Proliferation of Axillary Shoots of Chestnut in Temporary Immersion Systems.

Shoots from European chestnut and hybrids of European and Asian chestnuts can be efficiently proliferated in liquid medium by temporary immersion (TIS) using RITA® and Plantform™ bioreactors. The main challenges of applying TIS to these species were the lack of growth and hyperhydricity; problem solutions included the manipulation of the hormone concentration, the explant type, immersion frequency, and a support for maintaining shoots in a vertical position. After protocol optimization, explants cultured by TIS produced more rootable shoots than explants growing in semisolid medium, enabling increased number of rooted and acclimatable shoots. In this chapter, we will describe the protocols for proliferating chestnut by TIS in RITA® and Plantform™ bioreactors, together with tips for avoiding the main pitfalls of the technique. The strategies applied to chestnut can be useful for culturing other woody plants in bioreactors.

Effects of Monochromatic Light on Growth and Quality of Pistacia vera L.

Light-emitting diodes (LEDs) are popular as a light source for in vitro plants because they save energy and allow the morphology of the plant to be altered. The purpose of this study was to show that switching from classical fluorescent light (FL) to LED light can have both beneficial and adverse effects. Pistacia vera plantlets were exposed to FL, monochromatic Blue LED light (B), monochromatic Red LED light (R), and a 1:1 mixture of both B and R (BR). R increased the total weight, shoot length, number of shoots ≥ 1 cm, and proliferation. It also reduced hyperhydricity (HH), but also dramatically increased shoot tip necrosis (STN) and leaf necrosis (LN). B cured plants of HH and STN, but hardly enabled proliferation. It did not solve the problem of LN, but the plants were high in total chlorophyll and carotenoids. BR reduced HH but enabled limited proliferation, high STN, and LN. All three LED treatments reduced HH compared to FL. B induced both high total phenolic and flavonoid content and high DPPH-scavenging activity. These results show that switching from FL to LED can have a significant positive or negative effect on proliferation and quality. This suggests that finding an optimal lighting regimen will take a lot of trial and error.

Hyperhydricity in Plant Tissue Culture.

Hyperhydricity is the most common physiological disorder in in vitro plant cultivation. It is characterized by certain anatomical, morphological, physiological, and metabolic disturbances. Hyperhydricity significantly complicates the use of cell and tissue culture in research, reduces the efficiency of clonal micropropagation and the quality of seedlings, prevents the adaptation of plants in vivo, and can lead to significant losses of plant material. This review considers the main symptoms and causes of hyperhydricity, such as oxidative stress, impaired nitrogen metabolism, and the imbalance of endogenous hormones. The main factors influencing the level of hyperhydricity of plants in vitro are the mineral and hormonal composition of a medium and cultivation conditions, in particular the aeration of cultivation vessels. Based on these factors, various approaches are proposed to eliminate hyperhydricity, such as varying the mineral and hormonal composition of the medium, the use of exogenous additives, aeration systems, and specific lighting. However, not all methods used are universal in eliminating the symptoms of hyperhydricity. Therefore, the study of hyperhydricity requires a comprehensive approach, and measures aimed at its elimination should be complex and species-specific.

Hypolignification: A Decisive Factor in the Development of Hyperhydricity.

One of the characteristics of hyperhydric plants is the reduction of cell wall lignification (hypolignification), but how this is related to the observed abnormalities of hyperhydricity (HH), is still unclear. Lignin is hydrophobic, and we speculate that a reduction in lignin levels leads to more capillary action of the cell wall and consequently to more water in the apoplast. p-coumaric acid is the hydroxyl derivative of cinnamic acid and a precursor for lignin and flavonoids in higher plant. In the present study, we examined the role of lignin in the development of HH in Arabidopsis thaliana by checking the wild-types (Ler and Col-0) and mutants affected in phenylpropanoid biosynthesis, in the gene coding for cinnamate 4-hydroxylase, C4H (ref3-1 and ref3-3). Exogenously applied p-coumaric acid decreased the symptoms of HH in both wild-type and less-lignin mutants. Moreover, the results revealed that exogenously applied p-coumaric acid inhibited root growth and increased the total lignin content in both wild-type and less-lignin mutants. These effects appeared to diminish the symptoms of HH and suggest an important role for lignin in HH.

