Ex situ plant germplasm conservation revised at the light of mechanisms and methods of genetics / Conservación de germplasma Ex situ revisada a la luz de mecanismos y métodos de genética

ABSTRACT Plant genetic resources for food and agriculture are ex situ conserved in germplasm banks as samples (accessions) of natural or naturalized populations, either as the originally sampled propagules (mainly seeds) or their multiplications. The premises underlying ex situ conservation are that (a) it is the safest and cheapest alternative for germplasm preservation for future generations and (b) accessions are representative of the genetic diversity encountered in nature. In the past decades, ideas, alternatives and considerations have been put forward on the topic, and protocols have been devised for plant germplasm sampling, conservation and multiplication. However, limitations in the management efficiency of germplasm banks have been pointed out by international organizations. In our opinion, germplasm banks in general need to revise their functioning and management at the light of principles and methods of Genetics. To that end, it is necessary to consider the reproductive biology of higher plants -whose genetic consequences at both the individual plant and the population levels are not always either fully understood or taken into account in devising the protocols-, the genetic structures of wild and cultivated populations, and the course of the genetic material in the populations. In this paper, we discuss the three topics and provide an example of a national forage breeding program, from germplasm bank accessions as the germplasm of origin to the obtainment of commercial cultivars. Finally, we present a proposal as a base for discussion among curators, researchers and breeders.

RESUMEN Los recursos genéticos vegetales para la alimentación y la agricultura se conservan ex situ en bancos de germoplasma como muestras (introducciones) de poblaciones naturales o naturalizadas ya sea como propágulos originales (mayoritariamente semillas) o sus multiplicaciones. Las premisas subyacentes son que (a) es la alternativa más segura y barata de preservación de germoplasma para futuras generaciones y (b) las introducciones son representativas de la diversidad genética que se encuentra en la naturaleza En las últimas décadas, se han presentado ideas, alternativas y consideraciones sobre el tema y se han elaborado protocolos para el muestreo, conservación y multiplicación de germoplasma. Sin embargo, organizaciones internacionales han señalado limitaciones en la eficiencia del manejo de los bancos de germoplasma. En nuestra opinión, se necesita revisar el funcionamiento y manejo de dichos bancos en general a la luz de los principios y métodos de Genética. Para tal fin, es necesario considerar la biología reproductiva de las plantas superiores -cuyas consecuencias genéticas a nivel de planta individual y de población no se comprenden en su totalidad o no se consideran al idear los protocolos -, las estructuras genéticas de poblaciones naturales y cultivadas, y el curso del material genético en las poblaciones. En este trabajo discutimos los tres temas y proveemos un ejemplo de un programa nacional de mejoramiento de forrajeras, desde las introducciones como germoplasma de origen hasta la obtención de cultivares comerciales. Finalmente, presentamos una propuesta como base de discusión entre curadores, investigadores y mejoradores.

Pollen viability and meiotic irregularities in a collection of Stevia rebaudiana (Bertoni) Bertoni cultivated in Tucumán, Argentina / Viabilidad de polen e irregularidades meióticas en una colección de Stevia rebaudiana (Bertoni) Bertoni cultivada en Tucumán, Argentina

Stevia rebaudiana (Bertoni) Bertoni (2n=2x=22) (Asteraceae family) is a species of economic value due to the presence of steviol glycosides in leaves -mainly stevioside and rebaudioside A- which are non-caloric sugars. In 2013, a collection of plants (genotypes) from four contrasting environments was established in Tucuman, Argentina, for evaluation under local conditions and, eventually, breeding purposes. As a first step, pollen viability and meiosis was studied in a sample of 56 plants. The percentage of pollen viability varied from medium (69.4%) to high (99.6%) in 52 of them, but from low (36.5%) to relatively low (51.5%) in the remaining four. The latter four plants also presented pollen grains of heterogeneous size, which were classified as n (normal, the most frequent size in the sample),

