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Heredity 86 (2001) 167±176 Received 25 February 2000, accepted 17 October 2000 Androgenesis from Festuca pratensis ´ Lolium multi¯orum amphidiploid cultivars in order to select and stabilize rare gene combinations for grass breeding A. LESÂNIEWSKA , A. PONITKA , A. SÂLUSARKIEWICZ-JARZINA , E. ZWIERZYKOWSKA , Z. ZWIERZYKOWSKI , A. R. JAMESà, H. THOMASà & M. W. HUMPHREYS*à Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska 34, PL-60±479 Poznan, Poland and àInstitute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, U.K. Androgenesis using amphidiploid cultivars of Festuca pratensis ´ Lolium multi¯orum as parents, overcame earlier problems that gave rise to widespread plant sterility amongst androgenic Festulolium populations. Two Festuca pratensis ´ Lolium multi¯orum (2n 4x 28) cultivars, Sulino and Felopa, were highly amenable to androgenesis and 10% of plants, including some novel androgenic genotypes, had sucient fertility to produce progeny and further generations. The genomes of amphidiploid cultivars, which represent the F8 generation, were the result of considerable intergeneric chromosome recombination. Moreover, during cultivar development, natural and breeders' selection pressures had led to the assembly of gene combinations that conferred good growth characters and fertility with the removal of putative deleterious gene combinations. Over 80% of the androgenic plants derived from the amphidiploid F. pratensis ´ L. multi¯orum (2n 4x 28) had 14 chromosomes and were likely to be dihaploids with a single genome of Lolium and of Festuca. In contrast, hybrids of F. pratensis ´ L. multi¯orum (2n 2x 14) found naturally are invariably sterile. Structural reorganization within the genomes of the androgenic Festulolium plants had restored fertility in genotypes expected to contain the haploid genome of Lolium and Festuca. This provided opportunities for their future incorporation in breeding programmes and the development of fertile diploid Lolium±Festuca hybrids. Amongst the androgenic plants, Festulolium genotypes were recovered that conferred excellent drought resistance or freezing tolerance and were thought to be highly suitable for entry into plant breeding programmes. Keywords: amphidiploids, androgenesis, fertile genotypes, Festuca pratensis ´ Lolium multi¯orum hybrids, GISH, stress resistance. In recent years eective procedures have been established to generate large androgenic populations and diverse genotypes from single Lolium ´ Festuca hybrid plants (Humphreys et al., 1998a; Zwierzykowski et al., 1999). Lolium and Festuca species share valuable and complementary agronomic characters, hybridize naturally, and as hybrids regularly exchange genes at high frequency (Canter et al., 1999). Androgenesis was found to be an eective procedure for selecting Lolium±Festuca genotypes comprising gene combinations rarely or never recovered by conventional backcross breeding programmes (Humphreys et al., 1998a). These included androgenic plants with stress tolerance in excess of their parental species and also of their individual Introduction Androgenesis is a well-established component of plant breeding methodology for several crop species, and can make a signi®cant contribution to cultivar development. The principal advantage of androgenesis is that it rapidly achieves population uniformity and homozygous lines, and therefore circumnavigates the need for years of intercrossing by conventional breeding technology to achieve a similar outcome (Clapham, 1971). *Correspondence. E-mail: [email protected] Ó 2001 The Genetics Society of Great Britain. 