Morado De Huetor Research Articles

Cryopreservation of rhizome buds of Asparagus officinalis L. (cv. Morado de Huétor) and evaluation of their genetic stability

We describe an encapsulation–dehydration procedure with prefreezing steps for the cryopreservation of rhizome bud explants of Asparagus officinalis L. cv. Morado de Huetor. With this procedure, survival of Rhizome buds was at least 84 and 42% developed to complete plantlets at 8 weeks. Flow cytometry and EST-SSR molecular markers were used to assess genetic stability of the regenerated material. Effects of preculture time in a medium rich in sucrose and prefreezing treatments (0 °C or/and − 20 °C) on plant recovery were evaluated. Rhizome Buds of the “Morado de Huetor” landrace were incubated in preculture medium (MS + 0.3 M sucrose) for 48 h, encapsulated in alginate beads and desiccated until a water content of 35%, prefrozen for one hour at 0 °C plus one hour at − 20 °C, followed by cryopreservation in liquid nitrogen, and then were rewarmed and recovered in ARBM medium for 6 weeks and finally incubated in ARBM-0 for 4 weeks. Analyses of ploidy and molecular stability of plantlets recovered from cryopreserved rhizome buds of two selected genotypes showed no differences compared with the mother plants. Cryopreservation of RB explants of A. officinalis with this Encapsulation–Dehydration procedure will be useful in long-term preservation programs.

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

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 Huetor”, 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 EST-SRRs. We can establish that 50 % of PRACs obtained in this work were regenerated from diploid microspores of “Morado de Huetor”, 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 Huetor”.

Study of the somaclonal variation produced by different methods of polyploidization in Asparagus officinalis L.

Polyploid plants have been induced in different Asparagus officinalis L. breeding programs in order to obtain plants with improved agronomical traits, such as large spear diameter or segregation ratios with a higher number of males. The polyploidization methods can produce somaclonal variation in the polyploid plants obtained and, therefore, unwanted changes in the agronomical traits of the initial elite plants. We used two different polyploidization methods to induce polyploid plants from diploid genotypes of commercial varieties and tetraploid genotypes of the Spanish landrace “Morado de Huetor”. The first method was the culture of rhizome buds in the medium ARBM-3 (Asparagus Rhizome Bud Medium), supplemented with different concentrations of colchicine (0.1–0.75 g l−1) for 10 and 20 days. The best polyploidization rate obtained was 25 % (0.5 g l−1 colchicine for 10 days). The second method was the regeneration of polyploid plants from callus culture, resulting in a polyploidization rate of 40 and 12.5 % for the diploid genotype CM077 and the tetraploid genotype HT156, respectively. Additionally, we have developed a new protocol to separate the mixoploids generated into their different genetic components, obtaining plants with a unique ploidy level. EST-SSRs markers were employed to analyze the genetic stability of polyploidy plants. Somaclonal variation was not detected for polyploidy plants obtained through the culture of rhizome bud explants. Therefore, these polyploid plants should maintain the agronomical traits of the initial elite plants. However, somaclonal variation was detected in the polyploid plants regenerated from callus culture.

In vitro induction of autooctoploid asparagus genotypes

Octoploid genotypes of Asparagus officinalis L. cv. ‘Morado de Huetor’, a tetraploid Spanish landrace, were successfully induced by treating in vitro rhizome buds explants with colchicine. Pulses during 24 h with different concentrations of colchicine are able to induce polyploid plants, colchicine applied at 0.1 % induce a 7 % of octoploid genotypes with a stable ploidy level and a 3.5 % of mixoploid genotypes. The maximum rate of mixoploid genotypes (26 %) was obtained with 0.3 % colchicine. The plant survival and rooting rates of explants decrease for increasing doses of colchicine. All octoploid genotypes were micropropagated, rooted, transplanted and successfully acclimatized (100 %) in a glasshouse. The four octoploid plants recovered show significantly better agronomical parameters such as spear diameter, canopy area and shoot length than the original tetraploid plants and histological studies confirm the size increase of octoploid cells respect the original tetraploid cells.

