Influence of Silver Nitrate on Somatic Embryogenesis Induction in Arabica Coffee (Coffea arabica L.).

Plant somatic embryogenesis (SE) has been defined as the formation of embryos from a single or group of haploid or somatic cells [1, 2]. Low frequency (LFSE) and high frequency somatic embryogenesis (HFSE) have been described. In the first type, somatic embryos are induced directly from pro-embryogenic cells of explants, while in the second, they originate from embryogenic callus [1]. It has been suggested that in LFSE the origin of somatic embryos is unicellular, whereas in HFSE has been described as unicellular or multicellular [3]. SE is a powerful biotechnological tool used to propagate elite plants or to conserve important genotypes [4]. Moreover, SE offers an efficient in vitro regeneration approach as a fundamental step in plant genetic improvement for studying basic aspects of ontogenesis of somatic embryos [5]. In Coffea spp., the first studies of SE have been reported at the beginning of 1970 [6]. Since then, a large quantity of LFSE and HFSE protocols have been optimized demonstrating that coffee is not a recalcitrant species for SE [4]. In the LFSE the somatic embryos are obtained faster (approximately 70 days) using only one medium meanwhile in HFSE several media are used and somatic embryo formation takes 9-10 months [4]. Although, in LFSE small number of somatic embryos are obtained (around 10 per explant) compared to hundreds of somatic embryos obtained per gram of embryogenic calli [4], the unicellular origin of somatic embryos in LFSE represents an advantage for the chemical and physical mutagenesis, genetic transformation and genetic editing, since prevents or reduces the appearance of chimeras [7]. In C. arabica and C. canephora many factors (such as genotype, explant type, the physiological state, age and growth conditions of the donor plants, the season of collection, nutrient composition of the medium, the volume of dissolved CO2 or O2 in the culture flask, and plant growth regulators) that affect LFSE induction have been studied [3, 8, 9, 10, 11, 12, 13]. However, few studies reported the effect of silver nitrate on LFSE using leaf explants of C. arabica L. and to the best of our knowledge it has not been analyzed using Caturra and Catuaí, which are two economic important producer cultivars in Costa Rica. Since SE is genotype dependent, the culture medium need to be modified for the different genotypes [7].Therefore, the objective of this study was to determine the influence of the benzyladenine (BAP), indole-3-acetic acid (IAA), and silver nitrate (AgNO3) on low frequency somatic embryogenesis using leaf explants of Coffea arabica L. cultivars Caturra and Catuaí

In vitro plant regeneration system for common bean ( Phaseolus vulgaris ): effect of N6-benzylaminopurine and adenine sulphate

A method for regeneration of the commercially important common bean ( Phaseolus vulgaris ) using N6-benzylaminopurine(BAP) and adenine sulphate (AS) was established. Embryogenic axes of the Costa Rican common bean cultivars Bribr\ued, Brunca, Guaym\ued, Huetar and Telire were cultured on Murashige and Skoog medium supplemented with 100 mgl-1 myo-inositol, 1 mgl-1 thiamine, 30 gl-1 sucrose, BAP (0, 5 and 10 mgl-1), AS (0, 20 and 40 mgl-1) and 8 gl-1 agar. Regardless of the concentration of BAP and AS in the induction medium, the number of shoots and leaves differed significantly among the common bean cultivars evaluated. The higher average of shoots was obtained for Brunca > Telire > Bribr\ued > Guaym\ued > Huetar. Moreover, independently of the cultivar, the induction medium supplemented with 5 mgl-1 BAP and 20 or 40 mgl-1 AS resulted in the higher average of shoots formation. Culture of Bribr\ued, Brunca, Guaym\ued, Huetar and Telire embryogenic axes on induction medium supplemented with different BAP and AS resulted in a differential response. Successful acclimatization of common bean in vitro plants were achieved in the greenhouse, and plants appeared morphologically normal. The regeneration system developed in this investigation for this important crop could be a useful tool for the genetic modification through mutagenesis or genetic transformation

Effect of BAP and IAA on shoot regeneration in cotyledonary explants of Costa Rican melon genotypes

