M. Cristina Pedroso

Factors controlling somatic embryogenesis

Histological and ultrastructural, molecular and elemental distribution changes were investigated during the induction of direct somatic embryogenesis using theCamellia japonica leaf culture system. In this culture system, direct somatic embryogenesis is induced in a controlled way in a specific leaf region (leaf blade) within a leaf. Embryogenic and non-embryogenic leaf regions have characteristic energy-dispersive X-ray spectra already before induction. According to these results electron probe X-ray microanalysis (EPMA) can be a tool for early diagnosis of embryogenic competence. Histological studies showed that severe fluctuations in the number of calcium oxalate crystals and in starch accumulation occur after induction but only in induced tissues. Changes in the cell wall composition of competent cells occur shortly after the induction treatment. The induction of morphogenesis is linked to the appearance of callose covering the surface cells of induced leaves and calluses. A 2nd deposition of material (cutin) is necessary for normal somatic embryogenesis to occur. The involvement of lipid transfer proteins in the appearance of cutin in the embryogenic regions of the explant is suggested.

Nitric Oxide and Reactive Nitrogen Oxide Species in Plants

We now know that nitric oxide (NO) bursts are early and transient characteristics of plant stress under many biotic and abiotic conditions. NO originates non-enzymatically from nitrite (NO2-), as a by-product from nitrate reductase (NR). It arises enzymatically from nitrite:nitric oxide reductase (NI-NOR), and putative nitric oxide synthase (NOS) activity. NOS substrates (L-arginine, NADPH, and oxygen), and products (NO and L-citrulline) are ubiquitous in plants, and pivotal components of intermediary N metabolism. NO has both beneficial and harmful effects, depending on its concentrations and milieu. At low concentrations, it is a chemical messenger that directly integrates and differentiates time-dependent responses to stress and defence against pathogens. Prime direct targets of NO are the haems that shuttle three gases key to plant life, viz. -NO, carbon dioxide, and oxygen. -NO and thiols are nitrosated to S-nitrosothiols, stored, and probably shuttled by transnitrosylation of proteins. At high levels, and in the presence of reactive oxygen species (ROS), -NO produces

A scanning electron microscopy and X-ray microanalysis study during induction of morphogenesis in Camellia japonica L.

Abstract A scanning electron microscopy (SEM) study during the induction of direct and indirect morphogenesis (root and embryo formation) from leaves of Camellia japonica L. showed that the induction of morphogenesis is linked to the appearance of a layer of fibrillar material covering the surface cells of induced leaves and calli. Before embryo formation a 2nd deposition of material of smooth texture occured being the embryogenic regions of calli and globular embryos totally covered by a layer of this material. A positive reaction to aniline blue staining suggests that a deposition of ‘callose’ precedes embryo formation. Results of electron probe microanalysis (EPMA) during direct morphogenesis show higher levels of Ca, K, Na, Fe and S in leaf-induced parenchyma cells which decrease with the onset of morphogenesis. Results suggest a correlation between X-ray spectra and the in vitro response obtained (calli, embryogenesis, rhizogenesis). Identical X-ray spectra were obtained for both globular somatic and zygotic embryos. Roots also presented identical X-ray spectra independently of their origin. Can X-ray microanalysis be used to diagnose cell competence and determination?

Explant region-specific embryogenic competence and plant recovery inCamellia japonica

