Embryogenic Characteristics Research Articles

Embryo and endosperm development in Billbergia nutans H. Wendl. ex Regel (Bromelioideae - Bromeliaceae) and its implications for the embryonomy of Bromeliaceae

The proposition of patterns, types, and variations for embryogenic events is one of the objectives of embryonomy. Bromeliaceae is a diverse neotropical plant family for which this approach has been ambiguously and rarely discussed in detail. In this study, we analyzed the embryogeny and endospermogenesis of Billbergia nutans H. Wendl. ex Regel, a bromeliad worldwide known as ornamental and with no embryogenic studies recorded to date. Seeds of B. nutans were periodically collected after manual pollination and processed with standard methods for analysis under light microscopy. Among other embryogenic characteristics, we highlight the longitudinal division of ca and its major contribution to the formation of the embryo, the presence of hypophysis derived from m, and the formation of the first internal cells of the proembryo from q. Endospermogenesis is helobial, and the cellularization in each chamber occurs at specific stages of the embryogenesis. The embryonic number is 211 and the embryonomic formula ca = pco + phy + pvt + icc; cb = iec + ico + co + s, typical of the Onagrad Type. This classification is unprecedented for Bromeliaceae, although it can be contradictorily applied to other species of the family. Special embryogenic characteristics of B. nutans, as the contribution of n in the formation of the root cap and the longitudinal divisions in the suspensor cells, led to the proposal of a new variation for this type: the Billbergia Variation, which also can be applied to some other Bromeliaceae species.

INDUCTION AND MAINTENANCE OF EMBRYOGENIC CHARACTERISTICS OF CALLUS OF THE OIL PALM HYBRID MANICORÉ

ABSTRACT Large-scale oil palm propagation (Elaeis guineensis Jacq.) is difficult due to its unique apical meristem. In this context, micropropagation allows the multiplication of seedlings in vitro and the storage of germplasm elites. This study aimed to induce embryogenic calluses from leaves of oil palm plants in low concentrations of auxins and to observe the maintenance of these characteristics during in vitro cultivation. Calluses were induced in 0.5 cm leaf explants in Y3 culture medium supplemented with Picloram (4-Amino-3,5,6-trichloro-2-pyridinecarboxylic acid) or 2,4-D (2,4-dichlorophenoxyacetic acid), at concentrations of 0, 1, 3, 6, and 9 mg L-1. The callus with embryogenic appearance was subcultured and evaluated regarding maintenance of embryogenic characteristics by cytochemical analyses. The best treatment for induction of calluses was composed of 1mg.L-1 of Picloram, which led to 30% callus formation. The calluses were classified into4 types, based on color and morphology. The cells of calluses with nodular and beige appearance have embryogenic characteristics, and the embryogenic potential of the cell masses was maintained over the 20 months of cultivation. This differentiated adaptation to the protocol can allow the advance in the mass propagation of oil palm through tissue culture, indicating the importance of investigating the topics proposed by the research.

Somatic Embryogenesis in Centaurium erythraea Rafn-Current Status and Perspectives: A Review.

Centaurium erythraea (centaury) is a traditionally used medicinal plant, with a spectrum of secondary metabolites with confirmed healing properties. Centaury is an emerging model in plant developmental biology due to its vigorous regenerative potential and great developmental plasticity when cultured in vitro. Hereby, we review nearly two decades of research on somatic embryogenesis (SE) in centaury. During SE, somatic cells are induced by suitable culture conditions to express their totipotency, acquire embryogenic characteristics, and eventually give rise to somatic embryos. When SE is initiated from centaury root explants, the process occurs spontaneously (on hormone-free medium), directly (without the callusing phase), and the somatic embryos are of unicellular origin. SE from leaf explants has to be induced by plant growth regulators and is indirect (preceded by callusing). Histological observations and culture conditions are compared in these two systems. The changes in antioxidative enzymes were followed during SE from the leaf explants. Special focus is given to the role of arabinogalactan proteins during SE, which were analyzed using a variety of approaches. The newest and preliminary results, including centaury transcriptome, novel potential SE markers, and novel types of arabinogalactan proteins, are discussed as perspectives of centaury research.

