Determinación por AFLP de la estabilidad genética de plantas de yuca obtenidas por embriogenesis somática y organogenesis

AFLP techniques have been reported to determine the genetic stability at a molecular level of cassava plants propagated by several methods: somatic embryogenesis, organogenesis and field conditions. ‘CMC-76’, ‘CEMSA 74-725’ and ‘Señorita’ clones from the germplasm collection kept at the INIVIT were used. Two Kit for AFLPs were applied, one of them combined selection primers +2/+3 (AFLP Analysis System II GibcoBRL) and the other +3/+3 (AFLP® Analysis System I, GibcoBRL®). 44 combination primers (EcoRI + 2 ó EcoRI + 3) were tested and five were selected as the most efficient. Plants derived from different propagation methods within each clone resulted identical genetically and differences were only observed in the three studied clones. This result corroborates the efficiency of AFLPs to study the genetic stability of micropropagated material, storaged at medium term or in cryoconservation. Besides, plants obtained with these techniques can be used for the production of high quality planting material in this crop.Key word: genetic stability, organogenesis, somatic embryogenesisSe describe el empleo de la técnica de AFLP para determinar a nivel molecular la estabilidad genética de las plantas de yuca (Manihot esculenta, Crantz) propagadas por diferentes métodos: embriogénesis somática, organogénesis y tradicional en campo. Se utilizaron los clones CMC-76, CEMSA 74-725 y Señorita del banco de germoplasma del INIVIT. Se aplicaron dos Kit para AFLP, uno con una combinación de cebadores de selección +2/+3 (AFLP® Analysis System II, GibcoBRL®) y otro +3/+3 (AFLP® Analysis System I, GibcoBRL®). Se estudiaron 44 combinaciones de cebadores (EcoRI+2 ó EcoRI+3 x MseI+3) y se seleccionaron cinco como los más eficientes. Se encontró que las plantas procedentes de los diferentes métodos de propagación estudiados dentro de cada clon, resultaron genéticamente idénticas y sólo fueron observadas diferencias entre los tres clones estudiados. Este resultado confirmó la utilidad de los AFLP para el estudio de la estabilidad genética de los materiales micropropagados, conservados a mediano plazo o crioconservados. Además, las plantas obtenidas por estas técnicas pueden ser utilizadas para la producción de material de plantación de alta calidad en este cultivo.Palabras clave: embriogénesis somática, estabilidad genética, Manihot esculenta, organogénesi

Conservación in vitro de cultivares de Ipomoea batatas (L.) Lam por crecimiento mínimo con el uso de manitol

In vitro conservation of Ipomoea batatas (L.) Lam allows the exchange of germplasm and its availability for breeding programs. The objective of this work was to determine the effect of mannitol and abscisic acid in the conservation through minimum growth of I. batatas cultivars from INIVIT Germplasm Bank. Treatments included abscisic acid (ABA) (5 and 10 mg l-1) and mannitol (1.0, 1.5 and 2.0%) in a basal MS culture medium, the combination of ABA (10 mg l-1) and mannitol (1.0 , 1.5 and 2.0%). As controls were used the MS basal culture medium and MS with sorbitol (1.0%) and glucose (1.0%). In the cultivars 'Cautillo' and 'INIVIT BS 16-2006' the best results of survival, growth decrease and green but small leaves were obtained in the treatments that contained the basal culture medium with mannitol (10, 15 and 20%) . Growth of the explants was not achieved in culture media with abscisic acid. The MS culture medium with 2 mg l-1 of thiamine, 100 mg l-1 of myo-inositol and 1.0% of mannitol, allows the in vitro conservation of sweet potato cultivars between six and eight months. Keywords: osmotic agent, sweet potato, germplasm, culture mediumLa conservación in vitro de Ipomoea batatas (L.) Lam permite el intercambio de germoplasma y su disponibilidad para programas de mejoramiento genético. El objetivo de este trabajo fue determinar el efecto de manitol y ácido abscísico en la conservación mediante crecimiento mínimo de cultivares de I. batatas del Banco de Germoplasma del INIVIT. Los tratamientos incluyeron ácido abscísico (ABA)(5 y 10 mg l-1) y manitol (1.0, 1.5 y 2.0%) en un medio de cultivo MS basal, la combinación de ABA (10 mg l-1) y manitol (1.0, 1.5 y 2.0%). Como controles se emplearon el medio de cultivo MS basal y MS con sorbitol (1.0%) y glucosa (1.0%). En los cultivares ‘Cautillo’ e ‘INIVIT BS 16-2006’ se obtuvieron los mejores resultados de supervivencia, disminución del crecimiento y hojas verdes pero pequeñas en los tratamientos que contenían el medio de cultivo basal con manitol (10, 15 y 20%). En los medios de cultivo con ácido abscísico no se logró crecimiento de los explantes. El medio de cultivo MS con 2 mg l-1 de tiamina, 100 mg l-1 de mio-inositol y 1.0% de manitol, posibilita la conservación in vitro de cultivares de boniato entre seis y ocho meses Palabras clave: agente osmótico, boniato, germoplasma, medio de cultiv

