Partial hybridization in wide crosses between cultivated sunflower and the perennial Helianthus species H. mollis and H. orgyalis

To obtain introgressed sunflower lines with improved disease resistance, interspecific crosses were performed with foreign perennial species. We report on several unusual features displayed by these hybrid plants. The methods used to produce the kernels affected yield and genotypes of progeny. Phenotypic traits and DNA markers were investigated in 97 plants derived from cross-pollination between annual diploid cultivated sunflower (Helianthus annuus) and the perennial diploid species H. mollis or H. orgyalis, and the reverse reciprocal crosses. The level of hybridization in progeny was determined using RAPD and RFLP markers. Hybridization was performed by leaving embryos to develop normally on the head (classical crossing) or using embryo rescue. All observed plants derived from H. mollis were diploid (2n = 34). Phenotypes were predominantly similar to the female when cultivated sunflower was the female parent. Progeny from crosses using a wild species as the female parent resembled that parent. Thus, reciprocal crosses led to different progeny. F1 sister progeny shared different sets of molecular markers representing a few of those of the wild species used as the pollen donor. Our results indicate mechanisms leading to the unusual event of partial hybridization. Possible mechanisms behind these unusual events and their possible impact on evolution are discussed.     

Reversible male sterility in transgenic tobacco carrying a dominant-negative mutated glutamine synthetase gene under the control of microspore-specific promoter

Metabolic engineering was used to disrupt glutamine metabolism in microspores in order to block pollen development. We used a dominant-negative mutant (DNM) approach of cytosolic glutamine synthetase (GS1) gene under the microspore-specific promoter NTM19 to block glutamine synthesis in developing pollen grains. We observed partial male sterility in primary transgenic plants by using light microscopy, FDA, DAPI and in vitro pollen germination test. Microspores started to die in the early unicellular microspore stage, pollen viability in all primary transgenic lines ranged from 40-50%. All primary transgenics produced seeds like control plants, hence the inserted gene did not affect the sporophyte and was inherited through the female germline. We regenerated plants by in vitro microspore embryogenesis from 4 individual lines, pollen viability of progeny ranged from 12 to 20%, but some of them also showed 100% male sterility. After foliage spray with glutamine, 100% male-sterile plants were produced viable pollen and seed set was also observed. These results suggested that mutated GS1 activity on microspores had a significant effect on normal pollen development. Back-cross progenies (T2) of DH 100% male-sterile plants showed normal seed set like primary transgenics and control plants.     

The basis of natural and artificial postzygotic hybridization barriers in Arabidopsis species

The success or failure of interspecific crosses is vital to evolution and to agriculture, but much remains to be learned about the nature of hybridization barriers. Several mechanisms have been proposed to explain postzygotic barriers, including negative interactions between diverged sequences, global genome rearrangements, and widespread epigenetic reprogramming. Another explanation is imbalance of paternally and maternally imprinted genes in the endosperm. Interspecific crosses between diploid Arabidopsis thaliana as the seed parent and tetraploid Arabidopsis arenosa as the pollen parent produced seeds that aborted with the same paternal excess endosperm phenotype seen in crosses between diploid and hexaploid A. thaliana. Doubling maternal ploidy restored seed viability and normal endosperm morphology. However, substituting a hypomethylated tetraploid A. thaliana seed parent reestablished the hybridization barrier by causing seed abortion and a lethal paternal excess phenotype. We conclude from these findings that the dominant cause of seed abortion in the diploid A. thaliana x tetraploid A. arenosa cross is parental genomic imbalance. Our results also demonstrate that manipulation of DNA methylation can be sufficient to erect hybridization barriers, offering a potential mechanism for speciation and a means of controlling gene flow between species.     

Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.Key words: cytokinin, cytokinin receptor, female gametophyte, male gametophyte, stigma     

RECIPROCAL DIFFERENCES IN INTRASPECIFIC CROSSES OF TOBACCO RESULT FROM EMBRYO DEATH

Tetraploid tobacco plants (Nicotiana tabacum) derived from cultured cell lines (TC) are partially cross-incompatible with their diploid progenitors (C). C × TC crosses (TC-derived tetraploids as the pollen parent) yield only 2% viable seed. The remaining seeds are normal size but lack an embryo (apoembryonic seeds). Apoembryonic seeds do not occur in the reciprocal TC × C crosses. Sections of ovules from C × TC crosses revealed that an embryo formed but that embryo growth slowed at 5 days postpollination and that by the 12th day following pollination, the embryo had disappeared although the ovule continued to develop. Endosperm degeneration occurred concurrently with embryo death. Culturing ovules from C × TC crosses has increased the yield of F₁ plants from 2% to as much as 25% indicating that embryo rescue is possible. Surviving F₁ plants from TC × C crosses have close to triploid chromosome numbers and are fertile.     

Reproductive biology of a rare cactus, Opuntia spinosissima (Cactaceae), in the Florida Keys: why is seed set very low?

 Opuntia spinosissima (Martyn) Mill. (Cactaceae) is an extremely rare taxon with a single remaining wild population of 13 plants located on Little Torch Key, Florida. The plants rarely set viable seeds and most ovaries abscise without initiation of fruits. Pollination and breeding system were examined in Little Torch Key and in plant accessions located at Fairchild Tropical Garden. Four pollination treatments were carried out to test for apomixis, self-pollination, outcrossing, and autogamy; a control treatment was also monitored. Additionally, pollen viability, pollinators, and seed set and viability were investigated. On selfed, crossed, and open (control) stigmas pollen grains germinated, but the tubes usually did not reach the base of the style. This suggests that O. spinosissima has pre-zygotic self-incompatibility. None of the pairwise crosses set seed, so the extant plants were apparently not inter-compatible. Out of 173 manipulated and control flowers, only one set fruit. Although this flower was outcrossed, no pollen tubes germinated on the stigma; this suggests agamospermy, a process common in the Cactaceae. In those flowers where pollen tubes did reach the ovary they failed to penetrate ovules, suggesting ovarian inhibition or that this taxon has lost the ability to be fertilized. Most field-collected seeds were viable, but there is no seedling recruitment under natural conditions, and vegetative reproduction is common. Based on these findings, I hypothesize that O. spinosissima is a sterile polyploid and that the 13 extant plants are asexually derived from a single lineage. Received: 17 April 1998 / Revision accepted: 3 July 1998     

Starch biosynthesis in rice endosperm requires the presence of either starch synthase I or IIIa

Starch synthase (SS) I and IIIa are the first and second largest components of total soluble SS activity, respectively, in developing japonica rice (Oryza sativa L.) endosperm. To elucidate the distinct and overlapping functions of these enzymes, double mutants were created by crossing the ss1 null mutant with the ss3a null mutant. In the F(2) generation, two opaque seed types were found to have either the ss1ss1/SS3ass3a or the SS1ss1/ss3ass3a genotype. Phenotypic analyses revealed lower SS activity in the endosperm of these lines than in those of the parent mutant lines since these seeds had different copies of SSI and SSIIIa genes in a heterozygous state. The endosperm of the two types of opaque seeds contained the unique starch with modified fine structure, round-shaped starch granules, high amylose content, and specific physicochemical properties. The seed weight was ～90% of that of the wild type. The amount of granule-bound starch synthase I (GBSSI) and the activity of ADP-glucose pyrophosphorylase (AGPase) were higher than in the wild type and parent mutant lines. The double-recessive homozygous mutant prepared from both ss1 and ss3a null mutants was considered sterile, while the mutant produced by the leaky ss1 mutant×ss3a null mutant cross was fertile. This present study strongly suggests that at least SSI or SSIIIa is required for starch biosynthesis in rice endosperm.     

The use of rootless mutants for the screening of spontaneous androgenetic and gynogenetic haploids in Nicotiana tabacum: evidence for the direct transfer of cytoplasm

Summary In crosses between a homozygous rootless mutant line of Nicotiana tabacum used as female and other Nicotiana tabacum lines, androgenetic haploids can be directly selected by their ability to form plantlets with a normal rooting system, whereas hybrid plants are killed few weeks after sowing. These androgenetic plants have the nucleus of the male parent into the cytoplasm of the female parent. In crosses where the homozygous rootless mutant line is used as a pollen donor, gynogenetic haploids can also be directly selected. Haploids can therefore be derived from male sterile plants using this approach. A generalization of this system for direct cytoplasm transfer and for the screening of spontaneous haploids in dicotyledons is proposed.     

