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Journal of Heredity 2010:101(1):119–123
doi:10.1093/jhered/esp071
Advance Access publication August 12, 2009
Ploidy-Mediated Reduced Segregation
Facilitates Fixation of Heterozygosity in
the Aromatic Grass, Cymbopogon
martinii (Roxb.)
UMESH C. LAVANIA, SARITA SRIVASTAVA,
AND
SESHU LAVANIA
From the Department of Genetics and Plant Breeding, Central Institute of Medicinal and Aromatic Plants, Lucknow
226 015, Uttar Pradesh, India (U. C. Lavania and Srivastava); and the Department of Botany, University of Lucknow,
Lucknow 226 007, Uttar Pradesh, India (S. Lavania).
Address correspondence to U. C. Lavania at the address above, or e-mail: [email protected].
In most medicinal and aromatic plants, the vegetative tissue
(e.g., roots, stems, leaves) is the source of the economic
product. These plants are inherently heterozygous (natural
allelic hybrids) and maintain their genetic makeup in nature
by obligate vegetative propagation. Under seed cultivation,
these plants incur population heterogeneity that reduces
biomass and hampers product quality. Therefore, fixation of
heterozygosity is vital for maintaining uniformity in quality of
the economic product and quantity of biomass under seed
cultivation. Although seed-grown clonal progenies identical
to the mother plant can be obtained in certain plants that
show an unusual breeding system called apomixis, such
a breeding system is rare in medicinal and aromatic plants of
economic value. Here we show an effective experimental
strategy based on a polyploid model that facilitates fixation of
heterozygosity in obligate asexual species owing to tetrasomic
inheritance and low segregation in C1 progenies from highfertility C0 autopolyploids. Using an obligate asexual species of
aromatic grass—Cymbopogon martinii, we demonstrated that
progenitor diploids with distal chiasma localization and low
chiasmate association in meiosis, when changed into tetraploids, entail high gametic/seed fertility reflected in high
bivalent pairing and balanced anaphase segregation. Their seed
progenies evince crop homogeneity owing to reduced
segregation, indicating fixation of heterozygosity present in
the source diploids. Because C. martinii could be maintained
through obligate vegetative propagation, here is a unique
opportunity to utilize the polyploid advantage through C1 seed
progenies for commercial cultivation, as well as maintenance of
original C0 stock for raising seeds without losing polyploid
heterosis normally threatened in subsequent segregating
progenies on account of aneuploidy and gametic instability.
Key words:
Cymbopogon, diploidized polyploid, fixation of
heterozygosity, hybrid vigor, tetrasomic inheritance
The apomictic breeding system, if realized to its full
potential, could be quite rewarding for fixation of
heterozygosity and heterosis breeding in crop plants. It
is all the more important in inherently heterozygous
obligate asexual economic plants where seed-based
cultivation is desirable for commercial cultivation. Although, apomixis is known to be naturally occurring in
some 400 wild species from approximately 40 plant
families, including various grasses (Ozias-Atkins and van
Dijk 2007), the apomictic breeding system is rare in
medicinal and aromatic plants of economic value/crop
plants. Findings of Ravi et al. (2008) on a recessive mutation
‘‘Dyad’’ that promotes equational division leading to 2n egg
cells is an important milestone having far-reaching implications for hybrid fixation in crop plants. However, prospects
of these findings have inherent limitations because few 2n
egg cells are produced in those Dyad mutants. There are also
problems in proper development of triploid seeds owing to
endosperm nuclear balance. Additionally, the seeds produced
are not always genetically balanced and have a mix of
aneuploid variants.
Therefore, in the absence of a proper apomictic breeding
system, an alternative effective strategy that facilitates
fixation of heterozygosity in obligate asexuals is needed.
The autotetraploid tetrasomic mode of reduced genetic
segregation could be a prospective remedy if we could
obtain genetically balanced and high-fertility tetraploids. The
C0 autopolyploids could be maintained vegetatively in
obligate asexuals as a source stock for continual C1 seed
production for cultivation. Based on diploid–tetraploid
meiotic correlations, Lavania (1995) determined that progenitor diploids harboring distal chiasma localization, when
changed to the tetraploid state, are most likely to give rise to
high gametic/seed fertility consummated through high
bivalent pairing and balanced anaphase segregation. Taking
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Journal of Heredity 2010:101(1)
Table 1. Mean meiotic chromosome associations (±standard error) and pollen fertility in the progenitor diploid (2) and
corresponding autotetraploid (4) in Cymbopogon martinii (4 5 40)
Quadrivalents
Bivalents
Ploidy level
Ring
Chain
Trivalents
Ring
Rod
Univalents
Pollen fertility
Diploid
Tetraploid
2.4 ± 0.245
0.15 ± 0.031
0.45 ± 0.045
4.6 ± 0.236
5.2 ± 0.245
5.4 ± 0.248
8.1 ± 0.38
—
1.85 ± 0.245
98.4 ± 2.56
96.6 ± 2.33
SE, standard error.
cues from this, we planned to elucidate if the ploidy
intervention in obligate asexuals could help facilitate fixation
of allelic heterozygosity of the source diploids in the seedraised progenies.