Characterization of Aquilaria malaccensis Callus Cells using SEM and Somatic Embryogenesis Associated Genes Identification

Aquilaria malaccensis or gaharu is one of Malaysia’s top tropical resources that is being protected and conserved. Somatic embryogenesis is a plant tissue culture method used most extensively in commercial micropropagation systems and conservation purposes. This study aims to optimize callus induction techniques for somatic embryogenesis study using Scanning Electron Microscope (SEM) and Polymerase Chain Reaction (PCR) analysis. In this study, callus was induced on Murashige and Skoog’s (MS) medium supplemented with a combination of plant growth regulators and optimized parameters (explant used, sterilization techniques, media content, pH media, plant growth regulators, incubation condition, and incubation temperature). The cells were subcultured for long-term callus maintenance and subjected to SEM analysis for somatic embryogenesis confirmation. Four set of genes associated to somatic embryogenesis (SERK, BBM, LEC1, and WOX) were studied based on National Centre for Biotechnology Information database and literatures. The data obtained were used for primer design and gene amplification using 3 different tissues (leaf, stem, callus).The highest frequencies of callus induction were observed on Murashikage and Skoog medium supplemented with 6-Benzylaminopurine and 1-Naphthaleneacetic acid together with optimized growth parameters. SEM analysis showed embryogenic characteristics in cells of the yellow compact calli evidenced by the presence of small and isodiametric cells. Only SERK gene was successfully amplified and enable to proceed with in silico analysis. This study provides fundamental results for genetic conservation effort of A. malaccensis related to somatic embryogenesis study.

Vegetative propagation in vitro with content analysis of ginger oil from calluses of “Jahe Gajah” (Zingiber officinale) on treatments of types of mediums and carbohydrates

Rhizomes of “Jahe Gajah” were made to be explants in this research because of their contents of Ginger Oil were especially liked by consumers as they are not very spicy. The calluses of this research would also be developed for the propagation of the ginger “Jahe Gajah” itself. The purpose of this research was to discover the ideal type of carbohydrate to be added to the mediums MS and VW, in order to increase the biosynthesis process of Ginger Oil in the “Jahe Gajah” calluses. The methode of this research was using Complete Random Design with Factorials of two factors: Factor I : Mediums MS and VW, and Factor II : types of Carbohydrates : glucose, fructose and sucrose. So, there were 6 combinations of treatments, repeated 5 times, with 4 samples each. From those, were research the highest contents of Ginger Oil. The publications of this research were of the type of medium and carbohydrate that can increase the content of secondary metabolites of Ginger Oil. The results of this research were : [1] The highest quantity of calluses was found on the treatment of Medium MS and Glucose (C1M1); [2] The quality of calluses on Medium MS and Sucrose (C3M1) & Fructose (C2M1) were friable calluses with embryogenic characteristics, while on other treatments the calluses were compact and organogenic; [3] Embryogenic calluses are friable, and can be developed into ginger planlets; [4] The calluses of “Jahe Gajah” that were compact and organogenic (C2M2) had the highest content of Ginger Oil (Gingerol – Shogaol – Zingerone 0.184 – 0.118 – 0.982 %)

Arabidopsis LEC1 and LEC2 Orthologous Genes Are Key Regulators of Somatic Embryogenesis in Cassava