Efecto del BAP y el AIA en la regeneración de brotes a partir de explantes cotiledonares de genotipos de melón costarricense. Para establecer una metodología para la regeneración del melón criollo (Cucumis melo L), se investigó la influencia del genotipo (OSO-1, OSO-2, OSO- 3, PQRG-1, PQRG-2, PQRG-3, y EM-1) y la interacción de N6-bencilaminopurina (BAP) (0,1, 0,5 y 1,0 mg.l-1) con ácido indolacético (AIA) (0, 0,05 y 0,5 mg.l-1) en la inducción de brotes y regeneración de plantas. Independientemente de la concentración de BAP y AIA, el mayor porcentaje de formación de brotes se obtuvo en EM-1>OSO- 1>PQRG-3>OSO-2>PQRG-2>PQRG-1>OSO-3. Por otra parte, independientemente del genotipo, el mayor porcentaje de formación de brotes se obtuvo con 0,5 mg.l-1 BAP y 0,05 mg.l-1 AIA o 1 mg.l-1 BAP y 0 mg.l-1 AIA. El protocolo de cultivo in vitro establecido puede ser utilizado para la micropropagación de genotipos �criollos� de melón

Somatic embryogenesis, plant regeneration and acemannan detection in aloe (Aloe barbadensis mill.)

Embriogénesis somática, regeneración de plantas y detección de acemanan en aloe (Aloe barbadensis Mill). La presente investigación tuvo como objetivo la regeneración de plantas de aloe (Aloe barbadensis Mill.) vía embriogénesis somática. Para la desinfección de los explantes se evaluó 2, 3, 4, 5, 10 y 15 min de sonicación en combinación con 4% v/v de NaOCl. El mayor porcentaje de sobrevivencia (85%) y de menor contaminación (15%) se logró con 5 min de sonicación. Se obtuvo callos embriogénicos friables a partir de meristemos apicales, bases de hojas jóvenes y embriones cigóticos. El mejor explante para la inducción de callos embriogénicos fue la base de hojas jóvenes con 89%, cultivadas en el medio complementado con 2,5 mg.l-1 de 2,4-D, 2 mg.l-1 de BAP y 40 mg.l-1 de sulfato de adenina. El mayor número de brotes se obtuvo a partir de callos embriogénicos producidos de embriones cigóticos, en el medio con 0,05 mg.l-1 de 2,4-D y 2 mg.l-1 de BAP. El análisis de cromatografía líquida de alta resolución (HPLC) reveló que la concentración de acemanan en callos embriogénicos (0,8-2,1 mg.ml-1) fue menor en comparación con la de hojas frescas (85 mg.ml-1). El protocolo de cultivo establecido en la presente investigación puede ser utilizado tanto para la propagación como para la transformación genética

In vitro plant regeneration system for common bean ( Phaseolus vulgaris ): effect of N6-benzylaminopurine and adenine sulphate

A method for regeneration of the commercially important common bean ( Phaseolus vulgaris ) using N6-benzylaminopurine(BAP) and adenine sulphate (AS) was established. Embryogenic axes of the Costa Rican common bean cultivars Bribrí, Brunca, Guaymí, Huetar and Telire were cultured on Murashige and Skoog medium supplemented with 100 mgl-1 myo-inositol, 1 mgl-1 thiamine, 30 gl-1 sucrose, BAP (0, 5 and 10 mgl-1), AS (0, 20 and 40 mgl-1) and 8 gl-1 agar. Regardless of the concentration of BAP and AS in the induction medium, the number of shoots and leaves differed significantly among the common bean cultivars evaluated. The higher average of shoots was obtained for Brunca > Telire > Bribrí > Guaymí > Huetar. Moreover, independently of the cultivar, the induction medium supplemented with 5 mgl-1 BAP and 20 or 40 mgl-1 AS resulted in the higher average of shoots formation. Culture of Bribrí, Brunca, Guaymí, Huetar and Telire embryogenic axes on induction medium supplemented with different BAP and AS resulted in a differential response. Successful acclimatization of common bean in vitro plants were achieved in the greenhouse, and plants appeared morphologically normal. The regeneration system developed in this investigation for this important crop could be a useful tool for the genetic modification through mutagenesis or genetic transformation

