Evaluation of in vitro genotoxic effects induced by in vitro anther culture conditions in sunflower.

Sunflower is a globally important oilseed, food, and ornamental crop. This study seeks to investigate the genotoxic effects of tissue culture parameters in sunflower calli tissues belongs to two genotypes obtained via anther culture. Anthers were pretreated with cold for 24 hours at 4°C and heat for 2 days at 35°C in the dark and plated onto media supplemented with different concentrations and combinations of 6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid, α-naphthalene acetic acid and indole-3-acetic acid. Obtaining calli tissues were used to detect the DNA damage levels by Comet assay, evaluating changes on superoxide dismutase and guaiacol peroxidase activities derived from in vitro culture factors. 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 2 mg/L α-naphthalene acetic acid from plant growth regulators showed acute genotoxic effect while 0.5 mg/L indole-3-acetic acid and 0.5 mg/L α-naphthalene acetic acid showed no genotoxic effect. Total protein content analysis of antioxidant enzymes revealed that although superoxide dismutase activity did not increase, Guaiacol peroxidase (GPOX) activity decreased in comparison to control. The obtained results have indicated that in vitro culture factors apparently lead to genotoxicity and oxidative stress.

Arabinogalactan proteins mediate intercellular crosstalk in the ovule of apple flowers.

KEY MESSAGE: AGP-rich glycoproteins mediate pollen-ovule interactions and cell patterning in the embryo sac of apple before and after fertilization. Glycoproteins are significant players in the dialog that takes place between growing pollen tubes and the stigma and style in the angiosperms. Yet, information is scarce on their possible involvement in the ovule, a sporophytic organ that hosts the female gametophyte. Apple flowers have a prolonged lapse of time between pollination and fertilization, offering a great system to study the developmental basis of glycoprotein secretion and their putative role during the last stages of the progamic phase and early seed initiation. For this purpose, the sequential pollen tube elongation within the ovary was examined in relation to changes in arabinogalactan proteins (AGPs) in the tissues of the ovule before and after fertilization. To evaluate what of these changes are developmentally regulated, unpollinated and pollinated flowers were compared. AGPs paved the pollen tube pathway in the ovules along the micropylar canal, and the nucellus entrance toward the synergids, which also developmentally accumulated AGPs at the filiform apparatus. Glycoproteins vanished from all these tissues following pollen tube passage, strongly suggesting a role in pollen-ovule interaction. In addition, AGPs marked the primary cell walls of the haploid cells of the female gametophyte, and they further built up in the cell walls of the embryo sac and developing embryo, layering the interactive walls of the three generations hosted in the ovule, the maternal sporophytic tissues, the female gametophyte, and the developing embryo.

Effects of High Temperature on Embryological Development and Hormone Profile in Flowers and Leaves of Common Buckwheat (Fagopyrum esculentum Moench).

Common buckwheat is a valuable crop, mainly due to the beneficial chemical composition of its seeds. However, buckwheat cultivation is limited because of unstable seed yield. The most important reasons for the low yield include embryo and flower abortion. The aim of this work is to verify whether high temperature affects embryological development in this plant species. The experiment was conducted on plants of a Polish cultivar 'Panda' and strain PA15, in which the percentage of degenerating embryo sacs was previously determined and amounted to 32% and 10%, respectively. The plants were cultivated in phytotronic conditions at 20 °C (control), and 30 °C (thermal stress). The embryological processes and hormonal profiles in flowers at various developmental stages (buds, open flowers, and wilted flowers) and in donor leaves were analyzed in two-month-old plants. Significant effects of thermal stress on the defective development of female gametophytes and hormone content in flowers and leaves were observed. Ovules were much more sensitive to high temperature than pollen grains in both genotypes. Pollen viability remained unaffected at 30 °C in both genotypes. The effect of temperature on female gametophyte development was visible in cv. Panda but not in PA15 buds. A drastic reduction in the number of properly developed embryo sacs was clear in open flowers at 30 °C in both genotypes. A considerable increase in abscisic acid in open flowers ready for fertilization may serve as a signal inducing flower senescence observed in the next few days. Based on embryological analyses and hormone profiles in flowers, we conclude that cv. 'Panda' is more sensitive to thermal stress than strain PA15, mainly due to a much earlier response to thermal stress involving impairment of embryological processes already in the flower buds.