Determination of the genetic stability in cassava plants obtained by somatic embryogenesis and organogenesis using AFLP

AFLP techniques have been reported to determine the genetic stability at a molecular level of cassava plants propagated by several methods: somatic embryogenesis, organogenesis and field conditions. ‘CMC-76’, ‘CEMSA 74-725’ and ‘Señorita’ clones from the germplasm collection kept at the INIVIT were used. Two Kit for AFLPs were applied, one of them combined selection primers +2/+3 (AFLP Analysis System II GibcoBRL) and the other +3/+3 (AFLP® Analysis System I, GibcoBRL®). 44 combination primers (EcoRI + 2 ó EcoRI + 3) were tested and five were selected as the most efficient. Plants derived from different propagation methods within each clone resulted identical genetically and differences were only observed in the three studied clones. This result corroborates the efficiency of AFLPs to study the genetic stability of micropropagated material, storaged at medium term or in cryoconservation. Besides, plants obtained with these techniques can be used for the production of high quality planting material in this crop. Key word: genetic stability, organogenesis, somatic embryogenesi

Protocolo para la formación de microtubérculos de ñame (Dioscorea alata L.) en sistema de inmersión temporal

The following working objectives were defined for developing a protocol for ´Pacala Duclos´ yam clone (Dioscorea alata L.) microtuber formation in temporary immersion systems (TIS): time effect, immersion frequency and culture medium volume influence per in vitro cultivated plant. The results led to defining that the highest performance regarding microtuber number formed per plant was achieved with a 15-minute immersion time and six-hourly immersion frequency; the microtubers presented the greatest dry and fresh weight and largest diameter at such immersion time and frequency. Furthermore, the highest total number of microtubers per system and the largest number of microtubers usable for planting (vegetal propagation) material were obtained after 18 weeks� culture. Regarding culture medium volume per plant, the greatest amount of usable microtubers was achieved with 60 ml per in vitro plant, microtubers presenting the highest dry matter content. As microtubers obtained in this type of TIS had fresh weights higher than 2.40 gFW they could be used as direct planting material in field conditions.Con el propósito de desarrollar un protocolo para la formación de microtubérculos en el clon de ñame Pacala Duclos (Dioscorea alata L.) en sistema de inmersión temporal, que pudiera ser usado como alternativa para la propagación de esta especie, se definieron como objetivos de trabajo determinar el efecto del tiempo y la frecuencia de inmersión, así como la influencia del volumen de medio de cultivo por planta cultivada in vitro. Con el empleo de 15 min de inmersión y una frecuencia de inmersión cada 6 horas, se alcanzaron los mejores resultados en cuanto al número de microtubérculos formados por planta. Con este tiempo y frecuencia de inmersión los microtubérculos presentaron la mayor masa fresca y seca, así como el mayor diámetro. Además, a las 18 semanas de cultivo se obtuvo el mayor número total de microtubérculos por sistema y el mayor número de microtubérculos aprovechables como material vegetal de propagación. En cuanto al volumen de medio de cultivo por planta, con 60 ml de medio de cultivo por planta in vitro se alcanzó el mayor número de microtubérculos aprovechables, los cuales presentaron el contenido más alto de materia seca. Los microtubérculos obtenidos en este tipo de sistema de inmersión temporal presentaron una masa fresca superior a 2,40 gMF, lo cual podría permitir su uso como material de plantación directo a campo

Efect of sodium chloride on in vitro growth of Grande naine and FHIA 03 cultivars

The effect of Sodium Chloride levels on in vitro development of “Gran Enano” (AAA) and `FHIA 03`(AABB) cultivars was determined. Researches were carried out at “Instituto de Investigaciones en Viandas Tropicales (INIVIT)” from 1999 to 2001. Meristems (3 mm) were situated in M – 3 medium supplented with different sodium chloride concentrations at a range of 0 – 10 000 ppm. They were incubated during 40 days in cach subculture until 80 days when they were evaluated at 25 ± 27°C temperature, DFFF 45 µmol m -2 s -1 light intensity a photoperiod of 16 hours per day and 85% relative humidity. Tolerance levels to sodium chloride are highly influenced by genotypes used. “Gran Enano” cultivar had success up to 4 500 ppm with a surviving of 96.7% and 25% damaged leaves. “FHIA 03” Hybrid showed tolerance to high salt levels from 2 500 to 8 000 ppm with 80.9% surviving but leaves were affected at 72.5%. Studies are recommended to continue with target genotypes including main marketable clones and diploids for genetic improvement. Key words: bananas, micropropagation, Musa, tissue culture