Phytohormone abscisic acid boosts pentobarbital-induced sleep through activation of GABA-A, PPARß and PPARγ receptor signaling / O fito-hormônio ácido abscísico estimula o sono induzido por fenobarbital por meio de ativação da sinalização de receptores GABA-A, PPARß e PPARγ

ABSTRACT Background: Sleep disorders induce anxiety and forgetfulness and change habits. The chemical hypnotic drugs currently used have serious side effects and, therefore, people are drawn towards using natural compounds such as plant-based healing agents. Abscisic acid (ABA) is produced in a variety of mammalian tissues and it is involved in many neurophysiological functions. Objective: To investigate the possible effect of ABA on pentobarbital-induced sleep and its possible signaling through GABA-A and PPAR (γ and β) receptors, in male Wistar rats. Methods: The possible effect of ABA (5 and 10 µg/rat, intracerebroventricularly) on sleep onset latency time and duration was evaluated in a V-maze model of sleep. Pentobarbital sodium (40 mg/kg, intraperitoneally) was injected to induce sleep 30 min after administration of ABA. PPARβ (GSK0660, 80 nM/rat), PPARγ (GW9662, 3 nM/rat) or GABA-A receptor (bicuculline, 6 µg/rat) antagonists were given 15 min before ABA injection. Diazepam (2 mg/kg, intraperitoneally) was used as a positive control group. Results: ABA at 5 µg significantly boosted the pentobarbital-induced subhypnotic effects and promoted induction of sleep onset in a manner comparable to diazepam treatment. Furthermore, pretreatment with bicuculline significantly abolished the ABA effects on sleep parameters, while the amplifying effects of ABA on the induction of sleep onset was not significantly affected by PPARβ or PPARγ antagonists. The sleep prolonging effect of ABA was significantly prevented by both PPAR antagonists. Conclusions: The data showed that ABA boosts pentobarbital-induced sleep and that GABA-A, PPARβ and PPARγ receptors are, at least in part, involved in ABA signaling.

RESUMO Introdução: Os distúrbios do sono induzem a ansiedade e esquecimento e mudam hábitos. Os medicamentos hipnóticos químicos utilizados atualmente têm efeitos colaterais graves e, portanto, as pessoas são atraídas para o uso de compostos naturais, como agentes de cura à base de plantas. O ácido abscísico (ABA) é produzido em uma variedade de tecidos de mamíferos e está envolvido em muitas funções neurofisiológicas. Objetivo: Investigar o possível efeito do ABA no sono induzido por pentobarbital e sua possível sinalização por meio dos receptores GABA-A e PPAR (γ e β), em ratos Wistar machos. Métodos: O possível efeito do ABA (5 e 10 µg/rato, intracerebroventricularmente) no tempo de latência e duração do início do sono foi avaliado em um modelo de labirinto em V de sono. Pentobarbital sódico (40 mg/kg, intraperitonealmente) foi injetado para induzir o sono 30 minutos após a administração de ABA. PPARβ (GSK0660, 80 nM/rato), PPARγ (GW9662, 3 nM/rato) ou antagonistas do receptor GABA-A (bicuculina, 6 µg/rato) foram administrados 15 minutos antes da injeção de ABA. Diazepam (2 mg/kg, intraperitonealmente) foi utilizado como grupo de controle positivo. Resultados: ABA a 5 µg aumentou significativamente os efeitos sub-hipnóticos induzidos por pentobarbital e promoveu a indução do início do sono de forma comparável ao tratamento com diazepam. Além disso, o pré-tratamento com bicuculina aboliu significativamente os efeitos do ABA nos parâmetros do sono, ao passo que os efeitos amplificadores do ABA na indução do início do sono não foram significativamente afetados pelos antagonistas do PPARβ ou PPARγ. O efeito de prolongamento do sono do ABA foi significativamente prevenido por ambos os antagonistas do PPAR. Conclusões: Os dados mostraram que o ABA estimula o sono induzido por pentobarbital e que os receptores GABA-A, PPARβ e PPARγ estão, pelo menos em parte, envolvidos na sinalização ABA.