Reproductive consequences of mate quantity versus mate diversity in a wind-pollinated plant

Since most pollen travels limited distances in wind-pollinated plants, both the local quantity and diversity of mates may limit female reproductive success. Yet little evidence exists on their relative contribution, despite the importance of viable seed production to population dynamics.To study how variation in female reproductive success is affected by the quantity versus the diversity of surrounding mates contributing pollen, we integrated pollination experiments, data on natural seed set and seed viability, and AFLP genetic marker data in the wind-pollinated dioecious clonal forest herb Mercurialis perennis.Pollination experiments indicated weak quantitative pollen limitation effects on seed set. Among-population crosses showed reduced seed viability, suggesting outbreeding depression due to genetic divergence. Pollination with pollen from a single source did not negatively affect reproductive success. These findings were consistent with results of the survey of natural female reproductive success. Seed set decreased with the distance to males in a female plants’ local neighborhood, suggesting a shortage of pollen in isolated female plants, and increased with the degree of local genetic diversity. Spatial isolation to other populations and population size did not affect seed set. None of these variables were related to seed viability.We conclude that pollen movement in M. perennis is likely very limited. Both male proximity and the local degree of genetic diversity influenced female reproductive success.     

K, Mg-ATPase activity in guard cells of Commelina communis

Siliqua development was studied in the wild type line Landsberg erecta and the GA-sensitive mutant ga-1 of Arabidopsis thaliana (L.) Heynh. Reciprocal crosses between wild type and ga-l mutant, and self pollinations of either parent have shown that siliqua growth is determined by endogenous GAs originating from maternal tissues and embryo. The ga-1 mutant either self pollinated or cross-pollinated with wild type pollen showed reduced siliqua growth, which to a large extent could be overcome by exogenously applied GAs. The siliquae of the ga-1 mutant possessed very reduced ent -kaurene synthesizing capacity and no detectable endogenous GA-activity indicating an early block in the GA-biosynthetic pathway. Seed weight is not affected by GA-deficiency during development.     

Mating interactions between coexisting dipoloid, triploid and tetraploid cytotypes ofHieracium Echioides (Asteraceae)

Experimental crosses between diploids, triploids and tetraploids ofHieracium echioides were made to examine mating interactions. Specifically, cytotype diversity in progeny from experimental crosses, intercytotype pollen competition as a reproductive barrier between diploids and tetraploids, and differences in seed set between intra- and intercytotype crosses were studied. Only diploids were found in progeny from 2x × 2x crosses. The other types of crosses yielded more than one cytotype in progeny, but one cytotype predominated in each cross type: diploids (92%) in 2x × 3x crosses, tetraploids (88%) in 3x × 2x crosses, triploids (96%) in 2x × 4x crosses, triploids (90%) in 4x × 2x crosses, tetraploids (60%) in 3x × 3x crosses, pentaploids (56%) in 3x × 4x crosses, triploids (80%) in 4x × 3x crosses and tetraploids (88%) in 4x × 4x crosses. No aneuploids have been detected among karyologically analyzed plants. Unreduced egg cell production was detected in triploids and tetraploids, but formation of unreduced pollen was recorded only in two cases in triploids. Triploid plants produced x, 2x and 3x gametes: in male gametes x (92%) gametes predominated whereas in female gametes 3x (88%) gametes predominated. Cytotype diversity in progeny from crosses where diploids and tetraploids were pollinated by mixture of pollen from diploid and tetraploid plants suggested intercytotype pollen competition to serve as a prezygotic reproductive barrier. No statistically significant difference in seed set obtained from intra- and intercytotype crosses between diploids and tetraploids was observed, suggesting the absence of postzygotic reproductive barriers among cytotypes.     

