Mapping of QTLs for androgenetic response based on a molecular genetic map of x Triticosecale Wittmack.

Quantitative trait loci (QTLs) for androgenetic response were mapped in a doubled haploid (DH) population derived from the F1 hybrid of 2 unrelated varieties of triticale, 'Torote' and 'Presto'. A molecular marker linkage map of this cross was previously constructed using 73 DH lines. This map contains 356 markers (18 random amplified 5 polymorphic DNA, 40 random amplified microsatellite polymorphics, 276 amplified fragment length polymorphisms, and 22 simple sequence repeats) and was used for QTL analysis. The genome was well covered, and of the markers analysed, 336 were located in 21 linkage groups (81.9%) identified using SSR markers. The map covered a total length of 2465.4 cM with an average of 1 marker for each 6.9 cM. The distribution of the markers was not homogeneous across the 3 genomes, with 50.7% detected in the R genome. Several QTLs were found for the following variables related to the androgenetic response: number of embryos/100 anthers; plants regenerated from 100 embryos; number of green plants/total number of plants; and number of green plants/1000 anthers. Two were detected on chromosome 6B and 4R, which together had a 30% total influence on the induction of embryos. Another was found on 6B and on the unidentified LG1; these influenced the production of total plants from haploid embryo cultures. One QTL on chromosome 3R determined the photosynthetic viability of the haploid plantlets regenerated from microspores. Other QTLs were found on chromosomes 1B, 1R, 4R, and 7R, which helped the control of the final androgenetic response (the number of plantlets obtained for every 1000 anthers cultured).     

Genetic improvement of anther culture response in maize: relationships with molecular, Mendelian and agronomic traits

 Two cycles of androgenetic in vitro doubled haploid (DH) plant production and intermating were implemented in an experimental synthetic population of maize. In vitro traits, including androgenetic embryo production, the regeneration potential and the frequency of spontaneous chromosome doubling, were studied. The success of the regenerated plants to self pollinate was also observed. Impressive genetic progress is reported for all the steps of the androgenetic process including seed set. Differential genetic progress is recorded according to the trait measured. Using a set of Mendelian and molecular markers that mapped to the different maize chromosomes, we were able to characterize the variation in the genetic variability in the population. Progress in the in vitro response was not found to be associated with any noticeable decline in global genetic variability. In addition, the QTL chromosomic regions tested, which were involved in androgenetic response, were not found to be subjected to a strong variation during the breeding experiment. Some phenotypical and morphological traits were also evaluated, and these showed that there was no depreciation effect in the agronomic value of the population. DH plant production and intermating the regenerated plants may be considered for the introduction and use of androgenesis in material which responds poorly. Received: 3 October 1997 / Accepted: 25 November 1997     

Plastid ultrastructure and DNA related to albinism in androgenetic embryos of various barley (Hordeum vulgare L.) cultivars

In order to understand the occurrence of albinism during androgenesis in barley, a number of plastid parameters were analyzed in microspore-derived embryos and androgenetic plantlets, and the results were compared in albino and non-albino producing cultivars. In the winter cv. Igri, plastids in microspore-derived embryos are characterized by numerous divisions, differentiated thylakoids, low amount of starch and a high DNA content examined by immunoelectron microscopy. After regeneration, the androgenetic plantlets were mostly chlorophyllous. In contrast, in the spring lines tested, the plastids of microspore-derived embryos were rarely dividing amyloplasts in which thylakoids and DNA were scarce and albino plantlets were mainly regenerated. After 2 weeks on the regeneration medium, plastids of Igri chlorophyllous androgenetic plantlets were typical chloroplasts, whereas in spring lines plastids of albino androgenetic plantlets were proplastids with the same characteristics as those in the corresponding microspore-derived embryos. These results strongly suggest that the origin of androgenetic albinism differs in winter and spring cvs.: in the winter cv. Igri plastid alteration may take place during the regeneration step of androgenesis whereas in the tested spring lines plastids are already affected in the microspore-derived embryos meaning that albinism is not initiated during regeneration but originates earlier during the androgenetic process likely as early as the sampling stage.     

Genotypic variation of quantitative trait loci controlling in vitro androgenesis in maize.