Identification of pre-fertilization reproductive barriers and the underlying cytological mechanism in crosses among three petal-types of Jasminum sambac and their relevance to phylogenetic relationships.

Crosses among single-, double- and multi-petal jasmine cultivars (Jasminum sambac Aiton) are unable to easily generate hybrids. To identify the reproductive barriers restricting hybrid set, dynamic changes in jasmine pollen viability and pistil receptivity were compared at different flowering stages. Pollen-pistil interactions in six reciprocal crosses were also investigated to characterize pollen-stigma compatibility. Additionally, paraffin sections of pollinated embryo sacs were prepared for subsequent analyses of developmental status. Furthermore, pistil cell ultrastructural characteristics were observed to reveal cytological mechanism regulating pistil receptivity and the pollen-pistil interactions. We observed that pollen viability and stigma receptivity varied depending on petal phenotype and flowering stage and were easily lost during flowering. Different reciprocal crosses exhibited varied pollen-stigma compatibilities according to the pollen germination rates. Although some pollen grains germinated normally on maternal stigmas, the pollen tubes were arrested in the pistils and were unable to reach the ovaries. Additionally, the embryo sacs remained unfertilized until degenerating. Therefore, jasmine crosses are affected by pre-fertilization reproductive barriers. Low pollen fertility and poor stigma receptivity are detrimental to pollen germination and pollen-pistil compatibility, indicating they are two factors affecting hybrid set. Ultrastructural observation of the pistil cells revealed that cell death occurred during flowering. Thus, the early and rapid senescence of pistils is likely responsible for the decreased pistil receptivity and inhibited pollen tube growth. These findings may be relevant for future jasmine hybridizations. They provide new insights for the development of methods to overcome reproductive barriers and may also be useful for clarifying the phylogenetic relationships among jasmine cultivars with differing petal phenotypes.

Starch Turnover and Metabolism during Flower and Early Embryo Development.

The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed.

Floral morphology and embryology of Helwingia (Helwingiaceae, Aquifoliales): systematic and evolutionary implications.

Helwingia, a shrub or small tree of four species distributed in East Asia, has been assigned to various families, mainly Cornaceae. However, molecular analyses show that the genus belongs to its own family Helwingiaceae which is sister to Phyllonomaceae (Phyllonoma only) in the order Aquifoliales. On the basis of H. japonica, we investigated the poorly understood floral and embryological characters of Helwingia, and compared their features with those of other Aquifoliales, particularly those of Phyllonomaceae. Results showed that perianth leaves of Helwingia represent sepals, because in plesiomorphic pentamerous flowers, they agreed in position with sepals (not with petals) in pentamerous flowers of Phyllonoma. Overall comparisons based on available information show that, while sharing with Phyllonoma the epiphyllous inflorescence, the inferior ovary, and an epigynous disc nectary as syapomorphies, Helwingia is characterized by loss of petals, obhaplostemony, large recurved stigmas, poorly developed disc nectary, tenuinucellate ovules with a mature female gametophyte filled with densely stained cytoplasm, and a thin mature seed coat. Morphological evidence, like molecular evidence, confirms that Helwingia is sufficiently distinct to be placed in its own family. Morphological and field observations suggest wind and insect pollination in H. japonica, which is the first example of ambophily in Aquifoliales.

Maize germinal cell initials accommodate hypoxia and precociously express meiotic genes.