167 168 A. LESÂNIEWSKA ET AL. L. multi¯orum ´ F. arundinacea parent genotype (Zare et al., 1999). Unfortunately, these potentially agriculturally useful androgenic genotypes proved to be sterile, and fertility was not restored following chromosome doubling, despite expectations that homologous chromosome pairing would ensue and lead to improved chromosome disjunction. The sterility of the androgenic plants was explained, at least in part, by detailed cytological investigations (Humphreys et al., 1998a; Zwierzykowski et al., 1998a). The androgenic plants carried intact and recombinant chromosomes of both species and also of L. multi¯orum in dierent numbers with the consequence that their genomes were highly unbalanced. This paper reports the use of an alternative strategy to incorporate the advantages accrued from androgenesis from Festuca ´ Lolium hybrids that will provide rare or unique genotypes suitable for plant breeding. Materials and methods Procedures for androgenesis All androgenesis was carried out at the Institute of Plant Genetics, Poznan, Poland. Anthers were cultured from 24 glasshouse-maintained genotypes of amphiploid Polish Festulolium cultivars Sulino (11 plants) and Felopa (13 plants) which were F8 hybrids derived from F. pratensis (Fp) ($ parent) ´ L. multi¯orum (Lm) (# parent) (2n 4x 28) (Ponitka et al., 1998; Zwierzykowski et al., 1998b). General procedures for androgenesis were as described by Zwierzykowski et al. (1999). In brief, in¯orescences with microspores at mid to late uninucleate stage were collected and then maintained prior to culture in a solution containing mineral salts derived from N6 medium (Chu et al., 1975) at 4°C for 3±6 days. Two dierent media were used for embryo induction: C17 (Wang & Chen, 1983), and PII (Chuang et al., 1978) where 2,4-D was increased to 2 mg/L. Both media contained 90 g/L maltose, pH 6.0, and were solidi®ed with 6 g/L agarose (SERVA high EEO). Two regeneration media were used: MS (Murashige & Skoog, 1962) or 190-2 medium (Zhuang & Jia, 1983). Once green androgenic plants (labelled here FpLm) had been established, their chromosome number was determined using standard procedures of ®xation and chromosome staining for root-tip mitosis (Zwierzykowski & RybczynÂski, 1998). Fertility of the FpLm plants following vernalization was determined by scoring numbers of plants with dehiscing anthers, and by scoring under a light microscope percentage pollen stainability in a 1:1 mixture of acetocarmine and glycerol. Procedures for determining the stress resistance of androgenic plants A representative sample of 52 FpLm plants with 14 chromosomes derived from cultivars Sulino and Felopa were transferred to the Institute of Grassland and Environmental Research, Aberystwyth (IGER) for assessment of their stress resistance under simulated freezing and drought stress. Information in Table 1 describes the origin of the androgenic plants, dierentiates those Table 1 Ancestry of androgenic plants derived from Festuca pratensis ´ Lolium multi¯orum (2n 4x 28) amphidiploid cultivars Sulino and Felopa evaluated for freezing tolerance, drought recovery, or both Cultivars used for androgenesis Cultivar Number of androgenic FpLm plants evaluated Genotype code no. Population code no. Plant code nos Freezing treatment Drought treatment Freezing + drought treatment Sulino 1 2 3 4 5 6 FpLm FpLm FpLm FpLm FpLm FpLm 1/ 2/ 3/ 4/ 5/ 6/ 1±6 1±3 1±12 1±3 1±9 1±2 0 0 0 0 1 0 4 2 8 3 5 0 2 1 4 0 3 2 Felopa 7 8 9 10 FpLm FpLm FpLm FpLm 7/ 8/ 9/ 10/ 1±3 1 1±11 1±2 0 0 0 0 1 1 8 0 2 0 3 2 Total 10 10 52 1 32 19 Ó The Genetics Society of Great Britain, Heredity, 86, 167±176. ANDROGENESIS FROM F. PRATENSIS ´ L. MULTIFLORUM derived from dierent parent genotypes, and indicates which androgenic plants were tested for assessments of their freezing tolerance and which for their drought resistance. Freezing tolerance A sample of 20 FpLm androgenic plants, each with similar size and vigour, were selected for an assessment of freezing tolerance. Plants were maintained in potting compost in identical growth conditions prior to their transfer to a CE room to cold-acclimate at 2°C and 8 h light for 3 weeks. Groups of ®ve tillers of similar developmental stage were detached from each genotype and were subjected to freezing temperatures of )6°C, )9°C, )12°C, and )15°C (total of 20 tillers/genotype) in tubes suspended in a freezing-tank containing 50% (v/v) polyethylene glycol (as described by Zare et al., 1999). Following exposure to freezing, tillers were transferred to potting compost and given 3 weeks recovery at 15°C and 8 h light (Fuller & Eagles, 1978) and scored for survival. Drought resistance FpLm plants were subjected to a prolonged period of drought to assess dierences in drought resistance. The drought treatment followed well established procedures used at IGER (Thomas & Evans, 1989). The androgenic plants described in Table 