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Background: Chinese sprangletop (Leptochloa chinensis (L.) Nees) is an invasive grass weed severely infesting rice fields across China. In October 2020, a suspected resistant Leptochloa chinensis population HFFD3 that survived the acetyl-CoA carboxylase (ACCase)-inhibiting herbicide cyhalofop-butyl applied at its field-recommended rate was collected from a rice field in Feidong County, Anhui Province, China. This study aimed to determine the resistance profile of HFFD3 to ACCase inhibitors and to investigate its mechanisms of resistance to cyhalofop-butyl., Results: Single-dose testing confirmed that HFFD3 had evolved resistance to cyhalofop-butyl. Two loci encoding plastidic ACCase were each amplified from the susceptible (S) and resistant (R, HFFD3) individual plants. Target gene sequencing and derived cleaved amplified polymorphic sequence assay revealed all the R plants carried a Trp-2027-Leu substitution in their ACCase1,2 copies. Dose-response bioassays revealed that HFFD3 was highly resistant to cyhalofop-butyl and exhibited cross-resistance to metamifop, fenoxaprop-P-ethyl, quizalofop-P-ethyl, and clethodim. Pre-treatment with piperonyl butoxide and 4-chloro-7-nitrobenzoxadiazole considerably reversed the resistance of the R plants to cyhalofop-butyl, by 23% and 43%, respectively. Liquid chromatography-tandem mass spectrometry analysis suggested the metabolic rates of cyhalofop-butyl were significantly faster in the R than in the S plants., Conclusion: This study confirmed the first case of an arable weed species featuring cross-resistance to ACCase-inhibiting herbicides due to a novel Trp-2027-Leu mutation of ACCase. Target gene mutation and cytochrome P450s- and glutathione-S-transferases-involved enhanced metabolism may have simultaneously participated in the resistance of HFFD3 population to cyhalofop-butyl., (© 2021 Society of Chemical Industry.)

Polyhydroxybutyrate (PHB), the most well-known polyhydroxyalkanoate, is a bio-based, biodegradable polymer that has the potential to replace petroleum-based plastics. Lignocellulose hydrolysate, a non-edible resource, is a promising substrate for the sustainable, fermentative production of PHB. However, its application is limited by the generation of inhibitors during the pretreatment processes. In this study, we investigated the feasibility of PHB production in E. coli in the presence of inhibitors found in lignocellulose hydrolysates. Our results show that the introduction of PHB synthetic genes ( bktB , phaB , and phaC from Ralstonia eutropha H16) improved cell growth in the presence of the inhibitors such as furfural, 4-hydroxybenzaldehyde, and vanillin, suggesting that PHB synthetic genes confer resistance to these inhibitors. In addition, increased PHB production was observed in the presence of furfural as opposed to the absence of furfural, suggesting that this compound could be used to stimulate PHB production. Our findings indicate that PHB production using lignocellulose hydrolysates in recombinant E. coli could be an innovative strategy for cost-effective PHB production, and PHB could be a good target product from lignocellulose hydrolysates, especially glucose.