A new and efficient micropropagation method and its breeding applications in Asparagus genera

Cultivated asparagus (Asparagus officinalis L.) is an economically important plant worldwide. “Morado de Huetor” is a Spanish autochthonous landrace characterized by their longevity, organoleptic characteristics, differential biocompound content and high heterozygosity, resulting in heterogeneous plantations with limited productivity. Consequently, this landrace suffers high risk of extinction due the lack of productivity. The preservation of the genetic pool of asparagus requires the development of a reliable micropropagation method. A new, rapid and efficient method of micropropagation for asparagus using rhizome bud explants has been developed. The rate of disinfection reached 90 %, and the system for shoot development and rooting on Asparagus Rhizome Bud Medium took place in one step. Recovery of the full plantlets ranged between 65 and 90 %. The plantlets were ready to be transplanted by 8 weeks, with a successful acclimatization of 80 % in average. The micropropagated plants were normal in phenotype, and the genetic stability was verified using molecular markers expressed sequence tags–microsatellites or simple sequence repeats and Flow Cytometry and certified as true-to-type. Applying this method, an in vitro breeder collection of “Morado de Huetor” landrace, A. officinalis, wild asparagus relatives and hybrid progenies has been established.

Assessment of genetic diversity and phylogenetic relationships in Asparagus species related to Asparagus officinalis

Eight species of the genus Asparagus, members of the group of European species closely related to A. officinalis, were analysed using internal transcribed spacer (ITS), and expressed sequence tag-derived simple sequence repeat (EST-SSR) markers, as well as cytological observations of their hybrids, to study their phylogenetic relationships and the possibility of broadening the narrow genetic base of cultivated varieties. Phylogenetic analysis using ITS data revealed two major clades: clade I consisting of A. acutifolius and clade II (referred to in this study as the ‘officinalis group’) comprised of sequences derived from species closely related to A. officinalis; but the different species within the ‘officinalis group’ could not be clearly separated. In contrast, cluster analysis of EST-SSR marker data showed six major clades and clearly separated each population, grouping most of the genotypes from each population together. That is, EST-SSR markers were found to be more informative than ITS markers about the relationships within the ‘officinalis group’, indicating that EST-SSR markers are more useful than ITS sequences for establishing phylogenetic relationships at the intrageneric level. All the crosses carried out at the same ploidy level were successful. The high crossability, together with the regular meiotic behaviour and high pollen and seed fertility observed in the interspecific hybrids analysed, suggest relatively close relationships between the species studied. We conclude that the group of species classified in the ‘officinalis group’ are in the primary gene pool, indicating that these species could be used to increase the genetic diversity of the cultivated species. In addition, the tetraploid landrace “Morado de Huetor” could be employed as a bridge to generate new cultivated germplasm.

Development of triploid hybrids in asparagus breeding employing a tetraploid landrace

The development of triploid cultivars from crosses between the tetraploid landrace ‘Morado de Huetor’ and diploid commercial cultivars was studied in this work. Fertility of both 4x × 2x and 2x × 4x crosses, as measured by fruit set and number of seeds per fruit, and germination rate of triploid seeds, were studied in order to assess the viability of producing commercial seeds. Seven triploid hybrid progenies were included in a progeny test together with thirteen experimental tetraploid hybrids and four cultivars (‘Grande’, ‘UC157-F2’, ‘Purple Passion’ and ‘Morado de Huetor’). Marketable spear production and mean diameter of spears were evaluated in both 2007 and 2008. Crosses 4x × 2x were more successful than 2x × 4x, and a wide variation from high to very low fertility within tetraploid females was found. Germination rate of triploid seeds was similar to diploid ones. After 2 years of evaluation experimental hybrid triploids were more productive than tetraploids with values similar to the highest yielding diploid cultivar (‘Grande’). Spear diameter of triploid hybrids was in general thicker than the tetraploid hybrids and significantly thinner than the standard cultivars ‘Grande’ and ‘Purple Passion’ (tertraploid). These results suggest that the development of new triploid cultivars (4x × 2x) employing a tetraploid landrace from a different genetic background of current diplods could be interesting in asparagus breeding, broadening the spectrum of cultivated asparagus cultivars.

