Morphological and Genetic Aspects of Buckwheat Interspecific ...

Proceedings ofthe ](jh international symposium on buckwheat
Morphological and Genetic Aspects of Buckwheat
Interspecific Incompatibility Overcoming
Taranenko L.K., Yatsishen O.L., Taranenko P.P.
The science-research Institute ofAgriculture ofthe Ukrainian Academy ofAgricultural Sciences
Mashynobudivnykiv str; Zb, 08162 Chabany, Kyiv region, Ukraine
Message 1. Genetic affinity among cultural buckwheat and
some ofits wild species
Abstract: Narrow polymorphism of buckwheat regarding particular elements of yield formula and
adaptive value requires researches on creation of the carriers and using them as donors. The necessary
characteristics are related to some wild species . That is why involving these species into selection
process as donors owning the criteria required for buckwheat characteristics improvement is an
important question of the crop breeding.
There are some facts about the reasons of remote combining disability of different flora resources,
about full or particular sterility of young remote hybrids, very strong and hardly surmountable
anesthesia in hybrids, low probability of interspecies hybrids characteristics and abilities recombination,
and almost a total absence of generic hybrids recombination.
A lot of hypothesis related to the incompatibility mechanism discovering give the evidence of very
complicated processes, which are running at the moment of a plant pollination/fertilization together with
enzyme systems and other biochemical complexes.
Present-day development of genetic-and-selection science in conjunction with
physiologic-and-biochemical science achievements open the possibilities to overcome the difficulties
connected with interspecies incompatibility of many cultivated plants. Interspecies wheat-and-rye
hybrids, barley-and-rye hybrids, interspecies tomatoes, cotton plants, soya, aborigine, tobacco, pea, bean,
strawberry, pine hybrids and many other cultivated plants were created by using these achievements.
In addition to this, common nature of interaction was discovered in the systems of remote
interspecies and intergeneric incompatibility, and in the systems of intravariety incompatibility. At the
same time, different methods were suggested to overcome all these types of incompatibility. These
methods are based on suppression ofincompatibility mechanism at different stages of its onset.
Actually, all attempts to cross the cultivar with some wild species of buckwheat were not
successful. In 1976 E. Grishyna conducted some researches crossing Tartary buckwheat with ordinary
buckwheat. As a result, in some cases the ovary started to develop and reached about one-forth size of a
matured yield. However, after that it often became brown and dried up. Similar researches results were
collected by M. R. Morris in 1951 during reciprocal crossing between ordinary buckwheat and Tartary
buckwheat at different stages of fertility.
The only successful way to overcome interspecies incompatibility of buckwheat was achieved by E.
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Section B Genetic resources and breeding
T. Danilenko and A. S. Kotov, as they obtained amphidiploids E gigantenum - interspecies hybrids - by
crossing between wild species - E cymosum and E tatricum. General performance of the hybrid as well
as its particular characteristics proves its hybrid origin: the hybrid was superior to its both parents in
growth, leafiness, and yield. Its height reached up to 205m and the hybrid produced I02g of seeds,
whereas the best specimen of Tartary buckwheat was only 1.68m high and produced 28g of seeds. The
hybrid was more similar to its maternal plant in fetus shape and size as well as in the length of the
vegetation season. At the same time it was more like its paternal plant in color and formation of the
flower, way of pollination, lodging resistance, and branching habit.
The fact that the hybrid has 32 chromosomes in its somatic cell, has superior seed yield, and both
parents characteristics proves that it was developed by crossing between diploid sets of Tartary
buckwheat and ordinary buckwheat. Allotetraploid origin of the hybrid is also proved by its biologic
disintegration. If crossed with parents and ordinary buckwheat the amphidiploid does not seed properly
developed hybrid seeds.
Overcoming of buckwheat interspecies incompatibility by the examples of amphidiploids obtaining,
genetic-and-selection science development in conjunction with physiologic-and-biochemical science
achievements concerned about the methods how to overcome cross incompatibility of other crops
taxonomic units presume such possibility in case of some other wild and cultivar buckwheat species
hybridization.