Resource use efficiency of cotton in improved vs conventional planting geometry with exogenous application of bio-stimulant and synthetic growth retardant / Eficiência do uso de recursos do algodão na geometria de plantio melhorada vs convencional com aplicação exógena de bioestimulante e retardante de crescimento sintético

Abstract Plant growth regulators and improved planting density are the innovative techniques in the establishment of more productive cotton crop. A field study was planned to assess the role of growth regulators in the resource utilization efficiency of cotton cultivars under different row spacing at Agronomic Research Area, Bahauddin Zakariya University Multan and Usmania Agricultural Farm, Shujaabad during Kharif 2012. The study was comprised of cotton cultivars viz. CIM-573 and CIM-598, cultivated under conventional (75 cm), medium (50 cm) and improved ultra-narrow row spacing (25 cm) and foliar spray of growth regulators viz. moringa leaf extract (MLE) and mepiquat chloride (MC), either alone or in combination, distilled water as a control. The application of MLE alone and in combination (MLE + MC) showed the promoting effect on crop growth rate, net assimilation rate, leaf area index, leaf area duration, sympodial branches and number of bolls leading to higher seed cotton yield of both cotton cultivars grown under conventional row spacing. While application of MC averts the plant growth without considerably improving the productivity. MLE being rich source of growth promoting hormone and nutrients showed its potential to a far greater extent under conventional row spacing in efficient utilization of available resources compared to MC and distilled water.

Resumo Reguladores de crescimento de plantas e melhor densidade de plantio são técnicas inovadoras no estabelecimento de culturas mais produtivas de algodão. Um estudo de campo foi planejado com o objetivo de avaliar o papel dos reguladores de crescimento na eficiência de uso de recursos de cultivares de algodão sob diferentes espaçamentos na Área de Pesquisa Agronômica, da Universidade Multil de Bahauddin Zakariya, e na Fazenda Agrícola da Usmania, Shujabad, durante o Kharif 2012. O estudo foi composto de cultivares de algodão CIM-573 e CIM-598, cultivados em espaçamento de linhas convencional (75 cm), médio (50 cm) e superestreito (25 cm) e de pulverização foliar de reguladores de crescimento, a saber, extrato de folhas de moringa (MLE) e cloreto de mepiquat (MC), isoladamente ou em combinação, e água destilada como controle. A aplicação de MLE isoladamente e em combinação (MLE + MC) mostrou efeito promotor na taxa de crescimento da cultura, taxa de assimilação líquida, índice de área foliar, duração de área foliar, ramos simpodiais e número de cápsulas levando à maior produção de algodão nas cultivares com espaçamento de linha convencional. Em contrapartida, a aplicação de MC evitou o crescimento da planta sem melhorar consideravelmente a produtividade. O MLE, por ser uma rica fonte de hormônio promotor de crescimento e nutrientes, mostrou seu potencial em uma extensão muito maior sob o espaçamento convencional entre as linhas no uso eficiente dos recursos disponíveis em comparação com o MC e a água destilada.

Spinal protein kinase A and phosphorylated extracellular signal-regulated kinase signaling are involved in the antinociceptive effect of phytohormone abscisic acid in rats / A proteína quinase A da medula espinhal e a sinalização da quinase fosforilada regulada por sinal extracelular estão envolvidas no efeito antinociceptivo do ácido fito-hormônio abscísico em ratos

Abstract Objective: The phytohormone abscisic acid (ABA) as a signaling molecule exists in various types of organisms from early multicellular to animal cells and tissues. It has been demonstrated that ABA has an antinociceptive effect in rodents. The present study was designed to assess the possible role of PKA and phosphorylated ERK (p-ERK) on the antinociceptive effects of intrathecal (i.t.) ABA in male Wistar rats. Methods: The animals were cannulated intrathecally and divided into different experimental groups (n=6‒7): Control (no surgery), vehicle (received ABA vehicle), ABA-treated groups (received ABA in doses of 10 or 20 µg/rat), ABA plus H.89 (PKA inhibitor)-treated group which received the inhibitor 15 min prior to the ABA injection. Tail-flick and hot-plate tests were used as acute nociceptive stimulators to assess ABA analgesic effects. p-ERK was evaluated in the dorsal portion of the spinal cord using immunoblotting. Results: Data showed that a microinjection of ABA (10 and 20 µg/rat, i.t.) significantly increased the nociceptive threshold in tail flick and hot plate tests. The application of PKA inhibitor (H.89, 100 nM/rat) significantly inhibited ABA-induced analgesic effects. Expression of p-ERK was significantly decreased in ABA-injected animals, which were not observed in the ABA+H.89-treated group. Conclusions: Overall, i.t. administration of ABA (10 µg/rat) induced analgesia and p-ERK down-expression likely by involving the PKA-dependent mechanism.

