Anther culture in Citrus clementina: a way to regenerate tri-haploids

Abstract

Abstract. Regenerants from anther culture of Citrus clementina Hort. ex Tan. cvv. Nules, SRA 63, and Monreal were obtained in different experiments from 1994 to 2002. Genetic analysis of 37 such regenerants was carried out using 4 microsatellite markers that were heterozygous in the parental genotypes. The results showed that in all cases but one the regenerants carried only one or the other allele of the parental genotype, and were therefore homozygous and produced through a process of gametophytic embryogenesis. Ploidy analysis by flow cytometry of 94 regenerants showed that as many as 82% of them were tri-haploids, rather than haploids or doubled-haploids as expected, with other ploidy levels such as n, 2n, 4n, and 6n occurring at very low percentages (2, 10, 3, and 3%, respectively). Regeneration from anther culture is therefore proposed as a rapid, and attractive method of obtaining new triploid varieties in clementine, which could be of great interest for the fresh fruit market that now requires fruit to be seedless.

Gametic and somatic embryogenesis through in vitro anther culture of different Citrus genotypes

“…Since the first haploid embryogenic calli production and plantlet regeneration by anther culture in C. clementina cv. Nules was reported (Germanà et al 1994), many studies have been carried out in Citrus, and they focused on increasing the frequency of embryogenesis with responsive species and on developing new protocols for recalcitrant ones (Germanà & Reforgiato 1997;Germanà et al 2000aGermanà et al , 2005. This study is significant because it increased the number of genotypes that respond to microspore embryogenesis as well as the haploid induction rate (the frequency of anthers that recovery embryos).…”

Characterization of Leaf Essential Oil Composition of Homozygous and HeterozygousCitrus clementinaHort. Extan. and its Ancestors

“…ex Tan, cv. Nules 6 , and they resulted, as most of regenerants evaluated (82 % of 94 regenerants), triploids at the flow cytometry analysis, other than homozygous through microsatellite marker analyses 8 . The tri-haploid plants (HOMO1 and HOMO2), obtained in vitro by anther culture, were acclimatized in vivo and grafted in vivo in 2000, at the same time of the heterozygous plant (HETERO).…”

“…Considerable research has been carried out since the 1970s to obtain haploids for ISSN Print: 0972-060X ISSN Online: 0976-5026 fruit tree breeding through gametic embryogenesis, but they were not always successful 9,10,19 . Many haploids and DHs are under observations in many fruit tree crops such as citrus, apple, papaya and peach, but it takes considerable time to characterize them 7,8,13,20,21,22,25,26 . Often in vitro regenerated plants show differences in their morphological and biochemical characteristics, as well in chromosome number and structures.…”

“…ex Tan., cv. Nules 8 . Two of them, (HOMO1 and HOMO2), have been acclimatized and grafted in vivo in 2000.…”

Techniques for Haploid and Doubled Haploid Production

“…se lahko na hranilnih gojiščih regenerirajo poleg haploidnih tudi diploidne rastline in celo rastline višjih stopenj ploidnosti (Sunderland, 1974;Germanà 2005Germanà , 2006Germanà , 2009Košmrlj in sod., 2013;Murovec in Bohanec, 2013). V preteklosti so za določanje ploidnosti uporabljali posredne metode kot so spremljanje morfoloških lastnosti rastlin (velikost rastlin in listov, oblika cvetov), fertilnosti rastlin, bujnost rastlin, število kloroplastov v celicah zapiralkah in velikost listnih rež.…”