Martin Pfosser

The microspore: A haploid multipurpose cell

Abstract The microspore is at the centre of a variety of topics in modern plant science and breeding. Isolated microspore cultures have the remarkable quality of resembling the alternation of generations in the life cycle of angiosperms, i.e. the change between the diploid sporophytec and the haploid gametophytec generation. Although the natural destination of microspore development is to differentiate into mature pollen and accomplish fertilization, isolated and in vitro cultured microspores or young pollen grains can either differentiate into mature, fertile pollen (the male gametophytes) by culture in a rich medium without stress, or divide repeatedly and develop into embryos (sporophytes) after a stress treatment. As experimental systems, microspore cultures are used to investigate pollen development and pollination, embryogenesis, totipotency, cytodifferentiation, cell cycle, phase change and the role of stress in development. As a tool in genetic engineering, they can be used to produce doubled haploids (recombinant inbreds) for plant breeding and gene mapping, to overcome crossing barriers (male sterility and self-incompatibility), to induce and select for mutants and to create transgenic plants. In this review genetic, cell biological and molecular aspects of in vitro microspore development are presented and put in the context of current basic and applied plant science.

Alfalfa cyclins: differential expression during the cell cycle and in plant organs.

Cell division in eukaryotes is mediated by the action of the mitosis promoting factor, which is composed of the CDC2 protein kinase and one of the various mitotic cyclins. We have recently isolated a cdc2 gene from alfalfa. Here, we report the isolation of two cyclin genes, cycMs1 and cycMs2, from alfalfa. The cycMs2 gene shows highest similarity to type B cyclins. In contrast, the predicted amino acid sequence of the cycMs1 gene shows similar homology scores to cyclins of all types (25 to 35%). Both genes are expressed in dividing suspension cultured cells but cease to be expressed when the cells enter stationary phase. In synchronized alfalfa suspension cultured cells, the mRNAs of cycMs1 and cycMs2 show maximal expression in the G2 and M phases. Transcripts of cycMs2 are found only in late G2 and M phase cells, an expression pattern typical for cyclin B genes, whereas cycMs1 appears with the onset of G2. This pattern indicates that alfalfa cycMs1 and cycMs2 belong to different classes of cyclins. In young leaves, expression of both genes is high, whereas in mature leaves no transcripts can be detected, indicating that the two cyclin genes are true cell division markers at the mRNA level. In other organs, a more complex expression pattern of the two cyclin genes was found.

Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis

Specific stress treatments (sucrose starvation, alone or combined with a heat shock) applied to isolated tobacco (Nicotiana tabacum L.) microspores irreversibly blocked normal gametophytic development and induced the formation of embryogenic cells, which developed subsequently into pollen-derived embryos by culture at 25°C in a sugar-containing medium. A cold shock at 4°C did not inhibit microspore maturation in vitro and did not induce cell division activity, even when combined with a starvation treatment. In the absence of sucrose, microspores isolated in the G1 phase of the cell cycle replicated their DNA and accumulated in G2. Late microspores underwent miotosis during the first day of culture which resulted in a mixed population of bicellular pollen grains and uninucleate microspores, both embryogenic. After the inductive stress treatments the origin of the first multicellular structures, formed in the sugar-containing medium, could be traced to divisions of the microspore cell or divisions of the vegetative cell of bicellular pollen, indicating that the symmetry of microspore mitosis in vitro is not important for embryogenic induction. These results represent a step forward towards a unified model of induction of embryogenesis from microspores/pollen which, within a relatively wide developmental window, are competent to deviate from normal gametophytic development and initiate the alternative sporophytic programme, in response to specific stress signals.

Plant transformation by particle bombardment of embryogenic pollen

SummaryDirect delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 104 pollen grains were GUS+. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS+ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.

Inferences of biogeographical histories within subfamily Hyacinthoideae using S-DIVA and Bayesian binary MCMC analysis implemented in RASP (Reconstruct Ancestral State in Phylogenies)

