Protein Phosphorylation during Coconut Zygotic Embryo Development1

Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [γ-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species.

Dynamics of biochemical and morphophysiological changes during zygotic embryogenesis in Acca sellowiana (Berg.) Burr.

Acca sellowiana (Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a domestication and breeding program. Biotechnological tools have been used to assist in this program. The establishment of a reliable protocol of somatic embryogenesis has been pursued, with a view to capturing and fixing genetic gains. The rationale behind this work relies on the fact that deepening comprehension of the general metabolism of zygotic embryogenesis may certainly improve the protocol for somatic embryogenesis. Thus, in the present work we studied the accumulation of protein, total sugars, starch, amino acids, polyamines (PAs), IAA and ABA, in different stages of A. sellowiana zygotic embryogenesis. Starch is the predominant storage compound during zygotic embryo development. Increased synthesis of amino acids in the cotyledonary stage, mainly of asparagine, was observed throughout development. Total free PAs showed increased synthesis, whereas total conjugated PAs were mainly observed in the early developmental stages. IAA decreased and ABA increased with the progression from early to late embryogenesis. Besides providing basic information on the morphophysiological and biochemical changes of zygotic embryogenesis, the results here obtained may provide adequate strategies towards the modulation of somatic embryogenesis in this species as well as in other woody angiosperms.

A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 mu M benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 mu M 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog`s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.

Structural aspects of the zygotic embryogenesis of Acca sellowiana (O. Berg) Burret (Myrtaceae)

(Structural aspects of the zygotic embryogenesis of Acca sellowiana (O. Berg) Burret (Myrtaceae)). Acca sellowiana has anatropous, bitegmic and crassinucellate ovules. The outer and inner integuments are double-layered except in the micropyle, where they are composed of more layers; the micropyle is zig-zag shaped. The egg apparatus lies at the micropylar pole, and the zynergids present a conspicuous filiform apparatus. The antipodal cells are present in the chalazal region, persisting before the occurrence of double fertilization. The zygote is visible 21 days after pollination; nuclear endosperm is already present. The first mitotic division of the zygote occurs at 24(th) day. The globular, cordiform and torpedo embryo stages can be seen at 30, 45 and 60 days after pollination, respectively. The mature embryo characterized by the presence of a well-developed hypocotyl-radicular axis with two fleshy and folded cotyledons was observed 120 days after pollination. Endosperm is absent in the seeds, and the embryo has spiral form, characteristic of Myrtinae. The zygotic embryology studies of A. sellowiana indicate that this species has embryological characteristics which are in agreement with those reported for Myrtaceae (Myrteae, Myrtinae), and also broaden the knowledge about the sexual reproduction of this native species, whose commercial cultivation has been growing.

Changes in soluble carbohydrates and starch amounts during somatic and zygotic embryogenesis of Acca sellowiana (Myrtaceae)

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, myo-inositol, 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.

Evolution of polyembryony: Consequences to the fitness of mother and offspring

Polyembryony, referring here to situations where a nucellar embryo is formed along with the zygotic embryo, has different consequences for the fitness of the maternal parent and offspring. We have developed genetic and inclusive fitness models to derive the conditions that permit the evolution of polyembryony under maternal and offspring control. We have also derived expressions for the optimal allocation (evolutionarily stable strategy, ESS) of resources between zygotic and nucellar embryos. It is seen that (i) Polyembryony can evolve more easily under maternal control than under that of either the offspring or the ‘selfish’ endosperm. Under maternal regulation, evolution of polyembryony can occur for any clutch size. Under offspring control polyembryony is more likely to evolve for high clutch sizes, and is unlikely for low clutch sizes (<3). This conflict between mother and offspring decreases with increase in clutch size and favours the evolution of polyembryony at high clutch sizes, (ii) Polyembryony can evolve for values of “x” (the power of the function relating fitness to seed resource) greater than 0.5758; the possibility of its occurrence increases with “x”, indicating that a more efficient conversion of resource into fitness favours polyembryony. (iii) Under both maternal parent and offspring control, the evolution of polyembryony becomes increasingly unlikely as the level of inbreeding increases, (iv) The proportion of resources allocated to the nucellar embryo at ESS is always higher than that which maximizes the rate of spread of the allele against a non-polyembryonic allele.Finally we argue that polyembryony is a maternal counter strategy to compensate for the loss in her fitness due to brood reduction caused by sibling rivalry. We support this assertion by two empirical evidences: (a) the extent of polyembryony is positively correlated with brood reduction inCitrus, and (b) species exhibiting polyembryony are more often those that frequently exhibit brood reduction.

Somatic embryogenesis in Triticum aestivum L. Morphological observations on germination

Somatic embryos were induced from scutellar callus of immature zygotic embryos of T aestivum cv. Chinese Spring. Observations on precociously germinating somatic embryos revealed that: (i) In the initial stages the coleoptile is split, exposes the shoot apex and forms a green trichomatous leafy structure. In the germinating zygotic embryo, the coleoptile is tubular, (ii) Unlike what has been inferred earlier the leafy structure is the coleoptile and not the scutellum, (iii) Bipolarity of the embryoid is established later when root develops at the basal end.