Partial interfertility between independently originated populations of the neo-allopolyploid <Emphasis Type="Italic">Mimulus peregrinus</Emphasis>

The reduction in genetic diversity in polyploid lineages, formed from whole-genome duplication of a few individuals, can affect their long-term evolutionary potential. Because most polyploids originate multiple times, secondary contact and gene exchange among independently formed polyploids can create novel genetic combinations and reduce the severity of genetic bottlenecks. However, independently originated polyploids may be reproductively isolated from each other due to genetic and chromosomal differences predating the polyploidisation event, or evolving subsequently in the dynamic genomes of young polyploid populations. Here we conducted experimental crosses to investigate the phenotype and interfertility between two independently originated populations of the allopolyploid Mimulus peregrinus (Phrymaceae). We found that individuals from the two populations are phenotypically distinct, but that inter- and intrapopulation crosses are not statistically different. Interpopulation crosses produce viable and fertile offspring, although our results suggest the existence of partial reproductive barriers in the form of reduced pollen viability. We found no difference in pollen viability between F1 and F2 generations. In contrast, we detected a reduction in floral and vegetative size, and in the proportion of plants that flowered, between F1 and F2 generations for both intra- and interpopulation crosses. Together, our results indicate that populations of independent origin can partially exchange genes, producing variable offspring, and that the phenotype of M. peregrinus may be unstable in the early generations. Natural selection on genetically based variation may be required for the evolution of more stable and fertile individuals of this nascent allopolyploid.     

Sequential variation in the components of reproductive success in the distylousJasminum fruticans (Oleaceae)

A 2-year study of three natural populations of the distylousJasminum fruticans showed that mean fruit and seed production were significantly greater in shortstyled plants (thrums) than in long-styled plants (pins). In this study, we investigated the role of four sequential factors which may differentially influence fruit and seed set in the two floral morphs: (1) differences in flowering phenology, (2) a limitation of pollen transfer towards pins, (3) a differential capacity of the two morphs to act through famale and male function and (4) differential fruit abortion in the two morphs. Fruit set was significantly influenced by differences in flowering phenology although there were no differences in flowering time between the two morphs. supplementary pollinations in a natural population significantly increased fruit set and reduced the difference in fruit set between the two morphs in relation to controls, indicating a limitation on pollen transfer which was most severe towards pin stigmas. In reciprocal crosses, seed set was significantly dependent on the paternal and maternal identity of the pin parent. There was no significant variation among thrums for their performance as male or female parent. Furthermore, individual pin plants with relatively high percentage seed set as female parents gave poor seed set as male parents and vice-versa. Whereas fruit removal had no effect on seed number in thrum plants, a greater proportion of viable seeds were produced on pin plants which were left to naturally mature their fruits than on pins which had fruits artificially removed, suggesting the occurrence of selective fruit abortion in pins but not in thrums. The initially greater maternal fitness of thrums due to their greater success as pollen recipients may thus be opposed by increased viable seed set in the pins due to factors acting after the pollination stage. The relative reproductive success of floral morphs in the distylousJ. fruticans is thus differentially influenced by ecological factors occurring at different stages of the reproductive process.     

Does differential seed siring success change over time or with pollination history in wild radish, Raphanus sativus (Brassicaceae)?

Previous work with wild radish has shown that pollen donors sire different numbers of seeds and that the condition of the maternal tissue affects seed paternity, suggesting that both pollen donor characteristics and maternal tissue affect mating. However, because these results are from the greenhouse, it is difficult to know whether they would hold true in the field. Here, we performed hundreds of crosses on several maternal plants to simulate changes during the flowering season of field plants. During the experiment, maternal resource availability changed due to the costs of producing fruits, and we determined the pollination history of a plant by performing crosses in specific orders. Examination of seed paternity showed that there were small differences in pollen donor success at the beginning of the experiment when maternal resources were abundant. Differential pollen donor success was greatest slightly later in the flowering period, but declined toward the end of the experiment. Thus, maternal plants may distinguish most among pollen donors when they have both abundant resources and experience with the differences in quality of available pollen donors. In contrast, there were few significant effects of the recent pollination history of plants on pollen donor success. Finally, despite the changes in mating performance over time, there were strong overall differences in pollen donor success, suggesting that seasonal changes in the field will not eliminate the potential for nonrandom mating.     

