Adriana Pinheiro Martinelli Rodriguez

A NOVEL APPROACH FOR THE DEFINITION OF THE INORGANIC MEDIUM COMPONENTS FOR MICROPROPAGATION OF YELLOW PASSIONFRUIT (PASSIFLORA EDULIS SIMS. F. FLAVICARPA DEG.)

SummaryMineral deficiency symptoms were observed in leaves of yellow passionfruit plantlets grown in MS medium (Murashige and Skoog, 1962) with 1.0 mg l−1 (3.0 μM) gibberellic acid. Initially, leaves showed interveinal chlorosis, followed by bleaching of the leaves and retarded growth. Leaf mineral analysis was done and compared to mineral requirements suggested for passionfruit in the literature. Several modifications were made to the inorganic composition of MS medium, according to mineral deficiencies, mainly of Fe and Ca, and possible toxicity of Cl. The concentration of the elements in the new medium (MSM) was based on the mineral composition of leaves of healthy plants. The chemical equilibrium was checked using the software Geochem (Sposito and Mattigod, 1980) and final adjustments were made to ensure good availability of nutrients. To test the efficiency of the modified medium nodal segments were cultured in both MS and MSM supplemented with 3.0 mg l−1 (13.3 μM) 6-benzyladenine. After three subcultures mineral analysis of the leaves was done. Severe mineral deficiency was observed on the leaves of plantlets cultured in MS, while plantlets cultivated in MSM had green leaves. A comparison of the mineral analysis of plantlets in both media showed a fairly large increase in Ca, Cu, Fe, Mg and S and decrease in levels of B and Cl in plantlets cultivated in MSM. A slight increase or decrease in other elements was also observed. Subculture of the chlorotic plantlets into MSM showed that the visual symptoms of mineral deficiency disappeared in 2–4 wk.

Somatic embryogenesis in Citrus SPP.: Carbohydrate stimulation and histodifferentiation

SummarySomatic embryogenesis from nucellus-derived callus cultures of five cultivars, including three (Caipira, Seleta Vermelha, and Valencia) of sweet oranges (C. sinesis L. Osbeck), Rangpur lime (C. limonia L. Osbeck), and Cleopatra mandarin (C. reticulata Blanco) (lines I and II), were studied. Callus lines maintained on MT medium supplemented with 50 g l−1 sucrose were transferred to MT medium supplemented with different carbohydrate sources: galactose, glucose, lactose, maltose, or sucrose at 18, 37, 75, 110, or 150 mM, or glycerol at 6, 12, 24, 36, or 50 mM. Globular embryos were observed after approximately 4 wk, in several treatments. Cultures of Valencia and Caipira sweet oranges and Cleopatra mandarin (line I) showed high numbers of embryos on medium containing galactose, lactose, and maltose. Histological studies showed somatic embryos in all developmental stages with a normal histodiffeentiation pattern. The other two cultivars (Rangpur lime and Cleopatra mandarin, line II) formed very few embryos, which did not develop further following the globular stage. Some of the abnormalities observed were lack or dedifferentiation of protoderm and absence of apical meristems and procambial strands. Embryos that followed the normal sequence of development were easily converted into plants. Non-embryogenic cultures continued as proliferating callus cultures, eventually forming a few embryos which did not convert into plants. Statistical analyses of the callus response to carbohydrate treatments was done using an overdispersion Poisson model.

In vitro organogenesis in watermelon cotyledons

The objective of this work was to study the in vitro organogenesis of Citrullus lanatus, by the induction of adventitious buds in cotyledon segments cultured in medium supplemented with cytokinin. Explants were collected from one, three and five-day-old in vitro germinated seedlings, considering the distal and proximal cotyledon regions. The data obtained showed that in vitro organogenesis of watermelon occurred with higher efficiency, when cotyledon segments from the proximal region collected from three-day-old seedlings were cultivated in medium MS, supplemented with BAP (1 mg L-1) and coconut water (10%). The histological study showed that the organogenesis occurs directly, without callus formation, on epidermal and subepidermal layers of the explants. Adventitious shoots were characterized by the development of shoot apical meristem and leaf primordia. The formation of protuberances, that do not develop into adventitious buds, was also observed.

