Changes in Soluble Carbohydrates and Starch Amounts During Somatic and Zygotic Embryogenesis of Acca Sellowiana (Myrtaceae)

By Pescador, Rosete Kerbauy, Gilberto B; Kraus, Jane E; de Melo Ferreira, Wagner; Guerra, Miguel Pedro; de Cassia L Figueiredo- Ribeiro, Rita

Abstract Comparative analysis of zygotic and somatic embryogenesis of Acca sellowiana showed higher amounts of sucrose, fructose, raffinose, and myo-inositol in zygotic embryos at different developmental stages than in corresponding somatic ones. These differences were mostly constant. In general, glucose levels were significantly lower than the other soluble carbohydrates analyzed, showing minor variation in each embryo stage. Despite the presence of sucrose in the culture medium, its levels conspicuously diminished in somatic embryos compared with the zygotic ones. Raffinose enhanced parallel to embryo development, regardless of its zygotic or somatic origin. Analysis of the soluble carbohydrate composition of mature zygotic cotyledon used as explant pointed out fructose, glucose, myoinositol, sucrose, and raffinose as the most important. Similar composition was also found in the corresponding somatic cotyledon. Total soluble carbohydrates varied inversely, decreasing in zygotic embryos and increasing in somatic embryos until the 24th d, at which time they increased rapidly about sixfold in zygotic embryos until the 27th d, a period coinciding with the zygotic proembryos formation. Such condition seems to reflect directly the variation of endogenous sucrose level, mainly because glucose and fructose diminished continuously during this time period. This means that, in terms of soluble sugars, zygotic embryo formation occurred under a situation represented by high sucrose amounts, simultaneously with low fructose and glucose levels, while in contrast, somatic embryo formation took place under an endogenous sugar status characterized by a substantial fructose enhancement. Starch levels increased continuously in zygotic embryos and decreased in somatic ones, the reverse to what was found in fructose variation. Starch accumulation was significantly higher in somatic torpedo and cotyledonary embryos than in the corresponding zygotic ones.

Keywords Embryo development * Feijoa * Somatic embryo * Sugars * Zygotic embryo

Introduction

Zygotic and somatic embryogenesis are complex processes, the former initiating with the fusion of gametes and resulting in the formation of a mature embryo and the latter originating from single cells or a set of somatic cells (Dodeman et al. 1997), recapitulating the phenotypic characteristics of the former.

Embryonic development involves distinct but integrated processes such as mitosis, establishment of polarity, cellular differentiation, and synthesis of complex metabolites and the storage of reserve substances (Dodeman et al. 1997). These processes occur sequentially, following concatenated programs that finish with embryo dehydration (BrocardGifford et al. 2003).

Zygotic (ZE) and somatic embryos (SE) are bipolar structures, consisting essentially of a shoot apex and a radicular one. Both pass through several phases phenotypically coined as globular, heart, torpedo, and cotyledonary stages (Zimmerman 1993). In the heart stage, the protoderm and procambium are evident, as well the polarity and bilateral symmetry. SEs, in contrast to zygotic ones, develop in the absence of vascular connections with the mother plant, revealing the occurrence of an embryonic genetic program within the initial cell (Zimmerman 1993).

One of the potential applications of somatic embryogenesis is associated with the attainment of normal and vigorous plantlets, reflecting thus the same biochemical, physiological, and morphological similarities found in the corresponding ZEs. In general, SEs have high levels of starch, protein, and soluble carbohydrates producing excellent growth and more vigorous plants (Attree et al. 1992). The accumulation of reserves is vital for the development of SEs and their conversion into plantlets (Flinn et al. 1993).

Carbohydrates are energy sources for the cell and carbon frameworks for biosynthetic processes. They also act as osmotic agents and contribute to the maintenance of plasmic membrane integrity (Pareddy and Greyson 1989; Tremblay and Tremblay 1991). Raffmose is a soluble sugar that can exist in tissues as a reserve or can also prevent the crystallization of sucrose (Bruni and Leopold 1992). Thus, raffinose is associated with the process of tolerance to desiccation and to the survival of the embryos after the dehydration of the seeds (Keller and Ludlow 1993). Seed and embryo developments are also influenced by the metabolic levels of sugars and by nitrogen, acting as developmental signals in addition to their nutritional roles (Smeekens 2000; Rook and Bevan 2003).

Acca sellowiana (O. Berg) Burret (=Feijoa sellowiana) (Myrtaceae) is a native fruit-bearing species of southern Brazil. It has shown to be a recalcitrant reproductive species by means of both conventional methods of vegetative propagation and micropropagation organogenesis (Bhojwani et al. 1987; Canhoto and Cruz 1996a). Studies of somatic embryogenesis of this species indicated that this morphogenetic route in vitro is an efficient alternative for clonal mass propagation (Cruz et al. 1990; Canhoto and Cruz 1996a, b; Dal Vesco and Guerra 2001; Guerra et al. 2001; Stefanello et al. 2005). However, a limiting factor for the success of this reproductive method in feijoa has been the low percentage of embryos that maintain normal development following the cotyledonary phase (Cruz et al. 1990; Canhoto and Cruz 1996a, b; Guerra et al. 1997).