Poaceae (the grasses) is arguably the most successful plant family, in terms of its global occurrence in (almost) all ecosystems with angiosperms, its ecological dominance in many ecosystems, and high species richness. We suggest that the success of grasses is best understood in context of their capacity to colonize, persist, and transform environments (the "Viking syndrome"). This results from combining effective long-distance dispersal, efficacious establishment biology, ecological flexibility, resilience to disturbance and the capacity to modify environments by changing the nature of fire and mammalian herbivory. We identify a diverse set of functional traits linked to dispersal, establishment and competitive abilities. Enhanced long-distance dispersal is determined by anemochory, epizoochory and endozoochory and is facilitated via the spikelet (and especially the awned lemma) which functions as the dispersal unit. Establishment success could be a consequence of the precocious embryo and large starch reserves, which may underpin the extremely short generation times in grasses. Post-establishment genetic bottlenecks may be mitigated by wind pollination and the widespread occurrence of polyploidy, in combination with gametic self-incompatibility. The ecological competitiveness of grasses is corroborated by their dominance across the range of environmental extremes tolerated by angiosperms, facilitated by both C 3 and C 4 photosynthesis, well-developed frost tolerance in several clades, and a sympodial growth form that enabled the evolution of both annual and long-lived life forms. Finally, absence of investment in wood (except in bamboos), and the presence of persistent buds at or below ground level, provides tolerance of repeated defoliation (whether by fire, frost, drought or herbivores). Biotic modification of environments via feedbacks with herbivory or fire reinforce grass dominance leading to open ecosystems. Grasses can be both palatable and productive, fostering high biomass and diversity of mammalian herbivores. Many grasses have a suite of architectural and functional traits that facilitate frequent fire, including a tufted growth form, and tannin-like substances in leaves which slow decomposition. We mapped these traits over the phylogeny of the Poales, spanning the grasses and their relatives, and demonstrated the accumulation of traits since monocots originated in the mid-Cretaceous. Although the sympodial growth form is a monocot trait, tillering resulting in the tufted growth form most likely evolved within the grasses. Similarly, although an ovary apparently constructed of a single carpel evolved in the most recent grass ancestor, spikelets and the awned lemma dispersal units evolved within the grasses. Frost tolerance and C 4 photosynthesis evolved relatively late (late Palaeogene), and the last significant trait to evolve was probably the production of tannins, associated with pyrophytic savannas. This fits palaeobotanical data, suggesting several phases in the grass success story: from a late Cretaceous origin, to occasional tropical grassland patches in the later Palaeogene, to extensive C 3 grassy woodlands in the early-middle Miocene, to the dramatic expansion of the tropical C 4 grass savannas and grasslands in the Pliocene, and the C 3 steppe grasslands during the Pleistocene glacial periods. Modern grasslands depend heavily on strongly seasonal climates, making them sensitive to climate change., (© 2017 Cambridge Philosophical Society.)

Biological soil crusts (BSCs) can improve the stability and health of native plant communities in arid ecosystems. However, it is unknown whether BSCs can also inhibit invasions of exotic vascular plants on stabilized reclaimed sand dunes. To answer this question, we conducted a greenhouse experiment to test the effects of cyanobacteria-dominated BSCs on 1) seed germination and biomass of an exotic grass (Stipa glareosa P. Smirn.), and 2) individual biomass of the exotic S. glareosa growing with two native plants, Eragrostis poaeoides Beauv. and Artemisia capillaris Thunb. Our experiment included three BSC treatments (intact crust, disturbed crust, and bare soil) and five species trials (native E. poaeoides alone, E. poaeoides mixed with exotic S. glareosa, native A. capillaris alone, A. capillaris mixed with exotic S. glareosa, and S. glareosa alone). The results showed that cyanobacteria-dominated crusts can significantly reduce the cumulative percent germination of the exotic grass (P<0.001) and native plants (P<0.001). Maximum cumulative percent germinations of the exotic grass and two native plants were found in bare soil, and minimum in intact crusts. The interaction of crust treatment × species trials on shoot biomass of the two native plants was significant (P<0.05). These results indicate that the presence of BSCs on stabilized sand dunes may reduce the germination of the exotic and two native plants. The effect of reducing exotic and native plant seeds germination would maintain more diverse plant communities and contribute to the formation of clumped vegetation patterns. We conclude that BSCs act as a natural regulator for vegetation patterns and thus promote ecosystem stability and sustainability.

Apomixis is a naturally occurring mode of asexual reproduction in flowering plants that results in seed formation without the involvement of meiosis or fertilization of the egg. Seeds formed on an apomictic plant contain offspring genetically identical to the maternal plant. Apomixis has significant potential for preserving hybrid vigor from one generation to the next in highly productive crop plant genotypes. Apomictic Pennisetum/Cenchrus species, members of the Poaceae (grass) family, reproduce by apospory. Apospory is characterized by apomeiosis, the formation of unreduced embryo sacs derived from nucellar cells of the ovary and, by parthenogenesis, the development of the unreduced egg into an embryo without fertilization. In Pennisetum squamulatum (L.) R.Br., apospory segregates as a single dominant locus, the apospory-specific genomic region (ASGR). In this study, we demonstrate that the PsASGR-BABY BOOM-like (PsASGR-BBML) gene is expressed in egg cells before fertilization and can induce parthenogenesis and the production of haploid offspring in transgenic sexual pearl millet. A reduction of PsASGR-BBML expression in apomictic F1 RNAi transgenic plants results in fewer visible parthenogenetic embryos and a reduction of embryo cell number compared with controls. Our results endorse a key role for PsASGR-BBML in parthenogenesis and a newly discovered role for a member of the BBM-like clade of APETALA 2 transcription factors. Induction of parthenogenesis by PsASGR-BBML will be valuable for installing parthenogenesis to synthesize apomixis in crops and will have further application for haploid induction to rapidly obtain homozygous lines for breeding.

