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Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery. Key features • Two sgRNAs employed. • Takes 6-12 months to have an edited transgene-free plant. • Setup in tomato.

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Nidularium minutum is an ornamental bromeliad from the Brazilian Rainforest. The micropropagation of this species is essential for obtaining plants available for conservation programs or commercial use. Our study aimed to establish an efficient plant production method by in vitro sprouting. This bromeliad takes a long time to sprout in vitro, and 10% of the plants produce shoots in a culture medium without plant growth regulators (PGRs). When subcultured in a PGR-free medium, these individualized shoots can sprout like the mother plant. The Murashige and Skoog basal medium (MS) containing 1.0 mg L-1 of 6-benzylaminopurine (BAP) promoted the induction of adventitious shoots in greater than 90% of the plants after 240 days of culture with an average of more than eight shoots per plant. Approximately 100% of the in vitro-produced shoots survived after acclimatization, reaching the flowering stage. Therefore, our results showed that in vitro regeneration of N. minutum depends on the cultivation period and that plants with a higher sprouting capacity can be selected and used as micropropagation matrices, contributing to the production of this endangered bromeliad.(AU)

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Somatic embryogenesis is an in vitro plant morphogenetic process due to cell totipotentiality to induce shoot regeneration. To induce this proliferation pathway, we used auxins such as 2,4-dichlorophenoxyacetic acid in combination with cytokinins. There are numerous somatic embryogenesis protocols for a great diversity of plants, including orchids, but none has been yet reported in Vanilla planifolia. Vanilla (V. planifolia) is propagated mainly asexually through cuttings. Under in vitro conditions, it is propagated asexually through direct and indirect organogenesis involving the use of various plant growth regulators in different concentrations. The cell response depends on explant type, culture medium used, and incubation conditions. Direct organogenesis involves de novo formation from differentiated cells; the indirect pathway develops from cell dedifferentiation that produces an explant called "callus." In most cases, this type of cell regeneration uses Benzyladenine. The explants most used in this pathway are shoots, roots, and protocorms, although some studies report the use of other types of explants, including leaves and seeds. Somatic embryogenesis in vanilla has been poorly studied partly because of the recalcitrance of this species. This work mentioned the advances in the in vitro morphogenesis of V. planifolia, mentioning the advantages and disadvantages of each morphogenetic pathway and its characteristics.

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BACKGROUND: Piper hispidinervum is a species native from the Amazon region with great economic potential, given its scientifically proven insecticidal properties. In this study, an efficient protocol of plant regeneration via indirect somatic embryogenesis has been established for the first time. In a first experiment, for the induction of calluses, foliar explants of non-discriminated accesses of P. hispidinervum were inoculated in MS medium supplemented with α-naphtalenacetic acid (NAA) and 6-benzylaminopurine (BAP), in different combinations. For a second experiment, foliar explants from five different accesses of P. hispidinervum (PH17, PH21, PH28, PH37, and PH39) were analyzed regarding the formation of calluses when cultivated in MS medium with 5 mg L-1 NAA + 2.5 mg L-1 BAP. To obtain somatic embryos-like structures, calluses were cultivated in MS medium with 10 mg L-1 NAA + 2.5 mg L-1 of BAP. The somatic embryos-like structures obtained were inoculated in MS medium devoid of growth regulators and the plantlets were subjected to acclimatization. Calluses and somatic embryos-like structures were subjected to anatomical analysis and genetic stability of regenerated plants was analyzed by flow cytometry. RESULTS: The treatments 2.5 mg L-1 BAP and 5 mg L-1 NAA + 2.5 mg L-1 BAP, after 60 days of cultivation, provided each 32% of primary callus, not being verified the formation of calluses in medium devoid of BAP. It was found that accesses differed among them with respect to the formation of primary calluses, with emphasis on accesses PH28, PH37, and PH39, with mean percentage of 95.3%. Regarding the percentage of embryogenic calluses and formation of somatic embryos-like structures, there were no statistical differences between accesses, with mean values of 90.6% and 77.3%, respectively. The somatic embryos-like structures of P. hispidinervum have conspicuous morphoanatomical similarities with the zygotic embryo, and flow cytometry analysis showed no significant variation in nuclear DNA size among plants regenerated in vitro and plants coming from seed germination, which indicates ploidy level stability. CONCLUSION: This protocol is the first cited in the literature that demonstrates an efficient micropropagation process by somatic embryogenesis of P. hispidinervum. It can be used either to enable large-scale vegetative production or to subsidize germplasm conservation or genetic engineering of P. hispidinervum.