Keywords: Incompatibility; Hybridization; Recombination; Buckwheat; Growth-regulating substances;
Embryo crop
METHODS
Researches on development of a method how to overcome interspecific combining disability of buckwheat
where conducted at the National Scientific Centre of the Husbandry under the Ukraine Academy of Agricultural
Science, whereas biochemical, biotechnological and, partly, cytoembryological researches were doing at the
Institute ofPlant Physiology and at the Institute ofMolecular Biology under the National Academy of Sciences of
Ukraine.
Wild buckwheat species were involved during the researches.
Etataricum Ggearth /2n =16/
Etetratataricum /4n =32/
Egiganteum Krot /4n =32/
Ecymosum Meissn /4n =32/
They used diploid varieties of the cultivar of Astra, self-consistent types, and tetraploid types as ordinary
buckwheat. Cytoembryological, biochemical, and immunochemical characteristics were specified by different
methods to determine genetic relationship between wild and cultivar buckwheat species. Karyotype description of
the wild species was done according to literature data, whereas chromosomes in metaphase plates of the radicles
belonged to buckwheat cultivar types and E tataricum, which were fixed by the Karnua method and colored with
acetocarmin by the Feulgen method, were calculated.
In order to determine immunologic differences the
analysis method developed by A. D. Volodarsky for plant tissue was used.
Generative organs (pollen and pistils of buckwheat incompatible and compatible species) were analyzed in
terms of immunodetection.
For protein fractionation, disc electrophoresis in polyacrylamide gel method (The Ornstein-Davis
electrophoretic system) was used. Micromethod (the diameter of gel pellets was 2mm and 4mm, the length was
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Proceedings ofthe 1d h international symposium on buckwheat
4.5mm) was used because ofthe limited amount ofhistic material.
Buckwheat seeds, anthers, and pistils with embryo sac were used as the material.
RESULTS AND DISCUSSION
Karyotype analyses of Eesculentum revealed the presence of 8 chromosomes in its haploid chromosome set.
Their sizes varied from 3.78 to 6.97 mer. Its mapping was done by V. V. Mansurova in 1948.
Karyotype analysis of E tatarium revealed that the karyotypes of Eesculentum and Etatarium are similar to
each other in the number and shape of their large, small, and middle chromosomes. The essential difference of
Etatarium karyotype is a comparatively small size of its chromosomes, which vary from 2.0 mer to 3.3 mer, and
also slight difference in their lengths.
The karyotye of E cymosum Meissn is different from the karyotypes of Eesculentum and Etatarium. The
number of somatic chromosomes in this species is equal to 16 and 32. It can be assumed that this species has 4p as
the result of natural chromosome doubling. The essential differences between E cymosum, Etatarium and E
Tatarium karyotypes are the absence of accompanying chromosomes and the existence of supernumerary
chromosomes with secondary constriction.
The chromosomes of F. cymosum are bigger than the chromosomes of F.tataricum, but smaller than the
chromosomes of F.esculentum, and their size is equal to 2.6 - 4.6 mkr.
Karyotype analysis of E giganteum revealed that it has chromosomes, which are typical for the karyotypes of
initial species. This amphidiploid has 2 genomes ofE tatricum and 2 genomes ofordinary buckwheat.
Researches on the chromosome sets of the Astra, self-consistent type, and tetraploid buckwheat were done by
using metaphase plates of radicles, which were fixed by the Karnua method and colored by the Feulgen method ,
and were equal to 16 for diploid types and 32 for tetraploid buckwheat.
Due to the fact that kryotype characteristics are insufficient to deduce the relation degree of species,
researches on immunological and biochemical differences, which fully reveal this data, were conducted.
As a consequence of the immunological researches, titer and specificity of the antiserum /AT/, which were
obtained with the use of pollen and pistils of F. giganteum and F.esculentum /40/, revealed that after the reaction
between the AT of these species and homologous antiserum the precipitation line was not specific.
