Soybean (Glycine max L) seed quality treated with timbó (Magonia pubescens A. St.-Hil) seed coat / Qualidade de sementes de soja (Glycine max L) tratadas com diasporo do tegumento de sementes de timbó (Magonia pubescens A. St.-Hil)

The objective of this study was to evaluate the effect of timbó seed coat on the physiological and sanitary quality of soybean seeds. Timbó seeds were collected from mature fruits in the Cerrado. The external layer of the seed coat was removed and crushed in a mill and used in the treatment of soybean seeds. Three lots of soybean seeds were used, from high, medium and low vigor seeds. The treatments were: powder; gel; hydroalcoholic extract; synthetic fungicide (Vitavax® - Thiram 200 sc), and untreated seeds. The physiological quality of the soybean seeds was determined by standard germination, accelerated aging, length, fresh and dry mass of seedlings. Blotter test was used to determine the sanitary quality. The statistical analyze used was a completely randomized 3 × 5 factorial design, considering three seed lots and five treatments. Treatment with the coat seed extract and gel improves the physiological quality of soybean seeds from low vigor lots. The treatment of seeds with gel decreases the incidence of Fusarium and Penicillium genus; and treatment with the powder reduced the incidence of the Cercospora genus.(AU)

O objetivo deste estudo foi avaliar o efeito do tegumento das sementes de timbó sobre a qualidade fisiológica e sanitária de sementes de soja. As sementes de timbó foram coletadas de frutos maduros, no Cerrado. A camada mais externa do tegumento das sementes foi retirada e triturada em moinho e utilizada no tratamento de sementes de soja. Foram utilizados três lotes de sementes de soja, provenientes de sementes de alto, médio e baixo vigor. Os tratamentos foram: pó, gel, extrato hidroalcoólico; fungicida sintético (Vitavax® - Thiram 200 sc) e sementes não tratadas. A qualidade fisiológica das sementes de soja foi determinada pelo teste padrão de germinação, envelhecimento acelerado, comprimento, massa fresca e seca de plântulas. Para a determinação da qualidade sanitária utilizou-se o teste de borrão (blotter test). O delineamento estatístico foi em esquema fatorial 3 × 5, inteiramente casualizado, considerando-se três lotes de sementes e cinco tratamentos. Os tratamentos com o extrato e o gel do tegumento das sementes de timbó melhoraram a qualidade fisiológica das sementes de soja provenientes de lotes de baixo vigor. O tratamento com gel do tegumento das sementes de timbó diminuiu a incidência de fungos dos gêneros Fusarium e Penicillium; e o tratamento com o pó reduziu a incidência do gênero Cercospora.(AU)

The influence of ultra-high diluted compounds on the growth and the metabolites of Oryza sativa L

The ultra-high dilutions (UHDs) have been widely used in the field of human, animal and plant treatment. In the present research, the effects of the potentized ultra-high dilutions (UHDs) on physiological and biochemical variations in Oryza sativaL. (rice) were investigated. Methods: To study the effect of UHDs (Calendula officinalisCalen. and Arnica montanaArn.) on the physiological and biochemical variations of the Oryza sativaL. (rice), 28 experiments were designed and statistically analyzed using the Design Expert 7.0.1 software over the general factorial design methodology. Three qualitative factors were studied including the UHDs/placebo usage, sterile/non-sterile experimental condition and the type and timing of the UHDs usage. The validated analysis was subjected to more extended studies on the variations in physiological growth, carbohydrate, protein content, pigment production, and amino acid patterns. To evaluate the effects of UHDs on rice, a desirable response percentage was formed from a number of healthy seedling productions, and the height percentage of the aerial parts and main roots were studied. Results: The statistical analysis resulted in a prediction model which was more than 97% correlates with experimental results. The results showed that the UHDs increased the pH variations, carbohydrate, protein and pigment levels each by ~2.5, ~1.5, ~1.4, and ~1.4 folds, respectively. Also compared to placebo, the amount and proportionof amino acids has significantly varied, showing a statistical effect on the germination and seedling growth of the rice, as well as the stress conditions caused by the sterilization process, seedlings entrance into the light and their transition into hydroponic culture medium.Conclusion: The use of UHDs leads to an increase in the production of chlorophyll, as well as carbohydrate and protein content. Moreover, it causes significant variations in the amino acid profile and the production of amino acids along with the photosynthesis, germination, and metabolism processes.(AU)

Germination constraints of dicarpic cypselae of Bidens pilosa L / Restrições a germinação de cipselas dicárpicas de Bidens pilosa L

Abstract Bidens pilosa L. is a heterocarpic weed species with two cypselae types that present morpho-physiological differences, being the peripheral type smaller and slower to germinate than the central one. We aimed to verify how the germination mechanism varied between types. We focused on two mechanisms: (1) pericarp constraints (physical and chemical) and (2) hormonal stimulation (Abcisic acid [ABA] and Gibberellin [GA]). Both cypselae types are physically constrained by the pericarp, for when it is excised both seed types increase their germination, but behavioral differences still remain. The pericarp of the peripheral type also has chemical inhibitors that effectively inhibited germination of the intact central cypsela. To test the hormonal effects, we focused on the ABA:GA control. Both cypselae responded to an exogenous ABA concentration gradient, however there is no variation between types on the sensitivity to it. Also, both cypselae types were indifferent to Fluridone (ABA inhibitor), which indicates that the dormancy is not maintained by de novo ABA synthesis. Cypselae types had different sensitivity to an exogenous GA3 gradient, the central type being more sensitive to the treatment than the peripheral one. But when the endogenous GA synthesis was blocked by Paclobutrazol, both types responded equally to same GA3 concentrations. This indicates that endogenous GA synthesis may be related to differences observed on germination of cypselae types. To conclude, seed types differ on their growth potential to overcome the pericarp resistance: while the inhibitor in the peripheral pericarp reduces growth potential, GA increases it.