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The available methods for plant transformation and expansion beyond its limits remain especially critical for crop improvement. For grass species, this is even more critical, mainly due to drawbacks in in vitro regeneration. Despite the existence of many protocols in grasses to achieve genetic transformation through Agrobacterium or biolistic gene delivery, their efficiencies are genotype-dependent and still very low due to the recalcitrance of these species to in vitro regeneration. Many plant transformation facilities for cereals and other important crops may be found around the world in universities and enterprises, but this is not the case for apomictic species, many of which are C4 grasses. Moreover, apomixis (asexual reproduction by seeds) represents an additional constraint for breeding. However, the transformation of an apomictic clone is an attractive strategy, as the transgene is immediately fixed in a highly adapted genetic background, capable of large-scale clonal propagation. With the exception of some species like Brachiaria brizantha which is planted in approximately 100 M ha in Brazil, apomixis is almost non-present in economically important crops. However, as it is sometimes present in their wild relatives, the main goal is to transfer this trait to crops to fix heterosis. Until now this has been a difficult task, mainly because many aspects of apomixis are unknown. Over the last few years, many candidate genes have been identified and attempts have been made to characterize them functionally in Arabidopsis and rice. However, functional analysis in true apomictic species lags far behind, mainly due to the complexity of its genomes, of the trait itself, and the lack of efficient genetic transformation protocols. In this study, we review the current status of the in vitro culture and genetic transformation methods focusing on apomictic grasses, and the prospects for the application of new tools assayed in other related species, with two aims: to pave the way for discovering the molecular pathways involved in apomixis and to develop new capacities for breeding purposes because many of these grasses are important forage or biofuel resources.

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BACKGROUND: Gymnema sylvestre is a medicinal woody perennial vine known for its sweetening properties and anti-diabetic therapeutic uses in the modern and traditional medicines. Its over-exploitation for the therapeutic uses and to meet the demand of pharmaceutical industry in raw materials supply for the production of anti-diabetic drugs has led to considerable decline in its natural population. RESULTS: An efficient system of shoot bud sprouting from nodal segment explants and indirect plant regeneration from apical meristem-induced callus cultures of G. sylvestre have been developed on Murashige and Skoog (MS) medium amended with concentrations of cytokinins. Of the three growth regulators tested, N6-benzylaminopurine (BAP) was the most efficient and 2.0 mg L-1 gave the best shoot formation efficiency. This was followed by thidiazuron (TDZ) and kinetin (Kin) but, most of the TDZ-induced micro shoots showed stunted growth. Multiple shoot formation was observed on medium amended with BAP or TDZ at higher concentrations. The produced micro shoots were rooted on half strength MS medium amended with auxins and rooted plantlets acclimatized with 87% survival of the regenerates. CONCLUSIONS: The developed regeneration system can be exploited for genetic transformation studies, particularly when aimed at producing its high yielding cell lines for the anti-diabetic phytochemicals. It also offers opportunities for exploring the expression of totipotency in the anti-diabetic perennial vine.

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BACKGROUND: Gymnema sylvestre is a medicinal woody perennial vine known for its sweetening properties and antidiabetic therapeutic uses in the modern and traditional medicines. Its over-exploitation for the therapeutic uses and to meet the demand of pharmaceutical industry in raw materials supply for the production of anti-diabetic drugs has led to considerable decline in its natural population. RESULTS: An efficient system of shoot bud sprouting from nodal segment explants and indirect plant regeneration from apical meristem-induced callus cultures of G. sylvestre have been developed on Murashige and Skoog (MS) medium amended with concentrations of cytokinins. Of the three growth regulators tested, N6-benzylaminopurine (BAP) was the most efficient and 2.0 mg L-1 gave the best shoot formation efficiency. This was followed by thidiazuron (TDZ) and kinetin (Kin) but, most of the TDZ-induced micro shoots showed stunted growth. Multiple shoot formation was observed on medium amended with BAP or TDZ at higher concentrations. The produced micro shoots were rooted on half strength MS medium amended with auxins and rooted plantlets acclimatized with 87% survival of the regenerates. CONCLUSIONS: The developed regeneration system can be exploited for genetic transformation studies, particularly when aimed at producing its high yielding cell lines for the anti-diabetic phytochemicals. It also offers opportunities for exploring the expression of totipotency in the anti-diabetic perennial vine.

