Callus Type, Growth Regulators, and Phytagel on Indirect Somatic Embryogenesis of Coffee (Coffea arabica L. var. Colombia).

Coffee is a crop of global relevance. Indirect somatic embryogenesis has allowed plants of different coffee genotypes to be massively regenerated. The culture medium composition can affect the calli characteristics that are generated and their ability to form somatic embryos. This research aimed to determine the influence of the type of callus, growth regulators, and phytagel concentration on the embryogenic capacity of the Colombia variety. Leaf explants were cultured on Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5-1.0 mg L-1), benzylaminopurine (BAP, 1.0 mg L-1), and phytagel (2.3-5.0 g L-1). The explants generated two types of calli: friable (beige, soft, watery, easy disintegration, polyhedral parenchyma cells) and compact (white, hard, low water content, difficult disintegration, elongated parenchyma cells). About 68% of the total callus generated was compact; this type of callus produced a greater number of embryos (71.3) than the friable one (29.2). The number of differentiated embryos was significantly affected by the concentration of phytagel; higher concentrations (5.0 g L-1) resulted in larger quantities (73.7). The highest number of embryos (127.47) was obtained by combining 1.0 mg L-1 2,4-D, 1.0 mg L-1 BAP, 5.0 g L-1 phytagel, and compact callus.

In Vitro Mass Propagation of Coffee Plants (Coffea arabica L. var. Colombia) through Indirect Somatic Embryogenesis.

Coffea arabica is one of the two most consumed coffee species in the world. Micropropagation through somatic embryogenesis has allowed the large-scale propagation of different coffee varieties. However, the regeneration of plants using this technique depends on the genotype. This study aimed to develop a protocol for the regeneration of C. arabica L. var. Colombia by somatic embryogenesis for its mass propagation. Foliar explants were cultured on Murashige and Skoog (MS) supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), and phytagel for inducing somatic embryogenesis. In total, 90% of the explants formed embryogenic calli with a culture medium containing 2 mg L-1 of 2,4-D, 0.2 mg L-1 BAP, and 2.3 g L-1 phytagel. The highest number of embryos per gram of callus (118.74) was obtained in a culture medium containing 0.5 mg L-1 2,4-D, 1.1 mg L-1 BAP, and 5.0 g L-1 phytagel. In total, 51% of the globular embryos reached the cotyledonary stage when they were cultured on the growth medium. This medium contained 0.25 mg L-1 BAP, 0.25 mg L-1 indoleacetic acid (IAA), and 5.0 g L-1 of phytagel. The mixture of vermiculite:perlite (3:1) allowed 21% of embryos to become plants.

Haploid Induction in Tomato (Solanum lycopersicum L.) via Gynogenesis.

The generation of new hybrid varieties of tomato (Solanum lycopersicum L.) is the most widely used breeding method for this species and requires at least seven self-fertilization cycles to generate stable parent lines. The development of doubled haploids aims at obtaining completely homozygous lines in a single generation, although, to date, routine commercial application has not been possible in this species. In contrast, obtaining doubled haploid lines via gynogenesis has been successfully implemented in recalcitrant crops such as melon, cucumber, pumpkin, loquat and walnut. This review provides an overview of the requirements and advantages of gynogenesis as an inducer of haploidy in different agricultural crops, with the purpose of assessing the potential for its application in tomato breeding. Successful cases of gynogenesis variants involving in vitro culture of unfertilized ovules, use of 60Co-irradiated pollen, in vivo haploid inducers and wide hybridization are presented, suggesting that these methodologies could be implemented in tomato breeding programs to obtain doubled haploids.

Respuesta de Fragaria mexicana y comunidades microbianas rizosféricas al aumento de temperature / Response of Fragaria Mexicana and rhizophere microbial communitites to temperature increase

