telopea speciosissima</Emphasis>)

Seed predation may reduce recruitment in populations that are limited by the availability of seeds rather than microsites. Fires increase the availability of both seeds and microsites, but in plants that lack a soil- or canopy-stored seed bank, post-fire recruitment is often delayed compared to the majority of species. Pyrogenic flowering species, such as Telopea speciosissima, release their non-dormant seeds more than 1 year after fire, by which time seed predation and the availability of microsites may differ from that experienced by plants recruiting soon after fire. I assessed the role of post-dispersal seed predation in limiting seedling establishment after fire in T. speciosissima, in southeastern Australia. Using a seed-planting experiment, I manipulated vertebrate access to seeds and the combined cover of litter and vegetation within experimental microsites in the 2 years of natural seed fall after a fire. Losses to vertebrate and invertebrate seed predators were rapid and substantial, with 50% of seeds consumed after 2 months in exposed locations and after 5 months when vertebrates were excluded. After 7 months, only 6% of seeds or seedlings survived, even where vertebrates were excluded. Removing litter and vegetation increased the likelihood of seed predation by vertebrates, but had little influence on losses due to invertebrates. Microsites with high-density vegetation and litter cover were more likely to have seed survival or germination than microsites with low-density cover. Recruitment in pyrogenic flowering species may depend upon the release of seeds into locations where dense cover may allow them to escape from vertebrate predators. Even here, conditions suitable for germination must occur soon after seed release for seeds to escape from invertebrate predators. Seed production will also affect recruitment after any one fire, while the ability of some juvenile and most adult plants to resprout after fire buffers populations against rapid declines when there is little successful recruitment.     

Early in vitro flowering and seed production in culture in <Emphasis Type="Italic">Dendrobium</Emphasis> Chao Praya Smile (Orchidaceae)

Plantlets of Dendrobium Chao Praya Smile maintained in vitro were induced to flower, which produced viable seeds within about 11 months. A two-layer (Gelrite-solidified layer topped with a layer of liquid medium of the same volume and composition) culture system containing benzyladenine (BA) at 11.1 μM induced the highest percent of flowering (45%) in plantlets within 6 months from germination. The percentage of inflorescence induction was increased to 72% by pre-selecting morphologically normal seedlings prior to two-layer culture. Plantlets in culture produced both complete (developmentally normal but smaller than flowers of field grown plants) and incomplete flowers. Pollen and female reproductive organs of in vitro-developed complete flowers were morphologically and anatomically similar to flowers of field grown plants. In addition, 65% of the pollen grains derived from in vitro-developed flower were tetrad suggesting that regular meiosis occurred during microsporogenesis. The percentage of germination of pollen grains derived from in vitro-developed flowers and flowers of field grown plants, incubated on modified Knops’ medium for 8 days, were 18.2 and 52.8%, respectively. Despite a lower percentage of germination of the pollen grains derived from in vitro-developed flowers, flowers induced in culture could be self-pollinated and developed seedpods with viable seeds. Nearly 90% of these seeds developed into protocorms on germination in vitro. These seedlings were grown in culture and induced to flower in vitro again using the same procedure.     

Precocious floral initiation and identification of exact timing of embryo physiological maturity facilitate germination of immature seeds to truncate the lifecycle of pea

We propose herein a novel single seed descent protocol that has application across a broad phenotypic range of pea genotypes. Manipulation of key in vivo growing conditions, including light, photoperiod and temperature, combined with precocious in vitro germination of the embryo at full physiological maturity substantially shortened the pea lifecycle. We define full embryo physiological maturity as the earliest point in seed development when precocious in vitro germination and robust seedling growth can be reliably achieved without supply of exogenous hormones. Under our optimised conditions for accelerated plant growth, embryo physiological maturity was attained at c. 18 days after pollination, when seed moisture content was below 60?% and sucrose level under 100 mg g^?1 DW. No delay penalty in terms of time to flowering and plant development was caused by the culture of immature seeds 18 days after pollination compared to the used of mature ones. Determining the role embryo maturity plays in the fitness of the germinated plant has facilitated the truncation of the lifecycle across pea genotypes. The accelerated single seed descent system proposed within this research will benefit complex genetic studies via the rapid development of recombinant inbred lines (RIL) and multi-parental advanced generation intercrosses (MAGIC) populations.     