High genotype-dependent variation in friable embryogenic callus (FEC) induction and subsequent somaclonal variation constitute bottlenecks for the application and scaling of genetic transformation (GT) technology to more farmer- and industry-preferred cassava varieties. The understanding and identification of molecular factors underlying embryogenic development in cassava may help to overcome these constraints. Here, we described the Arabidopsis thaliana LEAFY COTYLEDON (LEC) LEC1 and LEC2 orthologous genes in cassava, designated as MeLEC1 and MeLEC2, respectively. Expression analyses showed that both, MeLEC1 and MeLEC2, are expressed at higher levels in somatic embryogenic (SE) tissues in contrast with differentiated mature tissues. The rapid expression increase of MeLEC genes at early SE induction times strongly suggests that they are involved in the transition from a somatic to an embryonic state, and probably, in the competence acquisition for SE development in cassava. The independent overexpression of the MeLEC genes resulted in different regenerated events with embryogenic characteristics such as MeLEC1OE plants with cotyledon-like leaves and MeLEC2OE plants with somatic-like embryos that emerged over the surface of mature leaves. Transcript increases of other embryo-specific regulating factors were also detected in MeLECOE plants, supporting their mutual interaction in the embryo development coordination. The single overexpression of MeLEC2 was enough to reprogram the vegetative cells and induce direct somatic embryogenesis, which converts this gene into a tool that could improve the recovery of transformed plants of recalcitrant genotypes. The identification of MeLEC genes contributes not only to improve our understanding of SE process in cassava, but also provides viable alternatives to optimize GT and advance in gene editing in this crop, through the development of genotype-independent protocols.

HISTOLOGICAL ANALYSIS OF INDIRECT SOMATIC EMBRYOGENESIS INDUCED FROM ROOT EXPLATS OF OIL PALM (Elaeis guineensis Jacq)

ABSTRACT Oil palm is economically important as a crop with high oil production. Indirect somatic embryogenesis in oil palm requires a long time for callus induction and plant formation, and it is important to study the embryogenic potential of calli and the mechanisms of somatic embryogenesis. The aim of this study was to test different growth regulators and spermine in induction of embryogenic calli in root explants of oil palm (Elaeis guineensis Jacq). Apex root explants of approximately 0.5 cm were isolated from plants cultivated in vitro and inoculated in Y3 culture medium in the following treatments: A - without growth regulators; B - 1 mg.L-1 picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid); C - 1 mg.L-1 picloram and 2 mg.L-1 2ip (2-isopentenyladenine); D - 2 mg.L-1 2ip; E - 1 mg.L-1 picloram and 2 mg.L-1 BAP (6-benzylaminopurine); F - 2 mg.L-1 BAP; and G - 14.5 mg.L-1 spermine. After six months of culturing, the calli induced in the treatments were analyzed by light microscopy. The calli induced in the treatments with 1 mg.L-1picloram (B) and treatment with 14.5 mg.L-1spermine (G) exhibited embryogenic characteristics, small and isodiametric cells, forming agglomerates, besides a large amount of starch. Calli of the best treatment (Y3 com 1 mg.L-1 de picloram) were inoculated in Y3 culture medium without addition of growth regulators. After eight months, calli were once more analyzed under light microscopy. All the treatments showed callus formation, except for treatments D and A. Calli of treatment B exhibited cells with embryogenic characteristics that developed somatic embryos.

Morpho and Cytological Differentiation of Calli of Eucalyptus grandis x Eucalyptus urophylla During Somatic Embryogenesis

The aim of this study was to induce and analyze embryogenic calli from two types of explants (leaves and meristems) of the hybrid Eucalyptus grandis x Eucalyptus urophylla. Leaves and meristems of plants kept in a nursery were disinfected and inoculated in Petri dishes containing MS culture medium supplemented with different concentrations of the growth regulator dicamba (1.13, 4.52, and 9.04 µM) and without it. At 60 days of culturing, the calli were analyzed by scanning electron microscopy and at 90 days were evaluated by light microscopy in regard to the embryogenic characteristics of the cells. Different type of calli were induced in leaf explants, designated as Type I with light yellow coloring, Type II with dark yellow coloring, and Type III of brown coloring; however, only Type I had embryogenic characteristics. In the meristematic explants, only one type of callus was induced, and it had embryogenic characteristics. At 90 days of culturing, the formation of somatic embryos in the different embryogenic stages was observed and the formation of procambium, protoderm, and ground meristem tissues. At 150 days of culturing, the concentration of 1.13 µM of dicamba was prominent in the formation of somatic embryos in the different embryogenic stages.

Histodifferentiation of oil palm somatic embryo development at low auxin concentration.