A quantitative trait loci (QTL) analysis for androgenetic capability has been conducted on three different crosses in maize, including very high and nonresponding lines for androgenesis. The doubled haploid lines derived by anther culture from the crosses DH5 x DH7, A188 x DH7, and R6 x DH99 showed a range of 0-70%, 0-40%, and 0-50% androgenetic responding anthers, respectively. The genotypic heritability of means for this trait is close to 0.90 for A188 x DH7 and 0.78 for R6 x DH99. The QTL analysis involved in each population the mapping of more than 100 loci covering a large part of the genome with reasonably spaced markers averaging 12 cM. Different measurements describing the androgenetic process were studied: AC, percentage of responding anthers; ELS, number of androgenetic embryos produced per 100 plated anthers; PLE, number of plantlets regenerated per 100 embryos; PLA, number of plantlets per 100 plated anthers. In each cross, three to four QTLs were found for AC, explaining 30-40% of the phenotypic variation. The QTL detected for PLA was also strong QTL for AC or ELS. This agrees with the observation that these last two traits are good predictors for final plantlet yield. The QTLs found were specific, although the same line DH7 was used in two crosses and DH99 derived from DH5 and DH7 in the third cross. These results suggest that the transfer of the androgenetic capabilities in elite germplasm will still involve a phenotypic evaluation of the androgenetic performances. A backcross-assisted selection based only on the genotype at the QTL is probably possible but only within the crosses used for this QTL analysis.     

In vitro androgenesis of triticale in isolated microspore culture

Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using microspore culture. Sporophytic development of isolated triticale microspores in culture is described in five winter hexaploid triticale genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators. The highest induction of microspore embryogenesis was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro development of triticale microspores. Albino and green plantlets were regenerated from embryo-like structures. More than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of candidate genes for in vitro androgenesis induction in maize

Extensive studies have been conducted to understand the genetic control of in vitro androgenesis, but little is know about the genes and the mechanisms involved in the switch that allows an immature pollen grain to develop as an embryo. We have developed two maize isogenic lines with high androgenetic aptitude, named AH5-44 and AH5-49, through backcross and selection from a high-responsive DH229 line on the non-responding A188 line genetic background. The genomic structure of these two lines was precisely described with microsatellite markers. Five regions retained from the parent DH229 highly responsive to androgenesis were localised in both AH5-44 and AH5-49. Sequences expressed on microspores extracted from the four lines were amplified using a cDNA-AFLP protocol. For each line, eight culture conditions were compared: microspores extracted after tassel recovery, after 7 or 14 days in cold room and after 1–4 days of in vitro culture. This genetic and developmental screening allowed us to identify four sequences, including a new HSP70-like candidate gene. Possible implication of the identified sequences in androgenesis response is discussed.Correspondence and request for material (near isogenic line seeds and pre-amplified cDNA-AFLP samples are available for other groups to identify new sequences potentially involved in androgenesis using a larger set of primer combinations on cDNA-AFLP samples) should be addressed to P.B. (e-mail: barret@clermont.inra.fr) Sequence data from this article have been deposited in the EMBL/GenBank data libraries under accession nos. AY222837 (HSP70-like), CD766057 (E9 M1-130), CD766058 (E13 M1-550), and CD766059 (E9 M5-320)     

Unilateral incompatibility as a major cause of skewed segregation in the cross between Lycopersicon esculentum and L. pennellii

Skewed segregations are frequent events in segregating populations derived from different interspecific crosses in tomato. To determine a basis for skewed segregations in the progeny of the cross between Lycopersicon esculentum and L. pennellii, monogenic segregations of 16 isozyme loci were analyzed in an F₂ and two backcross populations of this cross. In the F₂, 9 loci mapping to chromosomes 1, 2, 4, 9, 10 and 12 exhibited skewed segregations and in all cases there was an excess of L. pennellii homozygotes. The genotypic frequencies at all but one locus were at Hardy-Weinberg equilibria. In the backcross populations, all except two loci exhibited normal Mendelian segregations. No post-zygotic selection model could statistically or biologically explain the observed segregation patterns in the F₂ and backcross populations. A pre-zygotic selection model, assuming selective elimination of the male gametophytes during pollen function (i.e., from pollination to karyogamy), could adequately explain the observed segregations in all three populations. The direction of the skewed segregations in the F₂ population was consistent with that expected based on the effects of unilateral incompatibility reactions between the two species. In addition, the chromosomal locations of 5 of the 9 markers that exhibited skewed segregations coincided with the locations of several known compatibility-related genes in tomato. Multigenic unilateral incompatibility reactions between L. esculentum pollen and the stigma or style of L. pennellii (or its hybrid derivatives) are suggested to be the major cause of the skewed segregations in the F₂ progeny of this cross.     