Effective reversal of hyperhydricity leading to efficient micropropagation of Dianthus chinensis L.

The effective reversion of hyperhydricity (HH) in Dianthus chinensis L. facilitated efficient in vitro production of hyperhydricity-free plantlets. Under routine sub-culture practice, the problem of HH arises after third sub-culture in agar (0.85%) gelled Murashige and Skoog (MS) medium containing 2.5 µM 6-benzyladenine (BA). To confirm the role of ethylene on hyperhydricity induction, an ethylene releasing compound ethephon (5 µM) was used in combination with 2.5 µM BA and demonstrated 100% HH with reduced stomatal aperture. Supplementation of 10 µM silver nitrate (AgNO3) to 2.5 µM BA containing medium resulted HH reversion with reduced shoot number (19.0); however, addition of 5 µM cobalt chloride (CoCl2) produced highest microshoots (202.0). The combination effect of AgNO3 (10 µM), CoCl2 (5 µM), and BA (2.5 µM) showed complete HH reversion and upheld normal microshoots (55.0) with reduced relative water content (78.3%). The Ag and Co salts regulate ethylene biosynthesis and thereby 50% reductions in H2O2 content characterized by formation of green healthy shoots with proper stomatal morphology. The gene expression profile of 1-Amminocyclopropane-1-carboxylase synthase (ACS1) and 1-Amminocyclopropane-1-carboxylic acid oxidase (ACO1) showed reduced expression after the retroversion of microshoots in anti-ethylene reversion medium compared to hyperhydric shoot. In vitro raised shoots were rooted (93.3%) ex vitro by 10 mM IBA treatment and 92.2% plants were survived. The genetic stability of micropropagated plants were analyzed and proved that addition of low levels of heavy metal salt in the medium does not cause any variation in banding pattern. The protocol forwards a novel method to revert HH of in vitro cultures by adopting intermittent exposure of anti-ethylene compounds added in the medium and the procedure can be applied to many other plants facing similar HH problems. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02645-7.

Arabidopsis seedling establishment under waterlogging requires ABCG5-mediated formation of a dense cuticle layer.

Germination requires sufficient water absorption by seeds, but excessive water in the soil inhibits plant growth. We therefore hypothesized that tolerance mechanisms exist that help young seedlings survive and develop in waterlogged conditions. Many ATP-BINDING CASSETTE TRANSPORTER subfamily G (ABCG) proteins protect terrestrial plants from harsh environmental conditions. To establish whether any of these proteins facilitate plant development under waterlogged conditions, we observed the early seedling growth of many ABCG transporter mutants under waterlogged conditions. abcg5 seedlings exhibited severe developmental problems under waterlogged conditions: the shoot apical meristem was small, and the seedling failed to develop true leaves. The seedlings had a high water content and reduced buoyancy on water, suggesting that they were unable to retain air spaces on and inside the plant. Supporting this possibility, abcg5 cotyledons had increased cuticle permeability, reduced cuticular wax contents, and a much less dense cuticle layer than the wild-type. These results indicate that proper development of plants under waterlogged conditions requires the dense cuticle layer formed by ABCG5 activity.

Changes in the biochemical parameters of albino, hyperhydric and normal green leaves of Caladium bicolor cv. "Bleeding hearts" in vitro long-term cultures.

Caladium bicolor is an excellent landscape and pot plant grown for its ornamental value due to the varied shapes and multi-colored foliage. However, the ornamental value of in vitro clonally propagated plants is affected by the occurrence of morpho-physiological anomalies in the long-term cultures. During the long-term culture of rhizome-derived shoot cultures of C. bicolor cv. "Bleeding hearts", some incidences of hyperhydricity and albinism were observed. Hyperhydricity occurred at high frequency in the shoot cultures and was more prevalent in the liquid over solid medium cultivated plantlets. The addition of silver nitrate at 7.5 µM reduced prevalence of the condition over other treatment concentrations but, changes in the gelling agent concentration and photoperiodic incubation were ineffective. Albinism also occurred at a higher frequency but, was more prevalent in the cultures incubated below or above 12 h light/dark photoperiods. Evaluation of the physiological, biochemical parameters showed differences between the leaves of albino, hyperhydric and normal green C. bicolor micro shoots. The experimental results obtained suggested an effective role of pigment molecules, cellular osmolytes, and enzymatic and non-enzymatic antioxidant systems in the development of albino and hyperhydric leaves in C. bicolor micro shoot cultures. Photoperiodic incubation and culture conditions showed some effect on the incidences of hyperhydricity and albinism in the shoot cultures.

