Vol.7, No. 2 2007.pmd 
Artículo Científico                                                                                                                                                                                                                        Biotecnología Vegetal Vol. 7, No. 2: 115 - 117, abril - junio, 2007
ISSN 1609-1841 (Versión impresa)
 ISSN 2074-8647   (Versión electrónica)

 

Radiosensitivity to gamma rays (60Co) in shoot tips of henequen

Gerardo González Oramas1*, Silvia Alemán García1, Miguel Garriga1, Rodobaldo Ortíz2, Carlos de la Fe2. *Author for correspondence.

1Centro de Estudios Biotecnológicos. Universidad de Matanzas. Autopista a Varadero km 3 ½. CP 44 700. Matanzas. Cuba. e-mail: gerardo.gonzalez@umcc.cu

2Instituto Nacional de Ciencias Agrícolas. La Habana. Cuba

ABSTRACT

The current research was carried out with the objective of evaluating the radiosensitivity of shoot tips from henequen (Agave fourcroydes) to gamma radiations 60Co and to determine the optimum dose of radiation for breeding program in the cultivar Sac Ki. Shoot tips of 20 days of in vitro culture were selected and irradiated with five doses between 10, 20, 30, 40 and 50 Gy using a 60Co source. Evaluations were aimed to determine increasing of fresh weight of explants in relation to the control (no irradiated material), number of dead explants and average number of axillary shoots per explants. Percentage of mortality was bigger when irradiation dose was increased. Irradiation 20 dose of Gy was GR50, increasing fresh weight of explants in a 49.6% in relation to control. LD50 was 30 Gy and a 50% of irradiated explants died. When increasing irradiation dose the number of explants decreased, though starting of sprouting took place after 90 for both, irradiated and non irradiated explants, suggesting that this parameter was not radiostimulated nor radioinhibited. Results enable to suggest the application of 20 Gy in studies aimed to create genetic variability in breeding programs by mutation induction.

Key words: Agave fourcroydes, plant breeding, tissue culture

RESUMEN

El presente estudio se desarrolló con el objetivo de evaluar la radiosensibilidad de ápices de henequén (Agave fourcroydes) a los rayos gamma 60Co. Para su ejecución se seleccionaron e irradiaron ápices de la variedad Sac Ki con 20 días de sembrados in vitro. Las dosis de radiación fueron 10, 20, 30, 40 y 50 Gy. Las evaluaciones consistieron en determinar el incremento en masa fresca de los explantes en relación con el control (material no irradiado), el número de explantes muertos y el número promedio de brotes axilares por explante. Se comprobó que el porcentaje de mortalidad se elevó con el incremento de las dosis de radiación. La dosis de 20 Gy fue la GR50, con esta los explantes incrementaron su masa fresca en relación con el control en un 49.6%. La LD50 fue 30 Gy donde el 50% explantes irradiados murió. Con el incremento de las dosis de radiación se redujo el número de brotes por explante, sin embargo el inicio de la brotación fue posterior a los 90 días tanto para los explantes irradiados como los no irradiados, lo que sugiere que este parámetro no fue radioestimulado ni radioinhibido. Los resultados del estudio permiten sugerir la aplicación de la dosis de 20 Gy en los trabajos dirigidos a la creación de variabilidad genética en programas de mejora por inducción de mutaciones en esta especie.

Palabras clave: Agave fourcroydes, mejoramiento genético, cultivo de tejidos

INTRODUCTION

Henequen (Agave fourcroydes) is cultivated for its hard fiber which is used to make agricultural twin, saks, carpets and others products (Gonzalez et al., 2004). Furthermore it is used as a source of carbohydrates (inuline), spirits and steroidal sapogenin (hecogenin) (Infante et al., 2003). Due to its high levels of ploidy (5n) it is sterile and produces few seeds with very low viability at the end of its long life cycle (Piven et al., 2002). Moreover, it has undesirable characterists as: marginal thorne leaves. This problem is a handicap during its harvest (Eastmond et al., 2000). The problems mentioned above make very difficult to carry out a traditional cross breeding program, that is why henequen has never been genetically improved. Therefore, other methods to induce variability with gamma rays and in vitro culture are needed to be used (Nichterlein, 2000). Use of combination of both techniques may favor the program of genetic improvement (Pérez, 1998).

An assay aimed to apply different dose of gamma radiation on henequen shoot tips previously cultured in vitro to determine the optimum range to be used in the improvement program was carried out.

MATERIALS AND METHODS

Plant material

Bulbils were prepared and sterilized according to González et al. (1997) and Peña et al. (1997). Once isolated shoot tip was placed on 10 ml of MS culture medium (Murashige and Skoog, 1962) (slightly modified by Robert et al.,1992), supplemented with 1.6 mM naphtalenacetic acid (NAA) and 4.4 mM 6- benzylaminopurine (6-BAP) under 16 h illumination and 8h darkness at 22 ºC for 3 weeks.

Explants were irradiated with five doses (10, 20, 30, 40, 50 Gy) using a 60Co source in the irradiator Gammacell 500 with a dose potency of 13.7 Gy min-1. They were planted in vitro 24 h after being irradiated using the methodology developed by González et al. (2004). Explants without irradiation were used as control.

Explants were evaluated 45 days after the irradiation to measure the radiosensitivity index, the increase of fresh weight percentage in relation to control plant and mortality percentage (%). The rate production of new axillary shoots per explant was evaluated 45, 90 and 135 days after irradiation too.