Morphological events in cultures of mechanically isolated maize mcirospores

Summary In tis androgenic response, maize is considered to be a recalcitrant plant. We used mechanically isolated microspores of maize genotype A18 to establish a responsive microspore culture of maize. Morphological events occurring during the first days of maize androgenesis in a microspore culture were observed and described, and some morphological markers for distinguishing between embryogenic microspores and nonembryogenic microspores were identified. It was found that the enlargement of microspores during the first days in culture and the ‘star-like’ organization of the cytoplasm inside the microspore are connected with reprogramming of the developmental pathway in maize microspores. Some differences were also found in the surface wall architecture of embryogenic microspores. Fertile plants were successfully recovered from microspore-originated structures.     

RFLP analysis to identify putative chromosomal regions involved in the anther culture response and callus formation of maize

Summary RFLP analysis was performed with anther culture-derived callus lines developed from the maize F₁ hybrids Pa91 x FR16 (PF), H99 x Pa91 (HP) and H99 x FR16 (HF). Relatively evenly spaced RFLP markers were selected to cover the maize genome with 52, 58 and 35 RFLP markers for the PF, HP and HF callus lines, respectively. The results from populations PF and HP combined with limited information from HF showed that six chromosomal regions on chromosomes 1, 2 (two regions), 3, 6 and 8 appear to be associated with the formation of embryo-like structures (ELSs) from microspores or the subsequent formation of regenerable callus from the ELSs. Regions at the end of the long arm of chromosome 2 and on the long arm of chromosome 8 appear to be associated with ELS formation, and the other regions appear to be associated with either ELS or regenerable callus formation or both. Certain regions that we have identified are the same as those found in other studies to be important for friable, embryogenic callus formation (chromosomes 1 and 3 and near the centromere of 2) and for ESL formation (chromosomes 1 and 3). This study has provided evidence for the genetic basis of the maize anther culture response and callus formation.     

Androgenesis is a maternal trait in the invasive ant Wasmannia auropunctata

Androgenesis is the production of an offspring containing exclusively the nuclear genome of the fathering male via the maternal eggs. This unusual mating system is generally considered a male trait, giving to androgenetic males a substantial fitness advantage over their sexually reproducing relatives. We here provide the first empirical study of the evolutionary outcomes of androgenesis in a haplo-diploid organism: the invasive ant Wasmannia auropunctata. Some of the populations of this species have a classical haplo-diploid sexual mating system. In other populations, females and males are produced through parthenogenesis and androgenesis, respectively, whereas workers are produced sexually. We conducted laboratory reciprocal-cross experiments with reproductive individuals from both types of populations and analysed their progenies with genetic markers, to determine the respective contribution of males and females to the production of androgenetic males. We found that androgenesis was a parthenogenetic female trait. A population genetic study conducted in natura confirmed the parthenogenetic female origin of androgenesis, with the identification of introgression events of sexual male genotypes into androgenetic/parthenogenetic lineages. We argue that by producing males via androgenesis, parthenogenetic queen lineages may increase and/or maintain their adaptive potential, while maintaining the integrity of their own genome, by occasionally acquiring new male genetic material and avoiding inbreeding depression within the sexually produced worker cast.     

Genetic selection and liquid medium conditions improve the yield of androgenetic plants from diploid potatoes

Summary Solatium tuberosum L. diploid strains with superior androgenetic capacity have been selected for from androgenetic progenies of unselected diploid material. The paper also demonstrates that the use of a liquid medium for culturing potato anthers, instead of the conventional solid agar plates, improves the yield of androgenetic embryoids. The new method, associated with two successive cycles of selection for superior androgenetic response, allows the induction and regeneration of microspore derived plants on a large scale. The best genotype (clone 21 in this paper) regenerates androgenetic plants with a frequency around 30 per each anther plated. Over 80% of the regenerated plants are diploid. It is suggested that the androgenetic embryoids mainly originate from unreduced microspores by a mechanism which maintains a heterozygous or a partly heterozygous genetic situation.     

In vitro induction of androgenic plantlets inAesculus hippocastanum

Summary Horsechestnut anthers were isolated from flower buds in various stages of development and cultivated in the presence of auxin and kinetin. The anthers showed the capacity for androgenesis and evidence is presented that the plantlets developed from single microspores. Haploid karyotypes were observed in the root menstem cells in three plantlets.     

