Transcriptome analysis provides insight into venom evolution in a seed-parasitic wasp, Megastigmus spermotrophus.

One of the most striking host range transitions is the evolution of plant parasitism from animal parasitism. Parasitoid wasps that have secondarily evolved to attack plants (ie gall wasps and seed-feeders) demonstrate intimate associations with their hosts, yet the mechanism of plant-host manipulation is currently not known. There is, however, emerging evidence suggesting that ovipositional secretions play a role in plant manipulation. To investigate whether parasites have modified pre-existing adaptations to facilitate dramatic host shifts we aimed to characterize the expression of venom proteins in a plant parasite using a collection of parasitoid venom sequences as a guide. The transcriptome of a seed-feeding wasp, Megastigmus spermotrophus, was assembled de novo and three putative venoms were found to be highly expressed in adult females. One of these putative venoms, aspartylglucosaminidase, has been previously identified as a major venom component in two distantly related parasitoid wasps (Asobara tabida and Leptopilina heterotoma) and may have originated via gene duplication within the Hymenoptera. Our study shows that M. spermotrophus, a specialized plant parasite, expresses putative venom transcripts that share homology to venoms identified in Nasonia vitripennis (both superfamily Chalcidoidea), which suggests that M. spermotrophus may have co-opted pre-existing machinery to develop as a plant parasite.

Multinucleate storage cells in Douglas-fir (Pseudotsuga menziesii (Mirbel) Franco) and the effect of seed parasitism by the chalcid Megastigmus spermotrophus Wachtl.

Megagametophytes of Douglas-fir (Pseudotsuga menziesii (Mirbel) Franco) accumulated storage products following fertilization. As megagametophytes matured, the number of nuclei per cell rose, resulting in syncytial storage cells. Studies carried out on trees in France and Canada confirmed that such previously unreported, free nuclear cells were a normal part of late megagametophyte development. Unfertilized megagametophytes showed that some binucleate cells before degeneration resulted in empty seed. Insect parasitism prevented megagametophyte abortion in unfertilized ovules. Oviposition by a torymid chalcid wasp (Megastigmus spermotrophus Wachtl) early in megagametophyte development resulted in normal megagametophyte development. Around the time of plant egg maturation, binucleate and trinucleate cells were observed. As megagametophytes matured, multinucleate mature storage cells rich in proteins, lipids and starch were formed. The insect was able to induce identical nuclear behaviour in infested, unfertilized megagametophytes, as that of uninfested, fertilized megagametophytes.

Desiccation, cryopreservation and water relations parameters of white spruce (Picea glauca) and interior spruce (Picea glauca x engelmannii complex) somatic embryos.

Effects of drying and cryopreservation on survival of spruce (Picea glauca (Moench) Voss and Picea glauca x engelmannii complex) somatic embryos (SEs) were investigated with the aim of developing simple and robust protocols for embryo storage. Somatic embryos dried over salt solutions of known water potential (Psi) survived removal of virtually all free water, to a relative water content (RWC) of approximately 0.13, a value similar to that for spruce zygotic embryos from dry seed. Desiccated SEs also survived subsequent freezing in liquid nitrogen, without the addition of cryoprotectant or pre-culture steps. Highest survival (> 80%) after freezing in liquid nitrogen was in embryos pre-dried to Psi of -15 to -20 MPa, which yielded RWC close to predicted bound (apoplastic) water values. Low (< 35%) or no survival after freezing was observed in embryos pretreated at higher Psi (above -5 MPa) or at very low Psi (-540 MPa, using silica gel), respectively.

Influencing micropropagation and somatic embryogenesis in mature trees by manipulation of phase change, stress and culture environment.

This review focuses on clonal propagation of mature trees by tissue culture. Most trees have marked phase changes that result in a decline in their potential for somatic embryogenesis or micropropagation. By altering conditions of the source material ex vitro, or by changing in vitro conditions encountered by the explant, rejuvenation and increased propagation can sometimes be accomplished. Various methods of enhancing micropropagation are reviewed, with particular emphasis on manipulations that involve application of osmotic, temperature or hormonal stress.

Effects of endogenous ABA levels and temperature on cedar (Cedrus libani Loudon) bud dormancy in vitro.

Axillary and apical buds of in-vitro-propagated cuttings of Cedrus libani are unable to burst at 24 °C, but this inhibition was overcome at 30 °C. Here we have used cedar microcuttings to investigate whether the levels of endogenous hormones vary with bud dormancy and temperature. We analysed the levels of abscisic acid, indole-3-acetic acid, zeatin, isopentenyladenine and their major metabolites using HPLC purification and fractionation of the samples coupled to an ELISA method for hormonal quantitation involving several antibodies elicited against each hormonal family. Abscisic acid levels in microcuttings with dormant buds were higher than those in microcuttings with growing buds. At 24 °C, needles accumulated more abscisic acid than at 30 °C. In addition, when needles were removed, but growth release was achieved at 24 °C. Abscisic acid supplied at 30 °C induced the formation of dormant buds. These results suggest that abscisic acid accumulation in the needles can explain the bud dormancy of cedar microcuttings at 24 °C.

Diploidization in megagametophyte-derived cultures of the gymnosperm Larix decidua.