High accumulation of tilianin in in-vitro cultures of Agastache mexicana and its potential vasorelaxant action.

Agastache mexicana has gained importance during the last decade as a natural source of bioactive compounds, mainly due to the antidiabetic, antihyperlipidemic, and vasorelaxant effects derived from its flavonoids, particularly tilianin. The goal of this work was to evaluate the production of tilianin during the in-vitro process of morphogenesis leading to plant regeneration and to investigate the vasorelaxant activity of its methanolic extracts. The cultures were established from nodal segments and leaf explants, inoculated on Murashige and Skoog (MS) media supplemented with various concentrations of benzyl aminopurine (BAP) alone or in combination with 2,4-Dichlorophenoxyacetic acid (2,4-D). Callus inductions were obtained in all treatments from both types of explants, but the presence of auxin was essential. Maximal shoot multiplication and elongation was achieved with 0.1 mg/l 2,4-D and 1.0 mg/l BAP from nodal- segment explants. Shoots were rooted in 75% MS medium and the plantlets were transferred to a greenhouse with 33% average survival. Analysis of tilianin production in methanolic extracts from calli (0.15-2.01 ± 0.06 mg/g dry weight), shoots (4.45 ± 0.01 mg/g DW), and whole plants (9.77 ± 0.02 mg/g DW) derived from in-vitro cultured nodal segments reveals that tilianin accumulation is associated with high cell differentiation and morphogenetic response to the plant-growth regulators. All of the extracts showed strong vasorelaxant activity, as compared to those of wild plant extracts. These results indicate that plant-tissue cultures of A. mexicana possess vast potential as a source of tilianin and other bioactive compounds.

Tools for the ex situ conservation of the threatened species, Cycladenia humilis var. jonesii.

Ex situ conservation is critical for hedging against the loss of plant diversity. For those species (exceptional species) that cannot be conserved long-term in standard seed banks, alternative methods are required, often involving in vitro culture and cryopreservation, or storage in liquid nitrogen. Cycladenia humilis var. jonesii is a federally threatened perennial native to Utah and Arizona. It is classified as an exceptional species, because it produces few seeds, and, thus, in vitro propagation and cryopreservation were investigated as tools for its propagation and preservation. Shoot-propagating cultures were established from both seedling and wild-collected shoots, but cultures from both sources displayed an extreme form of the physiological disorder, hyperhydricity. This phenotype could be at least partially normalized by the use of vented closures, as well as by using agar, rather than gellan gum, in the medium. The hyperhydric (HH) phenotype had a lower dry weight, more branching, minimal leaf development and more poorly developed vascular tissue than the more normal (MN) phenotype. Only more normalized shoots could be rooted and the resulting plants acclimatized. Both HH and MN shoots also provided shoot tips capable of surviving cryopreservation using the droplet vitrification method. These in vitro and cryopreservation methods provide tools that can be used for propagating plants of C. humilis var. jonesii for research and restoration, as well as for supplying shoot tips for the ex situ conservation of this species. The two distinct phenotypes also provide a useful system for studying factors involved in the HH response of this dryland species in vitro.

Controlling Hyperhydricity in Date Palm In Vitro Culture by Reduced Concentration of Nitrate Nutrients.

Hyperhydricity (or vitrification) is a fundamental physiological disorder in date palm micropropagation. Several factors have been ascribed as being responsible for hyperhydricity, which are related to the explant, medium, culture vessel, and environment. The optimization of inorganic nutrients in the culture medium improves in vitro growth and morphogenesis, in addition to controlling hyperhydricity. This chapter describes a protocol for controlling hyperhydricity during the embryogenic callus stage by optimizing the ratio of nitrogen salts of the Murashige and Skoog (MS) nutrient culture medium. The best results of differentiation from cured hyperhydric callus are obtained using modification at a ratio of NH4+/NO3- at 10:15 (825:1425 mg/L) of the MS culture medium to remedy hyperhydric date palm callus and achieve the recovery of normal embryogenic callus and subsequent regeneration of plantlets. Based on the results of this study, nutrient medium composition has an important role in avoiding hyperhydricity problems during date palm micropropagation.