Stevia rebaudiana (Bertoni) Bertoni (2n=2x=22) (familia Asteraceae) es una especie de valor economico debido a la presencia de glucosidos de esteviol en sus hojas, principalmente esteviosido y rebaudiosido A, que son azucares no caloricos. En 2013, se establecio una coleccion de plantas (genotipos) provenientes de cuatro ambientes contrastantes en Tucuman, Argentina, para su evaluacion en condiciones locales y, eventualmente, con fines de mejoramiento genetico. Como primer paso, se estudio la viabilidad del polen y la meiosis en una muestra de 56 plantas. El porcentaje de viabilidad del polen fue de medio (69,4%) a alto (99,6%) en 52 de ellas, pero de bajo (36,5%) a relativamente bajo (51,2%) en las cuatro restantes. Las ultimas cuatro plantas tambien presentaron granos de polen de tamano heterogeneo, que se clasificaron como n (normal, el tamano mas frecuente en la muestra),

The importance of Arabidopsis seed mutants in the elucidation of the molecular basis of Endosperm Balance Number in tuber-bearing Solanum species

The Endosperm Balance Number (EBN) is an important concept for potato breeding and has evolutionary importance in tuber-bearing Solanum species. The EBN is part of the post-zygotic hybridization barriers in the group and represents a reproductive isolating mechanism. Few genes have been proposed to be involved in its genetic control; until now, however, neither specific genes nor its molecular basis have been well established. Histological observations of embryo and endosperm development in inter-EBN crosses in tuber-bearing Solanum revealed phenotypes similar to those recently described in Arabidopsis seed mutants. The common feature between them is that the endosperm nuclei become greatly enlarged and that embryos are arrested at the globular stage. The proteins encoded by the Arabidopsis TITAN genes are related to chromosome dynamics and cell division. Based on the sequence of titan mutants, genes in potato species related to cell cycle and microtubule assembly were isolated. In this article a perspective model is proposed to explore the utility of Arabidopsis mutants associated with cell cycle control as a tool to elucidate the molecular basis of EBN in potato. Further research focused on the expression pattern of these genes in intra- and inter-EBN crosses in potato species will be performed.

Plant regeneration after long-term callus culture in clones of Asparagus officinalis L

Callus growth and plant regeneration from long-term callus cultures were studied in two elite clones of Asparagus officinalis cv. Argenteuil, to establish a suitable protocol for a prospective in vitro selection program. Callus initiation and growth was evaluated on MS medium with 3

agar, 1 mg x l(-1) kinetin, and three levels of 2,4-D. The highest callus relative growth was obtained on medium with 1.5 mg x l(-1) 2,4-D and 1 mg x l(-1) kinetin. Shoot primordia (SP) induction from > 18-months-old calluses was evaluated on several media; the highest percentage of SP induction (89

) and average number of SP per callus (8.6) were obtained with clone [quot ]265[quot ] on MS medium with 5 mg x l(-1) 2iP, 1 mg x l(-1) IAA, 3

sucrose and 0.9

agar. The highest percentage of root induction (100

) was achieved with clone ’265’ on MS medium with 0.1 mg x l(-1) kinetin, 0.1 mg x l(-1) NAA, 1.32 mg x l(-1) ancymidol, 7

glucose and 0.8

agar. Important medium x genotype interactions were detected, pointing to the need of adjusting this and other in vitro protocols for specific asparagus genotypes.

Preliminary results on embryo rescue for circumventing hybridization barriers in Asparagus

Garden asparagus, Asparagus officinalis, is reproductively isolated from a related ornamental species with potential breeding value, Asparagus densiflorus cv. Sprengeri, by pre- and post-zygotic barriers. The latter barrier operates at the endosperm level five days after pollination in A. officinalis x A. densiflorus crosses. To try to circumvent this barrier, in vitro embryo rescue using ovule and ovary cultures was tested. Controlled interspecific crosses were made and 2,032 ovules and 826 ovaries were cultured three days after pollination under various culture media and incubation conditions. Ovaries cultured for 60 days became red (similar to mature fruits), but seed formation was incomplete. Transfer of ovules to other media was necessary to promote embryo development. The interspecific embryos increased their length from 35 microns at the initiation of culture to 1,900 microns after 120 days of culture, but seedlings were not obtained. Histological studies revealed differentiation of protoderm only. The possible causes of the failure of the embryos to complete differentiation and morphogenesis are discussed.