Germinação de sementes in vitro e desenvolvimento de plantas ex vitro de algodãozinho-do-campo

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

Difference in competence for in vitro proliferation and ex vitro growth of genetically identical mature and juvenile clones of apomictic Malus species

The apomictic system in Malus wits Used Is a model to examine rejuvenation by generating genetically identical tissue culture lines that had two entirely different developmental origins: either embryo-derived tissues (juvenile clones) or somatic tissue from the adult/mature tree (mature clones). These two lines were then subsequently used to examine in vitro difference between mature (M) and juvenile (J) tissues in potential for shoot, root proliferation and ex vitro (glasshouse) growth. The M clones of M. hupehensis and M. toringoides in vitro had significantly fewer total shoots and shoot more than 2 cm in length per proliferating explant than the J clones and also rooted less efficiently. Ex vitro (glasshouse) juvenile clones had shorter internodes, a greater number of leaves and more dry weight compared to their mature counterparts.

Quantitative determination of anti-fungal and insecticide amides in adult plants, plantlets and callus from Piper tuberculatum by reverse-phase high-performance liquid chromatography

A rapid, sensitive and reliable reverse-phase HPLC method was used for the quantitative determination of the anti-fungal and insecticide amides, dihydropiplartine (1), piplartine (2), Delta(alpha,beta)-dihydropiperine (3) and pellitorine (4) in plants in natura, in plantlets in vitro and ex vitro, and in callus of Piper tuberculatum. Well-resolved peaks were obtained with good detection response and linearity in the range of 15.0-3000 mug/mL. The plants in natura contained compounds 1-4, the plantlets ex vitro and in vitro accumulated compounds 1-2 and 1-4, respectively, while only amide 4 was found in callus. Copyright (C) 2003 John Wiley Sons, Ltd.

Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo

(Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo). The present work characterized and compared the anatomical structures of the leaves of Bactris gasipaes (Arecaceae) plants grown under different cultivation conditions (in vitro, ex vitro and in vivo) with the goal of identifying the origins of the difficulties encountered in acclimatizing micro-plants. The Quant program was used to determine leaf tissue thicknesses and areas, and histochemical tests were performed on leaf sections and analyzed using light microscopy. Stomatal and trichome densities were determined using the epidermal impression method and by scanning electronic microscopy. Our results indicated that there were no discernible alterations of the anatomical characteristics of the leaves of micro-plants cultivated under differing conditions and that the thickening of the mesophyll and the vascular fibers indicated adaptive responses to ex vitro conditions. As such, the observed difficulties in acclimatizing peach palm micro-plants to ex vitro conditions cannot be attributed to plant anatomical characteristics acquired during in vitro cultivation.

In vitro conservation and low cost micropropagation of Cochlospermum regium (Mart. Ex. Scharank)

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

In vitro propagation of Corymbia torelliana x C. citriodora (Myrtaceae) via cytokinin-free node culture

Hybrids between Corymbia torelliana (F.Muell.) K.D.Hill & L.A.S.Johnson and C. citriodora subsp. variegata (F.Muell.) A.R.Bean & M.W.McDonald are used extensively to establish forestry plantations in subtropical Australia. Methods were developed for in vitro seed germination, shoot multiplication and plantlet formation that could be used to establish in vitro and ex vitro clone banks of juvenile Corymbia hybrids. Effects of sodium hypochlorite concentration and exposure time on seed contamination and germination, and effects of cytokinin and auxin concentrations on shoot multiplication and subsequent rooting, were assessed. A two-step surface sterilisation procedure, involving 70% ethanol followed by 1% sodium hypochlorite, provided almost no contamination and at least 88% germination. A novel method of cytokinin-free node culture proved most effective for in vitro propagation. Lateral bud break of primary shoots was difficult to induce by using cytokinin, but primary shoots rooted prolifically, elongated rapidly and produced multiple nodes in the absence of exogenous cytokinin. Further multiplication was obtained either by elongating lateral shoots of nodal explants in cytokinin-free medium or by inducing organogenic callus and axillary shoot proliferation with 2.2 µm benzyladenine. Plantlets were produced using an in vitro soil-less method that provided extensive rooting in sterile propagation mixture. These methods provide a means for simultaneous laboratory storage and field-testing of clones before selection and multiplication of desired genotypes.

Crescimento in vitro e aclimatização de Cattleya loddigesii Lindley (Orchidaceae) com carvão ativado sob dois espectros luminosos

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

Concentrações de sacarose no desenvolvimento in vitro e na aclimatização de Cattleya loddigesii Lindley

This work aimed to study the influence of sucrose in the culture medium for in vitro growth and acclimatization of the epiphytic orchid Cattleya loddigesii. Five sucrose treatments (absence, 10, 20, 30 and 40g L-1) were used in a randomic experimental design. Mature seeds were sowed in 1/2 MS culture medium and after 90 days the plantlets (1.0 +/-0.2 cm) were inoculated between the treatments, whereby they were remained more 90 days. After 180 days of the beginning of the experiment the plantlets were removed from the flasks and evaluated the number of roots, shoot length, number of leafs, total dry weight and photosynthetic pigments. Survival percentage was evaluated after 75 days of acclimatization. The data of biometric variables were analyzed by Anovaand polynomial regression (p<0.05). Theothers data were submitted to the Anova and the means compared by the Tukey test (p<0.05). The sucrose concentration of 20g L-1 favored the in vitro growth in all evaluated parameters, showed higher production of chlorophyll a, total chlorophyll and carotenoids, in addition to increased survival under ex vitro condition. The sucrose concentration of 20g L-1 in the culture medium was the most efficient among the tested concentrations both for in vitro growth and ex vitro establishment of Cattleya loddigesii.