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ABSTRACT The present study reports a shoot organogenesis-based system for in vitro regeneration of Passiflora miniata, an Amazonia passion fruit species. Root segments were cultured in Murashige and Skoog (MS) medium supplemented with different concentrations (range 2-9 µM) of 6-benzyladenine (BA); thidiazuron (TDZ) or kinetin (KIN). Plant growth regulators were not added to the control treatment. Root explants have showed a high regenerative potential. After 30 days of in vitro culture, the root explants showed several shoots formed direct and indirectly. TDZ provided the best response in the differentiation adventitious shoots, mainly in the presence of 6.8 µM. The cytokinins BA and KIN responded producing a reduced number of shoots. After 120 days, rooted regenerated plants were transferred to a greenhouse for acclimatization. This regeneration system opens new perspectives for micropropagation and conservation of this wild Amazonic passion fruit species.

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Common bean Phaseolus vulgaris L. has been shown to be a recalcitrant plant to induce somatic embryogenesis (SE) under in vitro conditions. An alternative strategy to yield SE is based upon the use of a cytokinin (benzyladenine) coupled with osmotic stress adaptation instead of the auxin-inducing SE in common bean. Here we described the induction of proembryogenic masses (PEM) derived from the apical meristem and cotyledonary zone of zygotic embryos, from which secondary SE indirect embryogenesis emerged. Maturation of SE was achieved by using osmotic stress medium and converted to plants. Long-term recurrent SE was demonstrated by propagation of PEM at early stages of SE. This protocol is currently being applied for stable genetic transformation by means of Agrobacterium tumefaciens and biobalistics as well as basic biochemical and molecular biology research.

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Harsh environmental conditions in arid ecosystems limit seedling recruitment to microhabitats under nurse structures, such as shrubs or rocks. These structures, however, do not necessarily afford the same benefits to plants because nurse rocks provide only physical nurse effects, whereas nurse plants can provide both physical and biological nurse effects. Nevertheless, if the nurse plant is a conspecific, the benefits it provides may be outweighed by higher mortality due to negative density-dependent processes; consequently, negative density-dependence is expected to limit plants from acting as nurses to their own seedlings. The degree to which an abiotic nurse may be more beneficial than a conspecific one remains largely unexplored. Here, we examine the role and elucidate the mechanisms by which conspecific plants and rocks promote plant establishment in a hyper-arid desert. For 4 years, we examined establishment patterns of Myrcianthes coquimbensis (Myrtaceae), a threatened desert shrub that recruits solely in rock cavities and under conspecific shrubs. Specifically, we characterized these microhabitats, as well as open interspaces for comparison, and conducted germination, seed removal and seedling survival experiments. Our results revealed that conspecific shrubs and nurse rocks modified environmental conditions in similar ways; soil and air temperatures were lower, and water availability was higher than in open interspaces. We found no evidence on negative density-dependent recruitment: seed removal was lowest and seedling emergence highest under conspecific plants, moreover seedling survival probabilities were similar in rock cavities and under conspecific plants. We conclude that the probability of establishment was highest under conspecific plants than in other microhabitats, contrasting what is expected under the Janzen-Connell recruitment model. We suggest that for species living in stressful environments, population regulation may be a function of positive density-dependence and intraspecific facilitation may be a process that promotes the persistence of some plant species within a community.

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The Translationally Controlled Tumor Proteins, or TCTP, is a superfamily of exclusively eukaryotic proteins essential in the regulation of proliferation and general growth. However, it is clear that these are multifunctional proteins given (1) the pleiotropic effects of its mutations, and (2), the multiple processes in which this protein is involved. TCTP function in general is conserved, since Arabidopsis AtTCTP1 can rescue a Drosophila mutant, and vice versa. It has become clear, however, that these proteins may have "taxon-specific" functions. In the case of plants, mRNA and/or proteins have been found in the phloem translocation stream of different species, suggesting a role in long-distance signaling. We have found that a second Arabidopsis TCTP gene, AtTCTP2, codes for a protein that moves long-distance through a graft union in tobacco. Interestingly, the mRNA is also transported long-distance. Both mRNA and protein move long-distance; interestingly, the movement, while more efficient from source to sink tissues, also occurs in the opposite direction. The protein reaches the nuclei of parenchyma cells and adventitious roots. Furthermore, it is clear that the long-distance delivery of AtTCTP2 protein and mRNA is required for the induction of adventitious roots. A model is presented that accounts for these observations.