Moso bamboo [Phyllostachys heterocycla var. pubescens (Mazel ex J. Houz.) Ohwi] is one of the most important forest crops in China and the rest of Asia. Although many sympodial bamboo tissue culture protocols have been established, there is no protocol available for plantlet regeneration as indicated by callus induction for monopodial bamboos, such as Moso bamboo. In the present report, embryogenic callus induction, embryoid development, and germination were established for Moso bamboo from zygotic seed embryos. Callus was initiated from zygotic embryos after 10-20 d culture on MS media supplemented with 4.0 mg/L 2, 4-D and 0.1 mg/L zeatin (ZT). About 50% of the explants produced calli, and nearly 15% of the calli were found to be embryogenic in nature. These embryogenic calli can be subcultured for proliferation in the Murashige and Skoog media (MS) supplemented with 0.5-2.0 mg/L 2, 4-D. These calli were found to have maintained their capacity for regeneration even after one year of subculture. The viable somatic embryoids regenerated in medium containing 5.0-7.0 mg/L ZT. Nearly 5% of the calli were found capable of regenerating into plantlets directly in MS medium containing 5.0-7.0 mg/L ZT. Root growth was more pronounced when the plantlets were transferred to medium containing 2.0 mg/L NAA. After 30 days of subculture, the plantlets were transferred to a greenhouse.

Miscanthus × giganteus is a popular energy crop, which due to its hybrid origin is only vegetatively reproduced. Asexual embryogenesis in anther and microspore culture leading to double haploids production could allow to regain the ability for sexual reproduction and to increase the biodiversity of the species. Therefore, the goal of this paper was to investigate the requirements of androgenesis in Miscanthus. The standard protocols used for monocotyledonous plants were applied with many modifications regarding the developmental stage of the explants at the time of culture initiation, stress treatment applied to panicles and isolated anthers as well as various chemical and physical parameters of in vitro culture conditions. Our results indicated that the induction of androgenesis in M. × giganteus is possible. However, the very low efficiency of the process and the lack of regeneration ability of the androgenic structures presently prevent the use of this technique.

Survival of seeds in the seed bank is important for the population dynamics of many plant species, yet the environmental factors that control seed survival at a landscape level remain poorly understood. These factors may include soil moisture, vegetation cover, soil type, and soil pathogens. Because many soil fungi respond to moisture and host species, fungi may mediate environmental drivers of seed survival. Here, I measure patterns of seed survival in California annual grassland plants across 15 species in three experiments. First, I surveyed seed survival for eight species at 18 grasslands and coastal sage scrub sites ranging across coastal and inland Santa Barbara County, California. Species differed in seed survival, and soil moisture and geographic location had the strongest influence on survival. Grasslands had higher survival than coastal sage scrub sites for some species. Second, I used a fungicide addition and exotic grass thatch removal experiment in the field to tease apart the relative impact of fungi, thatch, and their interaction in an invaded grassland. Seed survival was lower in the winter (wet season) than in the summer (dry season), but fungicide improved winter survival. Seed survival varied between species but did not depend on thatch. Third, I manipulated water and fungicide in the laboratory to directly examine the relationship between water, fungi, and survival. Seed survival declined from dry to single watered to continuously watered treatments. Fungicide slightly improved seed survival when seeds were watered once but not continually. Together, these experiments demonstrate an important role of soil moisture, potentially mediated by fungal pathogens, in driving seed survival.

