Production of “super-males” of asparagus by anther culture and its detection with SSR-ESTs

Abstract

Anther culture is used to develop asparagus ''super-male'' (di-haploids) in asparagus, which can be used to develop ''all-male'' varieties, by crossing them with suitable females; their progenies will be formed only by males which is advantageous for producers. This report describe a new anther culture protocol adapted to ''Morado de Huétor'', a Spanish tetraploid landrace, and studied the different factors involved in callus proliferation success from anther explants such as the microspore development stage, or the type of stress used to induce the symmetric division of the microspores, to obtain a high success rate (90 %). For plantlets regenerates from anther culture (PRACs) regeneration we develop a proliferation media supplemented with a combination of pCPA and BA able to induce callus proliferation and plantlet regeneration in the same step in a 50 % of calli, simplifying the procedure. The high percentage of heterozygous male recovery, originated from somatic cells, is an important problem in the anther culture, and to elucidate the origin of PRACs we have combined different tools: ploidy analysis, characterization with the linked sex-marker Asp1-T7 and with ESTSRRs. We can establish that 50 % of PRACs obtained in this work were regenerated from diploid microspores of ''Morado de Huétor'', regenerating diploid, di-diploid and tetra-diploid plantlets. The di-diploids males (MMmm) would generate a ratio male:female of 5:1 (83.3 %) and the tetra-diploid males (MMMMmmmm) a ratio male:female of 69:1 (98.6 %), so the tetra-diploid males could be considered ''super-males'' and be used to develop ''all-male'' varieties of ''Morado de Huétor''.

Genetic Variability Assessment of a Diploid Pre-Breeding Asparagus Population Developed Using the Tetraploid Landrace ‘Morado de Huétor’

“…With this aim, Riccardi et al [25], using the in vitro anther culture technique, developed new diploid (di-haploid) germplasm carrying introgressions from two wild tetraploid populations (A. acutifolius, A. maritimus) and a tetraploid landrace ('Violetto d'Albenga'). Additionally, diploid plants carrying germplasm from the tetraploid landrace 'Morado de Huétor' were obtained by backcrossing using the diploid cultivars as a recurrent parent by Castro et al [26] and using in vitro anther culture by Regalado et al [27]. More recently, Plath et al [28] detected AV-1 resistant plants in the progenies resulting from the first backcrosses with different interspecific hybrids, which were obtained between A. officinalis and wild related species (A. maritimus (6x), A. pseudoscaber (6x), A. prostratus (4x)).…”

Origin of purple asparagus cultivar ‘Pacific Purple’ based on the sequence of sex determination gene

“…Asp1-T7sp marker can be used to identify the sex of various cultivars of A. officinalis and several dioecious Asparagus species, such as A. kiusianus , A. maritimus , A. pseudoscaber and A. schoberioides ( Nakayama et al., 2006 ; Kubota et al., 2012 ; Kanno et al., 2020 ). Furthermore, this marker is applicable in ‘Morado de Huetor’ (MH), which is tetraploid landrace in Spain ( Regalado et al., 2014 ; Regalado et al., 2016 ). ‘Pacific Purple’ was bred from VA, tetraploid landrace in Italy, and VA and MH are likely an interspecific hybrid between A. officinalis and A. maritimus ( Falloon and Andersen, 1999 ; Moreno et al., 2008a ).…”

“…MSSTS710 is only available for use in A. officinalis and cannot be used for sex identification in other Asparagus species, while Asp1-T7sp has been shown to be available in garden asparagus and some related Asparagus species ( Nakayama et al., 2006 ; Kubota et al., 2012 ; Kanno et al., 2014 ; Kanno et al., 2020 ). This marker is also applicable in ‘Morado de Huetor’ (MH), which is tetraploid landrace in Spain ( Regalado et al., 2014 ; Regalado et al., 2016 ).…”

Embryo Rescue in Plant Breeding

“…officinalis L. is a diploid species (2n = 2x = 20), but within the Asparagus genus, several wild species exhibit different ploidy levels: A. prostratus, A. acutifolius, A. maritimus, and some landraces of A. officinalis are tetraploids (4x); A. maritimus is hexaploid (6x); and A. macrorrhizus is dodecaploid (12x) [291,292]. The hybridization of genotypes with different ploidy levels in asparagus [293][294][295][296], as well as the induction of autotetraploid, auto-octoploid, or triploid genotypes [297][298][299], have occasionally resulted in plants with valuable agronomical traits. However, different ploidy levels also involve diverse challenges in asparagus breeding, such as crossing incompatibility, overcoming by ER, and genetic instability [299][300][301].…”