Origin of tetraploid cultivated asparagus landraces inferred from nuclear ribosomal DNA internal transcribed spacers’ polymorphisms

AbstractThe genus Asparagus includes a group of wild species that are closely related to the cultivated Asparagus officinalis (2n = 2× = 20). The narrow genetic background present in the asparagus cultivars shows the importance of asparagus landraces and the wild related species. The study of both genetic resources becomes necessary to facilitate their effective use in the breeding programmes. ‘Morado de Huetor’ (MH) and ‘Violetto d’Albenga’ (VA) are tetraploid asparagus landraces (2n = 4× = 40) cultivated in Spain and Italy, respectively, and whose origin remains unknown. To discover the origin of these landraces, a phylogenetic study was conducted based on restriction fragment length polymorphism (RFLP) of nuclear ribosomal DNA (nrDNA). The sequence of the two internal transcribed spacers (ITS) flanking the nrDNA5.8S gene (ITS1‐5.8S‐ITS2) were analysed for RFLP in 11 populations including both landraces (MH and VA), A. officinalis (wild and cultivated) and a group of closely related wild species (Asparagus maritimus, Asparagus prostratus, Asparagus pseudoscaber and Asparagus tenuifolius) with a European distribution. Restriction fragment patterns of both cultivated asparagus (2×) and two populations of A. maritimus (6×) from the Adriatic Sea area were present in the MH landrace. However, VA showed a similar pattern to A. officinalis. This study revealed that MH seems to be a hybrid between A. officinalis and A. maritimus that may have occurred in the Adriatic Sea region where hybridisations between cultivated diploid and wild species may have taken place. The origin of another tetraploid landrace (VA) might have had a similar origin but followed a different evolutionary path. Therefore, these landraces constitute a valuable genetic resource that could be used to enlarge the genetic background of modern cultivars. The ploidy levels of the populations employed in this study were analysed and levels not described previously were detected: A. maritimus (12×), A. tenuifolius (6×) and A. pseudoscaber (2×).

Collection and conservation of ‘Morado de Huetor’ Spanish tetraploid asparagus landrace

A cultivated tetraploid asparagus landrace ‘Morado de Huetor’ growing in the South of Spain has been collected and conserved ex situ in two experimental plots. This landrace has been reported to have an interspecific hybrid origin (A. officinalis × A. maritimus). A 59% of the total farmer plots cultivated in the region were visited and ripe fruits collected from 10 female plants per plot. Fruits from a total of 300 plants were collected and two half sibs descendents from each one were sown separate in two plots. Of the 88 farmer plots visited, the landrace was growing only in 34, in the rest the crop had been removed. Hence, ‘Morado de Huetor’ is in a very high risk of disappearing. Now this landrace is conserved ex situ in two experimental plots with sufficient number of plants to represent their maximum variability in order to be used in plant breeding.

Ploidic and Molecular Analysis of ‘Morado de Huetor’ Asparagus (Asparagus officinale L.) Population; A Spanish Tetraploid Landrace

A study has been conducted into the genetic variation analysis of the tetraploid asparagus landrace ‘Morado de Huetor’ and its relationship with current commercial cultivars using Random Amplified Polymorphic DNA (RAPD) molecular markers. The presence of different ploidic levels in the ‘Morado de Huetor’ landrace has been also studied using cytogenetic analysis. Ten decamer oligonucleotides were used to obtain RAPD markers and to characterise 52 individuals of ‘Morado de Huetor’, 7 of the tetraploid cultivar ‘Purple Passion’, and 55 of 5 diploid hybrid commercial cultivars. Jaccard similarity index was calculated and a cluster analysis using UPGMA (Unweighted Pair-Group Method using Arithmetic Average) was performed. Tetraploid asparagus, ‘Morado de Huetor’ and ‘Purple Passion’, were well differentiated from the diploid ones showing a higher genetic variability. This result suggests a different origin for tetraploid and diploid asparagus varieties. Within the diploid cultivars a variety distribution was obtained. A specific monomorphic band (OPB20883) was found in tetraploid varieties. Within the diploid cultivars, two bands (OPB20830, OPC15705) can be used to distinguish between the hybrid cultivars used in this study. Tri-, penta-, hexa-, and octoploid plants were found in ‘Morado de Huetor’. The origin of these ploidic levels is discussed and their use in the development of new varieties is proposed. In conclusion, ‘Morado de Huetor’ is a genetic resource that could be used to increase the narrow genetic background reported in diploid asparagus cultivars.