Assay for doubled haploid sunflower (Helianthus annuus) plant production by androgenesis: fact or artifact? Part 1. In vitro anther culture

Sunflower anthers placed on solid medium developed calli and embryos after 12 days. Embryogenesis was improved by the addition of 0.1% polyvinylpyrrolidone (PVP) that alleviated anther and medium browning. As in other species, genotypic variability was an important parameter in the anther response and a medium genotype interaction was suggested with a different PVP effect depending on the genotype. Embryo germination was largely increased by the successive use of germination media with decreasing sucrose concentrations (10%6%3%). Histological examination of the anthers during the first ten days of culture showed that, under our conditions, the embryos were of somatic origin, arising directly from the anther wall on the outside or inside of the anther loculus, or indirectly from proliferating anther wall- or connective tissue-derived callus. Finally, the ploidy status of 78 embryo-derived plants was determined by Feulgen stain or flow cytometry: all plants were diploid (2n=34).Abbreviations PVP polyvinylpyrrolidone     

Strain specificity in transformation of alfalfa by Agrobacterium tumefaciens

Production of transgenic alfalfa plants by Agrobacterium-mediated transformation requires Agrobacterium infection and regeneration from tissue culture. Variation in alfalfa (Medicago sativa L.) germplasm for resistance to oncogenic and disarmed strains of A. tumefaciens (Smith & Townsend) Conn was tested in plant populations representing the nine distinct sources of alfalfa germplasm introduced into North America and used to develop modern varieties. For each of the virulent strains there was a positive correlation (p=0.05) of resistance to tumorigenesis with the trait for fall dormancy. There was also a significant correlation between plants selected for ineffective nodulation and resistance to tumorigenesis suggesting that the genetic loci required for successful symbiosis are also involved in tumorigenesis. Tissue explants of seedlings from the nine diversity groups were tested for transformation by three disarmed strains containing a plasmid with the scorable marker -glucuronidase. The strong correlation between dormancy and resistance to oncogenic strains was not observed with disarmed strains. However, there was a strong germplasm-strain interaction or transformation and embryogenesis in a highly embryogenic genotype. Thus, transformation at the whole plant level is germplasm dependent while in tissue culture the bacterial strain used is the critical variable for successful transformation.Abbreviations pTi tumor-inducing plasmid - GUS -glucuronidase     

Evidence for widespread pollen limitation of fruiting success in Cape wildflowers

We used spot checks of stigmatic pollen deposition and hand-pollination experiments to test whether fruit production in Cape wildflower populations is limited by pollen availability. Natural levels of stigmatic pollen deposition were very low (median = 30.0% of flowers) in populations of 33 orchid species. We found similarly low levels of fruit set (median = 32% of flowers per plant) in six Orchidaceae and four Amaryllidaceae species. Experimental hand pollination at the whole plant level caused significant increases in fruit production in 11 of the 12 study populations. These results indicate that pollen limitation of fruit set may occur frequently among some plant families in the Cape flora. Received: 15 April 1996 / Accepted: 10 November 1996     

Free and conjugated polyamines in embryogenic and non-embryogenic leaf regions of camellia leaves before and during direct somatic embryogenesis

Contents of endogenous free and conjugated polyamines were determined in embryogenic and non-embryogenic leaf regions of Camellia japonica leaf explants, before (day 0), and 20 and 45 days after the induction of direct somatic embryogenesis, to clarify whether or not polyamines are linked to the specific morphogenic responses previously reported in the leaf regions. The analysis was carried out by high-performance liquid chromatography. The results showed that there were no significant differences in the endogenous free and conjugated putrescine (Put), spermidine (Spd) and spermine (Spm) contents between the embryogenic and non-embryogenic leaf regions of the same leaf. Thus, leaf region-specific embryogenic response in C. japonica is not being determined/conditioned by the endogenous levels of Put, Spd and/or Spm. However, soluble and insoluble conjugated Put and soluble conjugated Spd seem to be related to the formation and development of globular embryos.     