Resumo Objetivo: O ácido fito-hormônio abscísico (ABA) existe como molécula sinalizadora em vários tipos de organismos, de multicelulares a células e tecidos animais. Foi demonstrado que o ABA tem efeito antinociceptivo em roedores. O presente estudo foi desenhado para avaliar o possível papel da PKA e da ERK fosforilada (p-ERK) nos efeitos antinociceptivos do ABA intratecal (i.t.) em ratos Wistar machos. Métodos: Os animais foram canulados por via i.t. e divididos em diferentes grupos experimentais (n=6‒7): controle (sem cirurgia), veículo (veículo ABA recebido), grupos tratados com ABA (recebeu ABA em doses de 10 ou 20 µg/rato), grupo tratado com ABA mais H.89 (inibidor de PKA) que recebeu o inibidor 15 minutos antes da injeção de ABA. Os testes de movimento da cauda e placa quente foram utilizados como estimuladores nociceptivos agudos para avaliar os efeitos analgésicos da ABA. A p-ERK foi avaliada na porção dorsal da medula espinhal por imunotransferência. Resultados: A microinjeção de ABA (10 e 20 µg/rato, i.t.) aumentou significativamente o limiar nociceptivo nos testes de movimento da cauda e placa quente. A aplicação de inibidor de PKA (H.89, 100 nM/rato) inibiu significativamente os efeitos analgésicos induzidos por ABA. A expressão de p-ERK diminuiu significativamente em animais injetados com ABA que não foram observados no grupo tratado com ABA+H.89. Conclusões: No geral, a administração i.t. de ABA (10 µg/rato) induziu a analgesia e expressão negativa de p-ERK provavelmente envolvendo mecanismo dependente de PKA.

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.

De novo in vitro shoot morphogenesis from shoot tip-induced callus cultures of Gymnema sylvestre (Retz.) R. Br. ex Sm

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.

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%).

Carbon and nutrient stocks of three Fabaceae trees used for forest restoration and subjected to fertilization in Amazonia

ABSTRACT Amazonia is crucial to global carbon cycle. Deforestation continues to be one of the main causes of the release of C into the atmosphere, but forest restoration plantations can reverse this scenario. However, there is still diffuse information about the C and nutrient stocks in the vegetation biomass. We investigated the carbon and nutrient stocks of Fabaceae trees (Inga edulis, Schizolobium amazonicum and Dipteryx odorata) subjected to fertilization treatments (T1 - no fertilization; T2 - chemical; T3 - organic; and T4 - organic and chemical fertilization) in a degraded area of the Balbina Hydroelectric Dam, AM - Brazil. As an early successional species, I. edulis stocked more C and nutrients than the other two species independent of the fertilization treatment, and S. amazonicum stocked more C than D. odorata under T1 and T4. The mixed species plantation had the potential to stock 4.1 Mg C ha-1 year-1, while I. edulis alone could stock 9.4 Mg C ha-1 year-1. Mixing species that rapidly assimilate C and are of significant ecological and commercial value (e.g., Fabaceae trees) represents a good way to restore degraded areas. Our results suggest that the tested species be used for forest restoration in Amazonia.

Vegetative propagation of Plukenetia polyadenia by cuttings: effects of leaf area and indole-3-butyric acid concentration / Propagação vegetativa de Plukenetia polyadenia por estacas: efeitos de área foliar e concentração de ácido indol-3-butírico

Abstract The seeds of Plukenetia polyadenia have high levels of unsaturated fatty acids and are used as medicine and food for native people in the Peruvian and Brazilian Amazon. The objective of this study was to develop a method for vegetative propagation of Plukenetia polyadenia by rooting of cuttings. The experiment was laid out in a randomized complete block design with 12 treatments and 3 replications of 8 cuttings, in a 3 × 4 factorial arrangement. The factors were: 3 levels of leaf area (25, 50 and 75%) and 3 indole-3-butyric acid - IBA concentrations (9.84, 19.68 and 29.52mM) and a control without IBA. Data were submitted to analysis of variance and means were compared by Tukey test at 5% probability. Our results show that the use of cuttings with 50% of leaf area and treatment with 29.52mM of IBA induced high percentages of rooting (93%) and the best root formation. Vegetative propagation of Plukenetia polyadenia by cuttings will be used as a tool to conserve and propagate germplasm in breeding programs.