Variations in soluble carbohydrate and starch contents in SEs, in comparison with their zygotic counterparts, can provide important information on the conversion of SEs into plantlets (Chanprame et al. 1998). In this way, the objective of the present work was to analyze and compare the composition and levels of carbohydrates in the different stages of zygotic and somatic embryogenesis of A. sellowiana, to acquire a better understanding of the embryogenic process and the optimization of protocols for the attainment of SEs of this species.

Materials and Methods

Fertilized and unfertilized ovules, as well as ZE and SE of A. sellowiana (O. Berg) Burret (F. sellowiana Berg.), were used in this study. Ovules derived from unpollinated flowers at anthesis were considered to be at time zero in the analyses of zygotic embryogenesis. For the analyses of embryos in different developmental phases, about 500 flowers were emasculated, allowing manual pollination. During the first 30 d after pollination (DAP), plant material was harvested every 3 d and, then, every 10 d, until the physiological maturation of fruits, about 120 DAP (Guerra et al. 1997). Each harvest, representative of different developmental stages, consisted of ten replicates. Plant material was stored at – 2O0C until utilization.

For the induction of somatic embryogenesis, ZE expiants (0.4 mm long) obtained from mature seeds (approximately 120 DAP) were used. They were inoculated in test tubes (25 x 150 mm) containing 10 ml of culture medium. The medium consisted of full-strength macro- and micronutrients (Von Arnold and Eriksson 1981) and Morel vitamins (Morel and Wetmore 1951), supplemented with 30 g [mu]M sucrose, 0.7% agar (Sigma(R), St. Louis, MO A4550), 20 [mu]M 2,4- dichlorophenoxyacetic acid, and 4 mM glutamine. The pH of the culture medium was adjusted to 5.8 before autoclaving at 120[degrees]C, 0.15 MPa for 20 min. The cultures were maintained in the dark at 25+-3[degrees]C for 70 d.

For the analysis of carbohydrates, samples (1 g) of fresh matter were taken from the initial expiant. Equivalent samples from the ZEs and from representative SE cultures were also taken every 3 d during the first 30 d of incubation. On the 70th d of culture, embryos in the globular, heart, torpedo, and cotyledonary stages were isolated, frozen in liquid nitrogen, and stored at -20[degrees]C.

The extraction procedures for soluble carbohydrates and starch were the same used and optimized for in vitro plant material as described in Vaz et al. (1998). For the extraction of soluble carbohydrates, the samples were ground with mortar and pestle and subsequently submitted to an 80% ethanol extraction at 100[degrees]C for 5 min three times. The extracts were centrifuged at 3,000 x g, at 20[degrees]C, for 10 min and filtered through fiberglass. From the combined supernatant of the three alcohol extractions, a fraction of soluble sugars was obtained, which was quantified by the phenolsulfuric method (Dubois et al. 1956), using glucose as standard.

Following quantification of soluble sugars present in the samples, the extracts were concentrated at 35[degrees]C in a rotary evaporator until dryness and then dissolved in 1 ml of distilled water. After this, the deionization of samples was performed in ion exchange columns, in Dowex 50 x 8-200 cationic resin in the form of NA+, and Dowex 1 x 8-200 anionic resin in the form of CF-. Neutral sugars were eluted with ten volumes of distilled water and lyophilized until dryness and then resuspended in 1 ml of deionized water (Pollock and Jones 1979). Subsequently, concentrated samples were again quantified by the phenol-sulfuric method and analyzed by high-performance anion exchange chromatography, coupled to a pulsed amperometric detector, in Dionex DX-300 (Sunnyvale, CA), using a CarboPac PA-1 anion exchange column of 4 x 250 mm and a gradient of 150 mM sodium hydroxide and 500 mM sodium acetate in 150 mM NaOH for the quantification of individual sugars (according to Itaya et al. 2002) by the external standard method using authentic standards (Sigma, St. Louis, MO). The residue of the ethanolic extracts received the addition of 2 ml of cold distilled water and 2.6 ml of 52% perchloric acid and was maintained in an ice bath with occasional agitation. Subsequently, 20 ml of water was added, and the material was centrifuged at 1,500 x g for 15 min. The extraction was repeated three times and the starch estimated by the phenol- sulfuric method (Dubois et al. 1956), using glucose as a standard, according to the method proposed by McCready et al. (1950).