The pictures 1.1 and 1.2 show the fit of equivalent proportions of AT, related to pistils of E giganteum and
Eesculentum, to homologous antiserums.
As nonspecific precipitate was formed in pollen system AG-AT, further precipitation test was not conducted.
Equivalent proportion of AT-AG showed in the picture 1.1 is equal to 1:112, whereas in the picture 1.2 it is
equal to 1:118. The number of precipitation lines shows that the antigenic spectrum ofE giganteum pistils consists
of two precipitates, while the antigenic spectrum of Eesculentum is formed by one line. Therefore, the antigenic
spectrum ofE giganteum is somewhat wider than the spectrum ofEesculentum.
The data on partial similarity and partial difference between the antigenic mosaics of the two cultivars pistils
is also proved by the gemological and geterological method by Uhtbrolny (Picture 1.3). Presence of common
antigen is evident as horizontal precipitate was formed by immuno-diffusion reaction according to square scheme.
Consequently, if the indicated cultivars of buckwheat are pollinated with pollen from one cultivar, the percentage
of successful crossing will be markedly different. According to the empirical data, we can make the assumption
that protein-protein interaction of pollen-pistil system lies at the root of incompatibility. Therefore, in order to
improve crossing results, it is possible to use specific inhibitors or activators of protein synthesis, which can
inactivate or activate protein synthesis systems of every crossing mate.
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Section B Genetic resources and breeding
Picture 1.1 Comparison of buckwheat
pistil antigenic stnIctures according to
square scheme.
I-AG of pistils
2-AG of pistils
Picture 1.2 Comparison of buckwheat pistil
antigenic compositions.
I-AG of pistils
2-AG of pistils
3-AC opposite pistils
3-AC opposite pistils
4-AC opposite pistils
Picture 1.3 Precipitation reaction in agar between pistils AG and homologous
antisenun.In the centrallunula is AS being opposite pistils in peripherallunulas· 1·5
titrating /from 1 to 1I16,6-physiologicalsolution.
In order to explore the possibility of testing the degree of genetic relationship between initial species and
their hybrids, examination of protein synthesis of their reproductive organs as well as their protein composition
and quality was conducted.
The detailed research data prove that such analysis can be used to estimate the degree of genetic relationship
between buckwheat species (picture 1.4) where relationship coefficients are used as the criteria. These coefficients
should be estimated for one species against another one as the ratio of the number of electrophoretic strips, which
are common for both species electrophoregrams, to the number of electrophoretic strips of one species.
order of the most representative strips is also used as the criteria. For instance, segments with very large protein
amount IO.EP. 0.2-0.3/, and segments of easily mobile forms IO.E. 0-0.95-1.01 are considered to be representative.
It should be noted that in segment with high electrophoretic mobility the first kind of strip related to E
tatricum is comparatively more mobile than the same one related to Eesculentum. E tatricum has two clearly
defmed strips, which are close to each other, whereas Eesculentum has only one.
Arrangement of the strips with large protein amount in electrophoregrams of E giganteum seeds is more
similar to the same arrangement of E tatricum, whereas in segments with high electrophoretic mobility there is
only one strip (similar to Eesculentum).Therefore, such approach to the question gives the opportunity to estimate
the degree of genetic relationship between different buckwheat species, which is an important thing for hybrids
analysis and reproductive incompatibility forecasting during breeding.
Having analyzed the data of karyological, immunochemical, and biochemical analysis, we note the fact of genetic
relationship between E tatricum, E giganteum, and Eesculentum, Genetic relationship of E cymosum, whose
The
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Proceedings a/the ](jh international symposium on buckwheat
gene is a part of E giganteum, was implicitly determined by using the data of other researchers.
0.2$
0"'
0.15
I I
O.BS
0.90
O.9S
W
1.0 !.-
-
Picture 1.4 Electrophoregram of protein composition of
buckwheat species (F.escuientum, F.giganteum, F.tatricum) seeds
Message 2. Prospects ofInterspecific Hybridization in Buckwheat
Polymorphism Amplification
Taranenko L.K., Yatsishen O.L., Karazhbej P.P., Taranenko P.P.