Large-scale propagation of oil palm (Elaeis guineensis, Jacq.) is difficult due to its single apical meristem. Thus, obtaining plants is mainly through seed germination, and a long growing period is required before oil production is possible. An alternative to large-scale seedling production is indirect somatic embryogenesis. The aim of this study was to analyze the somatic embryogenesis process in oil palm (E. guineensis Jacq.) with amino acids and low concentrations of auxins. The Tenera hybrid was analyzed by cytochemical and ultrastructural methods and was used to regenerate oil palm plants. First, calli were induced in MS culture media supplemented with 2,4-D and picloram. Two types of calli were obtained, characterized by beige or translucent color. Beige calli had embryogenic characteristics, such as large nuclei with prominent nucleoli, and they were multiplied for 8months in MM culture (half strength MS, 1mgL-1 2,4-D, 2mgL-1 2iP, 1mgL-1 IBA, 250mgL-1 citric acid, 10mgL-1 cysteine, 100mgL-1 inositol, 1mgL-1 thiamine, 1mgL-1 pyridoxine, 1mgL-1 nicotinic acid, 1mgL-1 glycine, 200mgL-1 malt extract, and 100mgL-1 casein hydrolysate). After multiplication, the MCB culture medium (half strength MS, supplemented with 0.25mgL-1 NAA, 2mgL-1 BAP, MM vitamins and 200mgL-1 malt extract, and 100mgL-1 casein hydrolysate) was the most efficient for embryo formation, showing meristematic centers with totipotent cells in histochemical analyses. The somatic embryos were developed and germinated in MG medium (half strength MS, 0.45mgL-1 IAA, 0.25mgL-1 BAP, and MM vitamins), transplanted into polyethylene tubes containing pine bark substrates, and acclimatized in a greenhouse, achieving a 97% survival rate. The use of picloram for callus induction and somatic embryogenesis is advantageous and multiplication in MM medium is an important step for increasing cell mass. The calli with light beige color and nodular structures have meristematic cells with dense cytoplasm and totipotential features that later give rise to protoderm, procambium, and ground meristem during the globular, cordiform, and torpedo embryogenesis phases. In MCB medium, the concentration of vitamins and amino acids are crucial for somatic embryogenesis.

Somatic embryogenesis and metabolic differences between embryogenic and non-embryogenic structures in mangosteen

Somatic embryogenesis in mangosteen (Garcinia mangstana L.) was investigated using seed and leaf segments cultured on Murashige and Skoog medium with treatments of 6-benzyladenine (BA) [2.0, 3.0, 4.0 µM] and 2,4-diclorophenoxyacetic acid (2,4-D) [4.5, 9.0, 13.5 µM]. There were four types of structures (globular, nodular compact, friable and spongy) formed. Two treatments resulted in embryogenic characteristics from seed cultures; the highest percentage 46.67 % of globular structure (resembling somatic embryos) grown on 3.0 µM BA and 80 % of nodular compact structures on 4.0 µM BA + 13.5 µM 2,4-D. For the leaf culture, highest percentage, 93.33 % produced nodular compact structures on 2.0 µM BA + 4.5 µM 2,4-D. Histological analysis showed that the globular structure has well-defined protoderm and separated from the original explant. Nodular compact structure also showed the presence of densely cytoplasmic meristematic cells with a high nucleoplasmic ratio. These characteristics observed in globular and nodular compact structure indicates somatic embryo formation. The globular structures which were converted into shoots and roots (60.00 %) showed atypical somatic embryogenesis in mangosteen. Metabolite fingerprinting was carried out using gas chromatography–mass spectrometry. Amino acids, carbohydrates, organic acids and fatty acids were found in both the embryogenic structures and non-embryogenic structures tested. Multivariate discriminant analyses of the metabolic data revealed significant metabolites (P ≤ 0.05) for both types of structures. Principle component analysis suggested that amino acids and carbohydrates were the major compounds distinguishing embryogenic and non-embryogenic structures. Ornithine and mannose were present at significant level in embryogenic structures as compared to non-embryogenic ones while fructose was significantly higher in non-embryogenic structures.