Proteomic Analysis Reveals the Dynamic Role of Silicon in Alleviation of Hyperhydricity in Carnation Grown In Vitro.

The present study depicted the role of silicon in limiting the hyperhydricity in shoot cultures of carnation through proteomic analysis. Four-week-old healthy shoot cultures of carnation "Purple Beauty" were sub-cultured on Murashige and Skoog medium followed with four treatments, viz. control (-Si/-Hyperhydricity), hyperhydric with no silicon treatment (-Si/+Hyperhydricity), hyperhydric with silicon treatment (+Si/+Hyperhydricity), and only silicon treated with no hyperhydricity (+Si/-Hyperhydricity). Comparing to control morphological features of hyperhydric carnations showed significantly fragile, bushy and lustrous leaf nature, while Si supply restored these effects. Proteomic investigation revealed that approximately seventy protein spots were differentially expressed under Si and/or hyperhydric treatments and were either up- or downregulated in abundance depending on their functions. Most of the identified protein spots were related to stress responses, photosynthesis, and signal transduction. Proteomic results were further confirmed through immunoblots by selecting specific proteins such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), PsaA, and PsbA. Moreover, protein-protein interaction was also performed on differentially expressed protein spots using specific bioinformatic tools. In addition, stress markers were analyzed by histochemical localization of hydrogen peroxide (H2O2) and singlet oxygen (O21-). In addition, the ultrastructure of chloroplasts in hyperhydric leaves significantly resulted in inefficiency of thylakoid lamella with the loss of grana but were recovered in silicon supplemented leaves. The proteomic study together with physiological analysis indicated that Si has a substantial role in upholding the hyperhydricity in in vitro grown carnation shoot cultures.

Reversion of hyperhydricity in pink (Dianthus chinensis L.) plantlets by AgNO3 and its associated mechanism during in vitro culture.

Hyperhydricity occurs frequently in plant tissue culture and can severely affect commercial micropropagation and genetic improvement of the cultured plantlets. Hyperhydric shoots are charaterzized by high water content, but how this occurs is still a subject of investigation. Silver ion (Ag+) can reduce the extent of hyperhydricity in plants, but its effect on the reversion of hyperhydric plantlets and the underlying mechanism of reversion has not been clarified. In this study, about 67% of the hyperhydric Dianthus chinensis L. plantlets were found to revert to normal condition when the plantlets were cultured in medium supplemented with 29.4µmolL-1AgNO3. Water content and hydrogen peroxide (H2O2) content in the guard cells of these plantlets were reduced, while stomatal aperture and water loss rate were increased. AgNO3 also reduced the content of endogenous ethylene and expression of ethylene synthesis and ethylene signal transduction-associated genes. Reduced accumulation of ethylene consequently led to an increase in stomatal aperture mediated by decreased H2O2 content in the guard cells. These results adequately verified the role of AgNO3 in the reversion of hyperhydricity in D. chinensis L. and also provided clues for exploring the cause of excessive water accumulation in hyperhydric plants.

Induction of reactive oxygen species and the potential role of NADPH oxidase in hyperhydricity of garlic plantlets in vitro.

Hyperhydricity is a physiological disorder associated with oxidative stress. Reactive oxygen species (ROS) generation in plants is initiated by various enzymatic sources, including plasma membrane-localized nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, cell wall-bound peroxidase (POD), and apoplastic polyamine oxidase (PAO). The origin of the oxidative burst associated with hyperhydricity remains unknown. To investigate the role of NADPH oxidases, POD, and PAO in ROS production and hyperhydricity, exogenous hydrogen peroxide (H2O2) and inhibitors of each ROS-producing enzyme were applied to explore the mechanism of oxidative stress induction in garlic plantlets in vitro. A concentration of 1.5 mM H2O2 increased endogenous ROS production and hyperhydricity occurrence and enhanced the activities of NADPH oxidases, POD, and PAO. During the entire treatment period, NADPH oxidase activity increased continuously, whereas POD and PAO activities exhibited a transient increase and subsequently declined. Histochemical and cytochemical visualization demonstrated that specific inhibitors of each enzyme effectively suppressed ROS accumulation. Moreover, superoxide anion generation, H2O2 content, and hyperhydric shoot frequency in H2O2-stressed plantlets decreased significantly. The NADPH oxidase inhibitor was the most effective at suppressing superoxide anion production. The results suggested that NADPH oxidases, POD, and PAO were responsible for endogenous ROS induction. NADPH oxidase activation might play a pivotal role in the oxidative burst in garlic plantlets in vitro during hyperhydricity.

