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Agustin Huerta and Ramon Dolcet-Sanjuan

Adventitious shoots and viable plants were regenerated from bell pepper (Capsicum annuum L.) cultivars and dihaploid lines (DHLs) obtained from F1 hybrids via androgenesis (Dolcet-Sanjuan et al., in press). Hypocotil and cotyledon sections from in vitro-germinated seeds were used as explants. A modified MS medium (Murashige and Skoog, 1962) supplemented with IAA (0 to 3.2 μM) and BAP (0 to 100 μM) was used in a 3-week-long shoot primordia induction phase. Shoot elongation was best performed in the same basal medium, but supplemented with silver thiosulfate and GA3. Shoots were regenerated from eight selected DHLs (`C213', `C215', `C218', `C2123', `C2125', `C3111', `C3113', and `P493') and two cultivars (`Padrón' and `Yolo Wonder'). The percentage of cotyledon sections with shoot primordia after the induction phase was not genotype-dependent and always higher than with hypocotil sections (93.4% and 17.9%, respectively). The number of shoot primordia per responsive cotyledon section was also higher than with hypocotil sections (3.3 and 1.7, respectively). The genotype had a significant effect on the number of shoots regenerated per responsive cotyledon (1.1 to 5.5) or hypocotil (0.5 to 3.5) section. All adventitiously regenerated plants were fertile. This adventitious shoot regeneration protocol is being used to obtain transgenic plants from sweet bell pepper genotypes.

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Ramon Dolcet-Sanjuan, Elisabet Claveria, and Agustin Huerta

A new and simple protocol for androgenesis in bell pepper is described. The initial medium, a modification of Nitsch and Nitsch's H medium, consisted of a two-phase system of semi-solid and liquid medium and contained maltose as carbon source. The total number of embryos formed was greater with maltose at 40 g·L-1, but embryos developed better at 10 to 20 g·L-1. Depending on the genotype, the number of embryos and plants recovered ranged from 3 to 750 and 0.25 to 8, respectively, per 100 flowers. Further increases in the number of embryos (up to 3561 per 100 flowers) and plants (up to 23 per 100 flowers) could be attained by flushing cultures with air enriched with CO2 at 900 μL·L-1. The ploidy level and the microspore origin of the recovered plants were determined by flow cytometry and zymograms for isocitrate dehydrogenase. Nearly 65% of the acclimated plants had undergone spontaneous doubling of the chromosome number, as confirmed by flow cytometry of leaf nuclei. Isocitrate dehydrogenase zymograms demonstrated that plants originated from microspores and that the two parental alleles were equally represented among the haploid and dihaploid plants.

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Ramon Dolcet-Sanjuan, Elisabet Clavería, Alfonso J. Rodríguez, and Marta Llaurado

Callus and shoot organogenesis were obtained from anthers of Dianthus caryophyllus L. `Manon', `Amapola', `Elsy', and `IB212', harboring mid-uninucleated microspores. Significant differences between genotypes were observed on number of responsive anthers (10.4% to 72.1%) and rescued plants per responsive anthers (1.2% to 4.8%). A modified H medium (Nitsch and Nitsch, 1969) with 20 g/L maltose and 0.25% Gelrite, supplemented with 10 μM 2,4-D and 1 μM TDZ, was most appropriate for callus induction. Plants were regenerated after subsequent subculture to the same medium, but amended with 0.1 μM TDZ. Zymogram types for aminopeptidase (AAP) in polyacrilamide gel electrophoresis proved that all 40 plants regenerated from `Amapola', `Elsy', or `IB212' where heterozygous, and consequently not originated from the microspores but from somatic tissue. Alternatively, in situ-induced parthenogenesis through pollination with gamma-irradiated pollen and in vitro embryo rescue was tested. A total of 92 embryos, including normal and no cotyledonary embryos, were rescued from 38 fruits harvested out of 70 crosses between `Scania' and `Amapola'. Embryos were rescued 21 to 28 days after pollination by culture in a modified E20A (Sauton and Vaulx, 1987) medium. Phosphogluco isomerase (PGI) and Shikimic dehydrogenase (SDH) zymograms in starch gel electrophoresis, and AAP in polyacrilamide gel electrophoresis, indicated the parthenogenic origin of three of the regenerated plants. Flow cytometry of nuclei proved the early diploidization, during in-vitro micropropagation, of the parthenogenic carnation haploid plantlets.

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Ram K. Birhman, Sylvain R. Rivard, and Mario Cappadocia

Using restriction fragment length polymorphism (RFLP) analysis, the genetic architecture of some anther-culture-derived S. chacoense Bitt. plants was studied, and their origins were elucidated. Our RFLP analyses showed that 1) several plants, even of different ploidy but otherwise genetically identical (clones), can be regenerated from callus originating from a single microspore and, conversely, that 2) some plants regenerated from single callus can have different genetic constitutions and, therefore, must have originated from two different microspore. These findings imply that previous anther culture efficiency estimates might have to be reconsidered.