In flowering plants, anthers are the site of de novo germinal cell specification, male meiosis, and pollen development. Atypically, anthers lack a meristem. Instead, both germinal and somatic cell types differentiate from floral stem cells packed into anther lobes. To better understand anther cell fate specification and to provide a resource for the reproductive biology community, we isolated cohorts of germinal and somatic initials from maize anthers within 36 h of fate acquisition, identifying 815 specific and 1714 significantly enriched germinal transcripts, plus 2439 specific and 2112 significantly enriched somatic transcripts. To clarify transcripts involved in cell differentiation, we contrasted these profiles to anther primordia prior to fate specification and to msca1 anthers arrested in the first step of fate specification and hence lacking normal cell types. The refined cell-specific profiles demonstrated that both germinal and somatic cell populations differentiate quickly and express unique transcription factor sets; a subset of transcript localizations was validated by in situ hybridization. Surprisingly, germinal initials starting 5 days of mitotic divisions were enriched significantly in >100 transcripts classified in meiotic processes that included recombination and synapsis, along with gene sets involved in RNA metabolism, redox homeostasis, and cytoplasmic ATP generation. Enrichment of meiotic-specific genes in germinal initials challenges current dogma that the mitotic to meiotic transition occurs later in development during pre-meiotic S phase. Expression of cytoplasmic energy generation genes suggests that male germinal cells accommodate hypoxia by diverting carbon away from mitochondrial respiration into alternative pathways that avoid producing reactive oxygen species (ROS).

Immunolocalization of arabinogalactan proteins (AGPs) in reproductive structures of an early-divergent angiosperm, Trithuria (Hydatellaceae).

BACKGROUND AND AIMS: Trithuria is the sole genus of Hydatellaceae, a family of the early-divergent angiosperm lineage Nymphaeales (water-lilies). In this study different arabinogalactan protein (AGP) epitopes in T. submersa were evaluated in order to understand the diversity of these proteins and their functions in flowering plants. METHODS: Immunolabelling of different AGPs and pectin epitopes in reproductive structures of T. submersa at the stage of early seed development was achieved by immunofluorescence of specific antibodies. KEY RESULTS: AGPs in Trithuria pistil tissues could be important as structural proteins and also as possible signalling molecules. Intense labelling was obtained with anti-AGP antibodies both in the anthers and in the intine wall, the latter associated with pollen tube emergence. CONCLUSIONS: AGPs could play a significant role in Trithuria reproduction, due to their specific presence in the pollen tube pathway. The results agree with labellings obtained for Arabidopsis and confirms the importance of AGPs in angiosperm reproductive structures as essential structural components and probably important signalling molecules.

Mechanism of seedlessness in a new lemon cultivar 'Xiangshui' [Citrus limon (L.) Burm. F].

Seedlessness is an important economic trait of lemon. Understanding the cellular and molecular mechanisms of seedlessness in 'Xiangshui' lemon requires detailed data on pollen and embryo sac fertility, embryo development and compatibility mechanisms governing self- and cross-pollination. The results of the current study indicate that the fertility of pollen and mature embryo sac remains normal. When flowers were self- or cross-pollinated, pollen grains of 'Xiangshui' were able to germinate on the stigma. In the case of self-pollination, pollen tubes became twisted, tube tips enlarged and tubes ruptured in the bottom of stigma. Following cross-pollination, tubes were able to grow normally in the style and ovary and enter the embryo sac, where double fertilization took place. Embryonic development resulting from cross-pollination was normal. After cross-pollination, the zygote began to divide at 2 weeks post-pollination, with early globular embryos observed after 3 weeks, globular and heart-shaped embryos at 4 weeks, torpedo-shaped embryos at 5 weeks, cotyledonary embryos at 6 weeks and thereafter germinable seeds. After self-pollination, however, ovules began to abort at 2 weeks post-pollination, with ovules disappearing at 5 weeks, ultimately producing seedless fruits. Emasculated unpollinated flowers also developed into seedless fruits, indicating that seedlessness contributes to parthenocarpy. However, gametophytic self-incompatibility has a major role in seedlessness in 'Xiangshui' lemon by blocking fertilization at the bottom of the stigma.

Embryology of the Irvingiaceae, a family with uncertain relationships among the Malpighiales.