Developmental, nutritional and hormonal anomalies of weightlessness-grown wheat.

The behavior of water in weightlessness, as occurs in orbiting spacecraft, presents multiple challenges for plant growth. Soils remain saturated, impeding aeration, and leaf surfaces remain wet, impeding gas exchange. Herein we report developmental and biochemical anomalies of "Super Dwarf" wheat (Triticum aestivum L.) grown aboard Space Station Mir during the 1996-97 "Greenhouse 2" experiment. Leaves of Mir-grown wheat were hyperhydric, senesced precociously and accumulated aromatic and branched-chain amino acids typical of tissues experiencing oxidative stress. The highest levels of stress-specific amino acids occurred in precociously-senescing leaves. Our results suggest that the leaf ventilation system of the Svet Greenhouse failed to remove sufficient boundary layer water, thus leading to poor gas exchange and onset of oxidative stress. As oxidative stress in plants has been observed in recent space-flight experiments, we recommend that percentage water content in apoplast free-spaces of leaves be used to evaluate leaf ventilation effectiveness. Mir-grown plants also tillered excessively. Crowns and culms of these plants contained low levels of abscisic acid but high levels of cytokinins. High ethylene levels may have suppressed abscisic acid synthesis, thus permitting cytokinins to accumulate and tillering to occur.

Enhancement of germination, hyperhydricity control and in vitro shoot formation of Vasconcellea stipulata Badillo / Mejoramiento de la germinación, control de la hiperhidricidad y formación de brotes en Vasconcellea stipulata Badillo

Vasconcellea stipulata has great commercial importance because of its enzymatic activity and as a source for genetic improvement of papaya since it is resistant to the papaya ringspot virus. However, due to its low regeneration by seeds and limited knowledge of its genetic and pharmaceutical properties, this species is not widely cultivated. For propagation, in vitro culture of seeds has been used to address this problem, but hyperhydricity, a physiological disorder, mainly expressed in the developing embryonic axis and specifically associated with this species, is a significant constraint. In order to obtain elite material for culture of V. stipulata, the aim of this work was to increase germination, to control hyperhydricity in embryos and to evaluate the potential to induce morphogenic responses, i.e., shoot formation. Our results showed that it is possible to increase germination up to 53% under in vitro conditions within a short period in the presence of hydrogen peroxide. In addition, hyperhydricity was significantly reduced (50%) in vitro when gibberellic acid concentrations were included on a 1/2 Nitsch and Nitsch nutrient medium, resulting in approximately 80% recovery of viable seedlings. Finally, other plant growth regulators were evaluated and found to trigger shoot formation in axillary buds as well as induce the formation of callus in leaf sections derived of seedlings.

Vasconcellea stipulata posee una gran importancia comercial debido a su actividad enzimática y como fuente para el mejoramiento genético de papaya, debido a su resistencia al virus de la mancha anular de esta especie. Sin embargo, debido a su baja regeneración por semillas y al limitado conocimiento de sus propiedades genéticas y farmacéuticas, esta especie no es cultivada ampliamente. La propagación a través del cultivo in vitro de semillas se ha usado para contrarrestar este tipo de problema, pero la hiperhidricidad, un trastorno fisiológico, expresado principalmente en los ejes embrionarios en desarrollo y asociado específicamente a esta especie, es una restricción significativa. Con el fin de obtener material de élite para el cultivo de V. stipulata, el objetivo de este trabajo fue incrementar la germinación, controlar la hiperhidricidad en embriones y evaluar el potencial para inducir respuestas morfogénicas, es decir, la formación de brotes. Nuestros resultados mostraron que es posible aumentar la germinación hasta un 53% en condiciones in vitro, dentro de un período más corto en presencia de peróxido de hidrógeno. Además, la hiperhidricidad se redujo significativamente (50%) en condiciones in vitro cuando se incluyó ácido giberélico en bajas concentraciones en el medio 1/2 Nitsch y Nitsch. Esto permitió recuperar hasta aproximadamente el 80% de plántulas viables. Finalmente, otros reguladores de crecimiento vegetal evaluados, indujeron la formación de brotes en yemas axilares y la formación de callos en secciones de hoja derivadas de plántulas.