1 (including all the genotypes used for the freezing treatment, with the exception of FpLm 5/5), were split into four ramets and allowed to establish, prior to the drought treatment, for 8 weeks in 1 m ´ 1 m ´ 1 m polythene-lined brick bins ®lled at equal depth by a bottom layer of stones, a middle layer of grit, and a top layer of soil-based potting compost. The soil composition was designed to ensure rapid drainage, and following the commencement of the drought experiment, an early soil water de®cit ensured an early onset of drought stress on the plants. Two barrier rows comprising a mixture of L. multi¯orum cv. Tribune and F. pratensis cv. Fure plants were planted in the brick bins to surround the androgenic plants. As controls, four randomly selected genotypes of L. multi¯orum ´ F. pratensis (4x) cultivars Elmet (2), Felopa (1), and Punia (1) were included in the drought experiment. Genotypes were spaced evenly in rows, 7 cm apart. The experimental design was four replicate blocks, each a complete set of genotypes planted and fully randomised and subsequently given identical treatments throughout the duration of the experiment. Irrigation was withdrawn over a 54-day period from 28th May until the 21st July 1999. Plants were cut twice during the drought period to prevent over-shading of smaller plants. Droughting was ended and daily irrigation recommenced in all four bins once 90% of the androgenic plants and their controls had no visible living green Ó The Genetics Society of Great Britain, Heredity, 86, 167±176. 169 tissue. All bins were then rewatered and recovery evaluated after 3 weeks. Plants were compared on the basis of mean fresh weight gain (between the four replicates) during the recovery period. Procedures for cytological analysis using genomic in situ hybridization (GISH) Four FpLm androgenic plants with 14 chromosomes and good freezing tolerance and/or drought resistance were selected at IGER for detailed cytological investigation using GISH. Procedures for GISH carried out were as described by PasÏ akinskien_e et al. (1998). Total genomic DNA of F. pratensis was used as probe and total genomic DNA of L. multi¯orum was used as blocking DNA (at a ratio of 1:40). Probe DNA was labelled directly with rhodamine-4-dUTP (Amersham± Pharmacia) and chromosomes were counterstained with 4¢,6-diamidino-2-phenylindole (DAPI). Rhodamine and DAPI ¯uorescent chromosome images were captured with a monochrome CCD camera and merged as described by Thomas et al. (1996). Procedures for generating new plant populations from selected androgenic plants Four androgenic plants with good freezing tolerance and with some dehiscing anthers were interpollinated. These were also used as female parents in pair-crosses with L. multi¯orum cv. Atalja (2x). Results Androgenesis response Overall, 18083 anthers were cultured from 24 amphiploid F. pratensis ´ L. multi¯orum (2n 4x 28) initial plants (see Table 2). Both Polish cultivars Sulino and Felopa were very amenable to androgenesis but embryogenesis was more frequent from anthers of cultivar Felopa on both C17 and PII embryo-induction media. The frequency of embryogenesis from anthers of Felopa (13 parental genotypes) was similar on both induction media. Embryogenesis from plated anthers of the cultivar Sulino (11 parental genotypes) was lower than from Felopa and there was a dierent response on the two embryo induction media with more embryogenesis on C17 rather than PII medium. Despite the cultivar Felopa's high frequency of embryogenesis in culture compared with Sulino, more green plants were eventually recovered from Sulino. Green plants were obtained from nine genotypes of Sulino and 10 genotypes of Felopa. The total number of green androgenic plants was 1130 with 942 derived from 170 A. LESÂNIEWSKA ET AL. Mean (range) Anther culture Sulino No. of genotypes used for anther culture No. of anthers cultured 11 Embryos/100 anthers Medium C17 Medium PII 7973 24.8 (1±72) 14.3 (1±39) Total both cultivars Felopa 13 24 10 110 18 083 63.4 (1±253) 52.7 (1±236) 47.5 34.5 Green plants from androgenesis No. of successful genotypes Plants/100 anthers Plants/100 embryos Total no. of plants 9 11.8 60.8 942 10 1.9 3.2 188 19 6.2 15.1 1130 Albino plants from androgenesis No. of successful genotypes