The Irvingiaceae, one of 40 families of the Malpighiales, comprise a small woody family of 10 species in three genera distributed in Old World tropics. Its relationships with other families are unclear, although recent molecular analyses suggest affinities with Linaceae, Caryocaraceae, Erythroxylaceae, and Rhizophoraceae. To gain insight into family relationships, we investigated 63 embryological characters of two previously unstudied African species, Irvingia gabonensis and I. smithii, and compared them with other Malpighiales and the sister group Oxalidales. Embryologically, Irvingia is characterized by the absence of an integumentary tapetum and by having a non-multiplicative inner integument, a multiplicative testa, many discrete fascicles of vascular bundles running in the testa from the raphe to antiraphe (each fascicle comprised several strands arranged in a concentric manner), and a fibrous exotegmen. Comparisons showed that Irvingia did not resemble any of the Linaceae, Caryocaraceae, Erythroxylaceae, Rhizophoraceae, or any of the other malpighialean families for which embryological data are available. The genus rather resembled Huaceae and Connaraceae (Oxalidales) in seed coat structure. However, 18 families (45%) of the Malpighiales are still poorly understood embryologically, and therefore additional studies are required for further critical comparisons.

Anther and pollen development in some species of Poaceae (Poales).

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.

Disruption of endosperm development: an inbreeding effect in almond (Prunus dulcis).

A homozygous self-compatible almond, originated from self-fertilization of a self-compatible genotype and producing a reasonable yield following open pollination, exhibited a very high fruit drop rate when self-pollinated. To investigate whether fruit dropping in this individual is related to an abnormal development of the embryo sac following self-fertilization, histological sections of ovaries from self and cross-pollinated flowers were observed by light microscopy. Additionally, the presence of pollen tubes in the ovary and fruit set were determined for both types of pollination. Despite pollen tubes reached the ovary after both pollinations, differences in embryo sac and endosperm development after fertilization were found. Thus, while for cross-fertilized ovules a pro-embryo and an endosperm with abundant nuclei were generally observed, most self-fertilized ovules remained in a previous developmental stage in which the embryo sac was not elongated and endosperm nuclei were absent. Although 30 days after pollination fruit set was similar for both pollination types, at 60 days it was significantly reduced for self-pollination. These results provide evidence that the high fruit drop in this genotype is the consequence of a disrupted development of the endosperm, what could be an expression of its high level of inbreeding.

Anther and pollen development in some species of Poaceae (Poales) / Desenvolvimento da antera e do grão de pólen em espécies de Poaceae (Poales)

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.

O desenvolvimento da antera e do grão de pólen de 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) foi estudado visando caracterizar embriologicamente essas espécies de subfamílias consideradas basal, intermediária e derivada, respectivamente. As espécies são similares entre si e entre as demais Poaceae. Apresentam os seguintes caracteres: anteras tetrasporangiadas; desenvolvimento da parede da antera do tipo monocotiledôneo, endotécio com espessamento de parede anelar, tapete secretor; microsporogênese sucessiva; tétrades isobilaterais; grãos de pólen esféricos, tricelulares, monoporados, com anel e opérculo. Por outro lado, o padrão de ornamentação da exina do grão de pólen é distinto. Olyra humilis e Sucrea monophylla (Bambusoideae) apresentam padrão granuloso e as demais espécies padrão insular. Axonopus aureus e Paspalum polyphyllum (Panicoideae) apresentam espínulos densamente agrupados, enquanto Chloris elata e Eragrostis solida (Chloridoideae) espínulos esparsamente agrupados. A ornamentação da exina dos grãos de pólen pode ser considerada caráter importante a nível infrafamiliar.

Floral biology of Ziziphus mauritiana (Rhamnaceae).

Floral development of the synchronous dichogamous species Ziziphus mauritiana, as followed by light and scanning electron microscopy (SEM), was divided into 11 stages using a series of landmark events. Main cellular events happen synchronously in the female and the male structures, such as meiosis in micro- and macrosporocyte cells, tetrad microspore formation and appearance of the functional megaspore cell, and onset of embryo sac differentiation coinciding with mitosis in the microspores. The last stage was characterized by anthesis and continued development of the flower, beginning with anther dehiscence (male phase) and proceeding to the female phase, which was characterized by style elongation. Flowers exhibit synchronous protandrous dichogamy; anthesis takes place in the morning (group A, e.g., clone Q-29) and afternoon (group B, e.g., clone B5/4). Stigma receptivity started after the male phase and occurred synchronously and complementarily with pollen dispersal in the two clones. Pollen viability and production were similar in the two clones, but the pollen diameter of Q-29 was significantly larger than that of B5/4. This study provides the basis for understanding the biological mechanisms regulating floral development, thus expanding the prospects for Z. mauritiana breeding programs and for further molecular and genetic studies of this species.