Resumo Bidens pilosa L. é uma espécie de planta daninha heterocarpica com dois tipos de cipselas que possuem diferenças morfofisiológicas, sendo o tipo periférico de menor tamanho e com germinação lenta se comparado com o central. Nosso objetivo foi verificar como o mecanismo de germinação varia entre os tipos. Focamos em dois mecanismos: (1) restrição causada pelo pericarpo (física e química) e (2) estímulo hormonal (Ácido abscísico [ABA] e Giberelina [GA]). Os tipos de cipselas são fisicamente limitados pelo pericarpo, pois quando ambos os tipos de sementes são excisados há um aumento na germinação, contudo as diferenças no processo se mantém. O pericarpo do tipo periférico ainda possui inibidores que efetivamente retardam a germinação das cipselas centrais intactas. Para testar os efeitos hormonais, nós focamos no controle pelo ABA:GA. Ambas cipselas responderam ao gradiente de concentração de ABA exógeno, contudo não houve variação na sensibilidade entre os tipos. Ainda, ambos tipos de cipselas foram indiferentes à Fluoridona (inibidor de ABA), que indica que a dormência não é mantida pela nova síntese de ABA. Tipos de cipselas apresentam diferentes sensibilidades ao gradiente exógeno de GA3, com o tipo central sendo mais sensível ao tratamento que o periférico. Mas quando a síntese endógena de GA foi bloqueada pelo Paclobutrazol, ambos os tipos responderam de forma similar às concentrações de GA 3. Isso indica que a síntese de GA endógena pode estar relacionada com a diferença observada na germinação dos dois tipos de cipselas. Para concluir, os tipos de sementes diferem no potencial para superar a resistência do pericarpo, sendo o inibidor no pericarpo da cipsela periférica o redutor do potencial de crescimento, enquanto a GA aumenta esse potencial.

ß-Glucanase Addition in Brewing Malt Produced by Reduced Time of Germination

Abstract The β-Glucans content has straight influence on the quality of malt and beer, mainly during the filtration step. Barley presenting high β-Glucan content demands longer germination time at malting. The application of commercial β-Glucanase is an alternative to accelerate the process and preserve the quality of malt. This work aimed to evaluate the effect of commercial β-Glucanase addition in malt produced within reduced germination time (64 h). Micro-malting was conducted with BRS-Caue and Elis barley cultivars at germination time 64 h and 96 h. The β-Glucanase concentration applied were 0, 25, 50 and 100 mg.kg-1. Barley, malt and wort samples were analyzed to check their physical-chemical features. Beers were produced with BRS-Caue malt and the physical-chemical and sensory attributes were analyzed. The commercial enzyme addition in BRS-Caue and Elis (64 h), at concentration 25 and 50 mg.kg-1, resulted in wort presenting proper β-Glucan content (≤ 178 mg.L-1). The beer produced with malt germinated for 64 h and added with 50 mg.kg-1 of β-glucanase was the one showing the largest number of physical-chemical and sensory parameters similar to the beer made with malt germinated for 96 h (conventional process). Commercial β-glucanase application in malt allowed accelerating the malting process without affecting the quality of the malt for beer production.

Somatic embryogenesis in Carica papaya as affected by auxins and explants, and morphoanatomical-related aspects

ABSTRACT The aim of this study was to evaluate somatic embryogenesis in juvenile explants of the THB papaya cultivar. Apical shoots and cotyledonary leaves were inoculated in an induction medium composed of different concentrations of 2,4-D (6, 9, 12, 15 and 18 µM) or 4-CPA (19, 22, 25, 28 and 31 µM). The embryogenic calluses were transferred to a maturation medium for 30 days. Histological analysis were done during the induction and scanning electron microscopy after maturing. For both types of auxin, embryogenesis was achieved at higher frequencies with cotyledonary leaves incubated in induction medium than with apical shoots; except for callogenesis. The early-stage embryos (e.g., globular or heart-shape) predominated. Among the auxins, best results were observed in cotyledonary leaves induced with 4-CPA (25 µM). Histological analyses of the cotyledonary leaf-derived calluses confirmed that the somatic embryos (SEs) formed from parenchyma cells, predominantly differentiated via indirect and multicellular origin and infrequently via synchronized embryogenesis. The secondary embryogenesis was observed during induction and maturation phases in papaya THB cultivar. The combination of ABA (0.5 µM) and AC (15 g L-1) in maturation medium resulted in the highest somatic embryogenesis induction frequency (70 SEs callus-1) and the lowest percentage of early germination (4%).