The culture conditions for direct, indirect, and repetitive embryogenesis were established forCamellia japonica cv. Elegans and cv. Ville de Nantes. Direct embryo production from leaves averaged 15.3 embryos per responsive leaf on Murashige and Skoog medium (MS) with 1.0 mg·liter−1 N6-benzyladenine and 0.5 mg·liter−1 2,4-dichlorophenoxyacetic acid. Plantlet production was 7.1 (±1.5) plantlets per leaf. Direct embryo production from stems averaged 5.7 embryos per shoot, and 2.7 embryos per stem portion, on MS medium supplemented with 1.0 mg·liter−1 N6-benzyladenine and 0.1 mg·liter−1 indolbutyric acid (MS28). Conversion was only obtained after repetitive embryogenesis. Embryogenesis from leaf-derived callus occurred in all callus after transfer to MS/2–25 medium (half strength MS medium with 25 g·liter−1 D-glucose) (production stage). Plantlet production was 16.3 (±3.6) plantlets per callus. Repetitive embryogenesis increased embryo population by 2.3- to 3.6-fold every 4 wk. Conversion of secondary embryos was obtained on MS medium supplemented with 2.0 mg·liter−1 N6-benzyladenine, 0.2 mg·liter−1 indolbutyric acid, 5 mg·liter−1 gibberellic acid (MS56). Direct embryo formation from leaves, stems, and cotyledons, and embryogenic callus formation from leaves were restricted to specific regions of the explant.

Induction of microspore embryogenesis in Camellia japonica cv. elegans

Three methods of microspore culture were tested for the induction of microspore embryogenesis in Camellia japonica L. cv. Elegans. Culture was performed on 17 different media consisting of Murashige and Skoog (MS) and N6 basal media with different combinations of carbon, growth regulators, serine and glutamine. Microspore suspensions plated over solid MS medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid and 0.5 μM kinetin, with sucrose (MS6) or glucose (MS9) were seen as the best culture conditions for induction of embryogenesis. The development of microspore derived proembryos was obtained in MS medium supplemented with 2.2 μM N6-benzyladenine (MS10) and reached the highest level when the microspores were cultured in MS6 inducing medium. The development of microspore-derived embryos ceased at the maturation stage.

Early detection of embryogenic competence and of polarity in Camellia japonica L. by electron probe X-ray microanalysis

Abstract Elemental changes during the induction of embryogenesis in two defined leaf regions (embryogenic and non-embryogenic) were studied by electron probe X-ray microanalysis. Somatic and zygotic embryos were also analysed. Energy dispersive X-ray spectra from embryogenic and non-embryogenic leaf regions were already different before the induction treatment. Significant fluctuations on Ca, C, O, K, Na, P, Fe, S and Mg levels were detected after induction, in cells from both regions. Two weeks after induction, embryogenic and non-embryogenic leaf regions consistently displayed different spectra. In embryogenic leaf regions, variations in the spectra were recorded until embryo formation. Contrarily, non-embryogenic leaf regions revealed identical spectra during the same period. Significant differences in elemental composition were detected in the protoderms from zygotic and somatic embryos. In embryos, shoot pole cells consistently displayed a high S level while, in root pole cells, a high Fe level was always detected. The analysis of sections of leaf-derived globular embryos revealed differences in elemental distribution within the globular embryo. The results suggest that EPMA can be used for early detection of embryogenic competence and of polarity.

Plant regeneration from embryogenic suspension cultures ofCamellia Japonica

Two methods (I and II) for somatic embryo production from embryogenic suspension cultures ofCamellia japonica are presented. Method I, embryogenic suspension cultures, was established from suspension cultures initiated from leaf-derived callus. These cultures were maintained by reducing agitation and increasing subculture interval. Induction of somatic embryogenesis was achieved in MS28 medium, 6, 12, 24, and 36 mo. after culture establishment. Embryo production decreased after 1 yr of culture. Method II, suspensions of single embryogenic cells and proembryos, was obtained from leaves cultured in liquid MS13 medium 6 wk after culture initiation. Embryo production was 23 embryos/ml. Germination of cell suspension-derived embryos on MS56 medium was 16.7 % (±4.2%) for method I, and 35.4% (±5.1%) for method II. The embryos germinated into plantlets with 0 to 7 axillary shoots.