A wheat homolog of MOTHER OF FT AND TFL1 acts in the regulation of germination

Seed dormancy is an adaptive mechanism and an important agronomic trait. Temperature during seed development strongly affects seed dormancy in wheat (Triticum aestivum) with lower temperatures producing higher levels of seed dormancy. To identify genes important for seed dormancy, we used a wheat microarray to analyze gene expression in embryos from mature seeds grown at lower and higher temperatures. We found that a wheat homolog of MOTHER OF FT AND TFL1 (MFT) was upregulated after physiological maturity in dormant seeds grown at the lower temperature. In situ hybridization analysis indicated that MFT was exclusively expressed in the scutellum and coleorhiza. Mapping analysis showed that MFT on chromosome 3A (MFT-3A) colocalized with the seed dormancy quantitative trait locus (QTL) QPhs.ocs-3A.1. MFT-3A expression levels in a dormant cultivar used for the detection of the QTL were higher after physiological maturity; this increased expression correlated with a single nucleotide polymorphism in the promoter region. In a complementation analysis, high levels of MFT expression were correlated with a low germination index in T1 seeds. Furthermore, precocious germination of isolated immature embryos was suppressed by transient introduction of MFT driven by the maize (Zea mays) ubiquitin promoter. Taken together, these results suggest that MFT plays an important role in the regulation of germination in wheat.     

Modulating flowering time and prevention of pod shatter in oilseed rape

Floral induction is a key developmental switch in plants that leads to the production of flowers, fruits and seeds, which are of paramount importance for human life. To meet the demands of several crop harvests per year, or the growth of crop plants in regions with short vegetation times and for the production of ornamental plants, the timing of the floral transition is very important. The discovery of genes that are involved in flowering time control in model plants should allow the modulation of this developmental switch also in plants with economic value. By using a transgenic approach, we showed that a single MADS box gene accelerated flowering and seed ripening in summer rape plants. The MADSB transgene also partially substituted for the strict temperature requirements for flowering in winter rape plants. Transgenic winter rape plants expressing the MADSB transgene also produced more rigid siliques than wild type winter rape plants, and this prevented precocious seed dispersal.     

Seed after-ripening and dormancy determine adult life history independently of germination timing

? Seed dormancy can affect life history through its effects on germination time. Here, we investigate its influence on life history beyond the timing of germination. ? We used the response of Arabidopsis thaliana to chilling at the germination and flowering stages to test the following: how seed dormancy affects germination responses to the environment; whether variation in dormancy affects adult phenology independently of germination time; and whether environmental cues experienced by dormant seeds have an effect on adult life history. ? Dormancy conditioned the germination response to low temperatures, such that prolonged periods of chilling induced dormancy in nondormant seeds, but stimulated germination in dormant seeds. The alleviation of dormancy through after-ripening was associated with earlier flowering, independent of germination date. Experimental dormancy manipulations showed that prolonged chilling at the seed stage always induced earlier flowering, regardless of seed dormancy. Surprisingly, this effect of seed chilling on flowering time was observed even when low temperatures did not induce germination. ? In summary, seed dormancy influences flowering time and hence life history independent of its effects on germination timing. We conclude that the seed stage has a pronounced effect on life history, the influence of which goes well beyond the timing of germination.     