Anther and pollen development were studied in Olyra humilis Nees, Sucrea monophylla Soderstr, (Bambusoideae), Axonopus aureus P. Beauv., Paspalum polyphyllum Nees ex Trin. (Panicoideae), Eragrostis solida Nees, and Chloris elata Desv. (Chloridoideae). The objective of this study was to characterise, embryologically, these species of subfamilies which are considered basal, intermediate and derivate, respectively. The species are similar to each other and to other Poaceae. They present the following characters: tetrasporangiate anthers; monocotyledonous-type anther wall development, endothecium showing annular thickenings, secretory tapetum; successive microsporogenesis; isobilateral tetrads; spheroidal, tricellular, monoporate pollen grains with annulus and operculum. Nevertheless, the exine patterns of the species studied are distinct. Olyra humilis and Sucrea monophylla (Bambusoideae) show a granulose pattern, whereas in the other species, it is insular. In addition, Axonopus aureus and Paspalum polyphyllum (Panicoideae) have a compactly insular spinule pattern, while Chloris elata and Eragrostis solida (Chloridoideae) show a sparsely insular spinule pattern. The exine ornamentation may be considered an important feature at the infrafamiliar level.

Somatic embryogenesis could be achieved in Themeda quadrivalvis (Linn.) O. Ktze -fodder grass species on MS medium supplemented with 2,4-D. Incorporation of putrescine in the medium stimulated embryogenesis, however its lower concentration stimulated production of non-regenerative callus. Other polyamines such as spermine and spermidine could not evoke similar response. Ascorbic acid used as antioxidant could not prevent browning in embryogenic cultures, however it stimulated embryogenesis. Inhibition of auxin polar transport by use of TIBA and HFCA reduced the embryogenic response significantly and produced distorted or abnormal embryos. Antiethylene substances such as AgNO3 and CoCl2 added in the medium adversely affected the process of embryogenesis and counteracting the stimulatory role of ethylene.

Rhodes grass (Chloris gayana) is one of the most important warm-season forage grasses. It is cultivated in tropical and subtropical parts of the world and is mostly used for grazing and hay production. We have established a particle-bombardment transformation protocol for rhodes grass using multiple-shoot clumps (MSCs) as the target tissue. A vector pAHC25 containing a herbicide-resistance gene (bar) together with the beta-glucuronidase (GUS) gene was used in transformation experiments. The most efficient recovery of bialaphos-resistant tissue was achieved when the bombarded MSCs were first cultured for 15 d on bialaphos-free medium before being subjected to selection pressure. The resistant tissues regenerated transgenic plants that displayed GUS gene expression. Under optimized conditions, 251 target pieces yielded 46 transgenic plants from 4 independent transgenic lines.

Brachypodium distachyon is a novel model system for structural and functional genomics studies of temperate grasses because of its biological and genetic attributes. Recently, the genome sequence of the community standard line Bd21 has been released and the availability of an efficient transformation system is critical for the discovery and validation of the function of Brachypodium genes. Here, we provide an improved procedure for the facile and efficient Agrobacterium-mediated transformation of line Bd21. The protocol relies on the transformation of compact embryogenic calli derived from immature embryos using visual and chemical screening of transformed tissues and plants. The combination of green fluorescent protein expression and hygromycin resistance enables early identification of transformation events and drastically reduces the quantity of tissue to be handled throughout the selection process. Approximately eight independent fully developed transgenic Bd21 plants can be produced from each immature embryo, enabling the generation of thousands of T-DNA lines. The process--from wild-type seeds to transgenic T(1) seeds--takes approximately 8 months to complete.