The role of silicon in plant tissue culture.

Growth and morphogenesis of in vitro cultures of plant cells, tissues, and organs are greatly influenced by the composition of the culture medium. Mineral nutrients are necessary for the growth and development of plants. Several morpho-physiological disorders such as hooked leaves, hyperhydricity, fasciation, and shoot tip necrosis are often associated with the concentration of inorganic nutrient in the tissue culture medium. Silicon (Si) is the most abundant mineral element in the soil. The application of Si has been demonstrated to be beneficial for growth, development and yield of various plants and to alleviate various stresses including nutrient imbalance. Addition of Si to the tissue culture medium improves organogenesis, embryogenesis, growth traits, morphological, anatomical, and physiological characteristics of leaves, enhances tolerance to low temperature and salinity, protects cells and against metal toxicity, prevents oxidative phenolic browning and reduces the incidence of hyperhydricity in various plants. Therefore, Si possesses considerable potential for application in a wide range of plant tissue culture studies such as cryopreservation, organogenesis, micropropagation, somatic embryogenesis and secondary metabolites production.

Flooding of the apoplast is a key factor in the development of hyperhydricity.

The physiological disorder hyperhydricity occurs frequently in tissue culture and causes several morphological abnormalities such as thick, brittle, curled, and translucent leaves. It is well known that hyperhydric shoots are characterized by a high water content, but how this is related to the abnormalities is not clear. It was observed that water accumulated extensively in the apoplast of leaves of hyperhydric Arabidopsis seedlings and flooded apoplastic air spaces almost completely. In hyperhydric Arabidopsis seedlings, the volume of apoplastic air was reduced from 85% of the apoplast to only 15%. Similar results were obtained with hyperhydric shoots of statice. The elevated expression of hypoxia-responsive genes in hyperhydric seedlings showed that the water saturation of the apoplast decreased oxygen supply. This demonstrates a reduced gas exchange between the symplast and its surroundings, which will consequently lead to the accumulation of gases in the symplast, for example ethylene and methyl jasmonate. The impairment of gas exchange probably brings about the symptoms of hyperhydricity. Interestingly, stomatal aperture was reduced in hyperhydric plants, a previously reported response to injection of water into the apoplast. Closure of the stomata and the accumulation of water in the apoplast may be the reasons why seedlings with a low level of hyperhydricity showed improved acclimatization after planting into soil.

In vitro tuberization of Chlorophytum Borivilianum Sant & Fern (Safed musli) as influenced by sucrose, CCC and culture systems.

This study focuses on the establishment of in vitro tuberization of Chlorophytum borivilianum using solid and liquid culture systems. A high in vitro tuberization rate on solid and stationary liquid Murashige and Skoog media was observed in the presence of 60 g l⁻¹ sucrose with 950, 1,265 and 1,580 µM 2-chloroethyl-trimethylammonium chloride (CCC). Application of a higher sucrose concentration of 90 g l⁻¹ showed a negative interaction with CCC on in vitro tuber number and days to in vitro tuber induction. For economic feasibility, 950 µM CCC with 60 g l⁻¹ sucrose was chosen as the best combination for in vitro tuberization in both solid and stationary liquid media. For optimization of in vitro tuber production,a comparison between solid, stationary liquid and shake liquid culture was carried out. Liquid culture with shaking at 80 r.p.m. resulted in a >2.5-fold increase in in vitro tuber production compared with solid culture.