Histology of somatic embryogenesis in rice (Oryza sativa cv. 5272)

Rice (Oryza sativa cv. 5272) embryogenic calli were obtained from mature zygotic embryos culture on Murashige & Skoog (1962) medium supplemented with 2.5 mg/l 2,4- dichlorophenoxyacetic acid. Histological analysis of somatic embryogenesis revealed that after two weeks of culture of explants on the callus induction medium, somatic embryo development began with a cluster of proembryogenic cells in the peripheral region of the calli. The outer cell layer of embryogenic calli consisted of small and isodiametric cells with a dense cytoplasm and a prominent nucleus and nucleolus; whereas the inner cell layer is composed of large cells with small nucleus and large vacuole. These embryogenic cells underwent a series of organized divisions and formed the proembryo with a well-defined protodermis. Rev. Biol. Trop. 57 (Suppl. 1): 141-150. Epub 2009 November 30

In vitro plant regeneration system for tropical butternut squash genotypes (Cucurbita moschata)

An efficient and reproducible method for regeneration of commercial and pure lines of tropical butternut squash (Cucurbita moschata) plants via somatic embryogenesis was developed. The influence of genotype, explant source, N6-benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) concentration on somatic embryogenesis induction was investigated. Friable embryogenic calli was produced from zigotic embryos (53-56%) and cotyledons from seedlings (70%) of C. moschata cv. Sello de Oro cultured on callus induction medium (CIM) supplemented with 0.5 mg/l or 3.5 mg/l 2,4-D. No embryogenic calli was obtained from leaf segments of C. moschata cv. Sello de Oro cultured on CIM supplemented with different concentrations of BAP and 2,4-D and cotyledons from seedlings of C. moschata cv. PVG 04 cultured on CIM with BAP and 2,4,5-T. Embryogenic calli induction was achieved in 75% C. moschata pure lines evaluated and calli percentage frequency range from 5% to 34%. Successful acclimatization of squash in vitro plants was achieved in the greenhouse and in the field. Regenerated plants appeared morphologically normal and set flowers and fruits with seeds that could germinate normally. Rev. Biol. Trop. 57 (Suppl. 1): 119-127. Epub 2009 November 30

Responses of Arabica coffee (Coffea arabica L. var. Catuaí) cell suspensions to chemically induced mutagenesis and salinity stress under in vitro culture conditions

Crop improvement of Coffea arabica L. (coffee) via mutagenesis could accelerate breeding programs; thus, the present study aimed to develop an in vitro protocol using the chemical mutagens sodium azide (NaN3) and ethyl methanesulfonate (EMS) on embryogenic cell suspensions of Arabica coffee variety Catuaí and, subsequently, to evaluate the responses of the resulting mutagenized tissues to salinity stress. Embryogenic suspension cultures were incubated with 0.0, 2.5, 5.0, or 10.0 mM NaN3 or 0.0, 185.2, 370.5, or 741.0 mM EMS. As the concentration of NaN3 or EMS increased, the survival of embryogenic suspension cultures decreased compared to controls. The median lethal dose (LD50) for NaN3 was 5 mM for 15 min and for EMS it was 185.2 mM for 120 min. Embryogenic suspension cultures treated with NaN3 or EMS were cultured on selective medium supplemented with 0, 50, 100, 150, 250, or 300 mM NaCl showed that 50 mM NaCl could be used as selection pressure. Plantlet growth and total amino acid content were affected by NaCl stress; some mutants had longer shoots and higher amino acid content than controls. Random amplified polymorphic DNA (RAPD) analysis was performed to determine whether the NaN3 or EMS treatments could induce genetic variability and resulted in identifiable polymorphic markers. A total of 18 10-mer primers were used to amplify genomic DNA of putative mutant and non-mutant arabica coffee embryogenic cultures and produced 50 scorable bands, of which 22% were polymorphic.Universidad de Costa Rica/[111-B5-140]/UCR/Costa RicaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí