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Regalado J.J.,Institute Horticultura Subtropical y Mediterranea La Mayora | Carmona Martin E.,Institute Horticultura Subtropical y Mediterranea La Mayora | Castro P.,University of Cordoba, Spain | Moreno R.,University of Cordoba, Spain | And 2 more authors.
Plant Cell, Tissue and Organ Culture | Year: 2015

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 Huétor”. 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. © Springer Science+Business Media Dordrecht 2015.


Regalado J.J.,Institute Horticultura Subtropical y Mediterranea La Mayora | Martin E.C.,Institute Horticultura Subtropical y Mediterranea La Mayora | Madrid E.,CSIC - Institute for Sustainable Agriculture | Moreno R.,University of Cordoba, Spain | And 2 more authors.
Plant Cell, Tissue and Organ Culture | Year: 2016

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 EST-SRRs. 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”. © 2015, Springer Science+Business Media Dordrecht.


Regalado J.J.,Institute Horticultura Subtropical y Mediterranea La Mayora | Carmona-Martin E.,Institute Horticultura Subtropical y Mediterranea La Mayora | Castro P.,U.S. Department of Agriculture | Moreno R.,University of Cordoba, Spain | And 2 more authors.
Plant Cell, Tissue and Organ Culture | Year: 2015

An efficient micropropagation method for asparagus species was developed in this study. The method allows the fast cloning of the elite genotypes from different asparagus species and the interspecific hybrids obtained from these species. Rhizome bud explants were disinfected using 3 g l−1 of benomyl and 20 g l−1 of sodium hypochlorite. Then, they were cultured on Asparagus Rhizome Bud Medium 3 (ARBM-3) consisting in modified Murashige and Skoog medium with salts with EDDHA-Fe (85.7 mg l−1) instead of EDTA-Fe and vitamins, supplemented with 0.3 mg l−1 NAA, 0.1 mg l−1 KIN, 2 mg l−1 ancymidol and 6 % sucrose. Results showed that the method developed produced high disinfection rates (70–95 %). More than 70 % of the explants developed shoots and the rooting rate on ARBM-3 medium was 30–45 %. The rooting rate increased to 60–85 % when the unrooted shoots were subjected to an additional cycle of rooting, reaching 100 % after two cycles of rooting. The multiplication was achieved through mechanical division of rooted shoot clusters growing in ARBM-3. The acclimatization rate of the micropropagated plantlets was higher than 90 %. The micropopagated plantlets were screened for somaclonal variation using 12 expressed sequence tag derived simple sequence repeat markers. The results confirmed the character “true to type” of the plantlets, indicating that the method developed in this study can be used to successfully micropropagate asparagus species. © 2015, Springer Science+Business Media Dordrecht.


Regalado J.J.,Institute Horticultura Subtropical y Mediterranea La Mayora | Moreno R.,University of Cordoba, Spain | Castro P.,University of Cordoba, Spain | Carmona-Martin E.,Institute Horticultura Subtropical y Mediterranea La Mayora | And 6 more authors.
Genetic Resources and Crop Evolution | Year: 2016

Asparagus maritimus is a species distributed in sandy soils along the Mediterranean coast. It has been reported as salt tolerant and resistant to rust. The wild asparagus species are a very important genetic resources for asparagus breeding because the current commercial cultivars have a narrow genetic base. Until recently, the only population of A. maritimus catalogued in Spain was a small population, which is at high extinction risk, located around the coastal lagoon “Mar Menor” in the region of Murcia. Different studies carried out in the current work support the recent description of this population as a new species named Asparagus macrorrhizus. Plants from three populations of A. maritimus were used to carry out studies of characterization and the results were compared with plants of A. macrorrhizus. The morphological studies showed clear differences between the populations of A. maritimus and A. macrorrhizus. One of the differences found between these populations was at the ploidy level. The plants of A. maritimus were hexaploid (2n = 6x = 60), while the plants of A. macrorrhizus were dodecaploid (2n = 12x = 120). Also, the flavonoid composition showed that A. maritimus contains six different flavonoids while in A. macrorrhizus 90 % of the flavonoid content corresponds to only one flavonoid (Nicotiflorin) followed by minor quantities of other two. Another difference between these populations was supported by the principal coordinate analysis (PCoA) using data from 4 EST-SSRs markers amplified in plants of A. maritimus and A. macrorrhizus, and clearly separates the two species. The differences found in this work highlight the importance of A. macrorrhizus as a possible genetic resource for asparagus breeding. The distribution of A. macrorrhizus is limited to the area surrounding the “Mar Menor” lagoon. The prospections carried out in the last years indicated the high risk of extinction of this species due to the urbanization of this natural habitat. Therefore, we have included A. macrorrhizus in our germplasm bank in vivo and in vitro as well as in the breeding programs. © 2016 Springer Science+Business Media Dordrecht


Carmona-Martin E.,Institute Horticultura Subtropical y Mediterranea La Mayora | Regalado J.J.,Institute Horticultura Subtropical y Mediterranea La Mayora | Padilla I.M.G.,Institute Horticultura Subtropical y Mediterranea La Mayora | Padilla I.M.G.,Centro IFAPA Churriana | And 2 more authors.
Plant Cell, Tissue and Organ Culture | Year: 2014

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. © 2014, Springer Science+Business Media Dordrecht.

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