A statistical approach to study the dynamics of micropropagation rates, using banana (Musa spp.) as an example

Abstract The use of micropropagation to obtain large numbers of high-quality planting material has increased in recent years. Behavior in culture, mainly in terms of multiplication rate, varies among genotypes, directly affecting plant production planning. To study multiplication rates over time, suckers of banana, Musa spp., cv. Maçã, were collected in the field and the shoot apex introduced in vitro for micropropagation. The number of new shoots produced in each of the six multiplication cycles was recorded and the data analyzed statistically. Variability in total shoot production and differences in multiplication rates was considerable among families, which consisted of the initial explant and its progeny. Moreover, the adjusted Poisson regression models for the number of shoots showed that the multiplication rate in this cultivar tends to decrease with time: after the seventh subculture, new shoots may form at a very low rate. Interpretation of the first and second derivatives of the regression model allowed determination of the maximum speed of multiplication and the time at which the multiplication rate begins to decline.

In vitro morphogenesis of Cucumis melo var. inodorus

In vitro morphogenesis of C. melo L. var. inodorus was studied by the induction of adventitious buds and somatic embryos. Organogenesis was obtained from cotyledon segments and leaf discs in culture medium supplemented with benzylaminopurine (1 mg l−1) and somatic embryogenesis was induced in medium containing 2,4-dichlorophenoxyacetic acid (5 mg l−1) + thidiazuron (1 mg l−1). Through histological analysis it was possible to verify that in cotyledonary explants, protuberances that do not develop into well-formed shoot buds and leaf primordia are more frequently formed than complete shoot buds, resulting in a low frequency of plant recovery in the organogenic process. A high percentage of explants responded with the formation of somatic embryos; the microscopical analysis showed that the somatic embryos lacking well developed apical meristems had a low conversion rate into plants. Plant recovery was not obtained from leaf-disc explants, with high rates of contamination and formation of protuberances which did not develop into shoot buds. Histological sections showed the development of epidermis and leaf hairs, indicating those structures could be leaf primordia; however, these were not associated with a shoot apical meristem.In vitro morphogenesis of C. melo L. var. inodorus was studied by the induction of adventitious buds and somatic embryos. Organogenesis was obtained from cotyledon segments and leaf discs in culture medium supplemented with benzylaminopurine (1 mg l−1) and somatic embryogenesis was induced in medium containing 2,4-dichlorophenoxyacetic acid (5 mg l−1) + thidiazuron (1 mg l−1). Through histological analysis it was possible to verify that in cotyledonary explants, protuberances that do not develop into well-formed shoot buds and leaf primordia are more frequently formed than complete shoot buds, resulting in a low frequency of plant recovery in the organogenic process. A high percentage of explants responded with the formation of somatic embryos; the microscopical analysis showed that the somatic embryos lacking well developed apical meristems had a low conversion rate into plants. Plant recovery was not obtained from leaf-disc explants, with high rates of contamination and formation of protuberances which did not develop into shoot buds. Histological sections showed the development of epidermis and leaf hairs, indicating those structures could be leaf primordia; however, these were not associated with a shoot apical meristem.

EgLFY, the Eucalyptus grandis homolog of the Arabidopsis gene LEAFY is expressed in reproductive and vegetative tissues

The EgLFY gene cloned from Eucalyptus grandis has sequence homology to the floral meristem identity gene LEAFY (LFY) from Arabidopsis and FLORICAULA (FLO) from Antirrhinum. EgLFY is preferentially expressed in the developing eucalypt floral organs in a pattern similar to that described previously for the Arabidopsis LFY. In situ hybridization experiments have shown that EgLFY is strongly expressed in the early floral meristem and then successively in the primordia of sepals, petals, stamens and carpels. It is also expressed in the leaf primordia of adult trees. The expression of the EgLFY coding region under control of the Arabidopsis LFY promoter could complement strong lfy mutations in transgenic Arabidopsis plants. These data suggest that EgLFY plays a similar role to LFY in flower development and that the basic mechanisms involved in flower initiation and development in Eucalyptus may be similar to those occurring in Arabidopsis.