Tested haploid embryogenic lines (n=12) of Larix dedicua Mill, initiated from megagametophyte tissue were maintained on half-strength LM medium without growth regulators. The cultures were analyzed for ploidy level after 1-9 years. All lines tested were found to have doubled (2n=24) their chromosome number at the end of the experiment, though there were a few lines that still gave occasional haploid counts. Flow cytometric data of embryogenic tissue confirmed these results. Protoplasts were stained in ethidium bromide, and cultured human leucocytes and chicken erythrocytes were used as internal standards. Haploid megagametophytes from immature seeds of L. decidua and known diploid culture lines of a related hybrid (L. x eurolepis) were also analyzed by flow cytometry. Haploid reference material had 12.3-13.6 pg DNA per cell, whereas formerly haploid callus lines had an average of 25.0 pg DNA per cell. The one exception was a known, genetically unstable line of L. decidua (34.8 pg DNA per cell). The diploid cell line of L. x eurolepis had 27.6 pg DNA per cell. The results show that spontaneous diploidization of megagametophyte lines is relatively rapid and that both haploid and dihaploid lines are embryogenic in larch.

Plants from haploid tissue culture of Lavix decidua.

Haploid embryogenic tissue was initiated on 1/2 LM medium supplemented with 500 mg/l glutamine, 1000 mg/l casein hydrolysate, 100 mg/l inositol, 30000 mg/l sucrose, and 0.1 mg/l 2,4-D. The embryoids matured to produce plantlets. One plant from one of the two lines survived. The chromosome complement of tissue cultures, of the needle bases from the source plant, and of the plant produced in vitro were established by squashes. DNA content was assessed by DNA microdensiometry. In vitro tissues were haploid (n = 12). The plant produced was mixoploid, with a predominance of diploid cells (2n = 24).

Water relation parameters of embryogenic cultures and seedlings of larch.

Changes in the water relations parameters of developing somatic embryogenic and xygotic European larch (Larix decidua) were studied. Water release curves were generated by suspending tissue samples over unsaturated NaCl solutions until they reached vapor equilibration with the surrounding air. Twenty solutions were used whose water potentials ranged from -0.05 to -10 MPa. Water release curves were obtained by plotting paired values of tissue relative water content (RWC) and solution potential. Curves were derived for embryonic larch at various stages of development and for hypocotyls and roots from germinated zygotic and somatic embryos. The ability to resist dehydration increased markedly with development. Stage 1 tissue, which consisted of clusters of loosely associated nonchlorophyllous cells, had extremely low bulk elastic modulus (epsilon) (1.91 MPa) and apoplastic water content (A) (0.023), relatively high osmotic potential (Psi(pi)) (-0.53 MPa), and lost turgor at 0.56 RWC. In contrast, mature embryoids with primary roots, hypocotyl, and cotyledons (stage 3) had an almost 4-fold increase in A (0.089), significantly higher epsilon (3.49 MPa), and lower Psi(pi) (-0.88 MPa) and lost turgor at 0.66 RWC. Hypocotyl tissue from germinated somatic embryos lost turgor at 0.74 RWC and had higher epsilon, A, and solute accumulation than pregerminated tissue. Hypocotyl tissue resisted dehydration more strongly than root tissue, and differences between root and hypocotyl water relation parameters were more pronounced in xygotic than in somatic seedlings. Highest dehydration resistance was in zygotic hypocotyls. The characterization of the water relations of tissue cultures should allow the development of more consistent and reliable desiccation protocols to induce maturation of embryos and produce synchronously germinating seed.

Somatic embryogenesis and plant regeneration from immature embryos of western larch.

Somatic embryogenesis was initiated from immature embryos of western larch (Larix occidentalis Nutt.) on media containing 2,4-dichlorophenoxyacetic acid and N6- benzyladenine. The effects of explant type and ammonium nitrate and glutamine concentrations on initiation were tested. Although 21-93% of explants rendered cultures in various experiments, only 3% yielded sustainable embryogenic lines. Excised embryos at the early cotyledonary stage were optimal for initiation. Maturation of somatic embryos was promoted by abscisic acid. Response to abscisic acid concentrations and duration of exposure to abscisic acid varied with genotype. Maximal results were obtained with 0.025 µ M abscisic acid for 1 to 2 weeks followed by individual culture on medium without growth regulators. Mature somatic embryos developed into shoots with roots. Plantlets have been established in peat.

FORMATION OF HAPLOID EMBRYOIDS OF LARIX DECIDUA: EARLY EMBRYOGENESIS.

Callus initiation of sections of megagametophytes of Larix decidua occurs just below the cut surface and is followed by the formation of one or more long protruding cells. The long cells then divide transversely at their tips to yield small cytoplasmically-dense terminal cells. The latter divide, forming loose aggregates of dense cells, microcalli, which develop long cells that radiate in all directions and divide terminally to produce aggregates of small cells. This long/short cell alternation is repeated a few times. Eventually the aggregates divide in a polarized manner producing files of long cells predominantly in one direction. These loosely bundled long cells form a suspensor-like structure. The meristematic small cells continue dividing forming a mass of embryonal cells. This early embryoid eventually turns green and produces both a root and shoot. Haploid embryoids are also derived from long cells in which karyokinesis but not cytokinesis occurs, resulting in a four-nucleate coenocyte. The nuclei migrate to one pole and become surrounded by cell walls. The cells thus formed are the originators of a new embryoid.