Plants/100 anthers Plants/100 embryos Total no. of plants 9 8.6 44.3 687 11 5.9 10.0 593 20 7.1 17.1 1280 Sulino and 188 from Felopa. The overall frequency of albino plants (6% of anthers cultured) was as high as the number of green plants (7% of anthers cultured). The actual number of albinos derived from cultivars Sulino and Felopa was very similar but their frequency, in comparison with numbers of green plants, was higher from anthers of Felopa. The androgenic plants were very diverse in phenotype and growth rate. Variation was observed in vigour, ¯owering date, height, length and width of leaves, and type and length of in¯orescences. A random sample of 644 plants was analysed cytologically. A total of 532 (83%) androgenic plants had 14 chromosomes, 75 (12%) had 28 chromosomes, and 34 (5%) had 15 or 16 chromosomes. Three mixoploid 14/28 chromosome plants were also recorded. The chromosome constitution of four of the 14-chromosome plants selected for good stress tolerance (see below) is illustrated in Fig. 1. GISH distinguished Festuca and Lolium DNA and revealed large numbers of intergeneric recombinants in all four genotypes. Two single and two double recombination events that gave rise to four Lolium chromosome segments on a single Festuca chromosome are indicated in genotype FpLm 9/4 at metaphase (Fig. 1a) and anaphase (Fig. 1b). Within the four genotypes examined, only a very few chromosomes were complete with no visible alien chromosome segment. Whilst no attempt was made to make a comprehensive investigation of the numbers of Lolium and Festuca chromosomes present in the androgenic population, amongst the four plants investigated they were found in equal numbers (7 + 7). Table 2 Response from anther culture of Festuca pratensis ´ Lolium multi¯orum amphidiploid (2n 4x 28) cultivars Sulino and Felopa Freezing experiment The temperature range chosen was adequate to evaluate cold tolerance amongst the FpLm populations since the mean survival rate of all genotypes tested ranged from 98% at )6°C to 12% at )15°C (Table 3). The overall mean LT50 (lethal temperature to kill 50% of living plant tissue) for the androgenic populations was close to )12°C. At opposite extremes, genotype FpLm 1/4 showed 100% recovery following all four temperature treatments and could be considered to have a freezing tolerance in excess of )15°C. This contrasted with genotype FpLm 9/5 where a single tiller recovered following the )9°C treatment and no tillers recovered following exposure to any lower temperature. Dierences between genotypes for freezing tolerance was most noticeable at the most extreme temperature ()15°C), where only plants FpLm 1/4, FpLm 6/1, and FpLm 1/1 from Sulino (in decreasing order of freezing tolerance) and FpLm 9/2 and 9/4 from Felopa, demonstrated any capacity for survival and recovery. There was little consistency for freezing tolerance amongst dierent androgenic plants derived from the same parent plant and cultivar. However, over all androgenic plants, there was a marginally greater freezing tolerance amongst genotypes derived from Sulino. Indeed two of the three most freezing-tolerant genotypes were derived from Parent No. 1 of cultivar Sulino. The three mostfreezing-tolerant genotypes were examined cytologically by GISH (Fig. 1). Ó The Genetics Society of Great Britain, Heredity, 86, 167±176. ANDROGENESIS FROM F. PRATENSIS ´ L. MULTIFLORUM 171 Fig. 1 Genomic in situ hybridization of root tip chromosomes of androgenic plants derived from amphidiploid Festuca pratensis ´ Lolium multi¯orum (2n 4x 28) cultivars Felopa (cells a and b) and Sulino (cells c, d and e). In all cells, Festuca DNA is rhodamine 4-dUTP-labelled (pink), and Lolium DNA is DAPI-labelled (blue). All genotypes were 14-chromosome dihaploids (n + n) comprising approximately equal amounts of Lolium and Festuca DNA. Androgenic plants: FpLm9/4 (a) metaphase chromosomes, (b) anaphase chromosomes (a Festuca chromosome with 2 terminal and 2 intercalary Lolium recombinant chromosome segments is arrowed), (c) FpLm6/1, (d) FpLm1/1 and (e) FpLm 1/4. Bar, 10 lm. Fertility of androgenic plants Over 95% of the green androgenic plants (1076 plants) produced in¯orescences during the ®rst growing season. Of the plants that ¯owered, 10% (107 plants) produced dehiscing anthers with pollen stainability ranging from 8.5% to 87.6%. Male fertility was equally common in both 14- and 28-chromosome plants. Amongst the androgenic plants tested for freezing tolerance, only genotypes FpLm 7/2 and FpLm 9/2 (both with freezing tolerance >)12°C) produced dehiscing anthers and fertile pollen. However, either due to their incompatibility or low fertility, both failed to produce seed when intercrossed. However, 48 develÓ The Genetics Society of Great Britain, Heredity, 86, 167±176. oping embryos were recovered from 10 spikes of FpLm 9/2 used as female in a cross with the diploid L. multi¯orum cv. Atalja (2x). From these, seven vigorous green plants with L. multi¯orum-like phenotype were recovered following embryo rescue and were established successfully in soil. Drought experiment The soil/compost mixture used for the drought experiment dried rapidly with the consequence that all plants demonstrated an eect of the drought stress early into the drought treatment. Following the eight-week drought period, all L. multi¯orum plants in the border 172 A. LESÂNIEWSKA ET AL. Number of tillers out of 5 per temperature treatment that survived freezing Plant No. )9°C )12°C )15°C Total % cv. Sulino FpLm 1/4 5 FpLm 6/1 5 FpLm 1/1 5 FpLm 3/3 5 FpLm 5/8 5 FpLm 5/5 5 FpLm 2/3 5 FpLm 5/2 5 FpLm 3/11 5 FpLm 5/1 5 FpLm 6/2 5 FpLm 3/12 5 FpLm 3/9 5 Mean 12.46±62% 5 5 5 5 5 5 5 5 4 4 4 3 2 5 5 5 4 4 2 1 1 1 1 1 0 0 5 3 2 0 0 0 0 0 0 0 0 0 0 20 18 17 14 14 12 11 11 10 10 10 8 7 100 90 85 70 70 60 55 55 50 50 50 40 35 cv. Felopa FpLm 7/1 5 FpLm 9/2 5 FpLm 10/2 5 FpLm 7/2 4 FpLm 9/4 5 FpLm 10/1 5 FpLm 9/5 4 Mean 11.14±56% 5 5 5 5 4 1 1 4 3 4 4 1 1 0 0 1 0 0 1 0 0 14 14 14 13 11 7 5 70 70 70 65 55 35 25 83% 47% 12% % Overall recovery )6°C 98% rows had died and all androgenic genotypes either died or become quiescent. Amongst the FpLm populations, there was no indication of any living green tissue in around 90% of plants. However, despite the apparent heavy mortality due to drought, tiller buds in the majority of androgenic plants survived and recovery following drought was very high (93% of androgenic plants). There was generally good consistency between the four replicates of each genotype for survival and recovery after drought. The growth during recovery diered greatly between genotypes. Overall, the fresh weight mean for growth during recovery by the androgenic population was 3 g but 16% of androgenic plants produced signi®cantly more regrowth (P > 0.05), with the highest recorded in FpLm 2/3 (15.4 g). This androgenic plant was derived from genotype no. 2 of the cultivar Sulino. However, there was no relationship between regrowth during recovery and the parental genotype. Plants with good or poor recovery, were found in the androgenic populations (Table 1) derived from both cultivars Sulino and Felopa. The four representative genotypes of L. multi¯orum ´ F. pratensis Table 3 Numbers of surviving tillers of androgenic plants derived from Festuca pratensis ´ Lolium multi¯orum (2n 4x 28) amphidiploid cultivars Sulino and Felopa following freezing treatment 60% (4x) amphiploid cultivars Elmet (2 plants), Felopa (1 plant) and Punia (1 plant) used as controls, all produced inferior regrowth compared with the mean of the androgenic population. FpLm 1/4, FpLm 6/1 and FpLm 1/1, which were also the most freezing-tolerant genotypes, all produced more regrowth following drought than the overall mean, but only FpLm 6/1 produced signi®cantly more live weight gain (mean of 5.13 g, P > 0.05). Overall, there was absolutely no correlation between freezing tolerance and regrowth following drought. FpLm 6/1 was included in the GISH analysis described above (Fig. 1). Discussion Chromosome behaviour in Lolium ´ Festuca hybrids and its relevance in androgenesis An earlier report cited that androgenic plants (LmFa) derived from a L. multi¯orum ´ F. arundinacea (2n 5x 35) hybrid carried rare gene combinations conveying both drought and freezing tolerance in excess Ó The Genetics Society of Great Britain, Heredity, 86, 167±176. ANDROGENESIS FROM F. PRATENSIS ´ L. MULTIFLORUM of that of their parental genotypes (Humphreys et al., 1998a). The logical conclusion was that factors preventing the optimal expression of genes determining stress resistance in