Advances in Bioscience and Biotechnology, 2013, 4, 89-94 ABB Published Online October 2013 (
Influence of external conditions on the combinatorial
processes at agamospermy
Evgenii Vladimirovich Levites, Svetlana Sergeevna Kirikovich
Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
Received 10 August 2013; revised 10 September 2013; accepted 10 October 2013
Copyright © 2013 Evgenii Vladimirovich Levites, Svetlana Sergeevna Kirikovich. This is an open access article distributed under the
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
The estimation of the influence of external conditions
on marker enzymes phenotypic classes ratios in aga-
mospermous sugar beet progenies has been carried
out. It has been shown that different ways of flower-
ing sugar beet plant branches isolation lead to differ-
ent ratios of marker enzyme phenotypes in a devel-
oping agamospermous seed progeny. The obtained
data are an additional confirmation of the hypothesis
about the presence of differential chromosomes poly-
teny and its dependence on external conditions.
Keywords: Isozymes; Polyteny; Diminution;
Agamospermy; Sugar Beet
Different populational characteristics, such as the degree
of variability in sexual and asexual plant populations [1,
2], were analyzed in numerous experimental and theore-
tical research activities devoted to agamospermy; the le-
vel of apomixis in a plant population [3] was also evalu-
ated. Only in 1994-1996, there were contributions that ini-
tiated studying the mechanisms underlying agamosper-
mous progenies variability [4,5]. According to the con-
clusions made by the authors, the observed ratios are
conditioned by meiosis in polyploid cells of mother plant
capable of agamospermous reproduction. For the cases
when a mother plant was polyploid, such conclusion, on
the whole, can be thought of regular, though a way to
this understanding has turned quite long. In cases when a
mother plant was diploid, the phenotypic classes ratio
was determined by the meiosis that proceeds in tetraploid
cells present as an admixture among the bulk of diploid
cells [5]. For instance, it was shown that, in the agamo-
spermous progeny obtained from diploid sugar beet plants,
the phenotypic ratio on hypocotyl coloring was 11 color-
ed: 3 uncolored; and the phenotypic ratio on isozyme
markers was often equal to 3:8:3, which corresponded to
the tetraploid gametic segregation [5-7]. The process of
such progenies formation was called meiotic agamos-
permy [8].
Isozymes have become a convenient instrument for
studying agamospermy. A wonderful peculiarity of isozy-
mes is their codominant inheritance due to which the hy-
brid plant isozyme spectrum is different from each parent
isozyme spectrum. For instance, one isozyme with fast or
slow electrophoretic mobility, which corresponds to the
genotype of the given locus, is revealed in the electro-
phoregram for the homozygote on the gene controlling
this marker enzyme. But both enzyme allelic variants
(isozymes) and also hybrid isozymes [9,10] are revealed
in the heterozygote. This allows one to reveal all 3 phe-
notypic classes in the progeny of plant heterozygous on
the isozyme locus: two homozygous and one heterozy-
gous. Assisted with isozymes, the case of mitotic agamo-
spermy, at which the progeny developing in diploid sugar
beet plant was monomorphous on the marker enzyme he-
terozygous phenotype, was revealed. This monomorphi-
sm on the heterozygous enzyme phenotype was indica-
tive of the absence of meiotic genome transformations of
cells capable of agamospermous reproduction [11]. At
the same time, the polymorphism of this progeny on the
other marker enzyme indicated the presence of a specific
variability mechanism. To explain this phenomenon, a
hypothesis that implies: a) the presence of differential
chromosome polyteny, i.e. separate chromosome sites
polytenization including those that carry this marker lo-
cus alleles in plant generative organs cells, b) random
equiprobable attachment of two allelic copies (one copy
from each chromosome of two homologous in a diploid
plant) to the nuclear membrane or the nuclear matrix of
the cell before its entering embryogenesis (this allelic
pair then determines the genotype of a developing em-
E. V. Levites, S. S. Kirikovich / Advances in Bioscience and Biotechnology 4 (2013) 89-94
bryo), c) cell transition to embryogenesis and duplica-
tion of chromosomes attached to the nuclear membrane,
d) first division of embryogenesis and diminution (loss)
of unattached allelic gene copies [12,13] was proposed.
Occasional equiprobable attachment of allelic copies of
chromatide regions is a combinatorial process that im-
plies the existence of a mutual exchange by chromatide
regions among chromosomes.