Resumo As sementes de Plukenetia polyadenia têm altos níveis de ácidos graxos insaturados e são utilizadas como medicamentos e alimentos para as pessoas nativas da Amazônia Peruana e Brasileira. O objetivo do trabalho foi desenvolver um método de propagação vegetativa de Plukenetia polyadenia por meio do enraizamento de estacas em câmeras de sub-irrigação. Foi utilizado um delineamento de blocos ao acaso com 12 tratamentos e 3 repetições de 8 estacas, e esquema fatorial 3 × 4. Os fatores foram: 3 níveis de área foliar (25, 50 e 75%) e 3 doses de ácido indol-3-butírico - AIB (9,84; 19,68 e 29,52mM) e um controle sem AIB. Os dados foram submetidos à análise de variância e as médias foram comparadas pelo teste de Tukey a 5% de probabilidade. A maior taxa de enraizamento de estacas (93%) foi obtida com 29,52mM de AIB como indutor hormonal e estacas com área foliar de 50%. A propagação vegetativa de Plukenetia polyadenia por estacas será usada como ferramenta para conservar e propagar germoplasma em programas de melhoramento.

Influence of gibberellic acid, indole butyric acid, and methanol on morpho-physiological and phytochemical traits in Thymus vulgaris L.

Background: Plant growth regulators [PGRs] have important roles in many processes such as germination, seedling growth, nutrition uptake, morphogenesis, ripening, etc

Objective: This study aimed to evaluate the effect of gibberellic acid, indole butyric acid, and methanol as plant growth regulators on morpho-physiological and phytochemical features in Thymus vulgaris L

Methods: The farm experiment based on a randomized complete block design was performed at 2014. The treatments were included G1M1 [GA3 50 ppm + methanol 20 %], G1M2 [GA3 50 ppm +methanol 40 %], G2M1 [GA3 100 ppm + methanol 20 %], G2M2 [GA3 100 ppm + methanol 40 %], I1M1 [IBA 50 ppm + methanol 20 %], I1M2 [IBA 50 ppm + methanol 40 %], I2M1 [IBA 100 ppm + methanol 20 %], I2M2 [IBA 100 ppm + methanol 40 %], G1I2M2 [GA3 50 ppm + IBA 100 ppm + methanol 40 %], G1I2M2 [GA3 100 ppm + IBA 50 ppm + methanol 40 %], and control [distillate water]

Results: The results showed that the combination of GA3 or IBA along with methanol changed significantly leaf length and width, number of branches, leaf dry weight, stem dry weight, plant dry weight, seed weight, essential oil content and thymol amount. The highest values of the most traits were observed in I1M1 treatment

Conclusion: Methanol combination with one of the GA3 or IBA can improve morpho-physiological and phytochemical traits of thyme [Thymus vulgaris L.]. Thus, the most effective PGRs combination was related to GA3 50 ppm + Methanol 20 % and IBA 50 ppm + Methanol 20 %

In vitro propagation of the Garden Heliotrope, Valeriana officinalis L.: Influence of pre-chilling and light on seed germination.

Valeriana officinalis is an important medicinal herb commonly found in Kashmir valley. This study forms an important preliminary step for in-vitro micro propagation of V. officinalis from breaking the seed dormancy, inducing rapid seed germination and its subsequent micro propagation. We investigated the influence of pretreatment of V. officinalis seeds with reduced temperature and light on seed germination and in-vitro propagation. Culture of explants from cultivated seeds have demonstrated its potential for in vitro propagation and plantlet regeneration. Individual as well as combinations of treatments such as temperature and light availability influenced the germination of seeds variedly. Unchilled seeds of V. officinalis were given dip in GA3 (200 ppm) for 24, 48 and 120 h. Seeds treated with GA3 for 24 h and kept in darkness showed the best results, i.e. 48%. Seeds pretreated with GA3 for 120 h and incubated in dark showed 40% germination. Pre-chilling up to 72 h and kept in light showed maximum germination of 60% followed by 40% kept in darkness. Pre-chilling for 48 h resulted in 40 and 25% seed germination in light and darkness, respectively. GA3 pre-treatment for 72 h and 24 h pre chilling were most effective in inducing seed germination. Maximum shoot response was obtained on MS enriched with BAP (1mg/L) + IAA (0.1mg/L) combinations using shoot tips as explants. Multiple shoot regeneration from shoot apices was recorded on BAP (1mg/L) and BAP (1mg/L) + IAA (0.1mg/L).

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.