REPRODUCTIVE ISOLATION OF TRITICUM BOEOTICUM AND TRITICUM URARTU AND THE ORIGIN OF THE TETRAPLOID WHEATS

The diploid wheats Triticum boeoticum and T. urartu are sympatric with one another throughout the geographic range of the wild tetraploids. Reciprocal crosses between ecogeographic types within each diploid species gave viable seed, but interspecific crosses consistently gave viable seed only when T. boeoticum was the female parent. Apparently urartu cytoplasm in combination with the boeoticum genome resulted in nonviable seed. The endosperm failed to develop normally despite regular endosperm fertilization. The F₁ plants obtained were completely self sterile although they showed regular intergenomic pairing (7II) at meiosis. Presumably the accumulation of cryptic differences between the two closely related genomes under reproductive isolation accounts for this sterility. The same accumulated cryptic differences could largely account for the preferential diploid pairing in the tetrapolid wheats which presumably were derived from such hybrids by chromosome doubling. The behavior of reciprocal crosses between the diploids and tetraploids suggested that T. boeoticum contributed the cytoplasm to both of the wild tetraploid species.     

Paternal effects on seed germination: a barrier to the genetic assimilation of an endemic plant taxon?

We used a crossing experiment to investigate post-zygotic barriers that might limit introgression between a pair of closely-related, gynodioecious plant species--the widespread weed Silene vulgaris and the local Swedish endemic S. uniflora ssp. petraea. The study not only considered the effects of hybridization on conventionally-used (primary) fitness components such as seed set and progeny survival, but also provided a test for the effects of interspecific hybridization on characters with more subtle or habitat-specific effects on fitness. We detected highly significant paternal effects on seed germination properties, with the germination characteristics of hybrid seed resembling those of the species that served as the pollen donor. These paternal effects on germination represent a potentially strong barrier to interspecific introgression in the two species' natural habitats, where an inappropriate germination response in the habitat of the maternal parent may lead to the failure of seedling establishment. Interspecific crosses had weak or variable effects on progeny survival, flowering and sex ratio, but these effects could not be interpreted in terms of barriers to introgression. Our results indicate that nuclear restorers in S. vulgaris have the capacity to suppress cytoplasmic male-sterility genes in its endemic congener.     

Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants

Different transgenic tobacco lines down-regulated for either one or two enzymes of the monolignol pathway were compared for their lignin content and composition, and developmental patterns. The comparison concerned CCR and CAD down-regulated lines (homozygous or heterozygous for the transgene) and the hybrids resulting from the crossing of transgenic lines individually altered for CCR or CAD activities. Surprisingly, the crosses containing only one allele of each antisense transgene, exhibit a dramatic reduction of lignin content similar to the CCR down-regulated parent but, in contrast to this transgenic line, display a normal phenotype and only slight alterations of the shape of the vessels. Qualitatively the lignin of the double transformant displays characteristics more like the wild type control than either of the other transgenics. In the transgenics with a low lignin content, the transformations induced other biochemical changes involving polysaccharides, phenolic components of the cell wall and also soluble phenolics. These results show that the ectopic expression of a specific transgene may have a different impact depending on the genetic background and suggest that the two transgenes present in the crosses may operate synergistically to reduce the lignin content. In addition, these data confirm that plants with a severe reduction in lignin content may undergo normal development at least in controlled conditions.     

Optimisation of pollen viability tests for Acacia podalyriifolia and two ploidys of Acacia mearnsii

Acacia mearnsii (black wattle) is a commercially important forestry species in South Africa, grown for its timber as well as its bark. It is, however, also considered to be an alien invader of indigenous vegetation and for this reason the production of a sterile variety would be highly desirable for commercial forestry in South Africa. Previous research on crosses between diploid and tetraploid parent plants to produce triploid progeny has resulted in poor seed set. One possible barrier preventing seed set could be the viability of the pollen used in the cross pollination operations. Thus a study was conducted to test the pollen viability. In vitro agar media germination tests (ACIAR and Brewbaker and Kwack media) were optimised on Acacia podalyriifolia pollen and then used together with vital stain tests (Sigma® DAB peroxidase and p-phenylendiamine) to test pollen germination and viability of A. mearnsii pollen. These were then compared to in vivo pollen germination on the stigma, and were conducted on both diploid and tetraploid pollen mixes. Results showed that the vital stain tests gave significantly (p < 0.05) higher pollen viability than the agar germination tests and were more in agreement with the results from the pollen germination rate on the stigma. For both the diploid and tetraploid pollen mixes tested, there were no significant differences (p > 0.05) between the two agar media germination tests and between the two vital stain tests.