In vitro technique for selecting onion FOR white rot disease- resistance

In vitro selection is one of the most effective and efficient techniques for plant improvement. This is due to its ability to isolate plants with the desired character(s), either by applying a selection agent on the culture media to drive the selection of somaclones with the required character(s), or by establishing particular conditions that change in the genomes of somaclones toward the required character. The objective of this study was to identify a suitable protocol for in vitro selection of Allium white rot disease ( Sclerotium cepivorum ) tolerance in commercial Egyptian onion varieties, namely Giza 20, Giza 6 and Beheri Red. Oxalic acid (OA), the phytotoxin produced by Sclerotium cepivorum, was used as the selective agent. Seeds of the three Egyptian varieties were germinated on four concentrations (0.0, 0.02, 0.2, 2 and 20 mM) of Oxalic acid. Among the tested cultivars, Beheri Red had the highest germination frequency (52%) at all concentrations tested, followed by Giza 20 (42.6%), and Giza 6 at (32%). Cotyledon explants from the varieties were cultured on toxic MSBDK medium, supplemented with 0, 3, 6 and 12 mM OA. The survival of calli on MSBDK free toxic medium was 70.7% for all tested cultivars; however, MSBDK-stressed medium, with 3 mM OA reduced the viable calli to 42.1%. The highest OA concentration (12 mM) completely inhibited calli induction from cotyledons explants. A medium supplement with 3 mM OA retarded 80% of calli growth. Among 156 tested calli of Beheri Red, only 23 calli (14.7%) survived on toxic medium for 45 days. Similarly, there was 15.6% survival for Giza 20 calli, while 40.1% of the Giza 6 calli survived. Plantlets were regenerated from surviving calli and transplanted onto ex vitro, and formed bulb after acclimatisation.

Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana × C. citriodora

A protocol was developed for short-term preservation and distribution of the plantation eucalypt, Corymbia torelliana × C. citriodora, using alginate-encapsulated shoot tips and nodes as synthetic seeds. Effects of sowing medium, auxin concentration, storage temperature and planting substrate on shoot regrowth or conversion into plantlets were assessed for four different clones. High frequencies of shoot regrowth (76–100%) from encapsulated explants were consistently obtained in hormone-free half- and full-strength Murashige and Skoog (MS) sowing media. Conversion into plantlets from synthetic seeds was achieved on half-strength MS medium by treating shoot tips or nodes with 4.9–78.4 μM IBA prior to encapsulation. Pre-treatment with 19.6 μM IBA provided 62–100% conversion, and 95–100% of plantlets survived after acclimatisation under nursery conditions. Synthetic seeds containing explants pre-treated with IBA were stored for 8 weeks much more effectively at 25°C than at 4°C, with regrowth frequencies of 50–84% at 25°C compared with 0–4% at 4°C. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile planting substrates. Highest frequencies (46–90%) of plantlet formation from pre-converted synthetic seeds were obtained by transferring shoot tip-derived synthetic seeds onto an organic compost substrate. These plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion of non-embryonic synthetic seeds is a novel technique that provides a convenient alternative to somatic embryo-derived artificial seeds.

In vitro propagation of insectivorous plants for phytochemical purposes

Dissertação mest., Engenharia Biológica, Universidade do Algarve, 2009

In vitro spore germination of Polystichum drepanum, a threatened fern from Madeira Island

Polystichum drepanum (Sw) C. Presl is a threatened fern endemic to a few forest areas in the north-west of Madeira Island. The aims of this work were to establish suitable culture conditions for in vitro germination of spores, and to evaluate short-term storage conditions for P drepanum spores. The highest frequency of germination was obtained in Murishage and Skoog (MS) liquid medium, without agitation. However, gametophytes maintained in MS liquid medium did not grow and, after 4 weeks, became anoxic and died. Thus, after germination in liquid medium, gametophytes were transferred to an MS double-phase culture system for further growth. The effects of storage period, temperature, and relative humidity during storage on in vitro spore germination were studied. Spore viability was assessed after 2, 4 and 6 months, and high viability (> 94%) was observed in all the assays. However, germination capability decreased with increased storage periods. The number of sporophytes obtained also decreased with prolonged storage periods. The results indicate that spores of R drepanum stored for 4 months at 21 degrees C maintain high viability and high germination frequency. ne sporophytes obtained were acclimatised in a mixture of peat and vermiculite [2:1 (v/v)] under high relative humidity (90-95%). Seventy-five sporophytes were successfully acclimatised to ex vitro conditions and showed active growth in the glasshouse.

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)). Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA). Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l(-1)) were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l(-1) 2,4-D and 2 mg.l(-1) BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.