Effects of pollination intensity on Lesquerella fendleri seed set: variation among plants

 The amount of pollen arriving on a flower can be an important determinant of seed production. I investigated the effect of varying pollen loads on seed set of the perennial desert mustard Lesquerella fendleri. To do this, I quantified the dose response relationship between stigmatic pollen load and seed set per fruit using over 400 flowers from 13 greenhouse-grown plants. Seed set per fruit generally increased with pollen up to about 100 pollen grains, then reached a plateau. A negative exponential regression of seed set on pollen load for the pooled data explained less than 10% of the observed variation in seeds per fruit. However, accounting for variation among individual plants in the dose-response relationship increased explained variation to 40%, indicating that plants responded differently to the same amount of available pollen. Plants varied little in the initial slope of the dose-response curve, but differed substantially in the asymptote, which ranged from 3 to 16 seeds. This limit is not imposed by ovule number, and may instead result from variation among plants in vigor, propensity to abort seeds, or in gender specialization. Such variation among plants in dose-response relationships has important consequences for understanding pollination limitation and pollen competition. Received: 29 January 1996 / Accepted: 6 August 1996     

Morphogenetic effects of Brefeldin A on embryogenic cell cultures of Daucus carota L.

Brefeldin A, an inhibitor of protein secretion, caused typical alterations to the endomembrane system with limited effects on viability when given to unorganized carrot cells growing in suspension. When given to the same cells during particular stages of embryogenesis, it caused similar endomembrane lesions and an almost complete arrest of the embryogenic process. Addition of conditioned medium containing extracellular secreted proteins to the embryos during treatment with Brefeldin A allowed acquisition of polarity and the continuation of a quasi-normal embryogenic process. Received: 4 December 1996 / Accepted: 4 March 1997     

Growth inhibition and recovery in freeze-substitutedLilium longiflorum pollen tubes: structural effects of caffeine

Summary In an attempt to correlate structural effects with the known dissipation of the tip-focused Ca²⁺ gradient caused by caffeine, we have examined the ultrastructure of caffeine-treated lily pollen tubes prepared by rapid freeze fixation and freeze substitution. We show that treatment with caffeine results in a rapid rearrangement of secretory vesicles at the pollen tube tip; the normal cone-shaped array of vesicles is rapidly dispersed. In addition, microfilament bundles appear in the tip region, where they had previously been excluded. Delocalized vesicle fusion continues in the presence of caffeine but tube extension ceases. Removal of caffeine from the growth medium initially causes tip swelling, delocalized vesicle fusion and presence of microfilaments well into the tip before normal structure and growth resume, concurrent with the previously reported return to a normal Ca²⁺ gradient.Abbreviations ER endoplasmic reticulum - MES 2-[N-morpholino] ethanesulfonic acid - MFs microfilaments     

Loss of callose in the stigma papillae does not affect the Brassica self-incompatibility phenotype

As part of the Brassicaceae self-incompatibility response, callose is deposited in the stigma papillar cells. To determine if callose plays an important role in the rejection of incompatible pollen by the stigma, transgenic Brassica napus. L. plants were produced which express the tobacco β-1,3-glucanase cDNA (the enzyme which degrades callose) in the stigma papillae. Using aniline blue fluorescence, little or no callose was detected in the papillar cells of transgenic stigmas. However, the self-incompatibility system appeared to be unaffected based on the lack of pollen tube growth and the subsequent lack of seed set. The transgene had no effect on compatible pollinations. Thus, while callose deposition is associated with the B. napus self-incompatibility response, it is not required for the rejection of incompatible pollen. Received: 14 March 1997 / Accepted: 15 April 1997     

Main role of self-pollination rate on reproductive allocations in pseudogamous apomicts

 Most apomicts are hermaphroditic and pseudogamous (pollination is necessary to trigger parthenogenesis). In these plants, fitness depends on the number of progeny obtained by maternal reproduction. We determined the evolutionary stable strategy for male and female sex allocation. We show that the efficiency of pollination determines male and female resource allocations. Predictions are made of these allocations, of pollen/ovule ratio and of seed-set. We show that self-compatibility in apomicts is necessary for the maintenance of an apomictic population, and thus can account for the association between the loss of self-incompatibility and pseudogamous apomixis. In contrast to sexuals, male investment in pseudogamous apomicts increases with the rate of self-pollination. Received: 15 June 1996 / Accepted: 20 September 1996