[Histological and cytological study on the ontogeny of embryoid in pepper anther culture].

The characteristics of histology and cytology of embryogenesis in pepper anther culture were examined with fluorescence microscopy, scanning microscopy, and electron microscopy. Pepper was characterized by a strong asynchrony of pollen development within a single anther. With the change of culture period, the proportion of dead pollen increased drastically from 2 day after culture. Microspores that were cultured at the late-uninucleate stage followed one of two developmental pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be typical bicellular pollen. Embryogenic pollen was formed by repeated divisions of the vegetative nuclei. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture, the other showed cell cluster volume expansion over the entire microspore surface. Morphogenesis of microspore-derived embryos has been analyzed, at both light and electron microscopical levels. The changes in cell organization after embryogenesis induction, and the characterization of the time sequence of a set of structural events, had been also explained. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development.

Inflorescence proliferation for somatic embryogenesis induction and suspension-derived plant regeneration from banana (Musa AAA, cv. 'Dwarf Cavendish') male flowers.

Availability of explants with adequate embryogenic competence is one of the most important limitations for the development of regenerable cell suspensions in banana. To increase the number and ease of accessibility to potentially embryogenic explants, a novel methodology is described by which young male flower clusters isolated from adult plants are induced to form new flower buds and proliferate in vitro. Different concentrations of the plant growth regulator thidiazuron (TDZ) induced inflorescence proliferation, which could be maintained over time as a continuous source of young flower buds. Intensity of proliferation was evaluated during successive subcultures. At the third cycle of proliferation, the highest multiplication rate (2.89) was obtained on the medium containing 5 microM TDZ. Newly generated floral tissues were assessed for embryogenic competence, resulting in an average embryogenic frequency of 12.5%. The observed embryogenic capacity, together with the recurrent availability of immature flowers, allowed for the direct initiation of cell suspensions from bulked explant cultures. Regular observation and regeneration tests during the development of suspended cell cultures confirmed their embryogenic condition. Produced embryos successfully matured and germinated to regenerate hundreds of somatic in vitro plants.

[Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminata cv. Mas (AA)].