The science-research Institute ofAgriculture ofthe Ukrainian Academy ofAgricultural Sciences
Mashynobudivnykiv str; 2b, 08162 Chabany, Kyiv region, Ukraine
F.esculentum (2n =16) x E tataricum (2n =16)
/Astra species and incompatible species/
Eesculentum (4n) x E tetrataricum (4n =32)
/ tetraploid buckwheat x K-108/
Eesculentum (4n) x E cymosum (4n =32)
Eesculentum (4n) x E gigantueum (4n =32)
/ tetraploid buckwheat! x / amphidiploid!
METHODS
Wild buckwheat seeds were received from the VIR collection. Plants of different buckwheat species were
labeled according to flower formation. Individual legitimate pollinating of opposite heterostyled types was
provided. The pollinating was being carried out every day in two ways - by transferring the pollen of one
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Section B Genetic resources and breeding
buckwheat species to surfaces of clean, checked at magnification of x 7-10 stigmas belonged to plants with
castrated flowers, and also by transferring the pollen to the stigmas of flower buds, which were previously opened
up and castrated. In addition, thirty minutes before the hybridization, specific inhibitor of protein synthesis had
been transformed to the stigmas. In one case the pollen was soaked in growth-activating substance and in the other
case - growth stimulator was inserted after its transformation to stigmas. After having finished the crossing
process the rest of mature flowers and stigmas were moved away.
Crossing without physiologically active substances was considered as a standard for exemplified sachems.
But during the mentioned process such kinds of growth-activating substances as actinomycin D - in concentration
of 15mcglml, kinetin (0.1%) - IOmgIL, and also cadmium salt solutions in some concentrations were used.
Pollen germination and growth intensity of pollen tubes were examined by using temporary specimens
resulted from fixation of crossing products by the Karnua method after 1, 2, 4, 5, 6, 8, and 24 hoers after the
crossing. Cultivation of 6-10 days embryos was done in embryo culture with use of Murasige-Skoog growing
medium. True hybridism was cytologically and morphologically determined.
RESULTS AND DISCUSSION
In order to overcome cross incompatibility mechanism, growth-stimulating compositions were used. Most of
them were protein synthesis inhibitors and according to this fact incompatibility mechanism, which is admittedly
has the nature of protein, should probably be weakened. We used such inhibitors of protein complex as
chloramphenicol /chloromycetin/, antinomicin - D, and cadmium salt solutions - universal growth regulating
compositions, which can effect growth inhibition as well as activate their growth stimulating property, depending
on the time of the action. Kinetin is also a kind of growth stimulators, which was used during the experiment.
The researches on buckwheat interspecific incompatibility overcoming were being conducted during 10
vegetation periods as a field research and during 3 vegetation periods - as a greenhouse research.
During this period and according to the hybridisation schemes 4003 flowers were crossed, on 11682 of them
ovaries were formed, and, finally, 4934 complete seeds were formed as a result. Furthermore, 900 flowers were
pollinated for cyto-control (Table 1.5).
The largest amount of ovaries, when sampling was quite representational, were formed by the following
combinations: Astra x F. tataricum v. rotundatum ( K-7)(31.3 %), tetraploid buckwheat x F.gigantcum (K -109)
(27.2 %), during the hybridization tetraploid x F. tetratataricum (K - 108)(29.8 %) and tetraploid x perennial
buckwheat (p. cymosum) (K-4231) - 15 %. The largest inferior yield (14.2 and 16.2 %) was obtained by the
crossings where the Astra was used as a maternal component and two sorts of F. tataricum - as a paternal
component.
Results of the researches on efficiency of different physiologically active substances utilization in relation to
level of seed set during the remote hybridization of buckwheat are listed in the table 1.5. The present data show
that the largest amounts of ovaries and seeds are related to the following combinations: Astra x F. tataricum v.
tuberculatuml K - 21/, Astra x K - 17 with using of stimulant and protector of cadmium salt; and also when using
kinetin in such combinations as tetraploid buckwheat x K - 108 and as tetraploid buckwheat x K - 109.