Cytological and embryological studies on apomictic species Bothriochloa ischaemum L. were carried out. Our studies revealed that the chromosome number of its root apical cells was 40, indicating that it was a tetraploid cytotype. During the stage of microsporogenesis, meiosis seemed irregular, as the pairing chromosome number of microspore mother cell was more than 20. It was often found that some chromosomes did not assemble in the equatorial plane or moved to the two poles of the cell, a few laggards were seen. Multiporate pollens (22.3%) were often observed. The studies showed that a high frequency (87.8%) of 1-3 or more aposporous embryo sacs developed in one ovule of the species. The mature aposporous sac was usually characterized by an egg cell and one polar nucleus. The egg cell could develop spontaneously into a large proembryo (100-200 microm) mass prior to anthesis. When several aposporous sacs occurred in the same ovule, usually 2 aposporous sacs were involved in pseudogamy and developed into separate endosperm masses in the same ovary. In the low frequency of mature seed, 13.5% twin-embryo seedlings could be obtained after mature seeds germinated.

The success of calcareous grassland recreation following abandonment depends to a large extent on the composition of the soil seed bank. We studied the species richness and composition of the seed bank along a chronosequence from well-developed calcareous grassland to scrub and forest vegetation, which had developed on calcareous grassland over the last 225 years. The seed bank density was highest in the calcareous grassland soils (930 seeds/m(2)). However, the seed bank was mainly composed of rather common species of nutrient poor grassland, which were poorly represented in the surface vegetation. There were no significant differences in soil seed bank density and species composition between the calcareous grassland and scrub vegetation that was less than 15 years old, largely because several grassland species had persisted in the scrub vegetation and were therefore able to replenish the soil seed bank. In contrast seed density and species richness declined significantly after 40 years of grassland abandonment. Indeed, forest soils had the lowest seed densities (214 seeds/m(2)) with only a few grassland species represented. This reflects the lack of grassland species in the field layer of the forest, and, therefore, the lack of seed production and seed bank replenishment. It is clear that recreation of calcareous grassland on long abandoned sites cannot rely on germination of target species from the seed bank alone. Even in the calcareous grassland soil, seeds of target species are not abundant as the majority produces transient seeds, which decay rapidly if they do not germinate immediately. Successful grassland recreation on such sites therefore may require seeds of target species to be introduced artificially (e.g. as seed mixtures, green hay, etc.). Alternatively, reinstatement of traditional practices, such as grazing or mowing, will increase the natural dispersal potential of these species, allowing population reestablishment in the long term.

In this study, we investigated the potential of multispecies rhizoremediation and monoculture rhizoremediation in decontaminating polycyclic aromatic hydrocarbon (PAH) contaminated soil Plant-mediated PAH dissipation was evaluated using monoplanted soil microcosms and soil microcosms vegetated with several different grass species (Brachiaria serrata and Eleusine corocana). The dissipation of naphthalene and fluorene was higher in the "multispecies" vegetated soil compared to the monoplanted and nonplanted control soil. The concentration of naphthalene was undetectable in the multispecies vegetated treatment compared to 96% removal efficiencies in the monoplanted treatments and 63% in the nonplanted control after 10 wk of incubation. Similar removal efficiencies were obtained for fluorene. However, there was no significant difference in the dissipation of pyrene in both the mono- and multispecies vegetated treatments. There also was no significant difference between the dissipation of PAHs in the monoplanted treatments with different grass species. Principle component analysis (PCA) and cluster analysis were used to evaluate functional diversity of the different treatments during phytoremediation of PAHs. Both PCA and cluster analysis revealed differences in the metabolic fingerprints of the PAH contaminated and noncontaminated soils. However, the differences in metabolic diversity between the multispecies vegetated and monoplanted treatments were not clearly revealed. The results suggest that multispecies rhizoremediation using tolerant plant species rather than monoculture rhizoremediation have the potential to enhance pollutant removal in moderately contaminated soils.

Russian wildrye (Psathyrostachys juncea (Fisch.) Nevski) is a cool-season forage species well adapted to semi-arid climates. We are interested in developing biotechnological methods to improve this monocot forage species. Single genotype-derived embryogenic suspension cultures were established from the Russian wildrye cultivar Bozoisky-Select, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric beta-glucuronidase (gusA) gene was co-transformed with hph. Resistant calli were obtained from 29% of the bombarded dishes after selection with 200 mg/l hygromycin. Plants were regenerated from 45% of the hygromycin resistant calli. Thirty-six transgenic Russian wildrye plants were recovered after microprojectile bombardment of suspension cells and subsequent hygromycin selection. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. When a second gene (gusA) was co-transformed with hph, a reasonably high co-transformation frequency of 78% was observed. Transgenic expression of gusA was confirmed by GUS staining of shoot and leaf tissues. Fertile transgenic plants were obtained after two winters of vernalization under field conditions. This is the first report on the generation of transgenic plants in Russian wildrye.