For the anatomical studies, material was fixed in 1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2, Karnovsky’s fixative modified (Karnovsky 1965), for 4 h at room temperature. During this period, samples were placed under vacuum for 15 min. After fixation, samples were progressively dehydrated in a graded-ethanolic series (20-95%) and embedded in historesin (Leica(R), Wetzlar, Germany), according to the manufacturer’s instructions. Longitudinal sections (6 [mu]M wide) of the embedded samples were obtained with a rotary microtome, using a disposable tungsten blade. The histological sections were stained with 0.05% toluidine blue in 0.2 M phosphate buffer, pH 6.8 (O’Brien et al. 1965), for 5 min.

Results and Discussion

The time course of zygotic and somatic embryogenesis of A. sellowiana is shown in Fig. 1, indicating that embryo differentiation in the somatic cultures occurs in a faster rate. The most prominent histological and morphological features of zygotic and somatic embryogenesis of A. sellowiana are shown in Figs. 2A-G and 3A-D, respectively, indicating the main phases of zygotic embryogenesis (Fig. 2A-F), which include the anatropous ovule before fertilization (Fig. 2A). ZE in globular (Fig. 2B, C), heart (Fig. 2D), torpedo (Fig. 2E), and cotyledonary stages (Fig. 2F) are observed at 30, 40, 60, and 70 DAP, respectively. Figure 2G shows a seed. The histological and morphological characteristics of the SE are shown in Fig. 3A-D, indicating conspicuous phenotypic similarities with the ZE.

Total soluble carbohydrate amounts showed a slight tendency for inverse variation for the two embryogenesis routes during the first 24 d, increasing then sixfold by the 27th DAP in the zygotic one (Fig. 4), which coincided nearly with the first zygotic divisions and the formation of proembryos, before the development of globular embryos at 30 d DAP.

Regardless of the embryogenic route, analysis of soluble carbohydrates in embryos at cotyledon phase showed practically the same composition of fructose, glucose, sucrose, raffinose and wyo- inositol (Fig. 5). Similar sugar composition for zygotic and SEs have been found in other species, such as soybean, whose ZEs contain traces of fructose and galactose, and higher amounts of sucrose, glucose, myo-inositol, pinitol, raffinose, and stachyose (Chanprame et al. 1998). Blackman et al. (1992) previously reported that slow drying immature soybean ZE induced the accumulation of sucrose oligosaccharides of the raffmose family, mainly stachyose. In Picea abies, only glucose, fructose, and sucrose were detected in SE (Konradova et al. 2002). A gradient of sugars analyzed by bioluminescent assays in the cotyledons of beans was shown to be involved in the morphogenetic control, influencing cellular division and differentiation. Sugar carriers would also interfere with the regulation of the cell cycle, altering morphogenetic responses (Weber et al. 1997).

Figure 1. Schematic representation of the time course of zygotic and somatic embryogenesis of A. sellowiana, during 70 DAP and 40 d after inoculation (DAI), respectively. Ct cotyledonary stage, G globular stage, H heart stage, T torpedo stage; Z zygotic embryo.

Figure 2. Zygotic embryogenesis of A. sellowiana. (A) anatropous ovule (arrow), (B) globular embryo 30 DAP (arrow), (C) globular embryo (asterisk), (D) heart embryo 40 DAP (asterisk), (E) torpedo embryo 60 DAP (asterisk), (F) cotyledonary embryo 70 DAP, (G) seed. Co cotyledon, Hy hypocotyls, Ra radicule.

Figure 3. Somatic embryogenesis of A. sellowiana. (A) proembryo 12 DAI (arrow), (B) globular embryos 24 DAI (asterisks), (C) heart (black asterisk) and torpedo (white asterisk) embryos 30 DAI, (D) cotyledonary embryo 40 DAI.

According to Fig. 6A,B, and C, the levels of sucrose, glucose, and fructose varied during the first 30 d in the zygotic and somatic embryogenesis of A. sellowiana. With respect to sucrose levels, their variation was very similar to that found in total soluble carbohydrates (Fig. 4) and substantially higher than what was observed for glucose and fructose, mainly in the zygotic route. Considering the presence of sucrose in the medium, its decrease during somatic embryogenesis could be considered as an unexpected result. A sharp significant enhancement of sucrose occurred after the 24th DAP in zygotic embryogenesis, reaching 214.07 mg g^sup -1^ FW on the 27th d coinciding with zygotic proembryo formation. On the other hand, little significant difference was found in the somatic route along 30d culture.

In contrast to the variations observed for sucrose, the levels of glucose (Fig. 6B) and fructose (Fig. 6C) showed reciprocal tendencies to each other, being initially higher in zygotic embryogenesis, diminishing in the subsequent period.