The Effect of Rye Chromosomes on the Characteristic Features of Androgenesis in Wheat–Rye Substitution Lines Triticum aestivumL. (cv. Saratovskaya 29)/Secale cerealeL. (cv.Onokhoiskaya) and Triticale

The characteristic features of androgenesis in six wheat–rye substitution lines Triticum aestivumL. (cv. Saratovskaya 29)/Secale cerealeL. (cv. Onokhoiskaya) and triticale (2n= 56) using anther culture at different concentrations of 2,4-D in the growth medium were studied. Under variable cultivation conditions, the significant effect of genotypic diversity on the variability of such androgenesis parameters as the frequency of productive anthers, the frequency of embryoid formation, and the frequency of total regenerated plantlets, was shown. It was demonstrated that chromosomes 1R, 3R, and 7R stimulated the formation of androgenous embryoids, while chromosome 5R produced an opposite effect. In triticale and substitution lines, the regeneration ability of androgenous embryoids induced by elevated 2.4-D concentrations was inhibited. Chromosome 1R of the Onokhoiskaya cultivar was suggested to contain genes suppressing regeneration of green plantlets, while chromosome 3R, conversely, stimulated their formation. Chromosomes 1R, 2R, 3R, and 7R of the Onokhoiskaya cultivar did not inhibit the spontaneous formation of androgenous hexaploids in the substitution lines.     

Embryogenesis in suspension cultures of Datura innoxia Mill.

In vitro plant regeneration via embryogenesis was obtained in suspension cultures of Datura innoxia Mill. Embryogenesis was induced in suspension cultures raised from callus of androgenetic origin, using LS liquid medium supplemented with 0.22 mg/l 2,4-D. The total number of embryos formed was variable over time in culture. Embryos differentiated and matured in the liquid medium itself as also evidenced by histological observation. Embryos germinated to form plantlets on semisolid MS medium without growth substances. The regenerated plants had haploid number of chromosomes.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn Kinetin - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962)     

Dispermic androgenesis in sturgeons with the use of cryopreserved sperm: Production of androgenetic siberian sturgeon and androgenetic hybrids between Siberian and Russian sturgeons

Dispermic androgenesis was used to produce, for the first time, an androgenetic progeny of the Siberian sturgeon (Acipenser baerii) and the androgenetic nucleo-cytoplasmic hybrids (Siberian sturgeon, A. baerii × Russian sturgeon, A. gueldenstaedtii) using cryopreserved sperm. Microsatellite DNA analysis confirmed exclusively paternal inheritance in the androgenetic progeny of Siberian sturgeon. Heterozygotes for certain microsatellite loci were detected among the androgenetic hybrids, thereby confirming a dispermic nature of androgenesis. According to the data of comparative morphological analysis, the obtained androgenetic hybrid, by the age of 15 months old, was completely identical to the paternal species. Both a female and a male were detected in the androgenetic sturgeon progenies, which is of interest for producing bisexual progenies via androgenesis. The data of this study confirm the feasibility of dispermic androgenesis using cryopreserved sperm to preserve and recover the gene pools of endangered sturgeon species.     

<Emphasis Type="Italic">Secale cereale</Emphasis> inter-microsatellites (SCIMs): chromosomal location and genetic inheritance

The polymerase chain reaction (PCR) was used to locate Secale cereale (inter-simple sequence repeat ISSR) or Secale cereale inter-microsatellite (SCIM) markers using wheat–rye addition lines in order to develop a set of molecular markers distributed on the seven rye chromosomes. The number of SCIM markers located on 1R, 2R, 3R, 4R, 5R, 6R and 7R chromosomes were 4, 3, 12, 3, 2, 9 and 8, respectively. Therefore, a total of 41 new SCIMs were located on the seven rye chromosomes. The segregation of the 63 different SCIM markers in three different F₂ was studied. The observed ISSR segregations were the 3:1 (50.7%), the 15:1 (12.7%) and the 1:1 (14.2%). The linkage analysis carried out indicated that seven of the segregating SCIMs were linked to chromosome 7R and two were linked to chromosome 4R. The use of the SCIM markers as a source of molecular markers that could be linked to interesting genes or other important agronomic traits is discussed.     