A Floricaula/Leafy gene homolog is preferentially expressed in developing female cones of the tropical pine Pinus caribaea var. caribaea

In angiosperms, flower formation is controlled by meristem identity genes, one of which, FLORICAULA (FLO)/LEAFY (LFY), plays a central role. It is not known if the formation of reproductive organs of pre-angiosperm species is similarly regulated. Here, we report the cloning of a conifer (Pinus caribaea var. caribaea) FLO/LFY homolog, named PcLFY. This gene has a large C-terminal region of high similarity to angiosperm FLO/LFY orthologs and shorter regions of local similarity. In contrast to angiosperms, conifers have two divergent genes resembling LFY. Gymnosperm FLO/LFY proteins constitute a separate clade, that can be divided into two divergent groups. Phylogenetic analysis of deduced protein sequences has shown that PcLFY belongs to the LFY-like clade. Northern hybridization analysis has revealed that PcLFY is preferentially expressed in developing female cones but not in developing male cones. This expression pattern was confirmed by in situ hybridization and is consistent with the hypothesis of PcLFY being involved in the determination of the female cone identity. Additionally, mutant complementation experiments have shown that the expression of the PcLFY coding region, driven by the Arabidopsis LFY promoter, can confer the wild-type phenotype to lfy-26 transgenic mutants, suggesting that both gymnosperm and angiosperm LFY homologs share the same biological role.

Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments

ABSTRACT: Genetic transformation allows the release of improved cultivars with desirable characteristics ina shorter period of time and therefore may be useful in citrus breeding programs. The objective of this researchwas to establish a protocol for genetic transformation of Valencia and Natal sweet oranges ( Citrus sinensis L.Osbeck) and Rangpur lime ( Citrus limonia L. Osbeck). Epicotyl segments of germinated in vitro plantlets(three weeks in darkness and two weeks in a 16-h photoperiod) were used as explants. These were co-cultivated with Agrobacterium tumefaciens strain EHA-105 and different experiments were done to evaluatethe transformation efficiency: explants were co-cultivated with Agrobacterium for one, three or five days;explants were incubated with Agrobacterium suspension for 5, 10, 20 or 40 minutes; co-cultivation mediumwas supplemented with acetosyringone at 0, 100 or 200 µmol L -1 ; Explants ends had a longitudinal terminalincision (2-3 mm); co-cultivation temperatures of 19, 23 or 27°C were imposed. The experimental design wascompletely randomized in all experiments with five replications, each consisted of a Petri dish (100 x 15 mm)with 30 explants and resulted in a total of 150 explants per treatment. Longitudinal terminal incision in theexplant ends did not improve shoot regeneration. However, transgenic plants of all three cultivars were confirmedfrom explants that had been subjected to inoculation time of 20 minutes, co-culture of three days at 23-27°C,in the absence of acetosyringone.Key word

Regeneração in vitro de Passiflora suberosa a partir de discos foliares

In vitro regeneration of plantlets was obtained from Passiflora suberosa leaf discs. Leaves from plants germinated in the greenhouse were collected and immersed in commercial sodium hypochlorite solution (3:1), during 20 minutes. Leaf discs (0.5 cm in diameter) were obtained and placed with the adaxial side in contact with the culture medium in petri dishes. Organogenesis was obtained when MS medium was supplemented with 0.5 or 1.0 mg L-1 BAP (6-benzylaminopurine), after four to eight weeks, callus proliferated from the edge of the discs. After induction, calli were transferred to modified MS media supplemented with1.0 mg L-1 GA3, under 16-hour photoperiod. Development of adventitious shoots was obtained from the callus tissues and the shoots were elongated and rooted in the same culture medium and acclimatized in the greenhouse.

Organogenesis from internodal segments of yellow passion fruit

Organogenesis induction from internodal segments of yellow passionfruit (Passiflora edulis f. flavicarpa) was obtained in a medium supplemented with 1.0-4.0 mg L-1 benzylaminopurine (BAP). Callus proliferation and adventitious shoot development were analyzed by light and scanning electron microscopy. Callus proliferation and adventitious shoots were observed simultaneously and continuously, with asynchronous development of shoots in the explants. Proliferation of callus was observed in the explant cut ends in all treatments, including the control. The results show that concentrations of 1.0 or 2.0 mg L-1 BAP gave better rate of organogenesis. Higher concentrations were efficient for the induction of adventitious buds and, were, however, detrimental for subsequent shoot development. Growth and rooting of shoots were obtained in half strength medium without growth regulators.