Carbohydrates regulate gene expression in plants, and the enzymes of sucrose metabolism might play a pivotal role in sugar sensing and plant development (Koch 1996, 2004). Particularly, the relation between hexoses and sucrose was reported to be involved in the development of legume embryos. When the hexoses are favored, cell division and differentiation are promoted, whereas when sucrose is the predominating sugar, processes such as cell elongation as well storage of proteins and carbohydrates are stimulated (Borisjuk et al. 2002, 2003). In Vicia faba, higher levels of hexoses induced the formation of transfer cells that are involved in the nutrition of embryos, whereas high sucrose concentrations are inhibitory to this process (Borisjuk et al. 2003). The variations in the proportions of hexoses and sucrose observed in A. sellowiana suggest that they could be associated with metabolic and/or structural changes of SE and ZE, leading to differences in their growth and further development.

Figure 4. Changes in total soluble carbohydrates (mg.g^sup -1^ FW) during 30 d in zygotic (empty circle) and somatic (filled circle) embryogenesis of A. sellowiana after pollination and explant inoculation, respectively. In the former, time zero represents unfertilized ovules, while in the latter, it represents the situation of the initial explants (mature ZEs). Vertical bars indicate standard deviation and were not shown when they were lower than the symbol.

The variation in starch during ZE and in SE of A. sellowiana during 30 d showed significant and inverse tendencies, increasing after pollination and decreasing along the culture period (SE) (Fig. 7). These results point to a putative opposite importance of this carbohydrate for each embryogenic process.

Figure 5. HPAEC/PAD elution profile in CarboPac PA-I column of sugars present in both somatic and ZEs of A. sellowiana at cotyledonary stage. M myo-inositol, C glucose, F fructose, S sucrose, R raffinose.

Comparing total sugar contents in each embryonic stage in both routes (Fig. 8), we observed that ZE possessed more soluble carbohydrates than the corresponding SE, although SEs have developed completely in a sucrose-rich medium. The decrease in soluble carbohydrates observed in the cotyledonary phase of ZE suggests they are the main source of energy and/or synthesis of reserve polysaccharides, of which starch is an example (Fig. 12). Despite that the content of sucrose was about fourfold higher in ZE than in SE, no significant difference was observed among them within each embryogenic route (Fig. 9A). In comparison with sucrose, the amounts of glucose and fructose were conspicuously lower; the greatest levels of glucose were found in globular embryos and the lowest in the cotyledonary stage of SE (Fig. 9B). On the other hand, a significant enhancement of fructose content occurred in the ZE up to the torpedo phase, followed by a decline in the cotyledonary one. In SE, fructose contents decreased slowly from globular to cotyledonary stages (Fig. 9C). These variations in sucrose, glucose, and fructose levels suggest that they were coordinated during the studied processes and resulted from the metabolism of sucrose, as observed in other species (Hill et al. 2003). The decrease in hexoses during development of ZEs of avocado, for example, was attributed to the synthesis of sucrose and coincided with the maturation phase, with a simultaneous decrease in water content and accumulation of starch (Sanchez-Romero et al. 2002). The high and practically constant amounts of sucrose in every embryonic stage of A. sellowiana suggest the importance of this sugar to the embryo development, differently, for instance, from P. abies where sucrose acts as a regulatory factor for the maturation of SEs, in addition to signaling the synthesis of reserve proteins (Iraqui and Tremblay 2001).

Cloning of the gene vfsutl of beans and the localization of its transcription confirmed that sucrose carriers are strongly expressed in cells during embryonic differentiation, mainly between the cordiform and cotyledonary phases (Weber et al. 1997). On the other hand, Gaudin et al. (2000) showed that the expression of three D- cyclin genes in apical meristem of snapdragon was modulated by factors that regulate plant growth, particularly sucrose and cytokinins. It is interesting to highlight also that D-type cyclin gene CYCD4,1 could be highly expressed only with the presence of sucrose in various organs of A. thaliana, including embryo development in torpedo and heart stages, though decreasing in mature embryos (De Veylder et al. 1999). The relatively high levels of hexoses found in A. sellowiana during zygotic and somatic embryogenesis could be associated to the maintenance of cellular division and elongation, especially in embryos during the heart phase, as suggested by Hill et al. (2003) for Brassica napus. In this species, a decrease in glucose levels was observed in embryos only during the desiccation phase (Norton and Harms 1975). In seeds of Arabidopsis thaliana ecotype WS, the initial levels of glucose and fructose were also high but decreased during development (Baud et al. 2002). According to Borisjuk et al. (1995), the hexoses-to- sucrose ratio changes temporally and spatially during embryogenesis of V. faba and could act as a trigger, influencing cell differentiation and carbohydrate partitioning. The constant levels of sucrose and variable amounts of hexoses in A. sellowiana, would, in fact, lead to different ratios among them along the embryogenesis routes studied (Fig. 9A, B, and C).

