Undeveloped Ovules Research Articles

Histological, transcriptomic, and gene functional analyses reveal the regulatory events underlying gibberellin-induced parthenocarpy in tomato

Gibberellin (GA) is one of the major plant hormones that promote parthenocarpy, a highly valuable agronomic trait. Here, we demonstrated that exogenous GA3 application triggered the formation of parthenocarpic fruits with smaller size but unchanged shape in tomato (Solanum lycopersicum). These fruits exhibited a thicker pericarp, undeveloped ovules, and few jelly tissues, leading to smaller locules with empty cavities. Histological investigation showed that GA treatment produced more cell layers with larger cells in the pericarp, suggesting its promotion in both cell division and expansion. Transcriptomic analyses between GA3- and mock-treated unpollinated ovaries/fruits identified a large number of differentially expressed genes related to hormones, cell division, cell expansion, and transcription factors, implying that they coordinately regulated parthenocarpy conferred by GA. In particular, the downregulation of five reported repressors of tomato parthenocarpy, including two auxin signaling components, AUXIN RESPONSE FACTOR5 (SlARF5) and SlARF7, and three MADS-box genes, TOMATO APETALA3 (TAP3), TOMATO PISTILLATA (TPI), and AGAMOUS-LIKE6 (SlAGL6), after GA treatment might play a key role in this process. Furthermore, we found that the knockdown of a GA signaling factor SlMYB33, which was depressed by GA treatment, induced parthenocarpic fruit set in tomato, an effect that might have been achieved by enhancing GA biosynthesis and decreasing the expression of some repressors of tomato parthenocarpy. Thus, our results provide a basis for understanding the regulatory mechanism of GA in tomato parthenocarpy.

Citrus Cell Suspension Culture Establishment, Maintenance, Efficient Transformation and Regeneration to Complete Transgenic Plant.

Agrobacterium-mediated transformation of epicotyl segment has been used in Citrus transgenic studies. The approach suffers, however, from limitations such as occasionally seed unavailability, the low transformation efficiency of juvenile tissues and the high frequency of chimeric plants. Therefore, a suspension cell culture system was established and used to generate transgenic plants in this study to overcome the shortcomings. The embryonic calli were successfully developed from undeveloped ovules of the three cultivars used in this study, “Sweet orange”-Egyptian cultivar (Citrus sinensis), “Shatangju” (Citrus reticulata) and “W. Murcott” (Citrus reticulata), on three different solid media. Effects of media, genotypes and ages of ovules on the induction of embryonic calli were also investigated. The result showed that the ovules’ age interferes with the callus production more significantly than media and genotypes. The 8 to 10 week-old ovules were found to be the best materials. A cell suspension culture system was established in an H+H liquid medium. Transgenic plants were obtained from Agrobacterium-mediated transformation of cell suspension as long as eight weeks subculture intervals. A high transformation rate (~35%) was achieved by using our systems, confirming BASTA selection and later on by PCR confirmation. The results demonstrated that transformation of cell suspension should be more useful for the generation of non-chimeric transgenic Citrus plants. It was also shown that our cell suspension culture procedure was efficient in maintaining the vigor and regeneration potential of the cells.

The Nuclear Localization of the DnaJ-Like Zinc Finger Domain-Containing Protein EDA3 Affects Seed Development in Arabidopsis thaliana.

The DnaJ-like zinc finger domain-containing proteins are involved in different aspects of plastid function and development. Some of these proteins were recently reported to have dual subcellular localization in the nucleus and plastids. One member of this family, PSA2 (AT2G34860), was found to localize to the thylakoid lumen and regulate the assembly of photosystem I (PSI). However, PSA2 was also annotated as Embryo sac Development Arrest 3 (EDA3) from the observation that its embryo sac development was arrested at the two-nuclear stage. In this study, we characterized the eda3 mutant, and demonstrated that, as compared with the wild-type (WT) plants, the mutant has shorter siliques, fewer siliques per plant, and fewer seeds per silique. Both aborted and undeveloped ovules were observed in siliques of the mutant. By immunoblot analysis, we found that, different from the chloroplast localization in mature leaves, EDA3 localizes in the nucleus in seeds. A nuclear localization signal was identified from the deduced amino acid sequence of EDA3, and also proved to be sufficient for directing its fusion peptide into the nucleus.