Statistical analysis

A total of five replications with four explants were used for each treatment and the experimental trial was repeated twice. Significance of the treatment effects was determinated by non parametric analysis of variance Kruskal-Wallis test (Statgraphics ver. 5.0 Statistical Graphics Corp.), using a completely random design. Percentage data were subjected to arcsine transformation before analysis. Variations among treatment means were analyzed by Student-Newman-Keuls´s procedure.

RESULTS AND DISCUSSION

In the irradiated explants, different responses were observed in relation to the dose of radiation applied. The non irradiated explants (control explants) presented an increase of fresh weight of 6.31 g and 100% of survival, however, irradiated explants showed a decrease from increase of fresh weight and a greater mortality percentage as the dose of radiation increased.

In Musa spp., cultivar Grande Naine, the mortality was superior and the growth decreased as the dose increased (García et al.., 2000).

Figure 1 shows the decrease of fresh weight in relation to the control explant, and an increase of mortality percentage in irradiated explants. A reduction between 40-60% of their fresh weight in relation to control explants was noted in irradiated explants with 10, 20, 30 Gy. The dose that brought about a reduction between 40-60% of fresh weight in irradiated explants has been recommended for the improvement by in vitro mutation (Pérez, 1998), because these doses produce a proper frequency of mutation without multiple mutations (Afza et al., 1994).

A reduction of 49.6 % was reached in the increase of fresh weight with the dose of 20 Gy and for this reason it was considered as GR50. However, the 50% of mortality (LD50) was produced in the dose of 30 Gy.

The dose of 50 Gy produced a superior mortality percentage (over 90%) and 10% in the increase of fresh weight in relation to control explants. That is why this dose is considered as radioinhibiting.

Production of new axillary shoots per explant was influenced with the dose used, as it is shown in table 1, however, the onset of production of axillary shoots in all treatments was postponed to 90 days. This result evidenced that the beginning of the production of shoots is neither radiostimulated nor radioinhibited by the different doses used.

The number of axillary shoots regenerated from control explants was higher and statistically different than the rest of the treatments (Table 1). Decrease under 3.0 of production rate of new axillary shoots, in all dose used, evidenced the ruptuture of hormonal balance that allows a good proliferation of shoots. An either endogenous or exogenous hormonal balance, which allowed reaching a production rate of new axillary shoots superior to 3.0, was established in explants of henequen (Miguel et al., 2006).

The higher dose of gamma rays (50 Gy) was not acceptable due to the serious necrosis it causes to the explants furthermore only an axillary shoot was obtained. Phytotoxic action at this dose (50 Gy) was extremely harmful.

The parameters used in this work; increase of fresh weight percentage in relation to control explants, mortality percentage and production rate of new shoots were good indicators of the radiosensitivity. The dose of 20 Gy was selected to continue studies aimed to create genetic variability in breeding programs by mutation induction because it was considered the GR50 and it reached a production rate of new axillary shoots per explant near to the control explants.

All plants from the different doses grow properly in nursery condition. This will be studied in furthers experiments.

REFERENCES

Afza R, Roux N, Brunner M, van Duren M, Morpurgo R (1994) In vitro mutation techniques for Musa. Proceedings of the First Global Conference of international Musa Testing Program held at FHIA. Honduras

Eastmond A, Herrera JL, Robert ML (2000) Contribución de la biotecnología al henequen. En: Eastmond A, Herrera, JL, Robert, ML (Eds). La biotecnología aplicada al Henequén: Alternativas para el futuro. Centro de Investigaciones Científica de Yucatán. pp. 59-71. CICY. México DF.

García L, Bermúdez I, Orellana P, Veitía N, García L, Clavero J, Romero C (2000) Inducción de mutaciones por radiaciones Gamma en el cultivo in vitro de brotes del cultivar Gran Enano (AAA). Biotecnología Vegetal 1: 45-50

González G, Alemán S, Barredo F, Keb M, Ortiz R, Abreu E, Robert ML (2004) Una alternativa de la recuperación henequenera de Cuba, mediante el uso de técnicas biotecnológicas y moleculares. Biotecnología Aplicada 21 (1): 44-49

González G, Trujillo R, Darias R, Peña E (1997) Micropropagación del henequén: Aportes a una tecnología. Rev. Jardín Botánico Nacional 17:177-180

Infante D, González G, Peraza, L. And Keb-Llanes, M (2003) Asexual genetic variability in Agave. Plant Science 164 (2): 223-230

Miguel Garriga, Gerardo González, Silvia Alemán (2006) Comportamiento in vitro de la formación de brotes axillaries en Agave fourcroydes Lem. Biotecnología Vegetal 6(1): 3-7

Nichterlein K (2000) Workshop on mutation and in vitro culture techniques for the improvement of vegetatively propagated tropical food crops. Curso FAO/IAEA/UCR. Centro de Investigaciones Agronómicas. Universidad de Costa Rica.

Peña E, González G, Berrillo A, Sosa D, Arteaga M, Rittoles D, Pérez D, Torriente Z (1997) Tecnología para la micropropagación del Henequén a gran escala. Rev. Jardín Botánico Nacional 118: 169-176

Pérez JP (1998) Mutagénesis in vitro En: Pérez, JP (Eds) Propagación y Mejora Genética de Plantas por Biotecnología pp. 299-311. IBP. Santa Clara.

Piven N, Barredo-Pool F, Borges-Argáez I, Herrera-Alamillo M, Mayo-Mosqueda A, Herrera JL, Robert ML (2002) Reproductive biology of henequén (Agave fourcroydes) and its wild ancestor Agave Angustifolia (Agavaceae). i. Gametophyte development. American Journal of Botany 88:1966-1976

Robert ML, Herrera JL , Chan JL, Contreras F (1992) Micropropagation of Agave spp. Biotechnology in Agriculture and Forestry 19: 306-329



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