Indução, análises morfológicas e ultraestruturais de calos de maracujazeiro nativo

A análise de calos que apresentem características embriogênicas é importante para posterior regeneração, in vitro, de espécies com características agronômicas desejáveis, como o maracujazeiro nativo Passiflora gibertii. Diante do exposto, objetivou-se, com este trabalho, analisar a indução de calos oriundos de explantes foliares de Passiflora gibertiiN. E. Brown, bem como caracterizá-los, morfológica e ultraestruturalmente. Para obtenção de calos, folhas cotiledonares foram inoculadas, em meio de cultura, suplementado com picloram e 2,4-D, combinados com cinetina. Após 30 dias em meio de cultura, no escuro, os calos obtidos foram preparados para a visualização em microscopia eletrônica (transmissão e varredura) e microscopia de luz. Os resultados permitem afirmar que a adição de picloram e cinetina ao meio de cultura promove maior formação de calos em explantes foliares de P. gibertii que 2,4-D e cinetina. O regulador 2,4-D proporciona a obtenção de calos com células de formato isodiamétrico, pequenas e com pequeno espaço intercelular, sistema celular organizado e predominância de mitocôndrias de formato arredondado. Já com a utilização do regulador de crescimento picloram, observa-se a predominância de células grandes e de formato alongado, de espaços intercelulares, de sistema celular desorganizado e de mitocôndrias de formato alongado.

Cell Viability, Mitotic Index and Callus Morphology of Byrsonima verbascifolia (Malpighiaceae)

The combined analysis of cell viability, mitotic index and callus morphology of species of commercial interest may favor the identification of cell masses with embryogenic characteristics at early stages of cultivation, allowing the development or improvement of protocols for in vitro plant regeneration through indirect embryogenesis. Byrsonima verbascifolia L. (DC.) (Malpighiaceae) is a Brazilian medicinal species native to the Cerrado, which has a low seed germination rate due to tegument dormancy. This study aimed to identify cells with embryogenic characteristics during growth of callus of B. verbascifolia, by assessing cell viability, mitotic index and cell morphology. Callus was induced from leaf explants and inoculated on MS medium with 4.52 μM 2,4-D and 4.44 μM benzylaminopurine (BAP). Friable calli were obtained at four distinct growth phases: lag (until the 30th), exponential (from 31st to the 70th day), linear (from 71st to the 100th day) and decline (from 101st to the 120th day). Calli with 20 and 30 days showed cells with embryogenic characteristics, that is, isodiametric shape, juxtaposed, with small vacuoles, thin cell wall and presence of amyloplasts. After 120 days, calli were mostly elongated cells with lower cell viability and mitotic index.

Morphological characteristics and cell viability of coffee plants calli

The aim of this research was to characterize and compare two types of calli from leaf explants of Coffea arabica (cultivar Catiguá). Cells of different types of callus were successfully characterized regarding viability and internal and external morphological characteristics. It was obtained two morphologically distinct types of callus: (i) yellow friable and (ii) transparent watery. The yellow friable calli showed higher cell viability and embryogenic characteristics. Scanning and transmission electron microscopy showed embryogenic characteristics in cells of the yellow friable calli evidenced by the presence of small and isodiametric cells, while transparent watery calli showed elongated cells and large cytoplasm vacuolization.

Induction and characterization of oil palm (Elaeis guineensis Jacq.) pro-embryogenic masses

Oil palm is one of the most economically valuable oil seed plants, but the expansion of plantations has been limited by availability of seedlings, as the conventional propagation is through seeds, which have low germination rates. One possible solution for the large-scale production is the use of somatic embryogenesis. The aim of this study was evaluate the effects auxins 2,4-D and picloram on the induction of pro-embryogenic masses in E.guineenesis hybrid leaf explants and characterize, regarding embryogenic characteristics, with cytochemical and ultrastructural analysis. Specifically, in vitro plantlets leaves fragments were inoculated in Y3 culture medium supplemented by 2.4-D or picloram at different concentrations (0.0, 1.0, 3.0, 6.0 and 9.0 mg l⁻¹). After 90 days the presence/ absence of cell masses were evaluated. Both growth regulators efficiently induced cellular masses regardless of the concentrations applied. As the cell masses were not homogeneously formed, they were classified according to color and shape into four types: TYPE 1--elongated and translucent, TYPE 2--uneven and translucent, TYPE 3--globular and beige, TYPE 4--globular and white. Based on the anatomical and ultrastructural features, TYPE 2, 3 and 4 cell masses were considered to have the highest embryogenic potential and therefore may be most suited to large-scale vegetative propagation of oil palm.