Новые данные о пельтаспермовых птеридоспермах Ангариды в палеоэкологическом и таксономическом контексте

The paper deals with the morphological peculiarities of the species Peltaspermum morovii Naug., which is attributed to the peltaspermaceous pteridosperms, the completely extinct group of fossil gymnosperms probably closely related to ancestors of the flowering plants (angiosperms, or anthophytes). The material studied is originated from several localities of the fossil plants of Permian age, disposed in the Cis-Urals and the Volga River basin. One of the most important morphological features of the species Peltaspermum morovii was a presence of the long lobe-like outgrowths located on the seed-bearing disc margins. Most probably these lobe-like outgrowths were turned downwards the peltoid adaxial surface for partial covering and protecting of the undeveloped young ovules before their maturing. Some arguments for entomophily of the evolutionary advanced peltaspermaceous pteridosperms, to which the species Peltaspermum morovii can be assigned, are given. Detailed information about geographical and stratigraphical position of the localities cited is given as well. The leaves of callipterid morphology, which probably belonged to the same parent plants produced female fructifications of Peltaspermum morovii are discussed.

The genus Schizotechium (Caryophyllaceae) resurrected

Fenzl (1833, 1840) described the taxon Schizotechium as a section of the genus Stellaria Linnaeus (1753: 421) (Caryophyllaceae Juss.) to accommodate the Himalayan Stellaria crispata Wallich ex D.Don (1825: 215) [= Stellaria monosperma Buchanan-Hamilton ex D.Don (1825: 215)]. The section was morphologically characterized in having the calix 5-partite, 10 hypogynous stamens, the ovary 2–3-ovulate, (2–)3 styles, the capsule 1(–2)-seeded, and often without a central collumella. Bentham (1862), unaware of raised generic rank for the section by Reichenbach (1841), also pointed out the distinctness of this group, strongly supporting the generic rank. He concluded: “Schizotechium Fenzl, although only proposed as a section of Stellaria might have perhaps rather more claims than any of the preceding [subgenera/sections of Stellaria included in the said paper (Bentham 1862)] to be adopted as a genus. It consists of two Himalayan species with a scandent habit and diffuse panicles, almost as in Brachystemma D. Don (1825: 216) and only 3 ovules, of which one ripens. The ovary might thus be supposed to be reduced to uniovulate carpels, and to be brought technically nearer to that of Phytolaccaceae R.Br., but there is no central axis, and a slight comparison of actual specimens at once give the idea that it is an exceptional and irregular reduction in the ovules of a closely compound ovary, and not a normal conformity of the ovules with as many distinct or well-marked carpels. The leaves arrangement, inflorescence, and flowers are in all other respects those of Stellaria in which genus we continue to retain Schizotechium as a section.” Notably, members of Schizotechium differ from the other belonging to Stellaria by the following characters: sub-scandent habit, occurrence of tuberous or fusiform fleshy roots, large, diffuse, many-flowered panicle of compound cymes, central collumella of capsule extremely reduced or nearly absent and capsule with 1(–2) fertile enlarged seed and rest sterile, undeveloped ovules (Bentham 1862, Edgeworth & Hooker 1872).

Arabidopsis voltage-dependent anion channel 1 (AtVDAC1) is required for female development and maintenance of mitochondrial functions related to energy-transaction.

The voltage-dependent anion channels (VDACs), prominently localized in the outer mitochondrial membrane, play important roles in the metabolite exchange, energy metabolism and mitochondria-mediated apoptosis process in mammalian cells. However, relatively little is known about the functions of VDACs in plants. To further investigate the function of AtVDAC1 in Arabidopsis, we analyzed a T-DNA insertion line for the AtVDAC1 gene. The knock-out mutant atvdac1 showed reduced seed set due to a large number of undeveloped ovules in siliques. Genetic analyses indicated that the mutation of AtVDAC1 affected female fertility and belonged to a sporophytic mutation. Abnormal ovules in the process of female gametogenesis were observed using a confocal laser scanning microscope. Interestingly, both mitochondrial transmembrane potential (ΔΨ) and ATP synthesis rate were obviously reduced in the mitochondria isolated from atvdac1 plants.