Materials and Methods
Plant Materials
Obligate asexual aromatic grass species Cymbopogon martinii
(Palmarosa, family Poaceae, 2n 5 20) was used as a model
experimental system. This species can be maintained
vegetatively but is grown by seeds under commercial
cultivation, leading to crop heterogeneity. This species is
found naturally occurring throughout the semiarid plains of
Central India. It is highly cross-pollinated and is valued for its
essential oil obtained from its aboveground plant biomass.
A composite variety ‘‘PRC-1’’ of this species developed at the
Central Institute of Medicinal and Aromatic Plants (Lucknow,
India) by intermating selected clones in polycross nursery was
used as a starting experimental material. Seed progenies from
PRC-1 were scored for meiotic behavior and chiasmate
association to isolate 4 morphologically different plant types
harboring distal chiasma localization. Fast growing vegetative
tillers of the 4 selected plant types were used to produce
clonal autotetraploids by the colchicine immersion method.
Seed progenies from the source diploid clone (i.e., S1
progenies) and from their corresponding C0 tetraploid clones
(i.e., C1 progenies) were grown under field conditions and
scored for simple exomorphological appearance to elucidate
population homogeneity/heterogeneity from phenotypic
appearance. Clearly distinguishable and variable exomorphological characters such as width, length, orientation and
pigmentation of leaves, length and color of peduncle, bushy/
lax spikelet arrangement, spreading/erect growth habit, etc.,
were observed to determine gross plant population heterogeneity/homogeneity.
Meiotic and Fertility Analysis
Thirty well-analyzable cells at metaphase I and anaphase I
from each of the 4 progenitor diploid clones and their
derived autotetraploid clones were scored for meiotic
chromosome associations and anaphase segregation.
Pollen fertility was examined after the usual acetocarmine
staining; the fully stained pollen grains were considered as
fertile.
Analysis of Meiotic Configurations and Estimation of
Chiasmate Association Frequency
In order to assess uniformity for comparison of meiotic
configurations, the observed values were converted into
frequencies. This could be done by dividing the observed
values by the haploid chromosome number (i.e., 10 in this
case); however, for estimating univalent frequencies, the
observed values for univalents are correspondingly assigned
to univalent pairs and to trivalents as accompanying univalents. The simple approach of Sybenga (1975), as detailed
out in Lavania (1995) and Lavania et al. (2006), especially
suitable for species with predominantly distal chiasma
localization was followed, for the estimation of multivalent
pairing ( f ), and chiasmate association frequencies of average
long arms (a) and short arms (b). In the given equations, the
symbols t, rq, cq, r, o, and u represent the frequency of
trivalent þ accompanying univalents, ring quadrivalents,
chain quadrivalents, ring bivalents, open bivalents, and independent univalent pairs, respectively. Letters a and b represent the weighted average of chiasmate (bound arm)
association of average long arm and average short arm,
respectively. The simplified equations used are
Table 2. Frequencies of various meiotic configurations and their bound-arm chiasmate associations of the progenitor diploid (2)
and corresponding autotetraploid (4) in Cymbopogon martinii (4 5 40)
Quadrivalents
Ploidy
level
Trivalents þ
2-arm
Ring Chain accompanying Ring Open Univalent Multivalent pairing Long arm
Short arm
association
(rq) (cq)
univalents (t) (r)
(o)
pairs (u) frequency (f)
association (a) association (b) (a þ b)
Diploid
—
—
Tetraploid 0.24 0.015
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Bivalents
—
0.045
0.46 0.54
0.26 0.405
—
0.035
—
0.279
1.00
1.22
0.46
0.24
1.46
1.46
Brief Communications
Figure 1. Meiotic metaphase and anaphase in the diploid. 2 5 20 (A, C) and autotetraploid, 4 5 40 (B, D) of Cymbopogon
martinii. Note complete bivalent pairing and balanced anaphase segregation in the diploid as well as in the tetraploid.
For tetraploids:
2
Multivalent pairingðf Þ 5 ðt þ 2cq þ 4rqÞ =16rq
a b 5 rq þ 1=2cq þ 1=4t þ r
a þ b 2ab 5 1=2cq þ 1=4t þ o:
For diploids:
Frequency of ring bivalents ðrÞ 5 a b;
Frequency of open bivalentsðoÞ 5 a þ b 2ab:
Furthermore, the individual values of a and b in all cases
could be deduced using the quadratic equation:
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ða þ bÞ± ða þ bÞ2 4ab
:
a; b; respectively 5
2
Results
estimated as frequencies and are given in Table 2. The representative meiotic configurations depicting high bivalent
pairing and balanced anaphase segregation are shown in
Figure 1. Plant population from S1 and C1 seed progenies
representing source diploid and corresponding autotetraploid
are shown in Figure 2, depicting morphological crop heterogeneity in the diploid but homogeneity in the tetraploid.