Effectiveness of ants as pollinators of<Emphasis Type="Italic"> Lobularia maritima</Emphasis>: effects on main sequential fitness components of the host plant

The effectiveness of ants as pollinators of Lobularia maritima (Cruciferae) was experimentally analyzed by assessing (1) their quantitative importance at flowers; (2) their effect on host plant seed production; (3) their effect on the performance of host plant progeny, estimated as seed germination, seedling emergence, seedling survival to flowering, and (4) the overall effect of ants on a cumulative, more realistic measure of plant fitness related to the recruitment probability. Flowers of L. maritima were visited during the 2 years of study (1996 and 1997) by more than 50 pollinator species belonging to about 30 families of disparate taxonomic affiliation, notably ants and flies. There was significant seasonal variability in insect abundance and type. Ants, especially Camponotus micans (Formicidae), visited the flowers of L. maritima in summer, representing 81.2% of the visits during this season. This ant species acted as a pollinator of L. maritima, with flowers visited exclusively by ants producing significantly more seeds than flowers from which all pollinators were excluded, whereas flowers visited by only winged insects did not differ from self-pollination. Ant-pollinated flowers produced seeds with a germination rate comparable to the other treatments. Moreover, seedlings from these seeds emerged as fast, and survived at the same rate as controls. Consequently, both ants and winged insects had similar overall effects on host plant recruitment probability (0.6 and 0.7% of initial ovules produced flowering offspring, respectively), a result similar to that of open-pollinated flowers (1.0%). This study demonstrated that the overall effectiveness of the ant C. micans as a summer pollinator of L. maritima was as high as that of other winged insects, contributing not only to the seed production of this crucifer but also to the recruitment of new flowering offspring. Received: 17 February 1999 / Accepted: 26 August 1999     

Are mismatches the norm? Timing of flowering,fruiting, dispersal and germination and their fitness effects in Frangula alnus (Rhamnaceae)

The close morphological and temporal links between phases of plant growth and reproduction call for integrated studies incorporating several reproductive phases from flowering to recruitment, and associated plant‐animal interactions. Phenological strategies, as well as plastic phenological response to climate change, incorporate complex interactions between developmental constraints, pollination and seed dispersal. Relationships between reproductive phenology and components of fitness were studied for two years in the north‐temperate, self‐incompatible, insect‐pollinated, and bird‐dispersed shrub Frangula alnus (Rhamnaceae). Fruit set, dispersal, germination and juvenile survival, as well as seed mass and juvenile size were measured in relation to flowering, fruiting and germination time. The results suggest that effects of flowering and fruiting time prevailed in subsequent phases, to some extent as far as to the juvenile phase, but effects of timing were complex and had partly opposing effects on different fitness components. Early flowers had higher fruit‐set and experiments indicated that synchronous peak flowering increased fruit‐set, but later flowers had higher seed mass. Peak fruiting was not associated with peak dispersal. Late fruits derived from late flowers promoted dispersal. Juvenile recruitment was enhanced by increasing seed size. We conclude that the phenology of flowering and fruiting in F. alnus comprises several features, each with different and sometimes counteracting effects on fitness components. From a general perspective, this result implies that we should not expect to find finely tuned matches in timing specifically between flowering and pollinators, and fruiting and seed dispersing birds.     

Changes in Seed Constituents During Germination and Seedling Growth of Precociously Matured Soybean Seeds(Glycine max)

During 7 d of precocious maturation of soybean seed (Glycinemax), the starch content declined and soluble sugar levels increasedin patterns similar to natural seed dehydration and maturation.Total seed protein content and total seed dry weight increasedwhereas oil content remained relatively unchanged. Overall,the proportions of the constituents in precociously maturedseeds were comparable to naturally mature seeds. Precociouslymatured soybean seeds showed much the same germination and seedlinggrowth frequency patterns as naturally matured seeds. Duringgermination and seedling growth of precociously matured seeds,starch, soluble sugar, protein and oil levels followed patternssimilar to naturally mature, germinating seeds and seedlings.Therefore, precocious maturation may be used as a model systemto investigate the control of the physiological and biochemicalevents occurring during seed maturation which lead to germinationand subsequently, seedling growth. Glycine max (L.) Merr., soybean, cotyledons, maturation, germination/seedling growth     

Large plant size counteracts early seed predation during the extended flowering season of a Silene uniflora (Caryophyllaceae) population