Food from Earth: Sustainable Farming in Action

With a lurch, the motorized banana tram starts moving slowly along its simple track carrying 100 banana bunches—cornucopias of green fruits, cloaked in blue plastic bags to protect them from insects and sun damage. A Costa Rican worker in a hard hat sits at the helm guiding the tram—nicknamed “the spider”—toward the packing plant a couple miles away. The tram driver and other workers harvested these bananas with machetes on the 813-acre banana plantation at EARTH University (Escuela de Agricultura de la Region Tropical Humeda) in the Caribbean lowlands of Costa Rica.1 The banana is the world’s largest herbaceous flowering plant. Commercial bananas are sterile and are propagated by planting root stock from donor plants (the dark specks in the center of a banana are undeveloped ovules, the precursors to seeds). ... With its row upon row of genetically identical Cavendish banana plants stretching for acres, one might mistake EARTH’s plantation for a traditional banana farm. But through trial, error, and scientific research, EARTH University—funded largely by a U.S. Agency for International Development (USAID) endowment—has created a more ecologically friendly, lower-pesticide (but not organic) banana since the university was established in 1989. The university is also impacting the larger banana industry and nations around the world as alumni take sustainable agriculture initiatives and new business opportunities back to their home countries.

Somatic embryogenesis and plantlet regeneration from nucleus tissues of Local orange (Citrus sinensis (L.) Osbeck)

Somatic embryogenesis and plantlet regeneration were achieved in callus cultures of nucellus tissues derived from undeveloped ovules of immature fruits of local orange (Citrus sinensis (L.) Osbeck). Four types of culture media were used and all produced embryogenic callus. Somatic embryos were developed on MS medium supplemented with BA only. Embryo maturation took place on MS medium supplemented with BA and 2,4-D. Plantlets were regenerated from those somatic embryos on half strength MS medium free of plant growth regulators. The well-developed plantlets were transferred to a potting mix containing sand and peat moss (2: 1) and grown for 8 months, with a survival rate of 100 %.
 Abbreviation: BA: 6-benzyl amino purine; 2,4-D: 2,4-dichlorophenoxy acetic acid; NAA: α-naphthalene acetic acid

Arabidopsis thaliana MIRO1 and MIRO2 GTPases Are Unequally Redundant in Pollen Tube Growth and Fusion of Polar Nuclei during Female Gametogenesis

MIRO GTPases have evolved to regulate mitochondrial trafficking and morphology in eukaryotic organisms. A previous study showed that T-DNA insertion in the Arabidopsis MIRO1 gene is lethal during embryogenesis and affects pollen tube growth and mitochondrial morphology in pollen, whereas T-DNA insertion in MIRO2 does not affect plant development visibly. Phylogenetic analysis of MIRO from plants revealed that MIRO 1 and 2 orthologs in dicots cluster in two separate groups due to a gene/genome duplication event, suggesting that functional redundancy may exists between the two MIRO genes. To investigate this possibility, we generated miro1 (+/−)/miro2-2 (−/−) plants. Compared to miro1 (+/−) plants, the miro1 (+/−)/miro2-2 (−/−) plants showed increased segregation distortion. miro1 (+/−)/miro2-2 (−/−) siliques contained less aborted seeds, but more than 3 times the number of undeveloped ovules. In addition, reciprocal crosses showed that co-transmission through the male gametes was nearly absent, whereas co-transmission through the female gametes was severely reduced in miro1 (+/−)/miro2-2 (−/−) plants. Further investigations revealed that loss of MIRO2 (miro2 (−/−)) function in the miro1 (+/−) background enhanced pollen tube growth defects. In developing miro1 (+/−)/miro2 (−/−) embryo sacs, fusion of polar nuclei was further delayed or impaired compared to miro1 plants. This phenotype has not been reported previously for miro1 plants and coincides with studies showing that defects in some mitochondria-targeted genes results in the same phenotype. Our observations show that loss of function in MIRO2 in a miro1 (+/−) background enhances the miro1 (+/−) phenotype significantly, even though miro2 (−/−) plants alone does not display any phenotypes. Based on these findings, we conclude that MIRO1 and MIRO2 are unequally redundant and that a proportion of the miro1 (+/−)/miro2 (−/−) plants haploid gametes displays the complete null phenotype of MIRO GTPase function at key developmental stages.