BACKGROUND AND AIMSnSubfamily Hyacinthoideae (Hyacinthaceae) comprises more than 400 species. Members are distributed in sub-Saharan Africa, Madagascar, India, eastern Asia, the Mediterranean region and Eurasia. Hyacinthoideae, like many other plant lineages, show disjunct distribution patterns. The aim of this study was to reconstruct the biogeographical history of Hyacinthoideae based on phylogenetic analyses, to find the possible ancestral range of Hyacinthoideae and to identify factors responsible for the current disjunct distribution pattern.nnnMETHODSnParsimony and Bayesian approaches were applied to obtain phylogenetic trees, based on sequences of the trnL-F region. Biogeographical inferences were obtained by applying statistical dispersal-vicariance analysis (S-DIVA) and Bayesian binary MCMC (BBM) analysis implemented in RASP (Reconstruct Ancestral State in Phylogenies).nnnKEY RESULTSnS-DIVA and BBM analyses suggest that the Hyacinthoideae clade seem to have originated in sub-Saharan Africa. Dispersal and vicariance played vital roles in creating the disjunct distribution pattern. Results also suggest an early dispersal to the Mediterranean region, and thus the northward route (from sub-Saharan Africa to Mediterranean) of dispersal is plausible for members of subfamily Hyacinthoideae.nnnCONCLUSIONSnBiogeographical analyses reveal that subfamily Hyacinthoideae has originated in sub-Saharan Africa. S-DIVA indicates an early dispersal event to the Mediterranean region followed by a vicariance event, which resulted in Hyacintheae and Massonieae tribes. By contrast, BBM analysis favours dispersal to the Mediterranean region, eastern Asia and Europe. Biogeographical analysis suggests that sub-Saharan Africa and the Mediterranean region have played vital roles as centres of diversification and radiation within subfamily Hyacinthoideae. In this bimodal distribution pattern, sub-Saharan Africa is the primary centre of diversity and the Mediterranean region is the secondary centre of diversity. Sub-Saharan Africa was the source area for radiation toward Madagascar, the Mediterranean region and India. Radiations occurred from the Mediterranean region to eastern Asia, Europe, western Asia and India.

Searching for mechanisms leading to albino plant formation in cereals

Microspore embryogenesis is a method of great importance for plant production in many species. Its practical use in monocotyledonous plants, however, is limited due to the occurrence of albino plants. Molecular examinations of such albino plants showed an aberrant form of plastids and, frequently, deletions in their plastid DNA. Moreover, in our preceding work we found typical deficiencies in plastid transcription and translation. Nevertheless, there are also indications for an involvement of the nuclear genome in albino plant formation. To address these questions, we characterised, on the one hand, a plastid factor, which is possibly involved in the formation of plastid DNA deletions, and investigated, on the other hand, nuclear gene expression in albino plants. The ycf1 gene is present in the plastid genomes of dicots, while monocots only have a truncated pseudogene. The disruption of ycf1 in tobacco showed that it is essential for the survival of dicotyledonous plants. In addition, we replaced the tobacco ycf1 by the pseudogene of rice and found rearrangements in the plastid genomes of the resulting plants indicating that the ycf1 locus plays a role in the stability of plastid DNA. The study on nuclear gene expression showed that there is an extensive alteration of transcript levels in albino plants. We found that the respective genes encode proteins with a broad range of functions in plants. All these findings taken together suggest that albino plant formation is a complex phenomenon in which both, plastid and nuclear factors or their defective interactions are involved.

The Scilla plumbea Puzzle-Present Status of the Genus Scilla Sensu Lato in Southern Africa and Description of Spetaea lachenaliiflora, a New Genus and Species of Massonieae (Hyacinthaceae)

We have studied a taxon from South Africa that has been hidden for more than 150 years within Scilla plumbea (Hyacinthaceae). However, phylogenetic reconstruction based on trnL-F data, morphological evidence (including seed characters), and karyology suggest independent status. DNA data place it close to the Daubenya alliance but not close to any particular genus of Massonieae previously included within Scilla s.l. Unique characters of seeds indicate that this taxon does not belong to the Daubenyaclade. Analysis of the iconotype of Scilla plumbea Lindl., on the other hand, reveals morphological details suggesting membership in the Scilla natalensis clade (= genus Merwillain the recent classification of Speta). Merwilla, however, occupies a basal position among Massonieae and is not related to the Daubenyaclade. Based on all data, we describe this taxon as a new genus and species Spetaea lacheneliifloraWetschnig & Pfosser.

Out of Africa: Miocene Dispersal, Vicariance, and Extinction within Hyacinthaceae Subfamily Urgineoideae

Disjunct distribution patterns in plant lineages are usually explained according to three hypotheses: vicariance, geodispersal, and long-distance dispersal. The role of these hypotheses is tested in Urgineoideae (Hyacinthaceae), a subfamily disjunctly distributed in Africa, Madagascar, India, and the Mediterranean region. The potential ancestral range, dispersal routes, and factors responsible for the current distribution in Urgineoideae are investigated using divergence time estimations. Urgineoideae originated in Southern Africa approximately 48.9 Mya. Two independent dispersal events in the Western Mediterranean region possibly occurred during Early Oligocene and Miocene (29.9-8.5 Mya) via Eastern and Northwestern Africa. A dispersal from Northwestern Africa to India could have occurred between 16.3 and 7.6 Mya. Vicariance and extinction events occurred approximately 21.6 Mya. Colonization of Madagascar occurred between 30.6 and 16.6 Mya, after a single transoceanic dispersal event from Southern Africa. The current disjunct distributions of Urgineoideae are not satisfactorily explained by Gondwana fragmentation or dispersal via boreotropical forests, due to the younger divergence time estimates. The flattened winged seeds of Urgineoideae could have played an important role in long-distance dispersal by strong winds and big storms, whereas geodispersal could have also occurred from Southern Africa to Asia and the Mediterranean region via the so-called arid and high-altitude corridors.