Regeneration from anthers of adultCamellia japonica L

SummaryEmbryogenesis and callus formation inCamellia japonica L., cv. Elegans and cv. Ville de Nantes (>50 yr old), were higher when anthers at tetrad stage or early uninucleate microspore stage were used. Direct embryo formation was obtained, both in anthers and anther-petaloids. Embryogenesis only occurred under light on modified Murashige and Skoog medium supplemented with 6-benzylaminopurine (MS10). Embryo production was higher from petaloids (2.2 to 3.5 embryos/petaloid) than from anther locules (1.2 to 1.5 embryos/anther). However, petaloid-derived embryos aborted by Week 10 of culture. The embryogenic efficiency of anthers was 5.3% for cv. Elegans and 4.1% for cv. Ville de Nantes. Scanning electron microscopy images showed that anther-derived globular embryos were covered by a layer of material of smooth texture. Shoot regeneration efficiency by organogenesis was 6.8%. Regeneration by embryogenesis was 60% for cv. Ville de Nantes and 97.5% for cv. Elegans.

Anther and microspore culture in Camellia japonica

Haploid plants have the gametophytic chromosome number. They are of great importance for the production of homozygous plants and for mutation studies. The use of anther and microspore cultures for the induction of pollen embryogenesis hastens the production of haploids and gametoclonal variants. These systems can be useful tools for cultivar improvement. Camellia japonica L. (Theaceae family) is one of the most important species in the genus Camellia. Economically, it is valuable as an ornamental woody species and has a potential to be used for oil and wood production. The available Camellia varieties are genetically highly heterozygous. In this genus, frost and pathogens cause serious damage that results in drastic annual economic loses. So far, in vitro culture of this species, and other Camellia species, has been mainly concerned with the establishment of protocols for micropropagation from juvenile and adult materials (Bennet, 1977, 1978; Bennet & Scheibert, 1982; Carlisi & Torres, 1986; Creze, 1983; Kato, 1989a; Pedroso-Ubach, 1991; Samartin et al., 1984, 1986; Samartin, 1989; Vieitez et al., 1989a,b, 1992), and for mass propagation by somatic embryogenesis (Barciela & Vieitez, 1993; Kato, 1986, 1989b; Nakamura, 1988; Pedroso & Pais, 1993, 1994d,e; San-Jose & Vieitez, 1993; Vieitez & Barciela, 1990; Vieitez et al., 1991). Plant improvement has been obtained in some species by field selection and artificial crossing techniques (Yamaguchi et al., 1987). Anther culture, as a strategy for plant improvement, was reported for C. sinensis (Raina & Iyer, 1974; Chen & Liao, 1982, 1988) and C. japonica (Pedroso-Ubach, 1991). In C. sinensis, the production of haploid callus from anthers has been obtained in at least nine cultivars (Raina & Iyer, 1974; Chen & Liao, 1982, 1988) but haploid shoot regeneration was successful only in one of the cultivars (Chen & Liao, 1982, 1988). Embryo production has also been reported in this species by Shimokado et al.(1986) but apparently no plant regeneration was achieved. As far as we know, successful regeneration from anthers and microspore culture has been only reported in C. japonica (Pedroso-Ubach, 1991). Although pollen-derived plants have been obtained by anther culture in many species, plant regeneration from isolated microspores has only been successful in a few species (Bajaj, 1990). Microspore culture presents some potential advantages over anther culture, especially concerning in vitro selection strategies, genetic studies and genetic transformation. The culture of isolated microspores has become a valuable system for studying in vitro embryogenesis (Taylor et al., 1990). Complementing earlier work (Pedroso & Pais, 1992, 1993, 1994c,d), direct microspore embryogenesis in C. japonica would be valuable for studying gene expression during microspore embryogenesis and the factors that divert microspore development onto an embryogenic pathway.

Minituber production from immature seed suspension culture ofOrchis papilionacea

SummaryRipe and immature seeds ofOrchis papilionacea (method I and II, respectively) cultured on modified double strength Curtis medium were assayed for minituber production. Ripe seed germination both on solid and in liquid medium was low and the protocorms obtained developed into white calluses. Germination increased from, 9 to 33% when immature seed suspension culture was used. Protocorms obtained in suspension culture under light developed into minitubers, whereas those obtained on solid media developed into callus. A 30 s ultrasonication of immature seeds 1 wk after suspension, culture initiation further enhanced germination and minituber production. Minitubers had to be transferred and embedded in solid regeneration medium for normal growth.