Transcriptome analysis for discovering candidate genes involve in embryogenesis in coconut (Cocos nucifera L.) through 454 pyrosequencing

Coconut, Cocos nucifera L. is a major plantation crop, which ensures income for millions of people in the tropical region. Detailed molecular studies on zygotic embryo development would provide valuable clues for the identification of molecular markers to improve somatic embryogenesis. Since there is no ongoing genome project for this species, coconut expressed sequence tags (EST) would be an interesting technique to identify important coconut embryo specific genes as well as other functional genes in different biochemical pathways. The goal of this study was to analyse the ESTs by examining the transcriptome data of the different embryo tissue types together with one somatic tissue. Here, four cDNA libraries from immature embryo, mature embryo, microspore derived embryo and mature leaves were constructed. cDNA was sequenced by the Roche-454 GS-FLX system and assembled into 32621 putative unigenes and 155017 singletons. Of these unigenes, 18651 had significant sequence similarities to non-redundant protein database, from which 16153 were assigned to one or more gene ontology categories. Homologue genes, which are responsible for embryo development such as chitinase, beta-1,3-glucanase, ATP synthase CF0 subunit, thaumatin-like protein and metallothionein-like protein were identified among the embryo EST collection. Of the unigenes, 6694 were mapped into 139 KEGG pathways including carbohydrate metabolism, energy metabolism, lipid metabolism, amino acid metabolism and nucleotide metabolism. This collection of 454-derived EST data generated from different tissue types provides a significant resource for genome wide studies and gene discovery of coconut, a non-model species.

Endogenous abscisic acid and protein contents during seed development of Araucaria angustifolia

This paper describes a proteome analysis and changes in endogenous abscisic acid (ABA) contents during seed development of Araucaria angustifolia (Bert.) O. Ktze. Megagametophytes and embryonic axis tissues exhibited a similar ABA variation pattern during seed development, reaching maximum values at the pre-cotyledonary stage. The embryonic axis protein content increased until the cotyledonary stage with following stabilization at mature seed. The two-dimensional electrophoresis at the torpedo developmental stage showed approximately 230 polypeptides against 340 in the mature stage. Peptide mass fingerprinting analyses identified three polypeptides, corresponding to an AtSAC4, a late embryogenesis abundant (LEA) and a storage protein, respectively.

Detecção de polimorfismo em porta-enxertos para citros

Objetivando a verificação da existência de plântulas originadas de embrião zigótico em viveiros comerciais de mudas cítricas, realizou-se o presente trabalho, utilizando a técnica de fAFLP e quatro espécies de porta-enxertos para citros. Verificou-se uma base genética estreita entre as espécies testadas, além de grande variabilidade entre os materiais, independentemente do viveiro em que foram coletados, o que permite concluir que a seleção visual, comumente realizada nos viveiros, é ineficiente.

Self-fertility and polyembryony in South American yellow trumpet trees (Handroanthus chrysotrichus and H. ochraceus, Bignoniaceae): a histological study of postpollination events

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Apomixia em citros: expressão diferencial de mRNA e proteínas em plântulas e embriões zigóticos e apomíticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences

The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.

Coconut (Cocos nucifera) in vitro ecology: Modifications of headspace and medium additives can optimize somatic embryogenesis

Of those explants tested, immature zygotic embryo tissues proved to be the best for initiating callus with potential for somatic embryogenesis. Slicing of this tissue and use of the central sections (near to and including the meristematic tissue) gave the best embryogenic response. Slices that were placed under illumination necrosed more rapidly and to a greater degree than those incubated in the dark. Explant slice necrosis could be prevented or severely retarded by the addition of activated charcoal into the medium. Washing the explants for short periods of time prior to culture was also found to improve callus production. Prolonged washing resulted in low rates of callus production. In an attempt to prevent ethylene accumulation in the culture vessel headspace, AVG, an ethylene biosynthesis inhibitor and STS, a chemical which reduces the physiological action of ethylene, were successfully used to promote somatic embryogenesis. Spermidine, putrescine and spermine, polyamines that are known to delay plant senescence and promote somatic embryogenesis in some plant species, enhanced the rate of somatic embryogenesis when they were introduced into the callus induction medium. The use of polyethylene glycol in combination with abscisic acid helped promote somatic embryo formation and maturation as well as the subsequent formation of plantlets. The use of all of these improvements together has created a new and improved protocol for coconut somatic embryogenesis. This new protocol puts significant emphasis on improving the in vitro ecology of the explant, callus and somatic embryogenic tissues.

Effect of physical, chemical and light treatments on germination and growth of tissue-cultured coconuts

The acclimatization and ex vitro establishment of tissue cultured coconut plantlets regenerated either from zygotic or somatic embryos could result to serious losses. Although high germination rates can be achieved in vitro, the survival of zygotic embryo derived plantlets in soil is very low (0-30%). Hence, treatments that could promote development of good quality seedlings having well-developed shoot and root is needed to increase seedling survival ex vitro. The effect of physical, chemical and light quality treatments on germination and growth of coconut embryos and tissue-cultured seedlings respectively, was investigated. The germination of coconut embryos was promoted when placed in a liquid Euwens (Y3) medium and incubated using a roller drum. Gibberellic acid (GA3) significantly affected growth of seedlings as it promoted shoot elongation, shoot and root expansion, and fresh and dry weight increase. However, GA3 did not significantly affect germination. In addition, the blue, red and yellow light significantly affected growth of seedlings as it promoted leaf and shoot elongation, fresh and dry weight increase, and root and leaf production. These conditions could be used to improve the growth and survival ex vitro of tissue cultured coconuts.