40. Studies on Chromosomes Doubling of Tartary Buckwheat

Current Advances in Buckwheat Research (1995) : 313 - 321
40
<strong>Studies</strong> on Chromosomes Doubling of Tartary Buckwheat
Zhao Gang, Tang Yu and Qiu Shifeng
Department of Agriculture, Xichang Agricultural College, Sichuan, China
Abstract
The new strain of autotetraploid tartary buckwheat can be obtained by doubling the cellular
chromosomes of the three varieties of tartary buckwheat from the Liangshan area of China.
This is done by mixing with a solution ofcolchicine and dimethyl sulfoxide. Compared with its
parent stock, this new strain has a well developed root system, thicker and stronger stalks,
larger seeds, and, in general, a heavier and taller plant. Also, the protein, amino acid, vitamin
content and medicinal value are notably increased. Furthermore, the new strain exhibits stable
inheritance, higher seed-bearing rate and resistance to adverse environmental elements.
Key words : Tartary buckwheat, Chromosomes, Autotetraploid, Colchicine, Dimethyl
sulfoxide
Introduction
Tartary buckwheat is a natural health food, rich in nutrition and contains eight of the amino
acids with proper proportion required by the human body [5]. Most fatty acids in tartary
buckwheat are non-saturated oleic acid and linoleic acid. It is also rich in nutritional mineral
elements and vitamins, particularly in vitamin P. Tartary buckwheat can evidently reduce blood
sugar, blood fat and urine sugar, thus it offers protection against and treatment of the
deterioration ofheart blood vessels, vascular sclerosis ofthe brain, and hypertension. It is also a
very good medicinal food for people who have been exposed to radiation [6].
Because of its short growth span and easy cultivation, tartary buckwheat is an important
crop in the outlying cold mountain and remote upland areas inhabited by the minorities of
southwest China. Historically, its yield has been low and unstable. The practice of buckwheat
cultivation in China and foreign countries indicates that polyploid cultivation'is the important
way to improve the yield and quality. Good increases in yield and quality have been
experienced with Spark, Aimka, Pecard and the new strain of tetraploid tartary buckwheat 87-1
[2,4,8]. By doubling the chromosomes ofthe three varieties ofbuckwheat from the Liangshan
area, autotetraploid tartary buckwheat was obtained. This paper reports the inducing method
and main characteristics of autotetraploid tartary buckwheat, aiming at providing a theoretical
basis for polyploid cultivation oftartary buckwheat.

314
Materials and Methods
In the spring of 1989, the three varieties oftartary buckwheat, namely, Eluowuqie, Shichee and
Kuciqiao were cultivated in pots at the Agricultural Test Station of Xichang Agricultural
College. Forty pots were planted for each variety. After the seeds sprouted, four plants were
kept in each pot. The following method of treatment for the doubling of the chromosomes of
Jiujiang tartary buckwheat was used [9, 10]. After 2-3 leaves appeared, absorbent cotton balls
were placed on the growing point of the leafstalk. A solution mixture of colchicine (0.2%) and
dimethyl sulfoxide (1.5%) was dropped on the growing point. This was done twice a day (one
in the morning and the other in the evening) for six days.
The following tasks were completed : the botanical characteristics were identified and the
number ofchromosomes ofthe root tip cells were examined and counted. After the seeds of the
above mentioned plants were obtained and treated, they were sowed in the Autumn ofthe same
year. Good plants were selected when ripe. Each variety ofseed was sowed and harvested at the
same times.
In 1990, the seeds of the three varieties of autotetraploid tartary buckwheat and its diploid
parent stock were respectively sowed in large fields in holes 35 cm apart. After sprouting, four
plants were retained in each hole. During the seedling stage the peroxidase isozymes of their
leaves were electrophoreticly analyzed and stained with benzidine diaminodiphenyl.
Photographs were taken immediately [11]. The following tasks were completed: the botanical
characteristics were identified and recorded ; leaf guard cells, pollen grains and the sizes of
their root tip mitotic cells were examined, and measured during flowering period. The seeds
were dried when they were ripe and the amounts of amino acids in the seeds of both the
autotetraploid tartary buckwheat and its parent stock were measured with a Hitachi 835-50
High Rate Amino Acid Analyzer. The amounts of nutrients-crude proteins, fats, starches and
vitamins-were also measured according to the related methods ofthe International Association
ofSeed Testing.
Results and Analyses
1. The Effect ofTreatment by the Solution Mixture ofColchicine and Dimentyl Sulfoxide
Among the treated plants, some died ofpoisoning. In the surviving plants the variety SHICHEE
proved to be the most successful. It's doubling percentage was 62.81%, and the effect of the
treatment was also the best being 47.50. However, the doubling percentage and treatment effect
ofthe variety KUCHIQIAO were relatively poor (see Table 1).
The growing point on the main stalk ofthe successfully doubled plant expanded and stopped
growing taller. 7-12 days after being treated, 2-5 shoots grew from the swollen knot and the
plant appeared to be mosaic. Some shoots were creepers, and grew many wild roots from the
knot. The plant was shorter, leaves became thicker, leaf colour became deeper and there was a
marked change in the shape of the leaves. There were two types of flower on the plant. The
diameter ofthe corolla on one flower being larger than the other, and the resulting seeds being
larger from the larger flower. The small seeds from the small flower on the mosaic were similar