Aislados de Bacillus provenientes de la rizósfera de cactus incrementan la germinación y la floración en Mammillaria spp. (Cactaceae) / Bacillus isolates from rhizosphere of cacti improve germination and bloom in Mammillaria spp. (Cactaceae)

Las cactáceas son la vegetación característica de las zonas áridas en México, donde las lluvias son escasas, la evapotranspiración es elevada y la fertilidad de los suelos es baja. Las plantas han desarrollado estrategias fisiológicas como la asociación con microorganismos en la zona de la rizósfera para incrementar la captación de nutrientes. En el presente trabajo se obtuvieron 4 aislados bacterianos de la rizósfera de Mammillaria magnimamma y Coryphantha radians, los que fueron nombrados como QAP3, QAP19, QAP22 y QAP24 e identificados genéticamente como pertenecientes al género Bacillus. Estos aislados exhibieron in vitro propiedades bioquímicas como solubilización de fosfatos, producción de ácido indolacético y actividad ACC deaminasa, que se relacionan con la promoción del crecimiento de las plantas. Dicha promoción fue ensayada inoculando semillas de M. magnimamma y evaluando luego algunos parámetros. Se encontró que todos los aislados incrementaron la germinación desde un 17% hasta un 34,3% (con respecto a las semillas testigo sin inocular); el aislado QAP24 fue el que presentó el mayor efecto en este sentido y permitió la germinación de todas las semillas viables (84,7%) 3 días antes que en el testigo. La inoculación de este aislado en plantas de Mammillaria zeilmanniana mostró un efecto positivo sobre la floración: en 2 meses dentro del período de un año se detectó un incremento en el número de plantas en floración con respecto a las plantas testigo, de hasta el 31,0% en uno de ellos. Se concluye que los aislados de Bacillus spp. caracterizados poseen potencial para ser empleados en programas de conservación de especies vegetales de zonas áridas.

Cacti are the most representative vegetation of arid zones in Mexico where rainfall is scarce, evapotranspiration is high and soil fertility is low. Plants have developed physiological strategies such as the association with microorganisms in the rhizosphere zone to increase nutrient uptake. In the present work, four bacterial isolates from the rhizosphere of Mammillaria magnimamma and Coryphantha radians were obtained and named as QAP3, QAP19, QAP22 and QAP24, and were genetically identified as belonging to the genus Bacillus, exhibiting in vitro biochemical properties such as phosphate solubilization, indoleacetic acid production and ACC deaminase activity related to plant growth promotion, which was tested by inoculating M. magnimamma seeds. It was found that all isolates increased germination from 17 to 34.3% with respect to the uninoculated control seeds, being QAP24 the one having the greatest effect, accomplishing the germination of viable seeds (84.7%) three days before the control seeds. Subsequently, the inoculation of Mammillari zeilmanniana plants with this isolate showed a positive effect on bloom, registering during two months from a one year period, an increase of up to 31.0% in the number of flowering plants compared to control plants. The characterized Bacillus spp. isolates have potential to be used in conservation programs of plant species from arid zones.

Phytotoxic potential of young leaves from Blepharocalyx salicifolius (Kunth) O. Berg (Myrtaceae) / Potencial fitotóxico de folhas jovens de Blepharocalyx salicifolius (Kunth) O. Berg

Abstract The loss of leaves by plant species found in the Cerrado (Brazilian savanna) is an energetically expensive process due to adverse environmental conditions and predation by herbivory. The mature leaves have adaptations which minimize these events. However, the young individuals lack these structures and produce high leaf concentrations of secondary metabolites as a form of protection. These compounds can be used in bioprospection of natural herbicides. Thus, this study aimed to evaluate the phytotoxicity of hexane, ethyl acetate and aqueous extracts of young leaves from Blepharocalyx salicifolius (Kunth) O. Berg on the elongation of wheat coleoptiles (Triticum aestivum L.) and evaluate the potential phytotoxic of ethyl acetate extract on germination, growth and cell size of metaxylem of sesame (Sesamum indicum L.) seedlings. The hexane and ethyl acetate extracts inhibited the elongation of wheat coleoptiles at all concentrations; however, the most promising results were observed in coleoptile fragments treated with the ethyl acetate extract. This treatment changed the mean germination time and the synchrony of sesame seeds, inhibited the growth of shoots and roots, reduced the dry weight of seedlings, led to abnormalities in the seedlings and reduced the length of the metaxylem cells in the sesame seedlings. These results demonstrated the phytotoxic potential of young leaf extracts of B. salicifolius and the high phytotoxicity of the ethyl acetate extract in the initial development of S. indicum.

Resumo A perda das folhas pelas plantas do Cerrado é um processo energeticamente custoso devido às condições ambientais adversas e intensa predação por herbivoria. As folhas maduras possuem adaptações que minimizam esses eventos. Entretanto, os indivíduos jovens carecem dessas estruturas e produzem elevadas concentrações foliares de metabólitos secundários como forma de proteção. Esses compostos podem ser utilizados na bioprospecção de herbicidas naturais. Dessa forma, objetivamos com este estudo avaliar a fitotoxicidade dos extratos hexânico, acetato etílico e aquoso de folhas jovens de Blepharocalyx salicifolius (Kunth) O. Berg sobre o alongamento de coleóptilos estiolados de Triticum aestivum L. (trigo) e avaliar o potencial fitotóxico do extrato acetato etílico sobre a germinação, crescimento e comprimento das células do metaxilema de Sesamum indicum (gergelim). Os extratos hexânico e acetato etílico inibiram o alongamento dos coleóptilos em todas as concentrações testadas, entretanto os resultados mais promissores foram observados nos fragmentos submetidos ao extrato acetato etílico. Este extrato alterou o tempo médio de germinação e a sincronia das sementes de gergelim, inibiu o crescimento da parte aérea e subterrânea com redução da massa seca das plântulas, levou ao surgimento acentuado de anomalias e causou redução no comprimento médio das células do metaxilema de plântulas de gergelim. Assim, ficou comprovado o potencial fitotóxico dos extratos de folhas jovens de B. salicifolius e a acentuada fitotoxicidade do extrato acetato etílico sobre o desenvolvimento inicial de S. indicum.