Seeds of the endangered species Zizania texana are recalcitrant, making it difficult to preserve the remaining genetic diversity of this species in genebanks. Excised embryos can be cryopreserved using solution-based cryoprotection protocols. Survival following cryoexposure increased from less than 5% to about 75% by preculturing embryos in high concentrations of sugars, bathing them in cryoprotectant solutions, and partially drying them to water contents of about 0.6 g H2O/g dry mass.

This study compared the field performance of sugarcane plants originating from three different sources: control, non-cryopreserved embryogenic calluses, cryopreserved embryogenic calluses and macropropagated material of the same commercial hybrid. Several agronomic traits were evaluated on 100 plants per treatment over a 27-month period covering the growth of the stool and of the first ratoon. Significant differences between treatments were observed only during the first six months of field growth of sugarcane stools. Stems produced from in vitro cultured material, irrespective of their cryopreservation status, had a smaller diameter and a shorter height than those produced from macropropagated material. These differences disappeared by 12 months of stool field growth.

A relationship has been established between the expression of apomixis in natural polyploids of Tripsacum dactyloides and fertility as measured by percent seed set. Thus, fertility may be reliably used as a defining phenotype for apomixis when scoring the progeny from diploid (2n = 2x = 36) x tetraploid (2n = 4x = 72) crosses in Tripsacum. By exploiting the relationship between apomixis and fertility, as defined by seed set, analyses were performed on a set of related second-generation triploid populations segregating for apomixis. These populations were derived from sexual (diploid) x apomictic (tetraploid) crosses. Six out of 25 genome-dispersed restriction fragment length polymorphism (RFLP) markers co-segregate with fertility. Five of these markers were previously reported and include: php20855, tda48, tda53, umc62, and umc83, and are linked to Tripsacum genetic linkage groups F, I, H, L, and A, respectively. Significantly, we report here the syntenic relationships of the maize chromosome intervals to Tripsacum that segregate for numerous meiosis-specific and fertility-associated genes. Utilizing RFLP locus comparative mapping based on conservation of chromosome (genic) regions between related species, it may be concluded that the genes controlling fertility have been preserved in both Tripsacum and maize. A sixth marker, umc166, has also been shown to co-segregate with fertility and is conserved in both grass species. Specifically, umc166 is linked to Tripsacum linkage group D and, by syntenic comparison, to the short arm of maize chromosome 5. Encoded within this marked interval is the gene Ameiotic1 (Am1) whose function is required for the initiation of meiosis in both micro- and megaspore mother cells and whose absence of expression in the female is, in all likelihood, a prerequisite for the expression of apomixis.

Paspalum notatum is a subtropical forage grass, which reproduces by either sexuality or aposporous gametophytic apomixis. The objective of this work was to identify and isolate mRNA transcripts differentially expressed during the development of the megagametophyte from spikelets of apomictic and sexual P. notatum. Crossing of a sexual mother plant with an apomictic pollen donor generated a progeny family segregating for reproductive mode. Individuals from this F1 family were cytoembryologically classified as sexual or apomictic. Spikelet mRNA compositions from both groups of plants were compared by differential display using an RNA-bulked procedure. Fifty primer combinations were assayed to generate nearly 2,500 total bands in the fingerprints. Three transcripts expressed at higher levels in apomictic plants (apo417, apo398, and apo396) were identified, isolated and cloned. Sequencing showed a high level of homology among the isolated clones. Analysis by RT-PCR Southern blots followed by densitometric studies confirmed that expression reached a level around 30 times higher in apomictic than in sexual individuals and was probably induced at early stages of the megagametophyte development. Genomic DNA from the parental and the F1 progeny plants showed 4-5 bands when hybridised with apo417 in Southern blots. Comparisons to the sequence data banks revealed no identities to genes of known function. However, a putative deduced 3' protein fragment showed homology to the well-characterised KSP multi-phosphorylation domain previously detected in several cdc2-regulated cytoskeletal proteins.