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ABSTRACT Shoot regeneration, callus growth, and biosynthesis of shikonin in callus cultures of Onosma sericeum were examined. Plant tissue culture was used as an alternative method for increasing the production of shikonin, a secondary metabolite. The isolated cultures were subjected to abiotic factors such as light, plant growth regulators, and nutritional factors. Identification was carried out by High- Performance Liquid Chromatography (HPLC) after 10th subculture. Nodal explants were incubated in Murashige and Skoog (MS) medium along with different combination of growth hormones. Shoot regeneration from calli were achieved on MS basal medium supplemented with 3 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l Naphthalene acetic acid (NAA) under light cycle. Shikonin was formed in dark culture. Calli grown on MS (ammonium ion-free) medium supplemented with 3 mg/l BAP and 0.5 mg/l NAA contained the maximum shikonin level (15.26 µg/mg DW). Minimum shikonin content (9.85 µg/mg DW) was observed in calli cultured on MS (ammonium ion-free) medium supplemented with 3 mg/l BAP and 0.5 mg/l indole-3-acetic acid (IAA). In establishing cell culture, the ammonium ion, and light cycle inhibited shikonin formation. This is the first report on the establishment of isolated cultures of O. sericeum for shikonin production and callus growth.

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The Translationally Controlled Tumor Protein (TCTP) is a central regulator of cell proliferation and differentiation in animals, and probably also in plants. Arabidopsis harbors two TCTP genes, AtTCTP1 (At3g16640), which is an important mitotic regulator, and AtTCTP2 (At3g05540), which is considered a pseudogene. Nevertheless, we have obtained evidence suggesting that this gene is functional. Indeed, a T-DNA insertion mutant, SALK_045146, displays a lethal phenotype during early rosette stage. Also, both the AtTCTP2 promoter and structural gene are functional, and heterozygous plants show delayed development. AtTCTP1 cannot compensate for the loss of AtTCTP2, since the accumulation levels of the AtTCTP1 transcript are even higher in heterozygous plants than in wild-type plants. Leaf explants transformed with Agrobacterium rhizogenes harboring AtTCTP2, but not AtTCTP1, led to whole plant regeneration with a high frequency. Insertion of a sequence present in AtTCTP1 but absent in AtTCTP2 demonstrates that it suppresses the capacity for plant regeneration; also, this phenomenon is enhanced by the presence of TCTP (AtTCTP1 or 2) in the nuclei of root cells. This confirms that AtTCTP2 is not a pseudogene and suggests the involvement of certain TCTP isoforms in vegetative reproduction in some plant species.

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Celosia argentea (Var.) cristata (Amaranthaceae) is a widely cultivated ornamental plant, which has antibacterial, astringent, haemostatic, hypertensive, ophthalmic, and parasitic significance. This study describes a protocol for in vitro callus induction and plant regeneration from leaf and stem explants of C. argentea using Murashige and Skoog (MS) medium. Callus culture was initiated and established from seedling, leaf, and stem explants. Explants were cultured on MS medium supplemented with auxin alone (0.5 mg/L Naphthaleneacetic acid (NAA), 2, 4-Dicholorophenoxyacetic acid (2, 4-D). Green and red compact callus (98%) were induced using MS medium supplemented with 0.5 mg/L NAA and 1.0 mg/L Benzyladenine (BA). Two different concentrations (1.0 mg/L BA + 0.5 mg/L NAA and 1.0 mg/L BA + 1.0 mg/L NAA) successfully induced plant regeneration with multiple shoots (1.5 and 0.9 shoots per explants, respectively). Successful shoots were transferred to rooting medium supplemented with 1.0 mg/L Indole-3-acetic acid (IAA) (80%) at 35th day. Acclimatization was done, which resulted in 90% of the plantlets surviving in garden soil. This protocol could be used to micropropagate C. argentea for conservation, commercial natural product production. The high frequency of callus indicated potential of C. argentea for secondary metabolite production (celosin) in pharmaceutical industry.