Extratos de Brunfelsia uniflora na germinação de sementes de Crotalaria spectabilis / Brunfelsia uniflora extracts in the germination of Crotalaria spectabilis seeds / Extractos de Brunfelsia uniflora en germinación de semillas Crotalaria spectabilis

As sementes do gênero Crotalaria L., de um modo geral, apresentam dificuldades no processo germinativo. O objetivo da pesquisa foi avaliar a eficiência do extrato bruto de Brunfelsia uniflora na germinação de Crotalaria spectabilis. O delineamento foi inteiramente casualizado, com quatro tratamentos e quatro repetições. Os tratamentos foram extratos das folhas e flores de B. uniflora nas concentrações 25, 50, 100% e tratamento testemunha (0%). O experimento foi conduzido no laboratório de Fitopatologia da Universidade Paranaense ­ UNIPAR, Umuarama - PR. As folhas e frutos coletados foram expostos à secagem natural e posteriormente submetidos ao processo de maceração dinâmica com esgotamento e rota evaporação do etanol, obtendo o extrato bruto. As sementes foram expostas as diluições do extratos durante dois minutos, na sequência, foram dispostas para a secagem e distribuídas em rolo papel e armazenados na B.O.D., na temperatura de 26ºC. Na contagem, aos 4 e 10 dias, determinou-se a porcentagem de plântulas normais e anormais, sementes duras e dormentes. Os extratos brutos da folha e flor de B. uniflora não ocasionaram diferenças estatísticas significativas para as variáveis analisadas, demonstrando ausência de efeito antagônico.

Seeds of the Crotalaria L. genus present difficulties in the germination process. The purpose of this study was to evaluate the efficiency of crute extract of Brunfelsia uniflora in the germination of Crotalaria spectabilis. The experimental design was completely randomized, with four treatments and four replications. The treatments consisted in B. uniflora leaf and flower extract at concentrations of 25, 50, 100% and one control treatment (0%). The experiment was performed in the Laboratory of Phytopathology at Universidade Paranaense - UNIPAR, Umuarama - PR. The leaves and fruits collected were exposed to natural drying and then submitted to a dynamic maceration process with depletion and ethanol rotavaporation in order to obtain the crude extract. The seeds were exposed to the extract dilutions for two minutes, and then were arranged for drying, distributed on paper roll and stored in the B.O.D. at the temperature of 26 °C. At days 4 and 10, the percentage of normal and abnormal seedlings, hard and dormant seeds were determined. The crude extracts of B. uniflora leaves and flowers of did not cause significant statistical differences for the variables analyzed, demonstrating the absence of an antagonistic effect.

Las semillas del género Crotalaria L., en general, presentan dificultades en el proceso de germinación. El objetivo de la investigación ha sido evaluar la eficacia del extracto crudo Brunfelsia uniflora en la germinación de Crotalaria spectabilis. El diseño fue completamente al azar, con cuatro tratamientos y cuatro repeticiones. Los tratamientos fueron los extractos de las hojas y flores de B. uniflora a concentraciones de 25, 50 y 100%, y tratamiento testigo (0%). El experimento se llevó a cabo en el Laboratorio de Fitopatología de la Universidad Paranaense - UNIPAR, Umuarama - PR. Las hojas y frutos recogidos fueron expuestos a secado natural y después sometidos al proceso de maceración dinámica con agotamiento y ruta de la evaporación del etanol, obteniendo el extracto crudo. Las semillas fueron expuestas a las diluciones de los extractos durante dos minutos, enseguida, fueron dispuestas en rollo de papel y almacenadas en B.O.D., a temperatura de 26°C. En el recuento, al 4º y 10º día, se determinó el porcentaje de plántulas normales y anormales, semillas duras y latentes. Los extractos crudos de hojas y flor de B. uniflora no resultaron en diferencias estadísticamente significativas para las variables analizadas, demostrando ausencia de efecto antagónico.

Lead tolerance and accumulation in initial sporophytes of Regnellidium diphyllum Lindm. (Marsileaceae) / Tolerância e acumulação de chumbo em esporófitos jovens de Regnellidium diphyllum Lindm. (Marsileaceae)

Regnellidium diphyllum Lindm. is a heterosporous fern which grows in shallow waters and wetlands, and water pollution contributes to its vulnerability. Environmental lead contamination is mostly caused by industrial and agricultural residues as well as domestic sewage. Given its persistence in the environment, lead can cause important toxicity in living organisms. Megaspore germination and the initial growth of R. diphyllum sporophytes were assessed in Meyer's solution with lead nitrate (Pb(NO3)2) concentrations of 0 (control), 1, 5, 10 and 50 mg L–1. The study was conducted in a growth chamber at 25±1°C and a 12 hour photoperiod with a nominal irradiance of 100 μmol m–2 s–1, for 28 days. Lead concentration in sporophytes was assessed using atomic absorption spectrometry. In the absence of lead, 74% of spores germinated, while significantly lower germination percentages were observed in Pb(NO3)2 concentrations of 1, 10 and 50 mg L–1. The presence of lead did not significantly influence root growth. At 28 days, primary leaf development was significantly lower in Pb(NO3)2 concentrations of 5 mg L–1 and higher in relation to the control. The length of secondary leaves did not significantly differ between sporophytes exposed to different concentrations of lead and those of the control at 28 days. Sporophytes exposed to 10 and 50 mg L–1 Pb(NO3)2 accumulated 1129 mg kg–1 and 5145 mg kg–1 of Pb, respectively. The presence of high levels of lead in R. diphyllum sporophytes did not prevent initial development. Future studies should investigate the ability of the species to accumulate and tolerate high levels of lead in advanced stages of its development and in environmental conditions.