But at the same time, kinetin was less effective in combinations when the Astra was crossed with F. tataricum
sorts.
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Proceedings ofthe ](jh international symposium on buckwheat
Table 1.5 The level ofseed set during interspecific buckwheat hybridization with use ofgrowth-regulating substances
Crossing
combinations
Growth-activating
substances
Dowers
Average number of
ovaries
seeds
pieces % pieces
%
Control
4046
920
22,7
290
7,7
Astra x F. tataricum v.
tuberculatum
Kinetin + (DMSO (dimethyl
sulfoxide)
Cadmium salt +(DMSO)
1116
712
354
298
31,7
41,7
144
118
12,9
16,6
ACT-D+DMSO
836
320
38,2
86
10,2
Control
2128
358
16,8
154
7,2
Tetraploid buckwheat x
Kinetin +(DMSO)
340
172
50,4
68
20,2
F. tetratataricum Cadmium salt +(DMSO)
1160
244
21,0
100
8,6
ACT-D+ DMSO
Control
1354
532
19,6
202
7,5
Tetraploid buckwheat x
Kinetin +(DMSO)
682
278
40,7
128
18,7
F.gigantcum
Cadmium salt +(DMSO)
708
134
18,7
14
2,0
ACT-D+ DMSO
342
40
11,7
2
0,6
Control
1348
206
15,2
50
3,7
Tetraploid buckwheat x
Kinetin +(DMSO)
F. cymosum Cadmium salt +(DMSO)
ACT-D+ DMSO
596
156
26,4
34
5,7
Astra x
Control
1752
184
7,6
44
2,5
F. tataricum
Kinetin +(DMSO)
758
44
5,8
12
1,5
v.rotundatum
Cadmium salt +(DMSO)
626
134
21,4
101
16,3
It is important to note that the main part of the formed inferiors died on the 6-12 day. Therefore, at the
Institute of Microbiology and Hybridization 6-12 days ovaries were grown by use of embryo culture. Callus
culture was bred and cloned. The created clones had been multiplying and growing till the time they attained the
condition efreplanting to culture vessels of a plant house, and after that they were planted out into field conditions.
/pictures 1.6 and 1.7/.
138

Section B Genetic resources and breeding
Picture 1.6 Plant F (Astra lit Rtataricum v,rotundatum
K·17), grown from 10-12 days embryos, in embryo
culture.
Picture 1.7 Interspecific hybrids IAstra lit K·I71,
replanted from embryo culture to vials.
Picture 1.8
Picture 1.9
As a result of interspecific crossings, new morpho-phisio types of buckwheat were cultivated.
Short-growing plants (up to 20sm) with strong, almost bush-like footstalks and bushy, branching crowns,
little oval leafs, and short, nearly invisible petioles (picture 1.8) are of great value to buckwheat breeding.
Inflorescences of these plants are in the shape of compact and small whorls, their stigmatized flowers have
reduced petals and anthers, and considerable morphological anomalies.
Dwarf plants are also of great interest. They have defining characteristics, which are as follows: strong stalks,
stunting, well-developed foliage, which provides high photosynthetic potential (picture 1.9).
The plants with unusual architectonics (picture 1.10), whose typical characteristics are tall flower-bearing
stems, multiple inflorescences, consisted of 3-7 flower-bearing branches, are also ofinterest to the breeding
concerned about intensive generative weight. The inflorescences of these plants are only over their vegetative
weight and located within 15sm, whereas their foliage, in spite of being quite well-developed, is located within
10sm. Such generative potential and leaf formation were taken from wild tetraploid species ofF. tataricum K ­
108. As the result, inflorescence structure was altered and such characteristic as brachysm was obtained. Cyto
analyses proved that the plants have hybridous origin.
By crossing between the Astra and F. tataricum v. rotundantum ( K - 2l)a dwarf plant (picture 1.11), whose
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Proceedings ofthe 10 th international symposium on buckwheat
every branch is ended by a capitate inflorescence with stipules, was bred in second filial generation.