315
to the seeds from 'the parent stock (contrast) (see Table 2). After eXamination 'under a
microscope, it was found that the number of chromosomes in the root tip cell ofthe'largerseed
was 2n = 4x = 32. It was autotetraploid. The number ofchromosomes in. the root tip cell ofthe
small seed on both the parent stock and the mosaic was 2n = 2x = 16. They were diploids (see
Picture I, II)
Table I. Survival rate and chromosomes-doubling ofdifferent plants.
Variety Treated No. Survived No. Dead No, Successfully- Doubling-Rate Treatment Effect
Doubled No.
Eluowuqie 160 107 23 64 59.81 -<strong>40.</strong>00
Shichee 160 121 29 76 62.81 47.50
Kuochiqiao 160 110 34 61 55.45 38.13
Note: Any plant from which more than one doubled seed was harvested means that the plant was successfully doubled.
Doubling Rate = Successfully doubled No. I Survived No,
Treatment Effect =Successfully doubled No, I Treated No,
Table 2. A comparison on flower and seed between doubled plants and CK plants.
Variety Corolla Dia. (mm) Seed L.x W. (mm),
Eluowuqie diploid (CK) 3.29 5.2 x 3.7
Eluowuqie mosaic 2x 3.27 5.4 x 3.6
4x 4.41 6.9 x4,9
Shichee diploid (CK) 3.38 5.8 x 3.2
Shichee mosaic 2x 3.34 5.7 x 3.0
4x 4.75 7.9 x 3.6
Kuciquiao diploid (CK) 3.16 5.0 x 4.7
Kuciquiao mosaic 2x 3.17 4.8 x 4.6
4x 4.59 7.1 x 5.9
2. Main Characteristics ofthe Tetraploid Tartary Buckwheat
Compared with the diploid parent stock (CK), the tetraploid has a taller plant, thicker stalk,
many shoots on the main stalk. less leaves but larger in size. The seed-forming rates of aU the
tetraploid tartary buckwheat are decreased. The average seed-fonning rate of Eluowuqie
tetraploid is 24.12%, 16.05% less than the contrast rate 28.73%. The average seed-fonni!1g rate
of Shichee tetraploid is 27.1%, 10.56% less than the contrast rate. However, what merits
attention. is that, because of the smaller fJowers of the single plant of tetraplo.id tartary
buckwheat being notably increased (about 30-50%), its seeds from a single plant are still more
than that from diploid parent stock. In addition, the se~ds of tetraploid' tartary buckwheat are
remarkably enlarged. Thus the yield ofa single plant is higher than that of diploid tartary
buckwheat (see Table 3).

316
Table 3. A comparison between the botanical characteristics oftetraploid tartary buckwheat
and diploid tartary buckwheat.
Plant
Stem
Seed-
Height Diameter Shoots Leaves of LeafArea No. of Weight of fonning
Varietry (cm) (mm) No. a Plant (cm 2 ) Seeds Seeds rate
Eluowuqie 4X 84.3 6.8 5.1 101.3 1406.7 261.3 7.86 24.12
Eluowuqie 2X 71.7 4.2 3.9 120.7 1027.4 247.5 4.83 28.73
Shichee4X 67.5 5.9 4.7 89.5 1250.3 1250.3 307.1 27.10
Shichee2X 61.3 3.7 3.6 112.3 998.5 264.5 4.95 30.30
Kuciqiao4X 78.2 6.4 6.6 97.4 1320.8 175.5 5.15 23.16
Kuciqiao2X 70.1 4.7 4.1 127.1 1104.3 164.5 3.31 28.43
For instance, the average seeds from a plant of Shichee tetraploid is 307.1, which is 42.6
more than the contrast (264.5). The average weight ofthe seeds of Shichee tetraploid is 8.41 g,
which is 3.46 g heavier than the contrast (4.95 g). Microscopic examination shows that the leaf
guard cells, pollen grains and the root tip mitotic cells of tetraploid tartary buckwheat are
obviously larger than those ofdiploid parent stock (see Table 4).
Table 4. A comparison between cells oftetraploid and diploid tatrary buckwheat (UM) .
Root Tip Mitotic Cells
Pollen
LeafGuard Cells Cell Nucleus Cell
Variety L % W % Dia. % Dia. % Dia. %
Eluowuqie 4X 36.4 130.47 24.7 122.89 41.1 155.7 19.0 125.0 47.2 156.8
Eluowuqie 2X 27.9 100 20.1 100 26.4 100 15.2 100 30.1 100
Sbicbee4X 38.1 125.74 27.3 129.4 39.9 159.0 17.8 119.5 45.3 139.8
Sbicbee 2X 30.3 100 21.1 100 21.1 100 14.9 100 32.4 100
Note: Data in the table are the mean values of 100 cells.
3. Electrophoretic Analysis on Peroxidase Isozyme of Leaves of Tetraploid Tartary
Buckwheat
After electrophoretic analyses on the peroxidase isozyme of leaves being done during the
seedling stage, it is found that there are some differences between both zymobelt and zymeactivity
of tetraploid and diploid tartary buckwheat. For instance, in the belt with RF value of
0.85, the three varieties of tetraploid tartary buckwheat appear to be weak belt, but the three
varieties of diploids are all mid-strong belt. Numbers of all varieties of tetraploid tartary
buckwheat are respectively less than those of diploid tartary buckwheat (CK). For example, the
Eluowuqie tetraploid has 6 zymobelts while its diploid has 8 zymobelts (see Table 5).