Regnellidium diphyllum Lindm. é uma samambaia heterosporada que se desenvolve em águas rasas ou em solos úmidos, sendo que a poluição da água contribui para sua vulnerabilidade. A contaminação ambiental por chumbo ocorre principalmente por resíduos industriais e agrícolas, bem como por efluentes domésticos. Devido à sua persistência no ambiente, esse metal pode apresentar importante toxicidade aos organismos vivos. A germinação de megásporos e o desenvolvimento inicial de esporófitos de R. diphyllum foram avaliados em solução de Meyer com concentrações de 0 (controle), 1, 5, 10 e 50 mg L–1 de nitrato de chumbo (Pb(NO3)2). O estudo foi conduzido em câmara de germinação a 25±1 °C e fotoperíodo de 12 horas sob irradiância nominal de 100 μmol m–2 s–1, por 28 dias. A concentração de chumbo em esporófitos foi analisada por espectrometria de absorção atômica. Na ausência de chumbo, 74% dos esporos germinaram, enquanto que porcentagens de germinação significantemente menores foram observadas nas concentrações de 1, 10 e 50 mg L–1 de Pb(NO3)2. A presença de chumbo não influenciou significativamente o crescimento das raízes. O desenvolvimento das folhas primárias foi significativamente menor em relação ao controle a partir de 5 mg L–1 de Pb(NO3)2 aos 28 dias. O comprimento das folhas secundárias não diferiu significativamente entre esporófitos expostos às diferentes concentrações de chumbo e aqueles do controle, aos 28 dias. Esporófitos expostos a 10 e 50 mg L–1 de Pb(NO3)2 acumularam 1129 mg kg–1 e 5145 mg kg–1 de Pb, respectivamente. A presença de altas concentrações de chumbo nos esporófitos de R. diphyllum não impediu seu desenvolvimento inicial. Estudos futuros deverão investigar a capacidade de a espécie acumular e tolerar altas concentrações de chumbo em estádios avançados de desenvolvimento e também em condições ambientais.

Effect of carbon and nitrogen sources on in vitro flower and fruit formation and withanolides production in Withania somnifera (L.) Dunal.

We studied the influence of sucrose and nitrogen concentration on in vitro flowering and fruit setting in elongated shoots of Withania somnifera. BA (1.5 mg/l) and IAA (0.3 mg/l) on MS medium supplemented with 4% sucrose showed 67% of in vitro flower induction frequency, 9 flowers/shoot, 4 fruits/shoot and 11 seeds/fruit in elongated-shoots. Different concentrations of nitrogen sources (L-glutamine, adenine sulphate, ammonium nitrate, potassium nitrate and sodium nitrate 5-25 mg/l) were tested in combination with 4% sucrose and BA at 1.5 mg/l and IAA at 0.3 mg/l. Highest number of flowers (20 flowers/shoot; 2.2-fold) and fruits (16 fruits/shoot; 3.39-fold), fruit setting (12 seeds/fruit; 1.08-fold) at a higher frequency (88 %) were achieved on MS medium augmented with 15 mg/l adenine sulphate with same PGRs and sucrose concentration. The maximum production of withanolide A (0.68 mg/g DW) and withanolide B (0.77 mg/g DW) was recorded in in vitro fruits. Highest accumulation of withaferin A (2 mg/g DW) was quantified from in vitro flowers, whereas, it was low in in vitro fruits (0.49 mg/g DW withaferin A). However, withanone (0.23 mg/g DW) was found accumulated uniformly in both in vitro flowers and fruits compared to control.

Plant Growth Promotion and Root Colonization by EPS Producing Enterobacter sp. RZS5 under Heavy Metal Contaminated Soil.

The heavy metal resistant bacterium isolated from field soil and identified as Enterobacter sp. RZS5 tolerates a high concentration (100-2000 mM) of various heavy metal ions such as Mn2+, Ni2+, Zn2+, Cu2+, CO2+ and Fe2+ when grown in such environment and produces exopolysaccharides (EPS). Here, we have demonstrated EPS production by Enterobacter sp. RZS5 during 60 h of growth in yeast extract mannitol broth (YEMB). The yield increased by two fold after the addition of 60 M of Ca2+; 50 M of Fe2+ and 60 M of Mg2+ ions in YEMB, and the optimization of physico-chemical parameters. EPS was extracted with 30% (v/v) of isopropanol as against the commonly used 50% (v/v) isopropanol method. EPS-rich broth promoted seed germination, shoot height, root length, number of leaves and chlorophyll content of wheat (Triticum aestivum) and peanut (Arachis hypogaea) seeds. The higher colony-forming unit of Enterobacter sp. in soil inoculated with EPS rich broth of Enterobacter sp. indicated the root colonizing potential and rhizosphere competence of the isolate. The FTIR spectra of the EPS extract confirmed the presence of the functional group characteristics of EPS known to exhibit a high binding affinity towards certain metal ions. This overall growth and vigour in plants along with the effective root colonization, reflected the potential of the isolate as an efficient bio-inoculant in bioremediation.

