[Study on inducing embryogenic callus of Dioscorea zingiberensis by orthogonal design].

OBJECTIVE: To select the suitable medium to induce embryogenic callus of Dioscorea zingiberensis. METHODS: Plantlet of Dioscorea zingiberesis in vitro was obtained by using apical meristem as explant. The different parts of the plantlets were cultured to select the best explant used for inducing callus and embryoids. Growing rate and diosgenin content were calculated in orthogonal test to optimize combination of phytohormones for inducing embryogenic callus. RESULTS: The leaves were suitable explants to induce callus and embryoid. The inducing rate of callus and embryoids reached 92.5% and 42.5%, respectively. The optimal medium for inducing embryogenic callus was MS + 6-BA 2.0 mg/L + NAA 0.5 mg/L + 2,4-D 1.0 mg/L. CONCLUSION: The results of this study can be used for effective induction of embryogenic callus of Dioscorea zingiberensis, and lay the foundation for the subsequent research of artificial seeds.

Evaluación en campo de plantas de ñame (Dioscorea alata L) obtenidas de los microtubérculos formados en sistema de inmersión temporal / Field evaluation of yam plants (Dioscorea alata L) obtained from microtubers produced in a temporary immersion system

Los microtubérculos en algunas especies de plantas constituyen una importante alternativa como material vegetal de plantación. Se definió como objetivo de trabajo evaluar en campo la respuesta morfoagronómica de las plantas obtenidas de los microtubérculos de ñame formados en Sistema de Inmersión Temporal (SIT). Como variantes experimentales se plantaron tres categorías de microtubérculos, clasificados según su masa fresca (I. de 0,5 a 0,9 g; II. de 1,0 a 2,9 g; III. igual o mayor de 3,0 g), plantas in vitro previamente aclimatadas y corona de tubérculo. Se evaluó el efecto de la masa fresca de los microtubérculos sobre su brote, supervivencia y posterior desarrollo de las plantas derivadas de ellos en campo. Con los microtubérculos de ñame, con una masa fresca igual o superior a 3,0 g, se alcanzó el más alto porcentaje de brotación (91,30%) y supervivencia de las plantas (96,50%), así como las mejores respuestas en los caracteres cuantitativos que se evaluaron en campo. Estos resultados confirmaron la importancia de la masa fresca de los microtubérculos para ser empleados como material vegetal de plantación directo en campo.

Microtubers in some plant species represent an important alternative crop-planting material. The presentwork involved field work for evaluating the morphoagronomic response of plants obtained from yam microtubersproduced in a temporary immersion system (TIS). Three categories of microtuber were planted asexperimental variants; they were classified by fresh mass (1 - 0.5 to 0.9 g, 2 - from 1.0 to 2.9 g and 3 - equal to or greater than 3.0 g), previously in vitro-acclimated plants and tuber crowns. The effect of microtuber freshweight on their sprouting, survival and later development of the plants derived from them in the field were evaluated. The highest sprouting (91.30%) and plant survival percentages (96.50%) and the best response in quantitative traits evaluated in the field were obtained with yam microtubers having a fresh mass equal to or greater than 3.0 g. These results confirmed the importance of microtubers’ fresh weight for using them as plant material in direct planting in the field.

Cryopreservation of embryogenic callus of Dioscorea bulbifera by vitrification.

Cryopreservation of callus of Dioscorea bulbifera by vitrification was optimized. Calli of Dioscorea bulbifera were pretreated in liquid Murashige and Skoog (MS) medium supplemented with 2 mg L(-1) kinetin (KT), 0.5 mg L(-1) NAA, 0.5 mg L(-1) 2,4-D and 0.2 M sucrose for 5 d under continuous light (36 microM m(-2) s(-1)) at 25 + or - 1 degree C. The material was then loaded with 60 percent vitrification solution (PVS2) for 20 min at room temperature and dehydrated with 100 percent PVS2 for 30 min at 0 degree C. After changing the solution with fresh PVS2, the calli were directly immersed in liquid nitrogen and conserved for 1- 360 d. After rapid thawing in a water-bath at 35 degree C, the calli were washed three times with liquid MS medium supplemented with 2 mg L(-1) KT, 0.5 mg L(-1) NAA, 0.5 mg L(-1) 2, 4-D and 1.2 M sucrose and then transferred onto solid MS medium supplemented with KT 2 mg L(-1), NAA 0.5 mg per liter, 0.09 M sucrose and 0.75 percent agar. The cultures were kept in the dark for 2 days prior to exposure to the light (12 h light-dark cycle). The TTC test showed that 80-90 percent of the calli survived this cryoprocedure and there was a 60-70 percent regeneration of plantlets from the calli. The regenerated material did not exhibit any morphological variations.

Cryopreservation of embryogenic cultures of Dioscorea bulbifera l. by encapsulation- dehydration.

Embryogenic cultures of Dioscorea bulbifera were cryopreserved using an encapsulation- dehydration procedure with subsequent plant regeneration. Embryogenesis was induced by culturing in vitro grown axillary bud meristems on MS medium supplemented with 2.0 mg per liter 2,4-D. After cryopreservation, recovery growth of embryogenic culture up to 53.3 percent was recorded when excised proliferating embryogenic cultures of 1.5-2.0 mm in diameter were: encapsulated in 3 percent calcium alginate containing 0.15 M sucrose followed by preculturing with 0.5 M sucrose for 3 d; dehydrated in the laminar air flow for 4 h, thereby reducing the bead moisture content to 19.4 percent ( fresh weight basis); plunged into liquid nitrogen; thawed at 40 degree C; and cultured on recovery growth medium, i.e. MS supplemented with 2.0 mg per liter 2,4-D and 0.3 mg per liter BAP. However, preculturing for an extended period of 7 d increased the recovery growth further to 67.8 percent. During recovery growth the embryogenic tissue protruded out of the beads without loss of structural integrity of the cryopreserved embryos. Subculturing of these cultures on to embryo conversion medium, i.e. MS medium with 0.5 mg per liter zeatin and 400 mg per liter glutamine, resulted in production of plantlets through embryo conversion. The regenerated plantlets exhibited the same morphology as that of originally maintained in vitro plantlets and were established in vivo, in a net house with 80 percent success.

Genetic stability assessment of plants regenerated from cryopreserved embryogenic tissues of Dioscorea bulbifera l. Using RAPD, biochemical and morphological analysis.

Embryogenic tissues of Dioscorea bulbifera were cryopreserved using the encapsulation-dehydration technique. Genetic stability of plants regenerated from cryopreserved embryogenic tissues was assessed using molecular, biochemical and morphological analysis. The random amplified polymorphic DNA (RAPD) analysis of 60 cryopreserved-derived and 20 in vitro grown (control) plantlets showed that 10 primers produced 62 clear reproducible DNA fragment profiles. The amplification products were monomorphic for all the plantlets except one. A total of 4960 DNA fragments were obtained from this study showing no variation in RAPD profiles. The diosgenin content of cryopreserved-derived plants, analyzed using HPLC, was similar to that of control plants. Morphology and the ability to form microtuber were also found to be unaltered in cryopreserved embryo-derived plantlets. Thus, the D. bulbifera plants regenerated from cryopreserved embryogenic tissues were genetically stable at the molecular, biochemical and morphological levels.