A population of the alvar race of the perennial herb, Silene uniflora (Caryophyllaceae), which grows on thin soils in open alvar habitats on the Baltic island of Oland SE Sweden, was found to have an extended and more or less bimodal flowering phenology Large individuals produced flowers during both periods, while small, and presumably young, individuals only produced flowers in either of the two periods, or in part of both In the early flowering period plants were heavily infested by the seed-predatory larvae of a noctuid moth, Hadena confusa , but in the late flowering period only a small proportion of the fruits was attacked by the seed predators The proportion of flowers developing into fruits was consistently high throughout the season Both the number of seeds per capsule and the mass of seeds decreased over the flowering season However, the germination success of early and late seeds did not differ Thus, although differing in number of seeds, both early and late flowers contributed to individual reproductive success Large individuals started to flower early in the season and despite their high loss of seeds in the early part of the summer they contributed a larger number of seeds to the seed pool than smaller and later-flowering individuals Although selection was acting to favour later flowering during a year with high early seed predation, consistency of date of flower initiation and of relative predation impact on individuals across years was low suggesting that recurrent selection by seed predators is weak Seed predation, although heavy, is therefore judged to be unlikely to cause a significant evolutionary response on flowering phenology in this plant     

Offspring Performance in Oxalis acetosella, a Cleistogamous Perennial Herb

Abstract: Seed weight, seed germination, seedling survival, and juvenile/adult fitness in chasmogamously (CH) and cleistogamously (CL) derived offspring of Oxalis acetosella were compared during three growing seasons, to test hypotheses of fitness differences between the offspring types accounting for the maintenance of cleistogamy. In plots at three field sites, CH and CL seeds originating from all sites were sown to compare the performance of offspring growing in their habitat of origin and offspring growing in new habitats. Seeds were also sown in pots in a common garden, to test for effects of sibling competition. CL seeds had significantly lower germination than CH seeds in the field, possibly because of lower mean seed weight due to later flowering. Since the outcrossing rate in the CH flowers of O. acetosella is not known, it is uncertain whether the lower CL germination is a consequence of inbreeding depression. CH seeds had higher germination if sown at their home sites than at new sites, while for CL seeds this made no difference; this contradicts the local adaptation hypothesis for cleistogamy. No other fitness differences were found between the offspring types, and the findings did not support the sibling competition or local adaptation hypotheses. We suggest that the maintenance of the dimorphic reproductive system in O. acetosella is not explained by offspring characteristics, but rather by the two flowering phases complementing each other in maximizing annual seed production in the face of environmental variability. It is, therefore, important to include temporal and spatial variation in studies of reproductive strategies.     

Intra-inflorescence variation in floral traits and reproductive success of the hermaphrodite Silene acutifolia

BACKGROUND AND AIMS: Intraspecific variation in floral components and reproductive success is often located at the intra-individual level. The arrangement of flowers within inflorescences may explain a great deal of this variation. The variation in number of ovules, fruit set, number of seeds per fruit, seed set, seed weight and seed germination is investigated at different positions within the inflorescence of Silene acutifolia. METHODS: Data were obtained in natural populations, and germination experiments were conducted in a germination chamber. KEY RESULTS: The number of ovules, fruit set, number of seeds, seed set and seed weight, showed a significant decline from early (primary) position to later (tertiary) position. The patterns of intra-inflorescence variation were consistent in different populations and years of study. Seed germination showed an opposite pattern, seeds from primary position showed the lowest germination percentages and seeds from tertiary position the highest, although the effect of position on germination was only marginally significant. There was significant among-population variation in number of ovules per flower. Fruit set also varied significantly among populations, with lower fruit set in the smaller and more isolated population. No significant among-population differences were detected in number of seeds per fruit and seed set. Seeds from the smallest and more isolated population (Arnado) were the lightest. Seed germination showed strong differences between populations, seeds from Arnado started to germinate later, and showed the lowest final germination percentages. CONCLUSIONS: Architectural effects or resource competition are the most commonly proposed hypothesis to explain these patterns. Data suggest that there is less pollen available to pollinate tertiary flowers, and that there is not enough outcross pollen in Arnado. The germination percentages suggest that there is variation in the source of pollen within inflorescences, with high probability of receiving outcross pollen in flowers from primary position, and higher probability of geitonogamous crosses in tertiary flowers.