Morphogenetic potential of native passion fruit (Passiflora gibertii N. E. Brown.) calli

Some species of non-cultivated passion fruit plant have important contributions to genetic improvement. However, there are few studies concerning about embryogenic and organogenic calli mainly related with structural alterations during their development. The objective of this work was to characterize, structurally the callogenesis on leaf explants of Passiflora gibertii N. E. Brown. The cotyledons were inoculated in MS culture medium, containing half salt concentration, supplemented with sucrose (3 %), and picloram+kinetin for the calli formation. Different calli colors were obtained and used for structural analyses. The calli colors were translucent, white, dark-yellow, white-brown, light-yellow, and white-yellow. After 30 days of cultivation, the calli were fixed in Karnovsky and prepared for the visualization under the scanning and transmission electron microscope and optic microscope. Translucent and light-yellow calli did not present morphogenic characteristics. The cells had different shapes forming non-organized cellular system and the absence or reduced starch content. On the other hand, white and dark-yellow calli manifested embryogenic characteristics such as small isodiametric cells, an organized cellular, dense cytoplasm rich in mitochondria and endoplasmic reticulum, small vacuole and significant starch contend. The culture medium supplemented with 4.14 μM of picloram +0.46 μM of kinetin is the most suitable to induce embryogenic cells.

Morphological and polyamine content changes in embryogenic and non-embryogenic callus of sugarcane

Differences in competence acquisition and subsequent embryo maturation in embryogenic and non-embryogenic callus of sugarcane var. SP79-1011 were evaluated using histomorphological analysis, growth curves, numbers of somatic embryos, and polyamine contents. Embryogenic callus was formed by cells with embryogenic characteristics such as a rounded shape, prominent nuclei, a high nucleus: cytoplasm ratio, small vacuoles and organized globular structures. However, non-embryogenic callus presented dispersed, elongated and vacuolated cells with a low nucleus: cytoplasm ratio; these characteristics did not allow for the development of somatic embryos even upon exposure to a maturation stimulus. These results suggest that non-embryogenic callus does not acquire embryogenic competence during induction and that maturation treatment is not sufficient to promote somatic embryo differentiation. The use of activated charcoal (AC; 1.5 g L−1) resulted in a higher somatic embryo maturation rate in embryogenic callus but did not yield success in non-embryogenic callus. Embryogenic callus incubated with control (10 μM 2,4-dichlorophenoxyacetic acid) and maturation (1.5 g L−1 AC) treatments for 28 days showed similar patterns of total free polyamines; these results differed from the results observed with non-embryogenic callus, suggesting that embryogenic callus already exhibits a characteristic pattern of endogenous polyamine levels. At 28 days of culture with maturation treatment, embryogenic callus exhibited significantly higher levels of free Spm than embryogenic callus incubated with control treatment and non-embryogenic callus incubated with both treatments. This result suggests that Spm could be important for the acquisition of embryogenic competence and somatic embryo maturation in sugarcane var. SP79-1011.

Índice mitótico e viabilidade celular de calos embriogênicos de murici-pequeno

Byrsonima intermedia is a woody and medicinal species occurring in the Brazilian Cerrado. The objective of this research was to determine the pattern of division and viability of cells with embryogenic characteristics. Leaf segments were inoculated on MS medium supplemented with 1.0 mg L-1 2,4-D. Subcultures were performed three times, all at sixty days, resulting in three subcultures. We assessed cell viability and mitotic index of cells with embryogenic characteristics, the second subculture. The second subculture at 20 and 40 days, higher rates of cell division. To 80 days of incubation of calluses, the highest percentage of viable cells with embryogenic characteristics occurs.