Benzilaminopurina e ácido naftaleno acético na indução e multiplicação in vitro de gemas de abacaxizeiro da cultivar 'IAC Gomo-de-mel' / In vitro bud induction and multiplication of cv. 'IAC Gomo-de-mel' pineapple fruit with benzyl amino purine and n aphthalene acetic acid

O objetivo deste trabalho foi avaliar os efeitos de BAP (6-benzilaminopurina) e NAA (ácido naftaleno acético) na indução, na multiplicação in vitro de gemas, nas brotações de Ananas comosus da cultivar 'IAC Gomo-de-mel' e a correlação desses efeitos com a atividade de peroxidase e o teor de proteína solúvel total. Foram utilizadas gemas axilares retiradas da coroa de frutos sadios, inoculadas em tubo de ensaio contendo meio de cultura MS solidificado com ágar a 5 por cento, pH ajustado para 5,7, contendo os tratamentos que incluíam diferentes concentrações e combinações de BAP (0, 0,5, 1,0 e 1,5mg L-1) e NAA (0, 0,5 e 1,0mg L-1). Nessa fase, aos 65 dias, ocorreu a formação de 2,24 brotações, utilizando-se 1mg L-1 de BAP. Após o desenvolvimento, as gemas foram inoculadas em meio MS líquido associado a dois tratamentos (1,0mg L-1 BAP + 0,5mg L-1 NAA e 1,0mg L-1 BAP + 1,0mg L-1 NAA) e, aos 95 dias, o meio de cultura mais adequado foi aquele que continha 1,0mg L-1 BAP + 0,5mg L-1 NAA, proporcionando 7,42 brotações, menor porcentagem de hiper-hidricidade, maior número de brotações e indução de gemas. As proteínas solúveis apresentaram relação negativa com hiper-hidricidade e comprimento de brotações. A atividade da peroxidase foi maior em plantas com maior número de brotos e com maior porcentagem de hiper-hidricidade.

The objective of this research was to evaluate the effects of BAP (6-benzyl amino purine) and NAA (naphthalene acetic acid) on in vitro buds induction and multiplication on Ananas comosus cv. 'IAC Gomo-de-mel' shoots and its correlation with the peroxidase activity and soluble protein content. Axillary buds were excised from the healthy fruits crown and inoculated in MS solidified with agar at 5 percent and pH adjusted to 5.7 with the treatments that included different concentrations and combinations of BAP (0, 0.5, 1.0 and 1.5mg L-1) and NAA (0, 0.5 and 1.0mg L-1). At, 65 days stage, 2.24 shoots were formatted using BAP 1mg L-1. After bud development, which were inoculated in MS liquid medium associated with two treatments (1.0 mg L-1 BAP + 0.5mg L-1 NAA e 1.0mg L-1 BAP + 1.0mg L-1 NAA). At 95 days the medium more appropriate was 1.0mg L-1 BAP + 0.5mg L-1 NAA, providing 7.42 shoots and lower hyperhydricity percentage, more shoots induction and buds. The total soluble proteins showed negative relationship with hyperhydricity and shoots length. The peroxidase activity was higher in plants with more shoots and greater hyperhydricity percentage.

Caracterização isozimática e atividade de peroxidase em folhas de plantas hiperídrica, intermediária e normal de Bidens pilosa L. mantidas in vitro / Isoezymatic characterization and peroxidase activity in leaves of hyperhydric, intermediary and normal plants of Bidens pilosa L. grown in vitro

Foram caracterizadas as plantas: hiperídrica, intermediária e normal de um clone de Bidens pilosa mantido em cultivo in vitro por meio de isozimas e da atividade de peroxidase. Empregando-se a eletroforese em géis de amido a 12 por cento, testou-se seis isozimas, sendo detectado polimorfismo em peroxidase e fosfatase ácida, permitindo caracterizar cada tipo de planta. Não houve polimorfismo em fosfogluco isomerase, fosfoglucomutase, glutamato oxaloacetato transaminase e malato desidrogenase. A atividade da peroxidase foi maior nas plantas hiperídricas e intermediárias. Conclui-se que a variabilidade enzimática tem potencial como marcador de hiperidricidade em plantas mantidas in vitro.

Activity of peroxidase (EC 1.11.1.7) and isozymes analysis of a Bidens pilosa clone maintained in vitro culture were characterized in hyperhydric, intermediary and normal plants. Electrophorese in starch gels (12 percent) of six isozymes systems was tested, polymorphisms in peroxidase and acid phosphatase (EC 3.1.3.2) were detected. There was absence of polymorphism in phosphoglucoisomerase (EC 5.3.1.9), phosphoglucomutase (EC 5.4.2.2), glutamate oxaloacetate transaminase (EC 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37). Comparing the activity of peroxidase enzyme, it was higher in hyperhydric and intermediary plants in relation to normal ones. Enzymatic variability is a potential tool as hyperhydricity marker in plants grown in vitro.