the parental hybrid, were removed as a consequence of androgenesis. Unfortunately, the LmFa plants could not be used for further cultivar development due to high levels of male and female sterility. This remained even after chromosome doubling, which was intended to aid more regular chromosome pairing and development of better balanced and viable gametes (unpublished results). Hexaploid F. arundinacea comprises one genome of F. pratensis and two of F. glaucescens (Humphreys et al., 1995). With the aid of genomic in situ hybridization (GISH), it is possible to distinguish chromosomes of the two Festuca species, and this enabled Humphreys et al. (1998a) to demonstrate the unbalanced composition of the Festuca genomes within the LmFa androgenic population. This may have resulted in gene duplications and/or deletions and explains, at least in part, the high sterility found amongst the androgenic genotypes. Similar cytological and fertility problems were encountered by Zwierzykowski et al. (1998a) in androgenic populations derived from the reciprocal F. arundinacea ($) ´ L. multi¯orum (FaLm) hybrid. These authors reported chromosome variation among androgenic progeny to be much wider than that transmitted by conventional backcrossing. However, the low plant vigour and inherent sterility of genotypes of the FaLm population would indicate that this additional variation would be dicult to exploit in breeding. The genomes of L. multi¯orum and F. pratensis share close homology (PasÏ akinskien_e et al., 1998), and deletions of gene sequences of Lolium would be expected to be compensated for by the presence of homoeologous Festuca sequences or vice versa. Thus, less deleterious gene combinations might be expected in gametes and androgenic plants derived from hybrids between L. multi¯orum and F. pratensis than in those derived from the L. multi¯orum ´ F. arundinacea hybrid described above. The current work describes androgenic populations (FpLm) derived from F. pratensis ´ L. multi¯orum amphiploid cultivars (2n 4x 28). Over the years, the close homology between Lolium species and F. pratensis has been a problem for breeding stable amphiploid cultivars (Thomas & Humphreys, 1991). Strict preferential chromosome pairing between homologous sets of Lolium and Festuca chromosomes has been an unrealized ambition with high levels of intergeneric recombination leading to sterility or hybrid breakdown. For example, Canter et al. (1999) using GISH, reported that the L. perenne ´ F. pratensis amphiploid cultivar Prior, by the eighth generation (the same number of generaÓ The Genetics Society of Great Britain, Heredity, 86, 167±176. 173 tions used to develop the cultivars Sulino and Felopa used here), comprised predominantly L. perenne chromosomes. As a consequence of this and with the development of molecular markers, most eorts in recent years to combine Lolium and Festuca traits have concentrated on the introgression breeding approach using markers to target speci®c traits and to aid their recovery in backcross breeding programmes (e.g. Humphreys et al., 1998b). Zwierzykowski et al. (1993, 1998b) have developed Festulolium amphiploid cultivars that appear more stable than their earlier counterparts. Despite high levels of intergeneric recombination, the Polish amphidiploid F. pratensis ´ L. multi¯orum (2n 4x 28) cultivars Sulino and Felopa retain approximately equal numbers of Lolium and Festuca chromosomes after eight generations of development. In situ hybridization studies revealed that despite clear structural DNA dierences between Lolium and Festuca chromosomes that enabled their chromosomes to be distinguished, the chromosomes of the cultivars Sulino and Felopa were genetically equivalent. The outcome is almost perfect mixing of the genomes with no obvious deleterious eects with regard to hybrid fertility and stability. This combination of high levels of intergeneric recombination and balanced gametes, is an ideal starting point for androgenesis, where novel gene combinations and viable culture-responsive pollen microspores are desired. The earlier study by Zwierzykowski et al. (1998b) clearly demonstrated that between genotypes there was no regular pattern for recombination between Lolium and Festuca chromosomes and no preferential ®xing