Figure 6. Changes in the contents of sucrose (A), glucose (B), and fructose (C) in the zygotic (empty circle) and somatic (filled circle) embryogenesis of A. sellowiana during 30 DAP and explant inoculation. In the former, time zero represents unfertilized ovules, while in the latter, it represents the initial condition of the explants (mature ZEs). Vertical bars indicate the standard deviation.

Figure 7. Variations of starch contents (mg g^sup -1^ FW) in zygotic (empty circle) and somatic (filled circle) embryogenesis of A. sellowiana during 30 DAP and explant inoculation (mature ZEs), respectively. Vertical bars indicate the standard deviation.

The type of carbohydrate present in seeds is the determining factor of tolerance to desiccation, more than the accumulation of reserves (Sauter and van Cleeve 1991). However, the precise mechanism by which carbohydrates afford protection to the cells during desiccation still remains obscure. The possibility exists that sugars form hydrogen bonds that could substitute water in the maintenance of hydrophilic structures. Another proposed mechanism is the inhibition of formation of intercellular crystals of water (Hoekstra and Golovina 1999).

Raffinose was detected in all phases of SE of A. sellowiana, as well as in ZE, except at the globular stage of ZE (Fig. 10). Its levels increased in both routes; however, it was predominantly higher in zygotic embryogenesis, principally at torpedo and cotyledonary stages. This result reinforces the role of raffmose in protecting embryos against desiccation. In P. abies, raffinose was also found in all embryonic phases, increasing its concentration when the cultures were exposed to low temperatures (Konradova et al. 2003).

Figure 8. Amounts of total sugars in embryos of A. sellowiana in globular, heart, torpedo, and cotyledonary stages of zygotic embryogenesis (filled bars) and somatic embryogenesis (empty bars). The vertical bars indicate the standard deviation.

Figure 9. Contents (mg g^sup -1^ FW) of sucrose (A), glucose (B), and fructose (C) in embryos of A. sellowiana in globular, heart, torpedo, and cotyledonary stages, originated from zygotic (filled bars) and somatic (empty bars) embryogenesis. Vertical bars indicate the standard deviation.

Comparative studies of SE and ZE embryos of various species showed the presence of other oligosaccharides of the raffinose series, such as stachyose and verbascose. However, these sugars were not detected in embryos of A. sellowiana. Chanprame et al. (1998) found elevated concentrations of stachyose in the later phases of somatic and zygotic embryogenesis of soybean, although they were much lower in SE than in ZE. Stachyose and raffinose were also found in elevated concentrations in seeds of A. thaliana by about 20 d after fertilization, the levels of raffinose being higher than those of stachyose (Baud et al. 2002). In Glycine max, the high levels of verbascose and stachyose were associated with the ability of these seeds to tolerate desiccation (Koster and Leopold 1988).

Figure 10. Raffinose content (mg g^sup -1^ FW) in embryos of A. sellowiana in globular, heart, torpedo, and cotyledonary stages, originated from zygotic (filled bars) and somatic embryogenesis (empty bars). Vertical bars indicate the standard deviation.

The sugar alcohol myo-inositol was detected in lower amounts in somatic cultures of A. sellowiana than in ZE. The amounts found in ZE at globular, heart, torpedo, and cotyledonary stages are shown in Fig. 11. Its relation with raffinose synthesis (Peterbauer and Richter 2001) and part of phospholipid molecule of the plasma membrane (Moore 1982) were already observed. This cyclitol also conjugates with auxins forming auxin-inositol complex, inhibiting the action of this hormone (Cohen and Bandurski 1982).

In G. max, myo-inositol was present in precocious ZE and in the later phases of somatic embryogenesis (Chanprame et al. 1998). D- Pinitol, another cyclitol, was also present in ZE of soybean, while in SE, it was found only early during development, indicating the lack of its synthesis by these embryos. Pinitol is regarded as a possible drought protective agent during development of embryos, being synthesized in maternal tissues and transported to the embryos (Gomes et al. 2005). In A. sellowiana, pinitol was not detected in any phase of embryo development. However, the substantial levels of sucrose and raffinose found in the final stages of embryo development (torpedo and cotyledonary stage) could be associated with physiological desiccation tolerance, independently of its zygotic or somatic origin.

Figure 11. Concentration of myo-inositol (mg g^sup -1^ FW) in embryos of A. sellowiana in globular, heart, torpedo, and cotyledonary stages, originated from zygotic (filled bars) and somatic embryogenesis (empty bars). Vertical bars indicate the standard deviation.

Figure 12. Changes of starch contents (mg. g^sup -1^ FW) in zygotic (filled bars) and somatic (empty bars) embryos in globular, heart, torpedo, and cotyledonary stages. Vertical bars indicate the standard deviation.