Haploid plants regenerated via anther culture in wild barley (Hordeum spontaneum C. Kock)

Summary Androgenic plants have been obtained via anther culture in four natural populations of Hordeum spontaneum. Microscopic observations revealed that androgenesis started with the formation of two vegetative-type nuclei derived from the mitotic division of the uninucleate microspores. In this species androgenesis was affected by the type and concentration of the sugars added to the culture medium: the highest response (17% of callusing anthers) was observed on media containing 80 g l^–1 maltose. The highest production of androgenic plants (per 100 anthers, 5.9 green and 4.3 albino plants) was obtained from callus grown on these same media. About half of the green plants regenerated were haploid, while the others were diploid and set seed.Abbreviations IAA indolacetic acid - BAP 6-benzylaminopurine     

Characterization of chromosome instability in interspecific somatic hybrids obtained by X-ray fusion between potato (Solanum tuberosum L.) and S. brevidens Phil

Summary Asymmetric somatic hybrids between Solanum tuberosum L. and S. brevidens Phil. have been obtained via the fusion of protoplasts from potato leaves and from cell suspension culture of S. brevidens. The wild Solanum species served as donor after irradiation of its protoplasts with a lethal X-ray dose (200 Gy). Selection of the putative hybrids was based on the kanamycin-resistance marker gene previously introduced into the genome of Solanum brevidens by Agrobacterium-mediated gene transfer. Thirteen out of the 45 selected clones exhibited reduced morphogenic potential. The morphological abnormalities of the regenerated plantlets were gradually eliminated during the extended in vitro culture period. Cytological investigations revealed that the number of chromosomes in the cultured S. brevidens cells used as protoplast source ranged between 28–40 instead of the basic 2n=24 value. There was a high degree of aneuploidy in all of the investigated hybrid clones, and at least 12 extra chromosomes were observed in addition to the potato chromosomes (2n=48). Interand intraclonal variation and segregation during vegetative propagation indicated the genetic instability of the hybrids, which can be ascribed to the pre-existing and X-ray irradiation-induced chromosomal abnormalities in the donor S. brevidens cells. The detection of centromeric chromosome fragments and long, poly-constrictional chromosomes in cytological preparations as well as non-parental bands in Southern hybridizations with restriction fragment length polymorphism (RFLP) markers revealed extensive chromosome rearrangements in most of the regenerated clones. On the basis of the limited number of RFLP probes used, preferential loss of S. brevidens specific markers with a non-random elimination pattern could be detected in hybrid regenerants.     

Spontaneous versus colchicine-induced chromosome doubling in maize anther culture

The range of genetic variation of spontaneous chromosome doubling frequency of maize haploid plantlets derived from in vitro anther culture was evaluated. When regeneration is obtained by direct embryo-genesis, bypassing the callus phase, it appears that the frequency of spontaneous doubling may exceed 40 of the regenerated plantlets. This high frequency may be one consequence of the use of doubled haploid lines derived from anther culture and spontaneous chromosome doubling. We also report an increase, by more than 50, of the productivity of diploid fertile regenerated plantlets produced by colchicine supplemented medium during the cold shock pretreatment of the microspores inside the anthers. Optimization of the treatments and the anther culture procedure are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Observation of Androgenesis in Cultured Wheat Anthers at Meiosis,Tetrad, Early Uninucleate and Trinucleate Stage

The obtaining of calluses and plantlets from cultured wheat anthersat the stages from pollen mother cell to trinucleate microspore has been reported previously. Haploids as well as diploids existed among the regenerated plantlets derivedfrom anthers at these stages. Present paper reports the study on androgenesis patter-ns of cultured anthers at meiosis, tetrad, early mid- and late uninucleate and trinucleate stage. Cytological evidence of pollen-origin of calluses produced by anthers atthese stages was given. Observation showed that meiosis of wheat anthers was able tocomplete under culture conditions, resulting in releasing microspores, from which multinucleate and multicellular pollen grains formed. In meiosis anthers, abnormal cells,including syncytium and two kinds of binueleate calls were sometimes observed. Theymight be products of abnormal meiosis and abnormal development of tapetum cells. Itwas noted that failure and/or uncomplction of forming callus wall and/or pollen wallin in vitro anthers at meiosis, tetrad and early uninucleate stage occured often. Itmight lead to the low frequency of callus induction. Mature wheat anthers (trinucleate stage) contained both normal and abnormal pollen grains (pollen dimorphism); onlythe abnormal pollen grains developed into embryoids while all the normai trinucleatepollen grains degenerated rapidly. However, the date of the frequency of equal divisionof microspores suggested that abnormal pollen (N pollen, small pollen) could not be theonly source of androgenic pollens in cultured anthers at late uninucleate and other earlier stages.