A simple and cost effective liquid culture system for the micropropagation of two commercially important apple rootstocks.

The two commercially important apple rootstocks i.e., MM106 and B9 were micropropagated using a liquid culture system. Three different strengths of 0.8% agar solidified PGR free basal MS medium were first tested to optimize the culture media for both the rootstocks. Full strength medium (MS0) supported maximum in vitro growth, multiplication, rooting and survival under field conditions as opposed to quarter and half strength media. When three different volumes of liquid MS0 were tested, highest in vitro growth, multiplication, rooting and also survival under field conditions were achieved in 20 mL liquid MS0. The cost of one litre of liquid medium was also reduced by 8 times to Rs. 6.29 as compared to solid medium. The cost of 20 mL medium was further reduced to Rs. 0.125.

In vitro regeneration of Coelogyne nervosa A.Rich. and Eria pseudoclavicaulis Blatt., threatened orchids of Western Ghats, India.

The seeds of C. nervosa and E. pseudoclavicaulis were germinated asymbiotically on Knudson C (KC) and Schenk and Hildebrandt basal medium (SH). Growth regulators such as 2,4-Dichlorophenoxyacetic acid (2,4-D) individually and in combinations with benzyladenine (BA) and kinetin were used for callus induction from the protocorm like bodies. Coelogyne nervosa showed maximum (90%) callus induction in Knudson C medium supplemented with 2,4-D (2.26 µM) and Eria pseudoclavicaulis showed 60% callus induction in Schenk and Hildebrandt medium supplemented with 2,4-D (2.26 µM). Calli developed a route of production of protocorm-like bodies and eventually developed into plantlets on transfer to growth regulator free half strength basal medium. The well rooted plants were hardened successfully in the potting mixture containing coconut husk, charcoal, and brick pieces in the ratio 2:1:1.

Establishment of an in vitro plantlet regeneration protocol for unique varieties of brinjal (Solanum melongena L.) var. Mattu Gulla and Perampalli Gulla.

Brinjal (Solanum melongena L.) var. Mattu Gulla (MG) and var. Perampalli Gulla (PG) are unique varieties with distinct flavour cultivated in Udupi, Karnataka State, and are exposed to several biotic and abiotic stresses. An efficient and reproducible in vitro regeneration method is required to expedite the manipulation of these brinjal varieties to cope up with stress by tissue culture and gene transfer methods. The present study, reports a rapid and efficient in vitro regeneration protocol for these two varieties. The in vitro growth response was studied on Murashige and Skoog (MS) medium supplemented with 2, 4-D, BAP and IAA, and the plantlets were regenerated efficiently from callus cultures of leaf, cotyledon and hypocotyl explants. Among the three explants, the hypocotyl explants were found to have better callus induction and multiple shoot regeneration. High frequency of shoot initiation was achieved from hypocotyl derived calluses in MS media with 2.0 mg/L BAP and 0.5 mg/L IAA in MG and PG. Efficient and rapid shoot proliferation, and elongation were noted in MS medium with 1.0 mg/L BAP and 0.3 mg/L GA3. The in vitro regenerated shoots produced healthy roots when they were cultured on MS medium supplemented with 0.5 mg/L IBA. A significant difference was observed in percentage of callus induction, number of shoots per callus, shoot elongation and number of hardened plantlets of MG and PG. MG showed maximum response in all stages of culture than PG. Hardening of plantlets in tissue culture was achieved in three weeks. The hardened plantlets were grown in pots for further acclimatization in green house and finally transplanted to experimental garden where they developed into flowering plants and produced mature fruits with viable seeds.

Carotenoid genes transcriptional regulation for astaxanthin accumulation in fresh water unicellular alga Haematococcus pluvialis by gibberellin A3 (GA3).

The fresh water unicellular alga Haematococcus pluvialis is a promising natural source of astaxanthin. The present study investigated the transcriptional expression of carotenoid genes for astaxanthin accumulation in H. pluvialis using real-time fluorescence quantitative PCR (qRT-PCR). With treatments of 20 and 40 mg/L of gibberllin A3 (GA3), five genes ipi-1, ipi-2, psy, pds and bkt2 were up-regulated with different expression profiles. GA20 (20 mg/L of GA3) treatment had a greater effect on transcriptional expression of bkt2 than on ipi-1 ipi-2, psy and pds (>4-fold up-regulation). However, GA40 (40 mg/L of GA3) induced more transcriptional expression of ipi-2, psy and bkt2 than both ipi-1 and pds. The expression of lyc, crtR-B and crtO for astaxanthin biosynthesis was not affected by GA3 in H. piuvialis. In the presence of GA3, astaxanthin biosynthesis genes of ipi-1, pds and bkt2 were up-regulated at transcriptional level, psy at post-transcriptional level, whereas ipi-2 was up-regulated at both levels. The study could potentially lead to a scale application of exogenous GA3 in astaxanthin production with H. pluvialis just like GAs perform in increasing crops production and it would provide new insight about the multifunctional roles of carotenogenesis in response to GA3.