The observations suggest that the weighted average of
bound-arm association frequency of less than 2 in the
progenitor diploid inculcate high bivalent pairing in the
derived autotetraploids, as seen from the low frequency of
bound-arm association of the 2 arms in the autotetraploids
and also reduced multivalent pairing. This is accompanied
by balanced anaphase segregation followed by high gametic
fertility in the autotetraploids.
Meiotic Association and Gametic Fertility
Population Homogeneity versus Heterogeneity in Seed
Progenies
The C0 autotetraploids realized for all the 4 selected diploid
clones evinced high (.95%) gamete fertility. Such a high
fertility in C0 autotetraploids was realized from high bivalent
pairing and balanced anaphase segregation. Data recorded on
meiotic chromosome associations and gametic fertility for the
diploid vis-à-vis the autotetraploid (for a representative clone
no. 2) is given in Table 1. For uniformity in comparison for
bound-arm chromosome associations and multivalent pairing,
the observed values for various meiotic configurations were
Different specimens of seed progenies grown from the
selfed seeds obtained from the source diploid clone (S1
progeny) and its corresponding C0 clone (C1 progeny) are
shown in Figure 2. The C1 seed progenies exhibit
population homogeneity against the heterogeneity found
in the diploid seed progenies (S1). This clearly suggests that
the native allelic heterozygosity present in the progenitor
diploids is maintained (fixed) in C1 seed progenies through
ploidy-mediated reduced segregation.
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Journal of Heredity 2010:101(1)
Figure 2. Plant population from S1 and C1 seed progenies from source diploid and corresponding autotetraploid. (A)
Representative phenotypic variability in the diploid. (B) S1 seed progenies from source diploid. (C) C1 seed progenies from the
autotetraploid. Note morphological crop heterogeneity in the diploid but homogeneity in the tetraploid (depicting reduced
segregation/fixation of native heterozygosity in the tetraploid).
Discussion
Global duplication events are very common in plant
evolution (Adams and Wendel 2005) and the genomes of
most extant angiosperms retain evidence of one or more
ancient genome-wide duplications (Cui et al. 2006). Polyploidy is particularly widespread in angiosperms and,
frequently, involves unreduced gametes and interspecific
hybridization (Leitch AR and Leitch I 2008; Soltis PS and
Soltis DE 2009). Whereas incidence of allopolyploidy is
quite usual in plants, but autopolyploidy, although it does
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occur in nature, is found only in low frequency due to
maladaptive features associated with meiotic disturbances
(Ramsey and Schemske 2002; Otto 2007; Soltis et al. 2007).
Nevertheless, there are several instances where induced
autotetraploids are known to exhibit balanced anaphase
segregation leading to high gamete fertility and reasonably
good seed set in C0. A more regular anaphase segregation in
polyploids could be best achieved by increasing the
frequency of bivalents at the expense of other configurations, as observed in natural tetraploids of Lotus corniculatus,
Allium porum, and Dactylis glomerata (Lavania 1995). Weiss and
Brief Communications
Maluszynska (2000) observed almost complete bivalent
formation in established autotetraploids of Arabidopsis
attributing it to small size of the chromosomes and to a
process of partial diploidization.
Bivalent frequency was high in the autotetraploids on
account of a low number of autonomous pairing sites
(Santos et al. 2003)/low chiasmate association in diverse
species (Lavania et al. 1991; Srivastava et al. 1992). An
exhaustive account of quantitative genetic analysis of
meiotic configurations in highly fertile autotetraploids
compared with diploid progenitors, underpinned that
progenitor diploids with distal chiasma localization, caused
high bivalent pairing in polyploids and consequently
balanced anaphase segregation and high gamete and seed
fertility in derived polyploids (Lavania 1995). One of the
possibilities of the enhanced bivalent pairing observed in the
autoteraploid of Cymbopogon martini could be the gradual
shift in the point of pairing partner exchange toward one of
the chromosome ends during pairing just before chiasma
formation. This is reflected in the enlarged differences
observed in the estimated frequencies of bound associations
in the 2 chromosome arms. When the point of partner
exchange reaches the chromosome end in time, 2 bivalents
are formed, and these may be observed either as ring or as
rod (open) bivalents (Lavania et al. 1991). The almost
complete bivalent pairing observed in the present case
points to the possibility of obtaining high fertility in C0
tetraploids and the consequent reduced segregation/fixation
of heterozygosity in C1 seed progenies.
The cytological considerations mentioned above could
serve as possible basis for genotypic selection of prospective
diploid progenitors to develop high-fertility autotetraploids.
These findings have application in plant breeding where seeds
are required in cultivation. In addition to harnessing the
polyploid advantage per se in crop improvement, this
approach offers the opportunity of hybrid fixation in obligate
asexuals because the source C0 autotetraploids could be
maintained vegetatively and used as genetic stock for
continual seed production. The tetrasomic segregation pattern
would help maintain allelic hybridity from the source material
to a large extent (.95%) in C1 progenies in genetically
balanced high fertility autopolyploids, as demonstrated here.
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Funding
Received January 28, 2009; Revised June 25, 2009;
Accepted July 20, 2009
New Idea Fund Scheme of CSIR, New Delhi (to U.C.L.).
Corresponding Editor: Prem Jauhar
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