Resumen Para los próximos 100 años se pronostica un incremento en la temperatura del planeta de casi 4 °C, lo cual pondrá en riesgo las especies que no logren adaptarse. En esta investigación se determinaron las respuestas morfofisiológicas de F. mexicana y los cambios en la población de bacterias fijadoras de nitrógeno atmosférico (BFN) asociadas a sus raíces, debido a dos condiciones ambientales con diferencias medias de temperatura de 5.1 oC: invernadero (temperatura alta, TA) y campo abierto (temperatura baja, TB); y con cuatro tratamientos: sin inocular (T), inoculadas con la cepa ocho (CP8), cepa cuatro (CP4) y con ambas cepas (CP8 + 4). Las BFN fueron aisladas de la rizosfera de F. mexicana y transformadas genéticamente con reporteros, para cuantificar la población al final del experimento. Se midió el peso seco de la parte aérea y la raíz, la tasa de asimilación de CO2 (A), el inicio de la floración, el número de flores y frutos; y la persistencia de las bacterias fijadoras de N atmosférico (BFN). Además, se evaluó la concentración de NO3, NH4, P y materia orgánica (MO) en el sustrato, al inicio y final del experimento. Las plantas sometidas a alta temperatura presentaron mayor peso seco de la parte aérea y fotosíntesis; con una disminución en el peso seco de la raíz, y en el número de flores y frutos. La MO en el sustrato disminuyó, mientras que la disponibilidad de NO3, NH4 y P aumentó. El incremento de temperatura y la mayor presencia de N en el substrato provocaron reducción en la población de BFN. Estos resultados sugieren que temperaturas altas estimulan el crecimiento de F. mexicana y tienen un impacto negativo sobre su reproducción y en las BFN asociadas a sus raíces.(AU)

Abstract The earth could experience a warming of 4 °C in the next one hundred years. This would put at risk the plants that can´t adapt. Fragaria mexicana is an endemic plant of temperate forest of Mexico. The response of this wild strawberry to temperature increasing has not been studied and could play an important role for event of global warming. This study determinate the morphological and physiological responses of F. mexicana and changes in the N2-fixing bacteria (BFN) population on its roots, due to two environmental conditions with differences of temperature 5.1 °C: greenhouse (high temperature, TA) and open land (low temperature, TB); and with four free-living nitrogen-fixing bacteria (BFN) treatments: non-inoculated (T), inoculated with strain eight (CP8), strain four (CP4), and strains eight and four mixed (CP8 + 4). BFN were isolated from the rhizosphere of F. mexicana and were genetically transformed with reporters to quantify the population at the end of the experiment. NO3, NH4 and P and organic matter (MO) in the substrate were determined at beginning and finish of the experiment. Shoot and root dry weight, photosynthetic rate, flowering and fructification starting, flowers and fruits number, were measured. Shoot dry weight and photosynthetic rate were lower in TB than TA, decreasing 3.1 g and 0.94 µmol m-2 s-1, respectively. Root dry weight was 3.0 g less in TA compared with TB. Number flowers decrease in 40.89 % and number fruits in 38.11 % on TA than TB. F. mexicana plants start flowering 14 days previously in TB than TA. MO in the substrate decrease in TA, while the concentration of NO3, NH4 and P, increased. Population of BFN was lower in TA. Results obtained indicated that higher temperature promotes the growth of F. mexicana and reduce its reproduction and BNF population associated with its roots.(AU)

A reliable method for spectrophotometric determination of glycine betaine in cell suspension and other systems.

Glycine betaine is a quaternary ammonium compound that accumulates in a large variety of species in response to different types of stress. Glycine betaine counteracts adverse effects caused by abiotic factors, preventing the denaturation and inactivation of proteins. Thus, its determination is important, particularly for scientists focused on relating structural, biochemical, physiological, and/or molecular responses to plant water status. In the current work, we optimized the periodide technique for the determination of glycine betaine levels. This modification permitted large numbers of samples taken from a chlorophyllic cell line of the grass Bouteloua gracilis to be analyzed. Growth kinetics were assessed using the chlorophyllic suspension to determine glycine betaine levels in control (no stress) cells and cells osmotically stressed with 14 or 21% polyethylene glycol 8000. After glycine extraction, different wavelengths and reading times were evaluated in a spectrophotometer to determine the optimal quantification conditions for this osmolyte. Optimal results were obtained when readings were taken at a wavelength of 290 nm at 48 h after dissolving glycine betaine crystals in dichloroethane. We expect this modification to provide a simple, rapid, reliable, and cheap method for glycine betaine determination in plant samples and cell suspension cultures.

Callus and suspension culture induction, maintenance, and characterization.

Callus and cell suspension can be used for long-term cell cultures maintenance. This chapter describes procedures for the induction of somatic embryos of garlic, keeping a regeneration capacity for more than 5 yr, as well as the maintenance of a tobacco suspension culture (NT-1 cells), for more than 10 yr. Methods for plant regeneration and growth kinetics of garlic cultures are described, as well as for cell viability of NT-1 cells stained with 2,3,5 triphenyltetrazolium chloride. The packed cell volume determination as a parameter of growth is detailed.