Mature zygotic cotyledonary cells of A. sellowiana store proteins and lipids (Canhoto and Cruz 1996b). Amyloplasts were also found in all cotyledonary cells. After 2-3 wk of culture, somatic proembryos and globular embryos also possessed amyloplasts, but they were smaller and less numerous than those formed in non-embryonic cells of the explant. Cangahuala-Inocente et al. (2004) also observed that the cells of globular SE have few amyloplasts, but in torpedo and cotyledonary stages, they are present at their basal portion, suggesting that starch is rapidly metabolized in embryonic tissues, providing energy for the intense metabolic mitotic activities.

In the present work, an increase in the starch quantity was observed mainly in embryos at torpedo and cotyledonary stages, particularly in those of somatic origin (Fig. 12), concomitantly with the decrease of hexoses (Fig. 95, C), corroborating previous histochemical analysis. Similar results were obtained in avocado embryos (SanchezRomero et al. 2002) and in P. mariana and P. glauca (Iraqui and Tremblay 2001), in which starch accumulation also occurred specifically at final phases of embryonic development.

Taking into account the remarkable qualitative similarity in the composition of soluble carbohydrates in ZE and SE of A. sellowiana, it is plausible to suppose, at a first view, the occurrence of similar roles in both embryo types. On the other hand, the significant quantitative differences between them point to the existence of conspicuous sugar metabolic differences. The hexoses/ sucrose ratio could act as triggers influencing cell division and differentiation in the early development of ZE and SE, while starch seems to be involved in carbohydrate partitioning and storage, as they were present in greater concentrations in the cotyledonary stage of these embryos.

Received: 19 April 2007 /Accepted: 10 March 2008 /Published online: 5 June 2008 / Editor: Gregory C. Phillips

(c) The Society for In Vitro Biology 2008

Acknowledgments The authors thank the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) for the research grants awarded to R. C. L. F. Ribeiro, G. B. Kerbauy, and M. P. Guerra. Thanks are also due to Dr. Jean Pierre H. J. Ducroquet of the Epagri/Sao Joaquim, SC (Brazil) for supplying plant material.

References

Attree, S. M.; Pomeroy M. K.; Fowke L. C. Manipulation of conditions for the culture of somatic embryos of white spruce for improved triacylglycerol biosynthesis and desiccation tolerance. Planta 187: 395-404; 1992.

Baud, S.; Boutin J. P.; Miquel M.; Lepiniec L.; Rochat C. An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiol. Biochem 40: 151-160; 2002.

Bhojwani, S. S.; Mullins K.; Cohen D. Micropropagation of Acca sellowiana Berg. Acta Hort 212: 69-76; 1987.

Blackman, S. A.; Obendorf R. L.; Leopold A. C. Maturation proteins and sugars in desiccation tolerance of developing soybean seeds. Plant Physiol 100: 225-230; 1992.

Borisjuk, L.; Rolletschek H.; Wobus U.; Weber H. Differentiation of legume cotyledon as related to metabolism gradients and assimilate transport into seeds. J. Exp. Bot 54: 503-512; 2003.

Borisjuk, L.; Walenta S.; Rolletschek H.; Mueller-Klieser W.; Wobus U.; Weber H. Spatial analysis of plant metabolism: Sucrose imaging with Vicia faba cotyledons reveals specific development patterns. Plant J 29: 521-530; 2002.

Borisjuk, L.; Weber H.; Panitz R.; Manteuffel R.; Wobus U. Embryogenesis of Vicia faba L.: Histodifferentiation in relation to starch and storage protein synthesis. J. Plant Physiol 147: 203- 218; 1995. Brocard-Gifford, I. M.; Lynch T. J.; Finkelstein R. R. Regulatory networks in seeds integrating developmental, abscisic acid, sugar, and light signaling. Plant Physiol 1311: 78-92; 2003.

Bruni, E.; Leopold A. C. Pool of water in anhydrobiotic organism: a thermally stimulated depolarisation current study. Biophys. J 63: 663-672; 1992.

Cangahuala-Inocente G. C.; Steiner N.; Santos M.; Guerra M. P. Morphohistological analysis and histochemistry of Feijoa sellowiana somatic embryogenesis. Protoplasma 224: 33-40; 2004.

Canhoto, J. M.; Cruz G. S. Feijoa sellowiana Berg (pineapple guava). In: Bajaj Tree Y. P. S.I. V. (ed) Biotechnology in agriculture on forestry. Springer, Berlin, pp 156-171; 1996a.

Canhoto, J. M.; Cruz G. S. Histodifferentiation of somatic embryos in cotyledons of pineapple guava (Feijoa sellowiana Berg). Protoplasma 191: 34-45; 1996b.

Chanprame, S.; Kuo T. M.; Widholm J. M. Soluble carbohydrate content of soybean (Glycine Max (L.) Merr.: Somatic and zygotic embryos during development. In Vitro Cell. Dev. Biol.-Plant 34: 64- 68; 1998.

Cohen, J. D.; Bandurski R. S. Chemistry and physiology of the bound auxins. Ann. Rev. Plant Physiol 33: 403-430; 1982.

Cruz, G. S.; Canhoto J. M.; Abreu M. A. V. Somatic embryogenesis and plant regeneration from zygotic embryos of Acca sellowiana Berg. Plant Sci 66: 263-270; 1990.

Dal Vesco, L. L.; Guerra M. P. The effectiveness of nitrogen sources in Feijoa (Feijoa sellowiana Berg.) somatic embryogenesis. Plant Cell Tiss. Org. Cult 64: 19-25; 2001.

De Veylder, L.; Engler, J. A.; Burssens, S.; Manevski, A.; Lescure, B.; Van Montagu, M. et al. A new D-type cyclin of Arabidopsis thaliana expressed during lateral root primordia formation. Planta 208: 453-462; 1999.

Dodeman, V. L.; Ducreux G.; Kreis M. Zygotic embryogenesis versus somatic embryogenesis. J. Exp. Bot 48: 1493-1509; 1997.

Dubois, M.; Guilles A.; Hamilton J. K.; Rebers P. A.; Smith F. Colorimetric method for determination of sugars and related substances. Anal. Chem 28: 350-355; 1956.

Flinn, B. S.; Roberts D. R.; Newton C. H.; Cyr D. R.; Webster F. B.; Taylor I. E. P. Storage protein gene expression in zygotic and somatic embryos of interior spruce. Physiol. Plant 89: 719-730; 1993.

Gaudin, V.; Lunness P. A.; Fobert P. R.; Towers M.; Rioux- Khamlichi C.; Murray J. A. H. The expression of D-cyclin genes defines distinct developmental zones in snapdragon apical meristems and it is locally regulated by the Cycloidea gene. Plant Physiol 122: 1137-1148; 2000.

Gomes, C. I.; Obendorf R. L,; Horbowicz M; . myo-Inositol, D- chiroinositol, and D-pinitol synthesis, transport, and galactoside formation in soybean expiants. Crop Sci 45: 1312-1319; 2005.

Guerra, M. P.; DaI Vesco L. L.; Ducroquet J. P. H. J.; Nodari R. O.; Reis M. Somatic embryogenesis in goiabeira serrana: Genotype response, auxinic shock and synthetic seeds. Rev. Bras. Fisiol. Veg 13: 117-128; 2001.

Guerra, M. P.; Pescador R.; Dal Vesco L. L.; Nodari R. O.; Ducroquet J. P. H. J; . In vitro morphogenesis in Feijoa sellowiana: Somatic embryogenesis and plant regeneration. Acta Hort 452: 27-36; 1997.

Hill, L. M.; Morley-Smith E. R.; Rawsthome S. Metabolism of sugar in the endosperm of developing seeds of oilseed rape. Plant Physiol 131: 228-236; 2003.

Hoekstra, F. A.; Golovina E. A. Membrane behavior during dehydration: Implication for desiccation tolerance. Russ. J. Plant Physiol 46: 295-306; 1999.

Iraqui, D.; Tremblay F. M. Analysis of carbohydrate metabolism enzymes and cellular contents of sugars and proteins during spruce somatic embryogenesis suggests a regulatory role of exogenous sucrose in embryo development. J. Exp. Bot 52: 2301-2311; 2001.

Itaya, N. M.; Carvalho M. A. M.; Figueiredo-Ribeiro R. C. L. Fructosyl transferase and hydrolase activities in rhizophores and tuberous roots upon growth of Polymnia sonchifolia (Asteraceae). Physiol. Plant 116: 451-459; 2002.

Karnovsky M. J. A. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell. Biol 27: 137138; 1965.

Keller F.; Ludlow M. M. Carbohydrate metabolism in droughtstressed leaves of pigeon pea (Cajanus cajan). J. Exp. Bot 44: 1351-1359; 1993.

Koch, K. E. Carbohydrate-modulated gene expression in plants. Ann. Rev Plant Physiol. Plant Biol 47: 509-540; 1996.

Koch, K. E. Sucrose metabolism: regulatory mechanism and pivotal roles in sugar sensing and plant development. Curr. Opin. Plant. Biol 7: 235-246; 2004.

Konradova, H.; Grigova M.; Lipavska H. Cold-induced accumulation of raffmose family oligosaccharides in somatic embryos of Norway spruce (Picea abies). In Vitro Cell. Dev. Biol.-Plant 39: 425-427; 2003.

Konradova, H.; Lipavska H.; Albrechtova J.; Vreugdenhil D. Sucrose metabolism during somatic and zygotic embryogeneses in Norway spruce: Content of soluble saccharides and localisation of key enzyme activities. J. Plant Physiol 159: 387-396; 2002.

