To simplify further discussion, aneuploids generated either from aneuploid microspores or by chromosome instability during androgenesis itself will be referred to in this text as primary aneuploidy; aneuploidy due to meiotic instability of regenerated plants will be called secondary aneuploidy. A clear discrimination between the two possible origins of primary aneuploids cannot be easily made as their consequences are essentially identical: once the chromosome number is doubled, a plant becomes disomic for the chromosome aberration . If the type of aberration permits seed set, all progeny shares the same phenotype and the same chromosome aberration.The only exception here would be chromosome doubling by a somatic fusion of two nuclei, each with different chromosome numbers/constitutions sometime during the process of androgenesis. Fusion of sister nuclei in the early divisions of microspores pushed into the sporophytic pathway of development are well documented and appear responsible for the frequent occurrence of spontaneously DHs.The most plausible explanation for chromosomal differences between two sister nuclei is chromosome nondisjunction, in effect doubling the number of copies of this specific chromosome in one daughter nucleus, while leaving its sister nucleus deficient for one copy. Fusion of such sister nuclei automatically restores normal chromosome constitution, masking the nondisjunction event. On the other hand, fusions of non-sister/ different nuclei, if technically possible, would be more likely to occur in meiocytes where thick cell walls are not a barrier, and would have a chance of not only producing aneuploids but would almost always produce heterozygotes. Such heterozygotes would be quite unsuitable for the general purposes for which DH lines are made,grow table hydroponic and would be readily detectable upon the first seed increase by segregation of the characteristics of the two parents. They would also be difficult to distinguish from regenerants from somatic tissues of the anther.They seemed better explained by imperfect isolation during flowering and resulting spontaneous out crossing of the C0 plants rather than regeneration from somatic tissues or by fusion of non-sister nuclei.

Aneuploids produced in the process of out crossing would most likely create heterozygous aberrations, such as monosomics, telocentric plus completechromosome and translocation heterozygotes. Disomic aberrations, while statistically possible, would be quite unlikely. Essentially all aberrations observed here were disomic. Somatic chromosome instability following nuclear fusions, or after induced chromosome doubling, would result in single-dose aberrations . Aneuploids produced in this fashion would be difficult to detect by morphology; after harvest and in the subsequent generations they would be practically indistinguishable from secondary aneuploids resulting from imperfect chromosome pairing in the C0 plants. However, one line was observed here that raised suspicion of a possible fusion of non-sister nuclei producing an aneuploid. Most of its progeny appeared aneuploid and segregated for many morphological characters. However, its origin from somatic tissue, combined with a chromosome loss during regeneration, cannot be ruled out. Meiotic instability in C0 or later generation is most likely to produce a single-dose chromosome aberration and in individual progeny plants while a majority of progeny would maintain their uniform phenotype. This feature was used to discriminate between the primary and secondary aneuploidy. Secondary aneuploidy, that is the frequency of aneuploids generated by imperfect chromosome pairing in C0 or C1, was estimated here at ca. 2.0–3.6%, in different seasons and different combinations. The estimate is based on the frequency of single plants deviating from the morphology typical for a line in the C1 seed increase. Observations of the MI chromosome pairing confirmed considerable meiotic instability of triticale hybrids . In the most stable hybrid analyzed, Presto 9 NE422T, only 46% of PMCs had all chromosomes paired with no univalents present; the same parameter for the least stable hybrid, NE422T 9 Stan 1, was 13.3% . The numbers of univalents, when present, ranged from 2 to 10 per PMC. Most univalents were from the rye genome. This suggests that aneuploidy among regenerants is primarily a consequence of a high frequency of aneuploid microspores in the plated material. Apparently, aneuploidy does not preclude a microspore from the switch to sporophytic development under culture conditions. Given the proportions of PMCs with complete chromosome pairing and proportions of euploid and aneuploid regenerants in this study, it did not appear that aneuploidy per se predisposed microspores to the switch to the sporophytic pathway of development.

In the meiotically more stable combinations, the proportions of aneuploids were lower; conversely, in NE422T 9 Stan 1 with the lowest proportion of PMCs with complete chromosome pairing, aneuploids greatly outnumbered euploids among the regenerants. Moreover, higher frequencies of aneuploids among regenerants may be related to the general amenability of a combination to androgenesis. In the two most recalcitrant combinations, Presto 9 NE422T and NE422T 9 Stan 1, aneuploids predominated among the regenerants, frustrating even more all attempts to create populations of DH lines of sufficient sizes. Among aneuploid microspores producing regenerants, the possible role of chromosome 5R is intriguing. Among 67 C0 aneuploids analyzed, in 52 cases a rye chromosome was involved, but 23 of these cases involved chromosome 5R. This is very similar to the proportion of 40 aneuploids 5R among the total of 83 rye chromosomes involved in aneuploidy in androgenic triticales in the study of Charmet et al. . In three of the four hybrids analyzed at MI, there were no indications that chromosome 5R was more prone to pairing failure and hence, contributed to aneuploidy in an unduly high proportion. This suggests a possible role of chromosome 5R in the green plant regeneration. Both hybrids involving NE422T responded poorly to culture conditions, and both generated a large proportion of aneuploids. One hybrid was relatively stable in meiosis, the other was unstable. A majority of green plants recovered in both combinations were aneuploid, most of them were nulli 5R,grow table and the proportion of nulli 5R increased with time spent on the induction medium. The overall low yield of regenerants in these combinations was a consequence of a very low proportion of green plants; there were no obvious differences in the ability to form embryos and embryogenic structures . It is not clear at this point what effect an absence of a specific chromosome may have on the ability to form green plants. It is also unclear if the increase in 5R nullisomics among green plants over time reflects a delayed reaction of the 5Rdefificient microspores to culture conditions or a gradual loss of chromosomes 5R during different stages of the regeneration process. As there are no indications of an unusually high proportion of 5R deficient microspores in the two hybrids, such microspores were presumably selected for during androgenesis, suggesting some involvement of 5R in the regenerations process. Chromosome 5R was not identified as a carrier of QTLs for androgenic response in hexaploid triticale by Gonzalez et al. and cv. Presto was one of the parental lines in that study. On the other hand, selection for specific chromosome constitutions among microspores has been demonstrated in Brassica hybrids so it cannot be ruled out in these materials. If the two distinct aneuploid phenotypes, Curly and Narrow are eliminated from consideration , rye chromosomes still contributed to aneuploidy about twice as frequently as wheat chromosomes, on a per chromosome basis, and this parallels the contribution of rye and wheat chromosomes to univalency in MI.