of gene sequences. These Festulolium amphiploid cultivars therefore provide a perfect source of all gene combinations available within the two parental species. In the current study, both cultivars Felopa and especially Sulino proved to be very responsive to androgenesis, and it was possible to generate large numbers of green androgenic plants from dierent hybrid parental genotypes. Indeed, it may well be that F. pratensis ($) ´ L. multi¯orum or L. multi¯orum ($) ´ F. pratensis amphidiploid cultivars are particularly amenable to androgenesis, as success was readily achieved using alternative cultivars, namely the IGER cultivar Elmet, and the Lithuanian cultivar Punia, both of which yielded green plants (unpublished results). The use of androgenesis from Festuca ´ Lolium hybrids to select gene combinations governing stress resistance The two Polish cultivars are well adapted to a range of contrasting European growing conditions, performing well in maritime regions of Norway and the UK, as well 174 A. LESÂNIEWSKA ET AL. as the continental climate found in Poland (unpublished results). Thus the two cultivars would be expected to be an ideal source of genes for adaptations to diverse European growing conditions and a good starting point for any programme aimed at selecting gene combinations governing dierent components of stress resistance. Lolium multi¯orum is signi®cantly more sensitive to freezing than F. pratensis (Humphreys et al., 1997). Thus freezing tolerance among the FpLm genotypes would be expected to derive from F. pratensis gene combinations. In an earlier experiment, aimed at measuring freezing tolerance of androgenic genotypes derived from a L. multi¯orum ´ F. arundinacea hybrid, the majority of LmFa genotypes had inferior freezing tolerance (<)6°C) compared with L. multi¯orum (Zare et al., 1999). In the current study, the FpLm androgenic plants all survived )6°C and clearly retained the capability for cold-acclimation. For Lolium and Festuca this represents an exposure prior to freezing of low temperatures just above 0°C and short-day photoperiods (Harrison et al., 1997). Deleterious gene combinations which reduced the vigour and freezing tolerance of the LmFa androgenic plants derived from an F1 L. multi¯orum ´ F. arundinacea hybrid were clearly absent in the androgenic plants derived from the F8 generation of Sulino and Felopa. Aneuploidy was prevalent among the androgenic plants derived from the L. multi¯orum ´ F. arundinacea hybrid (Zare, 1996), and presumably contributed to the overall low vigour of the LmFa androgenic population. However aneuploidy was present in only 5% of the FpLm population, and indeed only 14-chromosome F. pratensis ´ L. multi¯orum plants were used in the drought and freezing experiment described here. If the small GISH study was representative of the whole androgenic FpLm population, then the androgenic plants used in the freezing and drought experiments were dihaploids and 1 each, like their parent genotype, comprised approximately equal amounts of Lolium and Festuca DNA. Over 80% of the androgenic plants had 14 chromosomes, and were likely to have resulted from regular disjunction of Lolium and Festuca chromosomes. The majority of the remainder of the androgenic plants had 28 chromosomes and were probably derived from either 14-chromosome genotypes that underwent spontaneous chromosome doubling during culture, or were the products of unreduced gametes. The combinations of Lolium and Festuca genes enhanced the stress tolerance of all the androgenic genotypes above that of diploid L. multi¯orum. Genotypes were recovered from the Polish cultivars with a freezing tolerance ranging from )9°C to below )15°C. The three most freezing-tolerant genotypes, siblings FpLm 1/1 and FpLm 1/4, and genotype FpLm 6/1 from another parent, were derived from the cultivar Sulino. There was considerable variation in growth rate during recovery following drought between genotypes of the androgenic populations. The drought resistance of representative genotypes of dierent Festulolium amphidiploid cultivars used as controls either did not dier signi®cantly or was inferior to the mean drought resistance of the androgenic population. All of the androgenic plants had superior drought resistance compared with