Improvement Germination and Conversion of Somatic Embryos and Production of Normal Plantlets in Ferula Assa-foetida L. (A Rare Medicinal Plant).

Secondary somatic embryogenesis leads to the formation of abnormal somatic embryos and produces abnormal seedlings. Normal plants are difficult to obtain from these embryos, due to the asynchronous maturation of the embryogenic tissues and low germination and conversion rates. The effects of some media additives and different strengths of MS medium on germination and plantlet formation of in vitro derived somatic embryos of Ferula Assa-foetida were studied. The highest number of normal embryos was observed in MS medium containing 30g/l sucrose with 0.5% or 1% AC and in MS medium supplemented with PEG and 0.5% or 1% AC. The treatments of MS medium with 30g/l sucrose and 0.5% AC × MS medium containing sorbitol and MS medium containing PEG and 1% AC × ½ MS had maximum number of normal germinated embryos without secondary somatic embryogenesis (SSE). In some of the treatments the embryos were converted better than the others, such as; the interaction effect of MS medium with 30g/l sucrose and 0.5% AC× MS, MS medium with 30g/l sucrose × MS medium with glutamine. Using different strength of MS medium and presence of some media additives is effective on germination and conversion of somatic embryos into normal plantlet. Presence of Activated Charcoal in the culture medium can reduce secondary somatic embryogenesis.

Mass propagation of Cymbidium giganteum Wall. ex Lindl. using in vitro seedlings.

In vitro seedlings were used as explants for protocorm like bodies (PLBs) production which in turn were used for regeneration purpose. PLBs were induced from the base of seedlings (1.0-1.5 cm in size) in MS + BAP (8.88 µM). After 90 days of inoculation, PLBs production rate started declining and most of the PLBs turned into plantlets. Preculture of seedlings in 1.0 µM thidiazuron (TDZ) for 7 days and transfer to BAP supplemented medium resulted in production of 16 PLBs per seedling within 90 days of culture. Increase of TDZ concentration to 2.5 µM and preculture time 15 days, resulted in induction of highest number of PLBs (19 PLBs per seedling) in the basal medium. The results emphasized the importance of thidiazuron (TDZ) concentration and preculture time for PLBs proliferation from the base of seedlings. The PLBs thus produced were used for regeneration studies. Irrespective of single, segmented or clumps of PLBs, the regeneration response was 100% in 2,4-D (4.52 µM) and KN (4.64 µM) but when KN was replaced by BAP (8.88 µM), response was observed only in clumps of PLBs, whereas in single and segmented ones it was 99 and 97%, respectively. Regenerants developed stout root system in half strength M medium supplemented with 2.84 µM of IAA and transferred to greenhouse with 90% survival. The present study holds tremendous potential as the mother plant is not destroyed and PLBs are produced as a continuous system.

Large scale propagation of an exotic edible bamboo, Phyllostachys pubescens Mazel ex H. De Lehale (Moso Bamboo) using seeds.

For ex vitro propagation, seeds of P.pubescens were treated with different concentrations of gibberellic acid (GA3) and germination of seeds was tested both in plastic pots as well as by direct sowing in the nursery beds. Maximum seed germination was achieved when treated with 200 mgL–1 (w/v) GA3. For in vitro propagation, an exposure of nodal explants from in vitro raised seedlings to 0.2 mgL–1 1–phenyl–3–(1,2,3–thiadiazol–5–yl) urea and 1 mgL–1 kinetin supplemented medium for 30 days and thereafter to hormone free Murashige and Skoog basal medium resulted in axillary shoot proliferation. For rooting, in vitro raised shoots were exposed to MS medium containing 2 mgL–1 indole-3-butyric acid for 15 days and then shifted to hormone free medium. On an average, 2.8 shoots were obtained in 75% of the cultures within 4 weeks. Such in vitro raised plants were successfully hardened and shifted to field conditions.

In vitro propagation of Cyathea atrovirens (Cyatheaceae): spore storage and sterilization conditions / Propagación in vitro de Cyathea atrovirens (Cyatheaceae): almacenamiento de esporas y condiciones de esterilización

Cyathea atrovirens occurs in a wide range of habitats in Brazil, Paraguay, Uruguay and Argentina. In the Brazilian State of Rio Grande do Sul, this commonly found species is a target of intense exploitation, because of its ornamental characteristics. The in vitro cultura is an important tool for propagation which may contribute toward the reduction of extractivism. However, exogenous contamination of spores is an obstacle for the success of aseptic long-term cultures. This study evaluated the influence of different sterilization methods combined with storage conditions on the contamination of the in vitro cultures and the gametophytic development of C. atrovirens, in order to establish an efficient propagation protocol. Spores were obtained from plants collected in Novo Hamburgo, State of Rio Grande do Sul, Brazil. In the first experiment, spores stored at 7oC were surface sterilized with 0.5, 0.8 and 2% of sodium hypochlorite (NaClO) for 15 minutes and sown in Meyer’s culture medium. The cultures were maintained in a growth room at 26±1ºC for a 12-h photoperiod and photon flux density of 100μmol/m²/s provided by cool white fluorescent light. Contamination was assessed at 60 days, and gametophytic development was scored at 30, 60, 120 and 130 days of in vitro culture, analyzing 300 individuals for each treatment. There was no significant difference in culture contamination among the different sodium hypochlorite concentrations tested, and all treatments allowed for the development of cordiform gametophytes at 130 days of culture. In the second experiment, spores stored at 7 and -20°C were divided into two groups. Half of the spores were surface sterilized with 2% of NaClO for 15 minutes and the other half was not sterilized. All spores were sown in Meyer’s medium supplemented with one of the following antibiotics: nystatin, Micostatin® and actidione. The culture conditions and the procedures used for evaluating contamination and gametophytic development were the same described for the first experiment. No contamination was observed in spores stored at -20°C and treated with NaClO and actidione. In all treatments, cordiform gametophytes presenting antheridia were observed at 120 days. The percentages of these gametophytes increased from 120 to 130 days and no significant differences were observed among treatments. Archegonia were observed on cordiform gametophytes at 130 days. The findings provide data relevant to in vitro propagation procedures of this species, which may increase the availability of plants for ornamental purposes, therefore contributing to the reduction of the exploitation of endangered tree ferns species. Rev. Biol. Trop. 62 (1): 299-308. Epub 2014 March 01.