Female gametophytic mutants of Arabidopsis thaliana identified in a gene trap insertional mutagenesis screen

In plants, the male and female gametophytes represent the haploid generation that alternates with the diploid sporophytic generation. Male and female gametophytes develop from haploid micro- and megaspores, respectively. In flowering plants (angiosperms), the spores themselves arise from the sporophyte through meiotic divisions of sporogenous cells in the reproductive organs of the flower. Male and female gametophytes contain two pairs of gametes that participate in double fertilization, a distinctive feature of angiosperms. In this paper, we describe the employment of a transposon-based gene trap system to identify mutations affecting the gametophytic phase of the plant life cycle. Mutants affecting female gametogenesis were identified in a two-step screen for (i) reduced fertility (seed abortion or undeveloped ovules) and (ii) segregation ratio distortion. Non-functional female gametophytes do not initiate seed development, leading to semi-sterility such that causal or linked alleles are transmitted at reduced frequency to the progeny (non-Mendelian segregation). From a population of 2,511 transposants, we identified 54 lines with reduced seed set (2%). Examination of their distorted segregation ratios and seed phenotypes led to the isolation of 12 gametophytic mutants, six of which are described herein. Chromosomal sequences flanking the transposon insertions were identified and physically mapped onto the genome sequence of Arabidopsis thaliana. Surprisingly, the insertion sites were often associated with chromosomal rearrangements, making it difficult to assign the mutant phenotypes to a specific gene. The mutants were classified according to the process affected at the time of arrest, i.e. showing mitotic, karyogamic, maternal or degenerative phenotypes.

Seed Production Enhanced by Antiauxin in the pat-2 Parthenocarpic Tomato Mutant

There is concern that high temperatures resulting from global warming could reduce fruit set of tomato (Solanum lycopersicum). However, fruit set of parthenocarpic tomato genotypes, which often bears seedless fruit, is not reduced when grown under a high temperature. The cause of seedless fruit development was studied with the aim of increasing the seed number in parthenocarpic tomato. Ovule number at anthesis in parthenocarpic and non-parthenocarpic fruit did not differ, but the proportion of undeveloped ovules increased with time after anthesis in parthenocarpic tomato, whereas most ovules in non-parthenocarpic tomato developed normally. Pollen grains germinated on the stigma and extruded pollen tubes in parthenocarpic and non-parthenocarpic tomatoes, but in parthenocarpic tomato, pollen tube elongation was markedly inhibited in the style base. Elongation of pollen tubes on agar containing indoleacetic acid (IAA) was depressed in parthenocarpic and non-parthenocarpic tomato plants. p-Chlorophenoxyisobutyric acid (PCIB), which inhibits auxin action, did not affect the fruit set and fresh weight in either type of tomato, although seed number per fruit in parthenocarpic tomato was significantly increased from 13 ± 2 to 74 ± 6 seeds by PCIB treatment. These results indicated that a high IAA concentration in the ovary of parthenocarpic tomato inhibited pollen tube elongation, and that poor fertilization resulted in failure of ovule development. Moreover, floral organs in parthenocarpic tomato were normally developed as in non-parthenocarpic tomato, and seed development could be induced in parthenocarpic tomato by PCIB treatment.

Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.

A New Species of Votomita (Melastomataceae) from Venezuela, with Thoughts on Ovule and Seed Number and Seed Size

