Download Androgenesis from Festuca pratensis Ч Lolium multiЇorum

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 ˆ 4x ˆ 28) cultivars, Sulino and Felopa,
were highly amenable to androgenesis and 10% of plants, including some novel androgenic
genotypes, had sucient 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 ˆ 4x ˆ 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 ˆ 2x ˆ 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 e€ective 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 e€ective 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 di€erent 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 ˆ 4x ˆ 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 di€erent 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, di€erentiates those
Table 1 Ancestry of androgenic plants derived from Festuca pratensis ´ Lolium multi¯orum (2n ˆ 4x ˆ 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 di€erent 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 di€erences 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 ˆ 4x ˆ 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 di€erent 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 ˆ 4x ˆ 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. Di€erences 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 di€erent 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 ˆ 4x ˆ 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 e€ect 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 ˆ 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 ˆ 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
di€ered 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 ˆ 4x ˆ 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 ˆ 5x ˆ 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 dicult 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 ˆ 4x ˆ 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 e€orts 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 ˆ 4x ˆ 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 di€erences
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 e€ects 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 di€erent 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 di€erent 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 di€erent Festulolium
amphidiploid cultivars used as controls either did not
di€er 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 e€ective 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 sucient 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 o€ers 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 e€ective 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.
Acknowledgements
This work was supported in part by the Agriculture
Agency of the State Treasury in Warsaw, Poland.
Agnieszka Les niewska acknowledges the support of
the European Science Foundation during her six months
stay at IGER, Aberystwyth, U.K.
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