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Davut Keleş, Hasan Pınar, Atilla Ata, Hatıra Taşkın, Serhat Yıldız and Saadet Büyükalaca

species. Positive results in pepper have been achieved with anther culture ( Al Remi et al., 2014 ; Buyukalaca et al., 2004 ; Comlekcioglu et al., 2001 ; Ercan et al., 2006 ; Gémesné Juhász et al., 2001 ; Niklas-Nowak et al., 2012 ; Olszewska et al

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Hatsuhiko Okada, Yoshitaka Ohashi, Mamoru Sato, Hideyuki Matsuno, Toshiya Yamamoto, Hoytaek Kim, Tatsuro Tukuni and Sadao Komori

, the first approaches were conducted to produce doubled haploids in apple by in vitro anther culture ( Nakayama et al., 1972 ). Since then, various homozygous genotypes induction methods such as in vitro anther culture and in situ parthenogenesis in

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Mark W. Farnham

Using anther culture to generate doubled-haploid (DH) homozygous lines for use as parents in F1 hybrid crosses has become a common practice in breeding broccoli (Brassica oleracea L. Italica Group). During anther culture and subsequent embryogenesis and plant regeneration, polyploidization of microspore-derived embryos may not occur or it may occur accompanied by a doubling, tripling, quadrupling, octupling, or irregular polyploidization of the genome. Thus regenerants from the process can be haploids, diploids, triploids, tetraploids, octaploids, or aneuploids. The objectives of this research were to 1) conduct repeat cycles of broccoli anther culture using a group of F1 hybrids as anther donors and develop populations of regenerants; 2) analyze resulting populations using DNA flow cytometry and determine the influence of F1 source on frequency of different ploidy levels among regenerants; and 3) compare seed set in broccoli inbreds developed in a traditional selfing program compared to seed set in DH broccoli derived from anther culture. In two cycles (1994 and 1995) of anther culture, anther-derived populations of regenerants were developed using the F1 hybrids `Marathon', `Everest', `High Sierra', and `Futura' as sources of anthers. In 1994, `Everest', `High Sierra', and `Futura' yielded populations that included 2% to 7% haploids, 53% to 56% diploids, 32% to 38% tetraploids, and 5% to 6% other types. `Marathon'-derived regenerants were 5% haploid, 78% diploid, 15% tetraploid, and 2% other, showing significantly more diploids. In 1995, `Marathon' regenerants again included significantly more diploids and fewer tetraploids than those derived from other F1 sources, confirming that the genotype of the anther source affects the frequency of a particular ploidy level among regenerants derived from culture. In manual self-pollinations of 1994 regenerants, only diploids and rare tetraploids set seed. When plants that set no seed were discounted, seed production following manual self pollinations of 1995 regenerants was not significantly different from that of traditional inbreds derived from the same F1 sources.

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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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Richard E. Veilleux

Anther culture has been one of the most successful techniques for generating haploid plants over a wide range of species. It is a reasonably simple procedure that can be accomplished successfully without sophisticated laboratory facilities; yet, the plants generated through anther culture can be used to demonstrate the application of many modern methods that have direct applicability to plant breeding. Anthers of diploid potato clones that have been selected for competence in anther culture can be cultured in a simple medium to yield androgenic embryos after 5 weeks. Plant regeneration requires an additional 3 to 4 weeks. Regenerated plants should be large enough 2 weeks after transfer to basal medium for ploidy determination by any of three methods depending on available facilities: chromosome counts in root tips; chloroplast counts in stomatal guard cells; or flow cytometry of nuclei released from in vitro plantlets. DNA can be extracted from anther-derived plantlets using a rapid extraction procedure to demonstrate segregation of PCR (polymerase chain reaction)-based markers such as RAPD (randomly amplified polymorphic DNA), RAMPs (randomly amplified microsatellite polymorphisms), or microsatellites. Microsatellite markers that were heterozygous in the anther donor can be used to verify haploidy in anther-derived plants. If an anther culture laboratory is scheduled early in a semester, such molecular analysis can be planned for late in the same semester.

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

development of homozygous lines in a single generation ( Ellialtıoğlu et al., 2002 ). Anther culture is one of the techniques that are used widely to obtain haploid plants. The advantage of this technique over other methods to obtain in vitro haploid plants is

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Richard E. Veilleux

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Dušica Ćalić, Nina Devrnja, Jelena Milojević, Igor Kostić, Dušica Janošević, Snežana Budimir and Snežana Zdravković-Korać

( Ducos et al., 2007 ), Cinnamomum camphora ( Shi et al., 2009 ), and Pterocarpus marsupium ( Husain et al., 2010 ). The aim of this research was to study the effect of ABA on SSE in horse chestnut microspore and anther culture with the goal of

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M.H. Aboul-Nasr and M.A. Ahmed

This experiment was performed at the Tissue Culture Laboratory of the Horticulture Dept. of the Faculty of Agriculture at Assiut Univ., Egypt. After several attempts to determine the proper stage of buds for collection of pollen, we determined that the tetrad stage was most suitable. The pollen was cultured on either MS or B5 liquid or solid media (7% agar). Both media were used as basic salts or supplemented with growth regulators. The four growth substances were BA, NAA, K, and 2,4-D. Each growth substance was added to the medium separately as follow: BA, NAA at 15, 10, or 5 ppm; K at 0.1, 1, 2, or 5 ppm; and 2,4-D at 0.5, 1, or 5 ppm. The solidified medium was superior to the liquid medium at all the treatments that were used for callus formation. Using B5 medium did not result in any callus. The highest value of callus formation was obtained when MS medium supplemented with BA at 5 ppm. Moreover, the callus that was grown on the MS medium that had BA at 5 or 10 ppm developed a merstim tip. The control treatment produced calluses but did not develop any meristem tips. This process can be used to develop haploid plants.