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Mark W. Farnham

Broccoli (Brassica oleracea L. Italica group) breeders are increasingly using anther or microspore culture to produce dihaploid (diploid), homozygous lines for use in making hybrids. During the process of anther culture and subsequent plant regeneration, wherein embryos develop from microspores and plants are regenerated from the embryos, polyploidization occurs and diploid regenerants can result. However, polyploidization may not occur at all, or it may involve a tripling or quadrupling of the chromosome complement, instead of a doubling. Thus, populations may contain haploids, triploids, or tetraploids, in addition to diploids. In two cycles (1994-95 and 1995-96) of anther culture, regenerated populations from different broccoli hybrid sources were evaluated using flow cytometry to facilitate efficient identification of diploids vs. haploids, tetraploids, or others and to determine if anther donor genotype has an effect on the frequency of different ploidy levels among regenerants. In the first cycle, five broccoli hybrids had anther-derived populations in which ≈33% were haploid, 55% diploid, 37% tetraploid, and 5% aneuploid or abherent types. The hybrid, `Marathon', was different; it's regenerants were 78% diploid and only 15% tetraploid. In the second cycle, anther-derived populations had a significantly different makeup with a most hybrids giving 30% to 40% diploids and 50% to 60% tetraploids. However, consistent with the previous cycle, `Marathon' gave significantly more diploids (68%) and fewer tetraploids (25%) than other hybrids. These results indicate that anther donor genotype affects ploidy frequency among regenerants. Genotypes producing a high frequency (>60%) of diploids may be relatively uncommon.

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Mark W. Farnham, Ellis J. Caniglia, and Claude E. Thomas

Broccoli (Brassica oleracea L. Italica group) breeders routinely use anther or microspore culture to produce dihaploid (diploid), homozygous lines. During the culture process, polyploidization occurs and diploid regenerants can result. However, polyploidization may not occur at all, or it may involve a tripling or quadrupling of the chromosome complement. Thus, regenerated populations must be screened to identify the diploids that are the regenerants most likely to set seed and serve as inbred lines. DNA flow cytometry has proven a useful procedure for determining ploidy of anther derived regenerants. This study was undertaken to evaluate the effect of leaf age and sampling procedures on ploidy determination via flow cytometry. Anther-derived plants were analyzed at a four- to five-leaf stage (transplant stage) and at time of heading (mature plant stage). In addition, leaves were sampled on a given date and stability of the flow cytometry preparations was evaluated over 7 days. Lastly, the stability of ploidy readings of leaves stored at 4°C was examined over a 7-day period. In only one case out of 123 comparative assays did leaf age affect ploidy determination. For that exception, a haploid at transplant stage was a diploid at the mature plant stage. Flow cytometry preparations and also leaves stored at 4°C gave consistent ploidy determinations up to four days after preparations were made or tissue was refrigerated, respectively. These results indicate that broccoli breeders can make flow cytometry preparations on site and send them offsite for flow cytometry analysis. Alternatively, leaves could be refrigerated, sent offsite, and then prepared and analyzed at another location.

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Md. Mizanur Rahim Khan, Mst. Hasnunnahar, and S. Isshiki

attributed to the allotetraploid status of the nuclear genomes. As a result of high pollen fertility, obtaining dihaploids of our synthetic amphidiploids might be possible by anther culture. This dihaploids might be backcrossed to recurrent eggplant to

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Les D. Padley Jr, Eileen A. Kabelka, Pamela D. Roberts, and Ronald French

hybrids obtained from crosses of the dihaploid S. tuberosum L. with wild and cultivated forms of Solanum 168 177 Kameraz, A.Y. Systemics, breeding and seed production of potatoes Amerind Publishing Co. Pvt. Ltd New Delhi, India

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Davut Keleş, Ceren Özcan, Hasan Pınar, Atilla Ata, Nihal Denli, Namık Kemal Yücel, Hatıra Taşkın, and Saadet Büyükalaca

. Başay, S. Şeniz, V. Ellialtıoğlu, Ş. 2011 Obtaining dihaploid lines by using anther culture in the different eggplant cultivars J. Food Agr. Environ. 9 188 190 Başay, S. Ellialtıoğlu, Ş. 2013 Effect of genotypical factors on the effectiveness of anther

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Mohammad Sadat Hosseini Grouh, Kourosh Vahdati, Mahmoud Lotfi, Darab Hassani, and Nejat Pirvali Biranvand

and di-haploids (DHs) are important for genetic and developmental studies as well as for plant breeding. They have potential use in mutation research, genetic analysis, transformation, and in the production of homozygous cultivar(s) that can be used to