It is therefore possible that the primary cause of high aneuploidy among DHs of triticale relates directly to incomplete chromosome pairing in MI and resulting aneuploid microspores. The most frequent aberrations here were nullisomy and tetrasomy; however, ditelosomy and most translocations can also be traced back to univalency in MI. Paired chromosomes do not misdivide to form telocentrics or centric translocations. In several instances, non-centric translocations were observed , but they were a clear minority. The mechanism by which they originated is unknown. On the one hand, the meiotic preparations were analyzed carefully enough to exclude a significant chance of homoeologous pairing. On the other hand, non-centric breakage of univalents and subsequent fusion has been noted so it appears more likely that the observed non-centric translocations resulted from chromosome breakage and fusion rather than by homoeologous recombination. Exceptions to all these considerations are three compensating nullitetrasomics in homoeologous group 1. All of them involved engineered chromosomes 1R; these chromosomes are six break point wheat-rye translocations . The nulli-tetrasomics observed here likely arose by multivalent pairing of the engineered 1R with their standard wheat and rye precursors, and uneven segregation from multivalents in anaphase I. These nulli-tetrasomics were practically indistinguishable from euploids, both by morphology and by seed characteristics. Secondary aneuploids were present in the analyzed material. This was judged by the frequency of single plants deviating from the standard phenotype of any given DH line. Among 844 and 488 plants scored for this feature in two different growing seasons, 21 and 14 were classified as aneuploid, respectively, for the frequencies of 2.5 and 2.9%, respectively. Of the first 21, six were tested cytologically and all were monosomic for various chromosomes. The 2.6% frequency of secondary aneuploidy among the DH lines is within the 2–3% range of aneuploid frequency assessed visually for the parental lines used in the study, and is at the lower end of perhaps the most recent data on aneuploidy in commercial hexaploid winter triticales but considerably below the ranges observed by Struss and Ro¨bbelen among F1 hybrids of triticale. These observations give no reason to suspect that the DH lines of hexaploid triticale will be less stable than the parental lines. On the other hand, if meiotic instability of triticale hybrids reflects differences in genetic systems of chromosome pairing control among parental lines, segregation among progeny is to be expected with an overall wider range of variation for various indices of chromosome paring than among the parents. Flow cytometry is often used to assess chromosome constitutions of regenerated DH progenies . This approach suffers certain methodological weakness in that any variation in the DNA contents between the parents may generate a range of variation among the regenerants that can be confused with aneuploidy. Among androgenic progenies from the Lolium 9 Festuca hybrids, Guo et al. 2005 found a 5–7% difference in the nuclear DNA contents. Given that these regenerants had 14 or 28 chromosomes, a 7% difference could easily be interpreted as nullisomy while in fact it may only reflect a general difference of the DNA contents between the two parents. The same may hold true for hexaploid triticale. Triticale is known for considerable differences in the amount of telomeric chromatin present on rye chromosomes. Telomeric C-bands in rye occupy about 12% of the total chromosome length and may contain as much as 18% of the total nuclear DNA . Given the amounts of DNA per A, B and R genomes, 18% difference in the amount of rye DNA present would be equivalent to ca. 7% difference in the total nuclear DNA contents, an equivalent to the absence or an extra dose of three chromosomes. Given that any two triticales may carry similar amounts of DNA and still differ in the presence/absence of certain heterochromatic blocks, flow cytometry results have to be interpreted with extreme caution and as a rule should not be used to asses the aneuploidy rates, or the general chromosome stability of the regenerated material . In this study, there was no significant difference in the DNA contents between the two parents in a cross and yet, there was 11.3% difference in the DNA contents among tested DH progeny , and these differences did not correlate with aneuploidy. While a majority of aneuploids observed here can be explained as a consequence of pairing failure in MI of meiosis of the donor plants, there were also some aneuploids with chromosome constitution that defied all efforts at explanation. Among them was a plant with 28 chromosomes where seven chromosomes were present in pairs and 14 were present in single dosage . Also interesting in this context was the 78 chromosome plant . It could not have originated by two rounds of chromosome doubling unless an assumption is made of somatic chromosome loss between the two rounds of doubling. Individuals with similarly impossible to explain origins were noted among androgenically derived progenies of Festuca arundinacea x Lolium multiflorum hybrids or haploids generated from tetraploid Festulolium .