Ultrastructural calli analysis of Inga vera Willd. subsp. Affinis (DC.) T.D. Penn

Subcellular changes are relevant to understand plant organogenesis and embryogenesis in the early stages of cell development. The cytology during cell development in tissue culture is however still poorly characterized. This study aimed to characterize the ultrastructural differences related to callogenesis of anthers, ovaries, leaf and nodal segments of Inga vera Willd. subsp. Affinis (DC.) T.D. Penn. Flower buds, nodal segments and leaves were disinfected and inoculated in test tubes containing MS medium with 3% sucrose and 4.5µM 2.4-D, except for leaf callogenesis, where 9µM of this auxin was used, and for the callogenesis of anthers and ovaries, where the culture medium was enriched with 0.25% activated charcoal and 90µM PVP. After 45 days in culture medium, the anther, ovary, leaf and nodal segment calli were fixed in Karnovisky and prepared for visualization by scanning and transmission electron microscopy. Ultrastructural differences were observed among the callus cells of anthers, ovaries, segments and leaves. There was no evidence of somatic embryo formation in the anther, leaf and nodal segment calli, in spite of some embryogenic characteristics in the cells. The ovary calli, with indications of embryo formation, seem to be the most responsive explant source for embryogenesis.

Somatic embryogenesis and the effect of particle bombardment on banana Maçã regeneration

A plant regeneration method with cell suspension cultures of banana, and the effect of biobalistic on regeneration potential are described in this report. Somatic embryos of banana were obtained from indirect embryogenesis of male inflorescence of banana cultivar Maçã (AAB group). Part of the calluses formed (40%) showed embryogenic characteristics (nonfriable, compact and yellow color). The cell suspension, originated from embryogenic calluses, contained clusters of small tightly packed cells with dense cytoplasms, relatively large nuclei and very dense nucleoli. After four months of culture, somatic embryos started to regenerate. The maximum number of regenerated plants was observed between 45 and 60 days after embryo formation.In the first experiment, 401 plants were regenerated from approximately 10 mL of packed cells. In the second experiment, 399 plants were regenerated from a cell suspension six months older than that of the first experiment. Cell transformation using particle bombardment with three different plasmid constructions, containing the uid-A gene, resulted in a strong GUS expression five days after bombardment; however, plant regeneration from bombarded cells was much lower than nonbombarded ones.

Embriogênese somática in vitro em couve-comum

O objetivo deste estudo foi obter calos e embriões somáticos de couve-comum (Brassica oleracea L., var. leucocephala) e caracterizá-los por meio de observações histológicas. Foram cultivadas, in vitro, explantes foliares, sob condições assépticas, em meio nutritivo MS, suplementado com várias combinações entre 2,4-D/BAP e 2,4-D/KIN. Na fase de indução de calos (fase I), houve o desenvolvimento de calos com características embriogênicas nos meios suplementados com 5,0 mg/L de 2,4-D; 1,0 mg/L de 2,4-D e 0,1 mg/L de BAP; 1,0 mg/L de 2,4-D e 1,0 mg/L de BAP e 2,0 mg/L de 2,4-D e 0,05 mg/L de BAP, que foram selecionados para o subcultivo em meio de regeneração (fase II). Nesta fase, foram utilizados meios de cultura suplementados com reduzidas concentrações de 2,4-D, BAP e AG3, ou totalmente desprovidos destes, em que os calos permaneceram por mais 30 dias em cultivo. Os calos subcultivados nos meios de regeneração com 0,1 mg/L de BAP (R3) e 0,01 e 0,001 mg/L de BAP e 2,4-D respectivamente (R8), provenientes dos meios para indução, acrescidos de 5,0 mg/L de 2,4-D (M16) e 1,0 e 1,0 mg/L de BAP e 2,4-D (M30), desenvolveram estruturas semelhantes a embriões. No entanto, a caracterização histológica revelou a superioridade da interação entre os meios M30 e R8, para a regeneração de calos e embriões somáticos.