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Recent evidence has shown that primates worldwide use agroecosystems as temporary or permanent habitats. Detailed information on how these primates are using these systems is scant, and yet their role as seed dispersers is often implied. The main objective of this study was to compare the activity, foraging patterns and seed dispersal role of black howler monkeys (Alouatta pigra) inhabiting shaded cocoa plantations and rainforest in southern Chiapas, Mexico. We gathered data on three monkey groups living in shaded cocoa plantations and three groups living in rainforest, using focal sampling, and collecting fecal samples. General activity and foraging patterns were similar in both habitats, with the exception that monkeys in the cocoa habitat spent more time feeding on petioles. Monkeys in shaded cocoa plantations dispersed 51,369 seeds (4% were seeds ≥3 mm width) of 16 plant species. Monkeys in the rainforest dispersed 6,536 seeds (78% were seeds ≥3 mm width) of 13 plant species. Our data suggest that the difference between habitats in the proportion of large versus small seeds dispersed reflects differences in fruit species abundance and availability in cocoa versus forest. Mean seed dispersal distances were statistically similar in both habitats (cocoa = 149 m, forest = 86 m). We conclude that the studied cocoa plantations provide all elements necessary to constitute a long-term permanent habitat for black howler monkeys. In turn, howler monkeys living in these plantations are able to maintain their functional role as seed dispersers for those native tree and liana species present within their areas of activities.

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Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supple mented Murashige & Skoog medium, and incubated at 25±2ºC under a light intensity of 61µmol/m2s from cool white fluorescent lamps and a 16h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10µM BAP in combination with 0.54µMNAA. Addition of 135.74-271.50µM adenine sulphate (Ads) and 0.72-1.44µM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10µM BAP in combination with 0.54µM NAA, 271.50µM Ads and 1.44µM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23µM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Vitex trifolia es una especie arbustiva de uso popular como planta medicinal, sus hojas, raíces y flores se han reportado para la cura de diferentes aflicciones. El aumento de la explotación de estas plantas ha puesto en peligro su conservación y ha justificado el uso de herramientas biotecnológicas para su propagación. El objetivo de esta investigación fue presentar un protocolo eficiente para la regeneración de estas plantas a través de la organogénesis, y analizar la homogeneidad genética de las líneas clonales establecidas por ADN polimórfico amplificado aleatoriamente (RAPD) mediante la repetición de marcadores de inter secuencia simple (ISSR). La regeneración de plántulas se logró en cultivos de callos derivados de explantes de tallo, hoja y pecíolo de V. trifolia en un medio diferenciado Murashige & Skoog, que se incubaron a 25±2ºC bajo una intensidad de luz de 61μmol/m2s con lámparas fluorescentes blancas y un fotoperíodo de 16h. La tasa de regeneración de brotes se correlacionó positivamente con la concentración de las hormonas en el medio nutritivo. Los brotes se regeneraron más rápidamente a partir de explantes de tallo y pecíolos en comparación con explantes de hoja. La mayor tasa de regeneración de brotes se obtuvo en los explantes de tallo utilizando 11.10μM BAP en combinación con 0.54μM NAA, 271.50μM Ads y 1.44μM GA3. Este protocolo puede ayudar a la propagación masiva y conservación de esta importante planta medicinal de gran potencial terapéutico.

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A callus induction and plant regeneration protocol was developed from leaf and thorn explants for the plant Ulex europaeus. Explants were incubated on 2 percent sucrose half-strength Murashige and Skoog Medium (MS) with various combinations of plant growth regulators and antioxidants. The best frequency of callus and shoot formation was obtained with 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/l x kinetin (Kin) 0.2 mg/l (DK Medium; callus induction) and zeatin (Z) 1 mg/l (DK medium; shoot induction). Both media were supplemented with ascorbic acid 200 mg/l to prevent browning and death of the explants. The regenerated shoots transferred to rooting medium (half-strength MS Medium, 2 percent sucrose) showed rapid growth and development of roots (100 percent). Rooted plantlets were successfully transferred to soil in pots containing a 3:1 mixture of soil and vermiculite.

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A callus induction and plant regeneration protocol was developed from leaf and petiole explants of the endemic Astragalus nezaketae. Explants were cultured on Murashige and Skoog medium (MS) supplemented with different plant growth regulators (PGRs) [a-naphthaleneacetic acid (NAA), benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kin), thidiazuron (TDZ)]. The combinations and concentrations of PGRs were shown significant variations for the frequency of callus formation, appearence of callus and the potential of callus differentiation. NAA x BA have been found highly affective in callusing and plant regeneration. Other PGRs have not resulted in callus differentiation for shoot formation. The highest number of shoots (6/explants) was obtained from leaf explants cultured on MS with 0.5 mg/l NAA and 4 mg/l BA. The regenerated shoots transferred to rooting medium (MS with 0.5 mg/l indole-3-butyric acid) were successfully rooted (100 percent) and showed rapid elongation. Rooted plantlets were acclimatized in pots containing 1:1 mixture of peat and perlite.