Picture 1.10. Picture 1.11
The hybrid plant which was obtained by crossing the Astra with F. tataricum v. rotundantum(picture 1. 12) is
also of interest to buckwheat breeding concerned about high photosynthetic potential. Its characteristic is a tall
and quite strong flower-bearing stem. It also has alternating and acuminate leafs with pronounced turning down
and venation. The branches are at an acute angle to the main footstalk. Such branch arrangement reduces shading.
Stunting and dwarf forms of interspecific hybrids with erectoid leaf and branch arrangement, with different
types of inflorescences and different breeding performance (pictures 1.13, 1.14, 1.15, 1.16) are a valuable starting
material for future breeding projects.
Picture 1.12 Picture 1.13
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Section B Genetic resources and breeding
Picture 1.14 Picture 1.15
Picture 1.16
SUMMARY
According to imrnunobiochemical analysis and karyological characteristics, genetic relationship between
wide species of buckwheat - F. tatricum, F. cymosum, their amphidiploid F. giganteum and cultural buckwheat
was finally deduced.
Among growth-activating and physiologically active substances used during the researches on buckwheat
interspecific incompatibility overcoming the most effective were cadmium salts.
It was proved by biological disintegration of the generation obtained after interspecific crossings and by
morphological and cytoembryological researches that some particular selected interspecies hybrids have true
hybrid origin.
References
[1] Kporos A. C., Ionyfeaa E. A . (1973). UHTOJlOIWIeCKOe asyseaae MeJKBH./lOBoro rn6pH./la F. tataricum x F. cymosum /I Tp. no
npHKJla,nHoR 6oraHHKe, reaeraxe H CeJleKUHH. - 11.: BI1P. - T.51. - Bun. 1.
[2] KPOTOB A . C., ):ij>aHeHKO E. T. (1973). AMcPH./lHnJlOH./l rpesaxa II EJOJlJI. BliP. - BhIII. 30. - c. 41-44.
[3] Maacypona B. B. (1948). CPaBHHTeJIhHlUI KaPHOJlOrIDlllBYX BH./lOB rpesaxa: F. esculentum, F. emargihatum II )l.OKJI. AH CCCP.
- T. 1. - Bsm. 6. - c. 119-122.
[4] CypHKOB H. M. (1987). MOPcP0JlOrnqeCKHe, reaerasecxae H 3BOJlJOUHOHHbie acnexrsr reopaa BHyrpHBH./lOBOR H MeJKBHllOBOR
nOJlOBOR HeCOBMeCTHMOCTH y UBeTKOBbIX pacreaaa II Ieaer-cenesn, acnexrsr CHCTeM pa3MHOJKeHIDI 3HTOM0cPHJlhHbIX BH./lOB
paCTeHHR. Marepaansi I Bcecoiosaoro pa6. COBeUJ;aHHJI no rener-cenexnaoa, acnexrast CHCTeM pa3MHOJKeHHJI y 3HTOMOcP.
BH./lOB pacreaaii /rpesaxa, XJI0nqaTHHK, mouepna/> )l.y1IlaH6e, 1-4oKT. 1985. - )l.yrnaH6e: )l.OHH1II. - c. 3-20.
[5] TapaHeHKO 11. K. H IIp. (1987). Hexoropsre acnexrsr MeJKBHllOBOR rn6pH./lH3aUHH rpesaxa II Teneraxa, CeJleKllHJI,
ceMeHOBOllCTBO H B03lleJlbIBaHHe spynsaux xynsryp, C6. aayxa. TPYllOB. - KH1IIHHeB:THnorpacPHJI KCXH. - c. 53-61.
[6] Tatiana N. Lasareva, Ivan N . Fesenko (2004). Electrophoresis Spectra of Total Seed Protein of Artificial Amphidiploid
Fagopyrum giganteum Krotov and its Parental Species F. tataricum ufaertn and F. cymosum Meisn. Proceedings of the 9 th
International Symposim on Buckwheat. - Prague,p. 229-301.
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