The data on marker enzymes phenotype ratios in aga-
mospermous progenies of tri- and diploid sugar beet
plants were the confirmation for the statements of this
hypothesis [14,15]. The existence of specific phenotypic
classes ratios in agamospermous progenies of diploid
sugar beet plants has become a weighty argument not
only in favor of the hypothesis about the presence of po-
lyteny, but it also pointed out a mutual exchange of chro-
matide regions among chromosomes in the cell before its
entering embryogenesis [15]. Initially, this hypothesis was
proposed to explain sugar beet seeds phenotypic ratios
obtained by mitotic agamospermy from nucellus somatic
cells or integuments [12,13]. However, it was noticed
that the same hypothesis is applicable also for the analy-
sis of meiotic agamospermy when polyteny occurs in
mother megaspore cells (MMC) [14,16].
To characterize the polytene state of this or that chro-
mosome site, we proposed the term “locus polygeno-
type” and the legend, e.g. FnSm, to describe its allelic
composition, number of chromosomes and chromatides
carrying each of the alleles [14]. The presented legend is
suitable for a diploid cell but, in the case of a tetraploid,
it can be FnFnSmSm.
The polyteny that affects the phenotypic ratio in aga-
mospermous progenies is the result of a big and compli-
cated DNA replication process which, as any enzymatic
process, depends on many conditions. In this connection,
studying the influence of external conditions on the phe-
notypic ratios in agamospermous progenies is of great
interest. Earlier we carried out investigation regarding
this point, and it was shown that colchicine or Triton X-
100 treatment [15,17] affects sugar beet plant pheno-
typic classes ratios.
Earlier, we also obtained the facts that evidence the
non-random distribution of isozyme phenotypes in the
seeds analyzed in the order they set on a sugar beet plant
branch. The non-random phenotypic distribution allowed
us to hypothesize on the influence of external conditions
on the degree of polyteny in the cells capable of entering
embryogenesis and, respectively, on the geno- and phe-
notype ratios of an agamospermous progeny [18]. In the
connection with the above-formulated, it was necessary
to widen the scope of the investigation devoted to the
influence of external conditions on the geno- and pheno-
typic ratios in agamospermous sugar beet progenies, and
that was the aim of this contribution.
The agamospermous progenies obtained from pollen-
sterile sugar beet plants in the pollenless regime were
involved in the investigation. Only plants of phenotype
ms0 and ms1 on Owen classification [19] were left in the
field to create the pollenless regime during flowering;
individual isolators were also used. Two types of isola-
tors were used to estimate the effect of environmental
conditions for each experimental plant; part of plant
branches was covered with an unbleached calico and part
of branches—with parchment isolators.
The ratios of enzyme phenotypic classes of alcohol de-
hydrogenase (ADH1, E.C., malic enzyme (ME1,
E.C., isocitrate dehydrogenase (IDH3, E.C. were analyzed with the method of electropho-
resis in starch gel with a further isozymes histochemical
staining in electrophoregrams [20,21].
Comparison of marker enzymes phenotypic classes ra-
tios in seeds obtained at various ways of isolation was
carried out using the G-criterion [22].
To determine locus enzyme polygenotypes, the reveal-
ed phenotypic classes ratios were compared to theoretical
calculations obtained for a different polytenized allelic
copies number in heterozygous enzyme loci. Theoretical
calculations were realized, implying the variation of po-
lytenized copies per allele in the interval of 2 - 8.
According to the accepted hypothesis, polymorphism
in agamospermous progenies was determined by the com-
binatorial process based on a random, equiprobable alle-
lic copies attachment to the nuclear membrane [12,13].
This combinatorial process can be described by hyperge-
ometrical probability distribution formulas [23,24]. Ac-
cording to these formulas, the portion of homozygotes is
determined by a number of combinations—two from the
number of the corresponding allelic copies accumulated
as a result of polytenization. For example, if one allele is
presented by n copies in the diploid cell, the other one—
by m copies; then the portion of homozygotes (homoal-
lelic genotypes) on the first allele is determined with the
formula: 22
, on the second-22
. Accord-
ingly, the portion of a heterozygous (heteroallelic) geno-
type is determined with the formula 11 2
nm nm
. These
formulas show the phenotypic ratio in portions from one.
However, these ratios can be expressed also with simple
numbers which are to be determined the following way:
for the first homoallelic genotype—as n(n-1):2, for the
second homoallelic genotype—as m(m-1):2 and for the
heteroallelic genotype—as nm.
The revealed experimental ratios were compared to the
theoretically expected using criterion χ2. The choice of
the polygenotype was made according to the minimal χ2
Copyright © 2013 SciRes. OPEN ACCESS
E. V. Levites, S. S. Kirikovich / Advances in Bioscience and Biotechnology 4 (2013) 89-94
Copyright © 2013 SciRes.