A new species of Votomita from southern Venezuela is described, illustrated, and distinguished from its relatives on the basis of fruiting material; certain useful floral characters can also be determined from the specimen. The new species illustrates a general condition in subfamily Memecyleae, i.e., a relatively large number of ovules is produced yet only one or a few large seeds are formed. Ovule and seed numbers are discussed, seed volumes are calculated for Mouriri and Votomita, and possible explanations for the differences are explored. Votomita ventuarensis Morley, sp. nov. TYPE: Venezuela. Terr. Fed. Amazonas: Dept. Atabapo, bosques medios y bajos inundables en el rio Ventuari-Macabana, 4?15'N, 66?20'W, 140 msnm, Sep. 1989, Luz Delgado 594 (holotype, MO; isotype, PORT). Figure 1. Arbor usque 15 m alta; pagina inferior costae mediae anguste 2-alata ad angulos, minute puberula; cryptae stomatatae unaquaeque cavitatibus 1-5; epidermes folii sine pigmento; hypodermis absens; stamina monadelpha; ovarium 4-loculare ovulis 22-25 axillaribus; semen unicum, globosum. Tree to 15 m high, glabrous except for the leaf midrib; young twigs rounded. Petioles 4.5-6 mm long; blades 7.9-12.4 cm long, 4.1-6 cm wide, ovate-elliptic to elliptic, acute at base, abruptly acuminate at the apex with an acuminum 0.7-1 cm long; midrib low-rounded to plane or slightly grooved adaxially when dry, prominent abaxially, flat and winged on the edges, the undermidrib minutely puberulent with hairs 20-40 pm long; lateral nerves when dry faintly visible or invisible adaxially, faintly visible abaxially. Midrib xylem tubular; stomatal crypts 30-36 per mm2 near the margin to 54 near the midrib, with (1-)2-5 cavities each, 50-60 /m high; adaxial epidermis of uniform thickness, 29-31 /tm thick including the cuticle, mostly one cell thick, occasionally two, mucilage walls none, the cytoplasm unpigmented and clear till stained, the inner walls straight and parallel with the outer ones; cuticle thin; abaxial epidermis also unpigmented; hypodermis none; foliar sclereids all terminal on the veinlets, irregularly stellate, often with an irregular horizontal central body 1-4 times as long as wide. Peduncles 1 per side at leafless nodes of twigs 3-4 mm thick below the leaf zone, 10.5-14 mm long with 2 internodes, the lower 0.5-1 mm long, the upper 10-13 mm, 1-flowered; bracts deciduous before fruit formation; pedicels 12-13 mm long in fruit; ovary locules 4, placentation axile, ovules 4-11 per placenta, 22-25 in all; fruits yellow to orange, ellipsoid, crowned with the calyx, 17-18 mm long including calyx by 12-14 mm diam. when dry, 20-21 mm long by 13-16 mm when boiled, the fruiting calyx 6.4-6.7 mm diam., 3-3.3 mm deep, the lobes low-triangular, 0.8-1.2 mm high, 4-5.2 mm wide; seed 1, spheroid, 9.79.9 mm high, 8.8-9.6 mm thick, with an irregularly elliptic raised area (function unknown) ca. 4 mm from the broken chalazal strand, the raised area 6 mm long, 4 mm wide, 0.5-1 mm high, with edges that overhang 0-0.5 mm; the raised area presumably includes the micropyle. Petal scars on the fruit rounded-triangular, 2.3-2.7 mm wide, 0.9-1.2 mm long; stamen scars 8, the filament scars broad, thin, and united, forming a continuous ring 3.2-3.7 mm in outside diam., 0.2-0.5 mm thick, the stamens thus monadelphous; thecae adaxial on the filaments and placed low so that their bases leave imprints 0.9-1.2 mm wide, 0.8-1.0 mm thick radially around the style base, the total stamen thickness at base 1.0-1.3 mm. Distribution. Known only from the type locality east of San Fernando de Atabapo, northwest of central Amazonas, Venezuela. Local name. Cometure tierra firmero. Although the type specimen bears only ripe fruits, the filament scars and anther imprints on the fruits yield useful floral information, and with careful dissection of the 1-seeded fruit (when boiled) all or most of the undeveloped ovules can be found and the nature of the placentation determined, in spite of the compaction and distortion that have taken place. The new species is unique in the genus in its unpigmented epidermises. Further distinctions of this plant from the other species follow, starting NovoN 9: 241-244. 1999. This content downloaded from 40.77.167.42 on Wed, 30 Nov 2016 05:02:53 UTC All use subject to http://about.jstor.org/terms

Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.

