Respuesta de cultivares de Musa spp. al estrés hídrico in vitro inducido con polietilenglicol 6000 / Responses of Musa spp. cultivars to in vitro water stress induced with polyethylene glycol 6000

RESUMEN Los plátanos y bananos son cultivos sensibles al déficit hídrico. Las sequías cada vez más prolongadas sugieren la necesidad de obtener plantas tolerantes a este factor; la selección temprana de estas plantas, comparada con la selección en campo, permite ahorrar tiempo y trabajar con mayores volúmenes de individuos. Para ello es conveniente contar con cultivares patrones cuya respuesta al déficit hídrico in vitro sea favorable. El objetivo del presente trabajo fue determinar la respuesta de cultivares de Musa spp. con diferente composición genómica al estrés hídrico inducido in vitro con polietilenglicol 6000 (PEG-6000). Se estudiaron los cultivares 'Pelipita' (ABB), 'Manzano' (AAB) y 'Grande naine' (AAA). El estrés se indujo con 30 g/L de PEG-6000 en medio de cultivo semisólido de multiplicación. A los 30 días se evaluaron variables indicadoras de estrés morfológicas (altura y número de brotes por explante), fisiológicas (masa fresca y masa seca) y bioquímicas (contenido prolina, peróxido de hidrógeno y malondialdehido). En el cultivar 'Pelipita' se afectó solamente la altura de las plantas, mientras que en los demás se afectaron todas las variables excepto la masa seca en el 'Manzano'. En este último y en el 'Grande naine' se incrementó la prolina, el peróxido de hidrógeno y el malondialdehido, lo que evidenció un mayor estrés oxidativo y daño en las membranas celulares. Los cultivares estudiados, pudieran emplearse como controles de tolerancia ('Pelipita') y sensibilidad ('Grande naine' y 'Manzano') en la selección in vitro de plantas tolerantes a la sequía, en futuros programas de mejoramiento genético.

ABSTRACT Bananas and plantains are crops very sensitive to water deficit. Increasingly prolonged drought condition suggests the need for tolerant plants to this factor. The early selection of these plants in in vitro conditions save time and allow working with large volumes of individuals. This requires having genotypes with favorable response to in vitro water deficit. The aim of this work was to determine the response of Musa spp. cultivars, with different genotype contribution, to in vitro water stress induced by polyethylene glycol 6000 (PEG-6000). Cultivars 'Pelipita' (ABB), 'Manzano' (AAB) and 'Grande naine' (AAA) were cultured in semisolid multiplication medium supplemented with PEG-6000 30 g/L. Different stress indicator traits were evaluated after 30 days (morphological: height and number of shoots per explant; physiological: fresh and dry weight; and biochemical: proline, hydrogen peroxide and malondialdehyde content). As results, osmotic stress affected only plant height in 'Pelipita' cultivar. However, in the other cultivars all variables were affected, except dry weight in 'Manzano'. Moreover cv. 'Grande naine' and 'Manzano' increased proline, hydrogen peroxide and malondialdehyde content. These results indicate that osmotic pressure could induce oxidative stress and cell membrane damages in these cultivars. The obtained results suggested that these genotypes could be used as a tolerant ('Pelipita') and sensitive ('Grande naine' and 'Manzano') controls in in vitro selection of drought-tolerant plants in future breeding programs.

Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter.

Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. 'Grande Naine' (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.

Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system.

Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species.