A novel combination of plant growth regulators for in vitro regeneration of complete plantlets of guar [Cyamopsis tetragonoloba (L.) Taub.].

A novel combination of plant growth regulators comprising indole-3-butyric acid (IBA), 6-benzylaminopurine (BA) and gibberellic acid (GA3) in Murashige and Skoog basal medium has been formulated for in vitro induction of both shoot and root in one culture using cotyledonary node explants of guar, (Cyamopsis tetragonoloba). Highest percentages of shoot (92%) and root (80%) induction were obtained in the medium containing (mg/L) 2 IBA, 3 BA and 1 GA3. Shoot regeneration from the cotyledonary node explants was observed after 10-15 days. Regeneration of roots from these shoots occurred after 20 to 25 days. The regenerated plantlets showed successful acclimatization on transfer to soil. This protocol is expected to be helpful in carrying out various in vitro manipulations in this economically and industrially important legume.

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.

Plant regeneration from callus cultures of Vitex trifolia (Lamiales: Lamiaceae): a potential medicinal plant / Regeneración de plantas a partir de cultivos de callos de Vitex trifolia (Lamiales: Lamiaceae): una planta medicinal potencial

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.

Interaction between cochleata and stipule-reduced mutations results in exstipulate hypertrophied leaves in Pisum sativum L.

In the wild type P. sativum, each of the adult plant stem nodes, bears a pair of sessile foliaceous stipules and a petiolated unipinnately compound leaf of 4 to 6 leaflets and 7-9 tendrils. The stipule-reduced (st) and cochleata (coch) single null mutants and coch st double null mutant differ fom the wild type in respectively having sessile stipules of much reduced size, petiolated simple and/or compound leaf-like stipules and no stipules. It is also known that coch leaves are somewhat bigger than st and wild type leaves. Here, pleiotropic phenotype of coch st double mutant was investigated. The morphologies of stipules and leaf were quantified in the field grown plants and microcultured shoots, latter in the presence and absence of gibberellic acid and N-1-naphthylphthalamic acid. The observations showed that as compared to the corresponding plants or shoots of COCH ST (WT) genotype, (a) coch st plants bore leaves in which all the organs were hypertrophied; (b) full complement of leaflets and 3-5 tendrils were formed on leaf; (c) the microcultured coch st shoots were taller despite lower number of nodes, and (d) they also produced leaves in which all the organs were bigger and the ratio of leaflets/tendrils was higher. It was concluded that in coch st double mutant (a) ST function is essential for stipule primordium differentiation, in the absence of COCH function and (b) absence of negative feedback loops between simple stipules and compound leaf for metabolite utilization allows hypertrophied growth in leaves.

Analysis of mRNA expression profiles of carotenogenesis and astaxanthin production of Haematococcus pluvialis under exogenous 2, 4-epibrassinolide (EBR)

The fresh-water green unicellular alga Haematococcus pluvialis is known to accumulate astaxanthin under stress conditions. In the present study, transcriptional expression of eight genes involved in astaxanthin biosynthesis exposed to EBR (25 and 50 mg/L) was analyzed using qRT-PCR. The results demonstrated that both 25 and 50 mg/L EBR could increase astaxanthin productivity and the eight carotenogenic genes were up-regulated by EBR with different expression profiles. Moreover, EBR25 induction had a greater influence on the transcriptional expression of ipi-1, ipi-2, crtR-B, lyc and crtO (> 5- fold up-regulation) than on psy, pds, bkt; EBR50 treatment had a greater effect on the transcriptional expression of ipi-2, pds, lyc, crtR-B, bkt and crtO than on ipi-1 and psy. Furthermore, astaxanthin biosynthesis under EBR was up-regulated mainly by ipi1־ and psy at the post-transcriptional level, pds, lyc, crtR-B, bkt and crtO at the transcriptional level and ipi-2 at both levels.