Somatic embryogenesis was induced and plant regeneration was obtained in 11 different genotypes of sweet orange navel group [Citrus sinensis (L.) Osb.] from cultures of stigma/style explants and undeveloped ovules. Explants were cultured on 3 different modifications of Murashige and Skoog medium: 500 mg l-1 malt extract; 500 mg l-1 malt extract and 4.6 μM kinetin; and 500 mg l-1 malt extract and 13.3 μM 6-benzylaminopurine. Sucrose (146 mM) was used as carbon source. Somatic embryogenesis occurred 1–3 months after culture initiation from undeveloped ovule and stigma/style cultures of all the genotypes tested. Somatic embryos developed into plantlets with a high frequency (74%) after transfer to Murashige and Skoog medium supplemented with 146 mM sucrose and 500 mg l-1 malt extract. Plants were successfully transferred to soil.

Seed set and germination in some wild species of Vicia from SW Europe (Spain)

We studied seed and ovule production, predation and abortion in 14 taxa of the genus Vicia. Germination capacity, seed vigour and germination kinetics after scarification were also evaluated, as well as the influence of light on these parameters. Pod size correlated with the number and weight of viable seeds, and with the total number of ovules and the number of undeveloped ovules per pod. Species with smaller seeds had lower rates of abortion than those with larger seeds, and seedling size correlated strongly with seed weight in this genus. In most taxa seed hardness was not great, and the scarifying agent used—sulfuric acid with different times of exposure—led in some cases to 100% germination. Light conditions did not notably influence germination. Germination speed was favored by scarification under both dark and light conditions, and vigour index was greater in scarified than in control seeds. In most taxa, germination after scarification fit an exponential curve, with the exception of Vicia lutea ssp. vestita, which showed very slow velocity.

Vaccinium Uliginosum L.

Section Vaccinium L. A deciduous shrub with horizontal subterranean rhizomes. Aerial shoots erect, up to 70-100cm in length (but usually much less), glabrous or puberulent, terete, brownish. Leaves 6-25 (35) x 4-12 (-20) mm, obovate, entire, obtuse to subacute, glabrous, glaucous, conspicuously reticulately veined; margin of the blade slightly revolute (Fl. Eur. 3). Vaccinium uliginosum has a sympodial branching system with the terminal bud producing flowers in spring while shoot growth continues from the uppermost lateral buds (Shevtsova et al. 1995). Flowers 4or 5-merous, in racemes of 1-4 which terminate short branches bearing only scale-leaves; peduncles glabrous (Warming 1908). The 4-5 calyx lobes are short, broad and obtuse; the calyx is persistent and accrescent (Fl. Eur. 3). The corolla is subglobose or ovoid, pale pink or white, white-greenish; 4-5mm long and 2.5-4.0mm broad with 4-5 reflexed very short lobes. The 8-10 stamens are apically dehiscent, appendages short (a little shorter than the anthers) and subulate, with glabrous filaments (Fl. Eur. 3). Pollen tetrad diameter ranges between 35.8 and 48.3 ,um (Oldfield 1959; Paquereau 1959; Hebda 1979; Hegi Fl. 1, ed. 3; Warner & Chinnappa 1986; Jacquemart 1993b). Style included, longer than the anthers. The number of ovules averages 84.9 + 6.5 per flower and the number of tetrads is about 550.0 + 86.6 per anther (Jacquemart 1993b). Flowers have a pleasant, sweet, aromatic perfume which is somewhat reminiscent of Asperula odorata (Warming 1908). Fruit a berry, bluish-black, with a glaucous bloom, globose to ellipsoid, sweet and edible, 5-9mm (Jacquemart 1993b) or 7-10 mm (Hegi Fl. 1, ed. 3), with a moderate number of viable seeds (mean + SD of 12.5 + 11.2), together with 16.7 + 10.4 enlarged ovules and 54.6 + 14.2 undeveloped ovules per berry (Jacquemart 1993b). Seeds dark brown, 1.3-1.7mm; seed coat reticulate-alveolate. Seed fresh mass 0.240.30 mg (Kondratiouk & Chabarova 1968; Guitian et al. 1994). Native, principally in acid upland, heaths and bogs; rarely on calcareous soils (McVean & Ratcliffe 1962).