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Natural regeneration of woody species in a Costa Rican plantation of Vochysia guatemalensis (Vochysiaceae) and the effect of P and NPK fertilization. Forest plantation management strategies, including the selection of species, may have positive or negative effects over plant regeneration in the tropics. in this case, understory woody plants density and richness were studied in Tabarcia de Mora, Costa Rica, in a monoculture of Vochysia guatemalensis (ten year old plantation). Nineteen 80 m²plots, with several fertilization treatments (0-0; 0-50; 50-0, 50-50 g/plant of P, and NPK, during the first years, P placed once at the hole) in a completely randomized factorial design, were analyzed. Afterwards, the NPK fertilizer was increased from 150 to 200 g/ plant/year until the plantation was six year old. The plots, established after the coffee plantation was eliminated, had a minimum management schedule, basically the elimination of herbaceous vegetation once or twice a year during the first three years, and a tree thinning when the plantation was four year old, to increase spacing from 2x2 to 4x4 m. All woody vegetation taller than 0.5 m was tallied. A total of circa 10 000 ind/ha, distributed in 90 species, were found, mostly native of the region, some identified for forestry use, others important for the fauna. The majority of the species had low relative densities and frequencies. Sixteen percent of the plants reached heights greater than 2.5 m. Several factors seem to explain this regeneration pattern: a canopy with an intermediate openness, a low intensity forestry management, the nearness of the plantation to a mature forest fragment, and that the Vochysia plantation substituted a coffee plantation where soil conservation strategies and an annual fertilization management plan were applied. Finally, plots with only P had significantly higher species richness and abundance (χ2=15.364, gl=3, p=0.002) probably because the trees in this treatment were less developed (when compared with the others). Rev. Biol. Trop. 57 (Suppl. 1): 111-118. Epub 2009 November 30.

Las estrategias de manejo y las especies seleccionadas en plantaciones forestales pueden tener efectos positivos o negativos sobre la regeneración vegetal en los trópicos. Esta investigación estudió la abundancia y riqueza de plantas leñosas bajo el dosel de un monocultivo de Vochysia guatemalensis de diez años. Se evaluaron 19 parcelas de 80 m²en Tabarcia de Mora, con varios tratamientos de fertilización (0-0; 0-50; 50-0, 50-50 g/ planta de P, y NPK, respectivamente, este ultimó se aumentó de 150 a 200 g hasta que la plantación alcanzó los seis años), en un diseño factorial totalmente al azar. Se contaron e identificaron todas las especies leñosas con más de 0.5 m de altura, con un total de aproximadamente 10 000 ind/ha en 90 especies, siendo éstas principalmente nativas de la zona (varias maderables, otras importantes para la fauna), la mayoría con bajos índices de importancia (suma de la densidad y frecuencias relativas). Un 16% alcanzaron alturas superiores a 2.5 m. Se considera que varios factores pudieron favorecer dicha regeneración, como un dosel con una apertura inter-media, un manejo forestal de bajo impacto, la cercanía de un fragmento boscoso maduro, y el establecimiento de la plantación en sustitución de un cafetal donde se aplicaban estrategias de conservación de suelos y se fertilizaba anualmente. Finalmente, se determinó una mayor abundancia y riqueza en las parcelas con solo P (χ²=15.364, gl=3, p=0.002), probablemente porque los árboles de Vochysia tendieron a ser menos desarrollados en comparación con los otros tratamientos.

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The regeneration of natural tetraploid T. pratense, originated from Erzurum-Turkey, is reported in this study. This plant has low seed setting and hard seed problems due to polyploidy. Hypocotyl, cotyledon, apical meristems, epicotyl and young primary leaves were inoculated on MS and PC-L2 media containing different concentrations of BAP and NAA as growth regulators. The best shoot formation has been observed on explants initiated from apical meristem placed on PC-L2 medium that includes 2 mg dm-3 BAP and 1 mg dm-3 NAA. 94.4 percent of the shoots originated from calli were rooted on PC-L2 medium with 1 mg dm-3 NAA. In vitro organogénesis has been accomplished in the natural tetraploid T. pratense regenerated plants successively transferred to the field.

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