Cyathea atrovirens (Langsd. & Fisch.) Domin (Cyatheaceae) se presenta en una amplia gama de hábitats en Brasil, Paraguay, Uruguay y Argentina. Debido a sus características ornamentales, la especie es objeto de intensa explotación. El cultivo in vitro es una herramienta importante para la propagación lo que puede contribuir a la reducción del impacto de las actividades extractivas. Sin embargo, la contaminación exógena de esporas es un obstáculo para el éxito de cultivos asépticos a largo plazo. Este estudio evaluó la influencia de diferentes métodos de esterilización en combinación con las condiciones de almacenamiento sobre la contaminación de los cultivos in vitro y el desarrollo gametofítico de C. atrovirens. En el primer experimento, las esporas almacenadas a 7°C se esterilizaron superficialmente con 0.5, 0.8 y 2% de hipoclorito de sodio (NaClO) durante 15 minutos y se sembraron en medio de cultivo de Meyer. Aunque no hubo diferencia en la contaminación de lós cultivos entre las concentraciones de hipoclorito de sodio de las diferentes pruebas, en el tratamiento con 2% NaClO se observó un mayor porcentaje de gametofitos cordiformes a los 130 días. En el segundo experimento, las esporas almacenadas a 7 y -20°C fueron divididas en dos grupos. La mitad de las esporas se esterilizaron con 2% de NaClO durante 15 minutos y la otra mitad no fue esterilizada. Todas las esporas se sembraron en medio de Meyer suplementado con uno de los siguientes antibióticos: nistatina, Micostatin® o actidiona. No se observó contaminación de las esporas almacenadas a -20°C y tratadas con NaClO y actidiona. En todos los tratamientos, se observaron gametofitos cordiformes con anteridios y arquegonios. Los resultados proporcionan datos relevantes para la propagación in vitro de C. atrovirens, que pueden aumentar la disponibilidad de las plantas para fines ornamentales, contribuyendo así a la reducción de la exploración de las especies de helechos arborescentes en peligro de extinción.

Assessment of somatic embryogenesis potency in Indian soybean [Glycine max (L.) Merr.] cultivars.

Majority of the Indian soybean cultivars are recalcitrant to tissue culture regeneration. The present communication reports the development of somatic embryogenesis in a liquid culture medium from immature cotyledons of G. max. Following induction with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthalene acetic acid (NAA), the number of somatic embryos and percentage of explants that responded were higher with 45.24 µM 2,4-D. The proliferation of somatic embryos for three successive cycles was achieved in 22.62 µM 2,4-D. Histodifferentiation of somatic embryos under NAA (10.74 µM) indicated that better embryo development and maturation was achieved without any growth regulator. The amino acids such as L-glutamine favoured the somatic embryo induction and histodifferentiation at 20 and 30 mM respectively, where as L-asparagine at 10 mM concentration enhanced the somatic embryo proliferation. In addition, somatic embryos that were desiccated (air-drying method) for 5 days showed better germination (40.88%). The Indian soybean cultivars also showed strict genotypic influence and cv. Pusa 16 was emerged as a best responding cultivar for somatic embryo induction with 74.42% of response.

Foliar application of Zn at flowering stage improves plant’s performance, yield and yield attributes of black gram.

Black gram plants subjected to varying levels of Zn supply (0.01 to 10 µM Zn) showed optimum growth and dry matter yield in plants receiving 1 µM Zn. The dry matter yield of plants decreased in plants receiving 0.01 and 0.1 µM Zn (deficient) and excess levels of Zn (2 and 10 µM Zn). The plants grown with Zn deficient supply showed delayed flowering, premature bud abscission, reduced size of anthers, pollen producing capacity, pollen viability and stigma receptivity resulting in poor pod formation and seed yield. Providing Zn as a foliar spray at pre-flowering stage minimized the severity of Zn deficiency on reproductive structure development and enhanced the seed nutritional status by enhancing seed Zn density, seed carbohydrate (sugar and starch content) and storage proteins (albumins, globulins, glutenins, and prolamines).