Koster, K. L.; Leopold A. C. Sugar and desiccation tolerance in seeds. Plant Physiol 88: 829-832; 1988.

McCready, R. M.; Guggolz A.; Silveira V; Owens H. S. Determination of starch and amylase in vegetables, application to peas. Anal. Chem 22: 1156-1158; 1950.

Moore, J. R. Phospholipid biosynthesis. Ann. Rev. Plant Physiol 33: 235-259;1982.

Morel G.; Wetmore R. H. Tissue culture of monocotyledons. Am. J. Bot 38: 138-140; 1951.

Norton, G.; Harms J. F. Compositional changes in developing rape seed (Brassica napus L.). Planta 123: 163-174; 1975.

O’Brien, T. P.; Feder N.; McCully M. E. Polychromatic staining of plant cell walls by toluidine blue. O. Protoplasma 59: 368-373; 1965.

Pareddy, D. R.; Greyson R. I. Studies on sucrose requirements of cultured maize tassel. Can. J. Bot 67: 225-229; 1989.

Peterbauer, T.; Richter A. Biochemistry and physiology of raffinose family oligosaccharides and galactosyl cyclitols in seeds. Seed Sci. Res 11: 185-198; 2001.

Pollock, C. G.; Jones T. Seasonal patterns of fructan metabolism in forage grasses. New Phytol 83: 8-15; 1979.

Rook, F.; Bevan M. W. Genetic approaches to understanding sugarresponse pathways. J. Exp. Bot 54: 495-501; 2003.

Sanchez-Romero, C.; Peran-Quesada R.; Barcelo-Munoz A.; PliegoAlfaro F. Variations in storage protein and carbohydrate levels during development of avocado zygotic embryos. Plant Physiol. Biochem 40: 1043-1049; 2002.

Sauter, J. J.; van Cleeve B. Biochemical and ultrastructural results during starch-sugar conversion in ray parenchyma cells of Populus during cold adaptation. J. Plant Physiol 139: 19-26; 1991.

Smeekens, S. Sugar-induced signal transduction in plants. Ann. Rev. Plant Physiol. Plant Mol. Biol 51: 49-81; 2000.

Stefanello, S.; Dal Vesco L. L.; Ducroquet J. P. H. J.; Nodari R. O.; Guerra M. P. Somatic embryogenesis from floral tissues of feijoa (Feijoa sellowiana Berg). Sci. Hort 105: 117-126; 2005.

Tremblay, L.; Tremblay F. M. Carbohydrate requirements for the development of black spruce (Picea mariana (Mill) (B.S.P) and red spruce (P. rubens Sarg.) somatic embryo. Plant Cell Tiss. Organ Cult 27: 95-103; 1991.

Vaz, A. P. A.; Kerbauy G. B.; Figueiredo-Ribeiro R. C. L. Changes in soluble carbohydrates and starch partitioning during vegetative bud formation from root tips of Catasetum fimbriatum (Orchidaceae). Plant Cell Tiss. Org. Cult 54: 105-111; 1998.

Von Arnold, S.; Eriksson T. In vitro studies of adventitious shoot formation in Pinus cordata. Can. J. Bot 59: 870-874; 1981.

Weber, H.; Borisjuk L.; Wobus U. Sugar import and metabolism during seed development. Trends Plant Sci 2: 169-174; 1997.

Zimmerman, J. L. Somatic embryogenesis: A model for early development in higher plants. Plant Cell 5: 1411-1423; 1993.

R. Pescador

Departamento de Ciencias Naturais,

Universidade Regional de Blumenau,

C. P. 1507,

Blumenau, SC CEP 89010-971, Brazil

G. B. Kerbauy * J. E. Kraus

Departamento de Botanica, Instituto de Biociencias,

Universidade de Sao Paulo,

C. P. 11461,

Sao Paulo, SP CEP 05422-970, Brazil

W. de Melo Ferreira

Nucleo de Estudos Ambientais,

Universidade Federal do Tocantins,

C. P. 111,

Porto Nacional, Tocantins CEP 77500-000, Brazil

M. P. Guerra

Departamento de Fitotecnia, Centre de Ciencias Agrarias,

Universidade Federal de Santa Catarina,

Rod. Admar Gonzaga, Km 3,

Florianopolis, SC CEP 88040-900, Brazil

R. d. C. L. Figueiredo-Ribeiro (S)

Instituto de Botanica,

Caixa Postal 3005,

Sao Paulo, SP 01061-970, Brazil

e-mail: ritarib@usp.br

Copyright Society for In Vitro Biology Jul/Aug 2008

(c) 2008 In Vitro Cellular & Developmental Biology; Plant. Provided by ProQuest LLC. All rights Reserved.