Conventional breeding for dual resistance of disease and pest of Musa cultivars remains a difficult endeavor, as the plant is polyploidic and high in sterility. Biotechnological techniques, eg., genetic engineering, in vitro mutation breeding, or protoplast fusion, may overcome the difficulties and improve the germplasm. Establishment of a stable embryogenic cell suspension (ECS) is a prerequisite for any of the biotechnological breeding methods. In this study an embryogenic cell suspension was established from immature male flower of Musa acuminata cv. Mas (AA), a popular commercial variety of banana in the South-East Asian region. After culture for 5-6 months on callus induction media, which consisted of MS salts, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 4.1 micromol/L biotin, 5.7 micromol/L indoleacetic acid (IAA), 5.4 micromol/L naphthaleneacetic acid (NAA), other vitamins, 87 mmol/L sucrose, and solidified with 7 g/L agarose, meristematic globules and yellow, friable embryogenic cultures were induced from the explants of 1-15th row young floral hands of immature male flowers. Of the four treatments of 2,4-D, 9 micromol/L was the most effective on the callus induction, it transformed 40.96% and 7.45% of the cultivated male floral hands into callus and embryogenic callus respectively. The explants to produce highest frequency of the embryogenic calli were floral hands of 6 to 12th rows, which generated 5.79% of the embryogenic calli. Suspension cultures were initiated from these embryogenic calli in liquid medium supplemented with 4.5 micromol/L 2, 4-D. After sieving selection of the cultures using a stainless steel metallic strainer with pore sizes of 154 microm at 15 day intervals for 3 months, homogeneous and yellow embryogenic cell suspensions, composed of single cells and small cell aggregates, were established. Based upon the growth quantity and growth rate of ECS, it was determined that the appropriate inoculum was 2.0 mL PCV ECS/30 mL medium in 100 mL flask, and the appropriate subculture cycle was 15 days. Planting of 6 months old ECS on semi-solid medium of somatic embryo induction and development (MSD) resulted in approximately 280 x 10(3) somatic embryos/mL PCV ECS. MSD contained SH macronutrients, micro-nutrients, Fe-EDTA and MS vitamins supplemented with 4.5 micromol/L biotin, 680 micromol/L glutamine, 2 mmol/L proline, 100 mg/L malt extract, 1.1 micromol/L NAA, 0.2 micromol/L zeatin, 0.5 micromol/L kinetin, 0.7 micromol/L N6-(2-isopentenyl) adenine, 29 mmol/L lactose, 130 mmol/L sucrose and solidified with 2g/L gelrite. After 3 months of maturity on MSD, 17.28% of the somatic embryos were germinated on germination media (MG), consisted of MS salt, Morel and Wetmore vitamins, 0.2 micromol/L 6-BA, 1.1 micromol/L IAA, 87 micromol/L sucrose and solidified with 2 g/L gelrite; and 14.16% of the somatic embryos could develop into normal plantlets on rooting media contained the same composition as that of MG but without auxin and cytokinin.

[The role of gene tepal-like bract (TLB) in the separation between the bracts and perianth in Fagopyrum esculentum moench].

The morphological and genetic studies of the tlb mutant of common buckwheat Fagopyrum esculentum from the collection of the All-Russia Research Institute of Legumes and Groat Crops have demonstrated that gene TLB controls an important stage of flower development-it determines the lower boundary of simple perianths. The loss of TLB gene function leads to changes in the structure of the bract from scale-like to tepal-like. Gene TLB is assumed to limit, on the basal side, the region of the expression of genes determining the development of flower organs as petals.

Somatic embryogenesis from leaf explants of Australian fan flower, Scaevola aemula R. Br.

Somatic embryogenesis from leaf explants of Scaevola aemula R. Br. was achieved. Somatic embryos were induced from explants cultured on MS medium supplemented with 0.2 mg/ 2,4-dichlorophenoxyacetic acid and 0.2-0.5 mg/l 6-benzylaminopurine (BAP). Various developmental stages of somatic embryos were found on this medium-from globular embryos to germinated embryos. The transfer of globular embryos to MS medium containing 0.5 mg/l BAP resulted in a high frequency of shoot regeneration. Leaf explants cultured on MS medium containing different combinations of BAP and alpha-naphthaleneacetic acid formed adventitious shoots and roots. Histological examination confirmed the process of somatic embryogenesis. Induction of somatic embryogenesis in Scaevola provides a system for studying embryogenesis in Australian native plants and will facilitate the improvement of these plants using genetic transformation techniques.

Production of haploids of neem (Azadirachta indica A. Juss.) by anther culture.

Androgenic haploids of the neem tree (Azadirachta indica A. Juss.) were produced by anther culture at the early- to late-uninucleate stage of pollen. Haploid formation occurred via callusing. The best medium for inducing callusing in the anther cultures was Murashige and Skoog's basal medium (MS) (9% sucrose) supplemented with 1 microM 2,4-D, 1 microM NAA and 5 microM BAP, while anther callus multiplied best on MS medium supplemented with 1 microM 2,4-D and 10 microM Kn. These calli differentiated shoots when transferred to a medium containing BAP; 5 microM BAP was optimum for young calli (75% cultures differentiated shoots), but older calli showed the best regeneration with 7.5 microM BAP. Shoots elongated at a lower concentration of BAP-0.5 microM. These shoots were multiplied by forced axillary branching and rooted in vitro. The plants were subsequently established in soil. Of the plants that regenerated from anther callus 60% were haploid, 20% were diploid and 20% were aneuploid.