Effects of nickel on the fern Regnellidium diphyllum Lindm. (Marsileaceae) / Efeitos do níquel sobre a filicínea Regnellidium diphyllum Lindm. (Marsileaceae)

The heterosporous fern Regnellidium diphyllum occurs in southern Brazil and some adjoining localities in Uruguay and Argentina. Currently it is on the list of threatened species in the state of Rio Grande do Sul. Anthropic alterations such as the conversion of wetlands into agricultural areas or water and soil contamination by pollutants may compromise the establishment and survival of this species. Nickel (Ni) is an essential nutrient for plants but increasing levels of this metal due to pollution can cause deleterious effects especially in aquatic macrophytes. Megaspore germination tests were performed using Meyer's solution, at concentrations of 0 (control), 0.05, 0.5, 1, 5, 10, 20, 30, 50 and 100 mg L-1 of Ni. The initial development of apomictic sporophytes was studied using solutions containing 0 (control) to 4.8 mg L-1 of Ni. A significant negative relation was observed between the different Ni concentrations and the megaspore germination/sporophyte formation rates. Primary roots, primary leaves and secondary leaves were significantly shorter at 3.2 and 4.8 mg L-1 of Ni, when compared with the treatment without this metal. At 4.8 mg L-1, leaves also presented chlorosis and necrosis. The introduction of pollutants with Ni in the natural habitat of Regnellidium diphyllum may inhibit the establishment of plants in the initial stage of development, a problem to be considered in relation to the conservation of this species.

A samambaia heterosporada Regnellidium diphyllum Lindm. ocorre no Sul do Brasil e em algumas localidades vizinhas no Uruguai e na Argentina. Atualmente, consta da lista de espécies ameaçadas do Estado do Rio Grande do Sul. Alterações antrópicas - tais como a conversão de áreas úmidas em áreas de agricultura ou a contaminação de água e solo por poluentes - podem comprometer o estabelecimento e a sobrevivência da espécie. Níquel (Ni) é um nutriente essencial às plantas, mas níveis aumentados desse metal, em razão da poluição, podem causar efeitos deletérios, especialmente em macrófitas aquáticas. Testes de germinação de megásporos foram realizados usando-se solução de Meyer, em concentrações de 0 (controle); 0,05; 0,5; 1; 5; 10; 20; 30; 50; 100 mg L-1 de Ni. O desenvolvimento inicial de esporófitos apomíticos foi estudado usando-se soluções contendo 0 (controle) a 4,8 mg L-1 de Ni. Relação negativa significante foi observada entre diferentes concentrações de Ni e as taxas de germinação de megásporos/formação de esporófitos. Raízes primárias e folhas primárias e secundárias foram significantemente mais curtas a 3,2 e 4,8 mg L-1 de Ni, quando comparadas ao tratamento sem esse metal. Em 4,8 mg L-1, folhas também apresentaram clorose e necrose. A introdução de poluentes com Ni no habitat natural de Regnellidium diphyllum pode inibir o estabelecimento de plantas em estádio inicial de desenvolvimento, um problemaa a ser considerado em relação à conservação dessa espécie.

Assessment of allelopathic properties of Aloe ferox Mill. on turnip, beetroot and carrot

Turnip (Brassica rapa var. rapa L.), beetroot (Beta vulgaris L.) and carrot (Daucus carota L.) are common vegetables in South Africa. The allelopathic potential of aqueous leaf and root extracts of Aloe ferox Mill.- a highly valued medicinal plant- was evaluated against seed germination and seedling growth of the three vegetables in Petri dish experiments. The extracts were tested at concentrations of 2, 4, 6, 8, and 10 mg/mL. Leaf extract concentrations above 4 mg/mL inhibited the germination of all the crops, while the root extract had no significant effect on germination irrespective of concentration. Interestingly, the lowest concentration of leaf extract stimulated root length elongation of beetroot by 31.71%. Other concentrations significantly inhibited both root and shoot growth of the vegetable crops except the turnip shoot. The most sensitive crop was carrot, with percentage inhibition ranging from 29.15 to 100% for root and shoot lengths. Lower percentage inhibition was observed for the root extract than the leaf extract against shoot growth of beetroot and carrot. The results from this study suggested the presence of allelochemicals mostly in the leaves of A. ferox that could inhibit the growth of the turnip, beetroot and carrot.

Role of H2O2 and cell wall monoamine oxidases in germination of Vigna radiata seeds.

Plant cell wall expresses monoamine oxidases (MAOs) that catalyze oxidation of secreted amines and produce H2O2 in the process. The H2O2, so produced is used by cell wall peroxidases for lignification of cell wall or for plant defense. The natural substrates for these MAOs are elusive, but polyamines and certain catecholamines have been proposed as candidates. Reactive oxygen species are also known to act as signaling molecules controlling plant metabolism. Mungbean (Vigna radiata) has long served as the plant model of choice while studying molecular programs followed during germination and seed development. In this study, we tested the effect of externally added MAO substrates epinephrine and H2O2 on storage protein mobilization in germinating seeds of Vigna radiata. The seeds were imbibed in the presence of 50 M epinephrine and 10 M H2O2. These low concentrations of the two compounds were used to exclude direct effects on proteolysis and were arrived at after testing a range of the two and choosing the most effective concentration. These seeds showed 11% and 7% decrease in fresh weight respectively, indicating greater storage mobilization and a corresponding 19% and 46% increase in axis length as compared to untreated seeds. Soluble protein in seeds treated with epinephrine and H2O2 decreased significantly by 34% and 33% as compared to untreated seeds. Electrophoretic analysis of seed proteins revealed a startling and selective depletion of storage proteins in treated seeds. The results indicated a clear involvement of H2O2 in storage protein mobilization in the cotyledons. We propose that H2O2 generated within cell walls of seeds serves as a signaling molecule guiding germination events, including protein reserve mobilization.