Embryo Rescue Techniques to Generate Variation in Citrus Crops

Although no longer as glamorous as it was a few decades past, the routine application of embryo rescue techniques, leading to plant recovery, is a valuable tool for citrus cultivar improvement. Embryo rescue approaches can be used to generate useful variation or to capture various kinds of spontaneous genetic variation. Embryo rescue, by in vitro culture of undeveloped, and presumably unfertilized, ovules in colchicine-supplemented media is a practical method of producing tetraploid clones, which are used then in crosses with diploids to produce seedless triploid hybrids. This same approach, i.e., in vitro culture of undeveloped ovules, is also used to recover plants from chimeric sectored fruit exhibiting economically important mutations for fruit characteristics, and for producing potentially variant somaclones. Seedlessness is an important objective for fresh citrus fruit cultivar improvement, and triploidy following 2x × 4x hybridizations is one approach being exploited for this objective. When monoembryonic diploid seed parents are crossed with tetraploid pollen parents, however, normal seed development is not usually possible. Embryos must be excised from abortive seeds fairly early in development and cultured appropriately to ensure the recovery of sufficient numbers of 3x offspring from these crosses, to increase the likelihood of identifying superior seedless hybrids. These applications will be described in some detail, and progress toward breeding objectives are highlighted.

Induction of somatic embryogenesis in pedunculate oak (Quercus robur L.)

Somatic embryogenesis of pedunculate oak (Quercus robur L.) has been successfully attempted. Internodes and leave pieces from seedlings, mature and immature embryos, undeveloped ovules and ovular tissues were used as initial explants. Globular and heart-shaped somatic embryos were obtained on hypocotyls and cotyledons of immature zygotic embryos cultures on solid WPM supplemented with 0.44 μM and 0.05 μM NAA, or 4.44 μM BA, or from embryo-derived callus cultured in liquid medium supplemented with glutamine (3.42 μM) after induction with 4.52 μM 2,4-D. Abbreviations: BA = 6-benzyl adenine 2,4-D = 2,4 dicholorophenoxyacetic acid IBA = indole-3-butyric acid (Potassium salt) Kin = kinetin NAA = α-naphthaleneacetic acid 2iP = 2-isopentenyl adenine WPM = Woody Plant Medium

Embryogenesis in Vitro and Nonchimeric Tetraploid Plant Recovery from Undeveloped Citrus Ovules Treated with Colchicine

A method was developed to produce nonchimeric, autotetraploid Citrus plants via in vitro somatic embryogenesis in the presence of colchicine. Undeveloped ovules from immature fruit of `Valencia' sweet orange (Citrus sinensis [L.] Osb.) and `Orlando' and `Minneola' tangelos (Citrus reticulata Blanco × Citrus × paradisi Macf.) were held on Murashige and Tucker medium with 500 mg malt extract/liter and 0.0090, 0.01%, or 0.10% colchicine for 21 days. Embryogenesis from tangelo ovules was suppressed by 0.10% colchicine, but no such effect was observed among sweet orange ovules. Colchicine treatments had no subsequent effect on embryo germination. The numbers of chromosomes in root tip cells showed that both tetraploid and diploid `Valencia' and `Orlando' plants were recovered from colchicine treatments. `Minneola' cultures produced only diploid plants. Tetraploid plant morphology was typical for Citrus tetraploids. Examination of chromosome numbers in root tip, shoot, and leaf meristems indicated that the regenerants were nonchimeric. Such nonchimeric tetraploids will be useful parents for interploid hybridization directed toward development of seedless triploid Citrus scion cultivars.

The effects of low levels of chloramphenicol and methotrexate on somatic embryogenesis inCitrus

The effects of the antibiotics methotrexate and chloramphenicol on somatic embryogenesis inCitrus were evaluated. Relatively low levels (0.1 to 1.0 μg/ml) of these antibiotics did not inhibit embryo production from undeveloped ovules of ‘Key’ lime [C. aurantifolia) (Christm.) Swing.]. Surprisingly, both antibiotics induced the formation of embryogenic callus in these cultures. This is usually a rare event in cultures of undevelopedCitrus ovules, and ‘Key’ lime is especially recalcitrant. The effects of these antibiotics on embryogenic callus appeared to be limited to the induction stage, because there was no consistent effect, either stimulatory or inhibitory, on established, lines of embryogenic callus.