the L. multi¯orum cultivar Tribune, used as a control. Twenty percent of androgenic plants were found with signi®cantly superior drought resistance compared with the population mean. The most drought-resistant genotype, FpLm 2/3, produced 300% more fresh-weight yield after recovery from drought compared with the population mean. The third most drought-resistant androgenic plant, FpLm 7/2 also demonstrated very good freezing tolerance and thus has the ability to coacclimate to extremes of both of the abiotic stress conditions. However, overall there was no correlation between genotypes for drought resistance and freezing tolerance. The second most drought-resistant androgenic plant, FpLm 9/4 (derived from the cultivar Felopa), was analysed cytologically using GISH, and like the three most freezing-tolerant genotypes, was dihaploid having maintained a 1:1 Lolium: Festuca genome composition. Although the numbers of androgenic genotypes assessed for stress resistance were quite small, there was little doubt that they were truly representative of the entire FpLm androgenic population and that most plants had high levels of stress resistance. The combinations of Lolium and Festuca genes in the dihaploid genomes complement each other well to maintain, or in many examples enhance, the stress resistance found in the parental amphiploid cultivar. The role of androgenesis in breeding Androgenesis is a very eective procedure for selecting gene combinations governing stress resistance in Lolium±Festuca hybrids. However, the procedure has no value to plant breeding unless it is later possible to produce fertile androgenic genotypes and new populations from selected stress-resistant genotypes. Fortunately, genotypes with both male and female fertility were found in the dihaploid androgenic F. pratensis ´ L. multi¯orum population, and it was possible to produce progeny following hybridization with L. multi¯orum. In the ®eld, diploid L. multi¯orum and F. pratensis hybridize naturally but all F1 hybrids are sterile. Fertility is only restored following chromosome doubling by generating amphiploids and restoring homologous Ó The Genetics Society of Great Britain, Heredity, 86, 167±176. ANDROGENESIS FROM F. PRATENSIS ´ L. MULTIFLORUM chromosome pairing and regular chromosome disjunction (Breese & Lewis, 1984). In contrast with the sterility found in naturally occurring diploid Festulolium hybrids, the dihaploid androgenic populations described here are fertile. Amphidiploid cultivars Sulino and Felopa represent the F8 generation and are the consequence of substantial intergeneric recombination and chromosome restructuring during their development. Ten percent of the green androgenic FpLm plants derived from Sulino and Felopa produce dehiscing anthers and have sucient fertility to generate new populations. The chromosome restructuring during cultivar development resulted in increased homology between homoeologous chromosome partners. The expected consequence, compared with androgenic plants derived from F1 or early generations of Festulolium hybrids, would be increased chromosome pairing between homoeologous Lolium and Festuca chromosomes. This in turn would give rise to better chromosome disjunction that lead to the development of viable gametes in FpLm genotypes. The production of fertile F. pratensis ´ L. multi¯orum dihaploid plants is an entirely new development in grass breeding and oers exciting future opportunities for the construction of new varieties combining Lolium and Festuca traits. One option is to ®x desirable gene combinations as homozygotes by chromosome doubling, thus generating new amphiploid cultivars. An alternative, which was found to be eective in the current study, was to backcross androgenic dihaploid plants onto Lolium (or Festuca). Genotypes with identical traits and closely associated molecular markers may subsequently be intercrossed to produce populations with isogenic sequences for targeted genes. The advantage of using androgenesis in grass breeding will be the potential for recovery of gene combinations governing important traits that are rarely recovered by conventional breeding and may include selection for complex suites of additive and recessive alleles. 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