Seed progenies were obtained from all three experiment-
tal sugar beet plants. We consider these progenies as aga-
mospermous on the base of the previous and numerous
data obtained with various methods, which are indicative
of the fact that the growth method we used for pollen-
sterile sugar beet plants in the pollenless regime allows
us to obtain an agamospermous progeny [14-17]. For
example, to prove the agamospermous origin of the pro-
geny, we used unopened buds castration with their fur-
ther isolation [13], natural sampling method consisting in
the analysis of seed phenotypes in the sequence they
were setting on plant branches [18], revealing specific
seed phenotypes ratios explained only by the agamosper-
mous origin of the analyzed progeny [15], obtaining the
genetic proofs for the absence cross-pollination between
growing sterile and semisterile sugar beet plants [17].
Polymorphism on two out of three marker enzymes
—alcohol dehydrogenase (ADH1), malic enzyme (ME1)
and isocitrate dehydrogenase (IDH3) (Table 1)—was re-
vealed in each investigated agamospermous sugar beet
progeny. The presence of polymorphism points out het-
erozygosity of the initial mother plants on the corres-
ponding enzyme loci. The phenotypic ratio on ME1 in
the progeny produced from plant 2 - 7 under the parch-
ment isolator is significantly different from ratio 1:2:1 (χ2
= 7.6; P < 0.05) typical of self-pollinated progenies. Be-
sides, the ratio of the total homoallelic phenotypes por-
tion to that of the heteroallelic phenotype is significantly
different from ratio 1:1 (χ2 = 7.2; P < 0.01) typical of the
progenies produced by self-pollination. This is one more
proof for the agamospermous origin of the progenies we
analyzed. This conclusion allowed us, further on, to eva-
luate the degree of heterozygous locus alleles polyteny in
mother plant cells.
The use of isolators in obtaining sugar beet seed pro-
genies from pollen-sterile plants leads to low seed pro-
ductivity. As a consequence of this, it is not always pos-
sible to obtain big samples (number of seeds) for the ge-
netic analysis.
G-criterion-assisted comparison of the phenotypic
classes ratios of seed progenies obtained at different
ways of isolation, in most cases, did not reveal signifi-
cant differences. This allowed us to integrate the samples
of one and the same plant and to determine marker locus
polygenotypes in mother plant cells on the integrated
Several values of polygenotypes, but with a different
probability, corresponded to the integrated ratios of phe-
notypes. For instance, several possible polygenotypes—
F4S6 (χ2 = 2.8244); F5S7 (χ2 = 1.9592); F6S8 (χ2 = 1.9120),
out of which the last is the most probable one-corres-
pond to the integrated ratio on ME1 being 22FF:59FS:45SS
in the KA-8 plant progeny. This polygenotype means that
locus Me1 is presented by two chromosomes in mother
plant cells, in one of which allele Me1-F, as a result of
polytenization, is presented by six copies and allele Me1-
S-by eight, respectively. In the same progeny, polyge-
notype F6S5 (χ2 = 0.0044) mostly corresponds to the in-
tegrated ratio on IDH3 (31FF:62FS:21SS).
In the progeny of plant 12 - 2, polygenotype F8S7 (χ2 =
1.6173) mostly corresponds to the integrated ratio ADH1
30FF:46FS:22SS and, in the same progeny, polygeno-
type F8S7 (χ2 = 0.5268) mostly corresponds to the inte-
grated ratio on ME1 equal to 27FF:49FS:22SS.
The sample size of the seeds for ME1 analysis in the
progenies of plant 2 - 7 obtained under different isolators
is also not big. However, the difference in phenotypic
classes ratios is so considerable (G = 220.8436; P <
0.001) that this allowed us to determine the polygeno-
types individually corresponding to each seed group.
Polygenotype F2S2 (χ2 = 2.2) mostly corresponds to the
ratio on ME1, being 1FF:16FS:3SS, revealed in the seeds
obtained under the parchment isolator, and polygenotype
F8S7 (χ2 = 1.4539) mostly corresponds to ratio 10FF:
Ta ble 1. Ratio of marker enzymes phenotypic classes in agamospermous sugar beet progenies and polygenotypes (FnSm) of mother
plant cells capable of embryogenesis.
of plant Type of isolator
Parchment 62:0:0 11:35:23 15:40:12
Unbleached calico 24:0:0
Parchment 21:22:9 13:27:11 10:0:0
12 - 2
Unbleached calico 9:24:13
Not analyz.
Parchment Not analyz. 1:16:3 F2S2 6:5:3
2 - 7
Unbleached calico 31:0:0
10:14:7 F8S7 5:16:5
E. V. Levites, S. S. Kirikovich / Advances in Bioscience and Biotechnology 4 (2013) 89-94
14FS:7SS in the seeds from the unbleached calico iso-
Drastic differences in cell polygenotypes belonging to
the plant branches, being under different isolators indi-
cate, that external (environmental) conditions affect poly-
tenization processes.
It is noteworthy that this influence depends on the ge-
notypic medium, as it was not found in other two plants.