We have examined the expression of four genes during somatic embryogenesis in two cultivars of sugarcane, Saccharum officinarum, one drought resistant (JA-605) and the other one sensitive (C-8751), as well as in embryogenic and nonembryogenic tissues treated with abscisic acid (ABA) or after drought stress. Three of the cDNAs probed, from plasmids pMA1049, pMA2005 and CM1, were lea genes and the other one, from a barley hemoglobin gene, was induced in other species under stress conditions. Only transcripts homologous to the pMA2005 and CM1 clones were differentially accumulated during somatic embryogenesis; moreover, the CM1 clone only appeared in somatic embryos of the non-resistant variety. All the lea cDNAs were dramatically increased in the embryogenic tissues treated with ABA, but we observed differences among the accumulation of these mRNAs in the nonembryogenic tissues treated with ABA, and only the pMA2005 transcript appeared. The mRNA homologous to the barley hemoglobin gene appeared in the nonembryogenic tissue and the expression increased after ABA treatment. The desiccation treatments had different effects on the varieties, and the pMA2005 transcripts only appeared in the sensitive variety.

Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell-division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division, divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all. Grant numbers: NAGW-3141, NAG10-0221.

Somatic embryos initiate and develop from single mesophyll cells in in vitro cultured leaf segments of orchard-grass (Dactylis glomerata L.). Segments were plated at time periods ranging from 21 to 0.9 d (21 h) prior to launch on an 11 d spaceflight (STS-64). Using a paired t-test, there was no significant difference in embryogenesis from preplating periods of 14 d and 21 d. However, embryogenesis was reduced by 70% in segments plated 21 h before launch and this treatment was significant at P=0.0001. The initial cell divisions leading to embryo formation would be taking place during flight in this treatment. A higher ratio of anticlinal:periclinal first cell divisions observed in the flight compared to the control tissue suggests that microgravity affects axis determination and embryo polarity at a very early stage. A similar reduction in zygotic embryogenesis would reduce seed formation and have important implications for long-term space flight or colonization where seeds would be needed either for direct consumption or to grow another generation of plants.

Ultra low temperature cryopreservation is one of the methods for preservation of biological material. Until now, a major problem of protoplast culture of Gramineae is the instability of state of the somatic embryogenic cell line. In our experiments, elements affecting the ultra low temperature cryopreservation of somatic embryogenic cell line were studied: components of cryopreserve solution, somatic embryogenic cell line of different subculture time, growth recovery of cryopreserved cell line, and their protoplast cultures. Results demonstrated that the ultra low temperature cryopreservation did not change the properties of protoplast culture, and by using the cryopreserve method, plating efficiency of protoplast culture of cryopreserved cell line was maintained or enhanced.

In cereal seeds, the aleurone layer and the embryo share several characteristics, including synthesis and accumulation of lipid bodies, desiccation tolerance and dormancy. A number of Balem transcripts present in both the barley aleurone layer and the embryo have been cloned by differential screening of a cDNA library from aleurone layers of immature barley grains. The Balem clones constitute two subgroups, one for which the transcripts are detectable in aleurone layers and embryos of developing seeds only (B23D and B15C), and another for which transcripts are present also in germinating embryos and in maternal tissues (B12D, B14E and B31E). Sequence analysis identified B23D and B15C as the barley homologues of the 18 kDa oleosin of maize embryos (72% amino acid identity) and the dormancy-associated transcript pBS128 from Bromus secalinus (95% identity), respectively. In situ hybridization experiments demonstrate that in the embryo, the B23D transcript is mainly present in the scutellum, whereas the B15C transcript is predominantly present in shoot and root apices. Using anther-derived embryos and embryogenic cell suspensions, it is demonstrated that the B23D and B15C transcripts can be used as molecular markers for somatic embryogenesis. The functions of the transcripts in the second Balem subgroup remain unknown. Further studies on the Balem transcripts may shed light on the molecular basis for the extensive similarities between the embryo and the aleurone layer of the endosperm in the grass family.