The role of genotypic medium manifests itself also in the
fact that, independent from the type of isolator, the phe-
notypic ratios on ME1 in the progeny of plants KA8 and
12 - 2 are significantly different in the G-criterion (G =
6.9666; P < 0.01) and, hence, these plant polygenotypes
are different (Table 1).
The sample size plays a considerable role in the de-
tection of a weak response to external impacts and, in
case of its (sample size) increase, the differences could,
apparently, have also been revealed in most cases, just as
e.g. in the progeny of plant 12 - 2 in the phenotypic ratio
on ADH1.
Outstanding is the fact that the calculated polyteniza-
tion degree of both locus Me1 alleles in plant 2 - 7 is
considerably lower under the parchment isolator than
that under the unbleached calico. Probably, this is condi-
tioned by the fact that the branch tissues and cells under
the parchment isolator experience a strong stress and
aberration of many biochemical processes as a conse-
quence of the breakdown of light regime, also gas and
temperature exchange. The conditions under the unblea-
ched calico isolator are, probably, a bit close to natural
ones, which contribute more to a normal procedure of bi-
ochemical processes including chromosome sites polyte-
A more considerable number of polygenotypes, among
which polygenotypes F8S7 (χ2 = 0.0156) and F6S5 (χ2 =
0.1041) are possible, may correspond to the integrated
ratio on IDH3 being 11FF:21FS:8SS in the progeny of
plant 2 - 7 due to a small sample size. Herein, due to a
small sample size, polygenotype F6S5 (χ2 = 0.1041) was
chosen, the same also for ratio 31FF:62FS:21SS on
IDH3 in plant К-8, in which this plant has no differences
in criterion G.
The calculated locus polygenotypes values with an
odd polyteny level, at least of one of the alleles, corre-
spond to the mitotic agamospermy at which the cell,
having not undergone meiotic genome transformations,
enters embryogenesis. This may be, for instance a nucel-
lus or integument cell. At the same time, the phenotypic
ratio that corresponds to polygenotype F6S8, may also
correspond to the other polygenotype: namely F3F3S4S4
which is typical of the mother megaspore cell (MMC). In
this case it is possible to hypothesize about the existence
of such tetraploid MMCs in which one allele is presented
by two chromosomes, each of which, as a consequence
of polytenization, has four chromatides, and the other
allele is presented by the other pair of chromosomes in
which there are only three copies as a result of under-
reduplication. In the entering of such MMC into em-
bryogenesis and a loss of excessive allelic copies, there
emerge diploid gametes developing into individual dip-
loid embryos (plants) at the same ratio as in mitotic aga-
mospermy. The only difference is that, in this case, this
will be a progeny obtained by meiotic agamospermy and
the combinatorial process of the excessive allelic copies
diminution is connected with a combinatorial process of
a two-fold decrease of chromosomes number in the cell.
The obtained data confirm the existing outlook on the
thing that many factors affect the polytenization process.
It was shown with many cytological investigations car-
ried out both in Drosophila and in plants that the level of
chromosome polytenization depends on both internal
conditions (level of inbreeding) [25,26] and external con-
ditions (temperature, daylight length) [27,28]. Our results
and analogous conclusions, unlike those of cited contri-
butions, were obtained only on the base of the data of ge-
netic analysis. Such an unusual approach is justified, on
the one hand, by the impossibility of any other explana-
tion for the specific sugar beet phenotypic classes ratios
and, on the other hand—confirmation of the hypothesis
we use with the data from the field of mammalian ge-
netic, including that of human. Abnormalities based on
the presence of one-parent disomy in an individual of
some chromosome pair or certain regions of homological
chromosomes obtained from only one parent were found
in both human and mice [29,30]. As we mentioned ear-
lier, such phenomenon could appear only as a result of
excessive reduplication of certain regions or whole chro-
matides which further led to an occasional equiprobable
diminution of the excess of genetic material.
The specificity of the problem we investigated consists
in the thing that it is difficult to determine the low po-
lyteny level with the help of cytological methods, but it
may be revealed in embryo sac cells or surrounding tis-
sues only on the base of genetic calculations. Therefore,
our further studies will be aimed at the use of additional
methods for evaluation of the degree of chromosome po-
lyteny in embryo sac cells and surrounding tissues.
The obtained proof for the agamospermous origin of
the progenies we analyzed and additional confirmation of
the hypothesis about the differential chromosomes poly-
teny, and its dependence on external conditions are in fa-
vor of the supposition regarding the role of polyteny in
the record of information about acquired traits.
The authors would like to express their gratitude to Alexander V.
Copyright © 2013 SciRes. OPEN ACCESS
E. V. Levites, S. S. Kirikovich / Advances in Bioscience and Biotechnology 4 (2013) 89-94 93
Zhuravlev for the English version of this article.
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