317
Table 5. RF value and zyme-activity ofperoxidase isozyme oftetraploid Tartary buckwheat.
RfValue
Variety 0.33 0.42 0.55 0.58 0.61 0.81 0.85 0.87
Eluowuqie 4X ++ ++ +++ + + ++
Eluowuqie 2X ++ ++ +++ ++ ++ + ++ +
Shichee4X ++ ++ +++ + + +
Shichee 2X ++ ++ +++ ++ + + ++ +
Kuciqiao 4X ++ ++ +++ + ++
Kuciqiao 2X ++ ++ +++ ++ + + + +
Note: +++ = strong belt; ++ = mid-strong belt; + = weak belt.
It is thus clear that the transcription RNA of tetraploid tartray buckwheat was changed
because ofthe increase of gene dosage, and there were differences both in variety and quantity
compared with the diploid tartray buckwheat.
4. Quality ofTetraploid Tartary Buckwheat
It is found, after laboratory tests, that the protein, fat and vitamins content of the tetraploid
tartary buckwheat are all higher than those ofthe diploid, especially vitamin P which increases
considerably. Except methionine, all other amino acids in the tetraploid buckwheat which are
needed by the human body are higher than those ofdiploid tartary buckwheat (see Table 6).
Table 6. A comparison between main nutrients oftetraploid and diploid tatary buckwheat.
Variety E.4X E.2X S.4X S.2X K.4X K. 2X
Crude Protein (%) 14.1 11.7 3.5 10.9 14.5 11.1
Crude Fat (%) 3.11 2.47 3.05 2.64 2.91 2.38
Vit. Bl (mg / g) 0.21 0.17 0.19 0.16 0.23 0.17
Vit. B2 (mg / g) 0.49 0.44 0.51 0.47 0.53 0.46
Vit. P(%) 4.17 2.99 4.43 3.16 4.26 3.04
Thr. (%) 0.54 0.36 0.50 0.33 0.57 0.39
Val. (%) 0.61 0.60 0.57 0.43 0.59 0.57
Met. (%) 0.05 0.06 0.04 0.05 0.03 0.03
Leu. (%) 1.01 0.74 0.95 0.63 1.17 0.81
lie. (%) 0.59 0.43 0.47 0.39 0.60 0.41
Try. (%) 0.17 0.14 0.15 0.10 0.19 0.11
Phe. (%) 0.65 0.50 0.57 0.43 0.69 0.47
Lys. (%) 0.87 0.61 0.74 0.53 0.83 0.44
Note: E. = Eluowuqie ; S. - Shichee ; K. =Kuciqiao.

318
ConclusioD
1. lJp. to now,. colchicine is still one of the 'best agents uS.ed for d.oublingchromosQmes. of
buckwheat. Previous experiments indicate that the best dilution is 0.2% if colchicine
solution only is used to treat buckwheat seedlings. The successful doubling-rate is 3'0-40%
when this kind of solution is used to treat Jiangxi Jiuijiang tartary buckwheat [9], and 20­
35% for common buckwheat [2]. Tests in this paper were conducted by using the mixed
solution ofcolchicine and dimethyl sulfoxide to treat the three varieties oftartary buckwheat,
and a better effect is achieved. The successful doubling-rate reaches 55-65%. This shows
that dimethyl sulfoxide can accelerate colchicine penetrating the cells of the plant quickly,
shortening the treatment time, and improving the effect ofthe treatment. This corresponds to
Bai Shouxin's result [1].
2. The tetraploid tartary buckwheat has a well developed root system, thicker and stronger stalk,
and wider leaves. Its flowerets and seeds are notably increased, and the seeds are much
enlarged. It is probable that this good relationship of co-ordination and balance among
source, flow and storage is one ofthe reasons that tetraploid tartary buckwheat has a higher
plant productivity than diploids.
3. The tetraploid tartary buckwheat has stable inheritance. The microscopic examinations for
three generations continued to exhibit normal gametes (n = 2x = 16) when the sexual cells
divide. The differences in the peroxidase isozymes of leaves between tetraploid and diploid
tartary buckwheats explains that the gene expressions of these two varieties of buckwheat
are not exactly the same. Most autotetraploid plants can not inherit stably. However, Mr.
Yian Yurui and Mr. Zhang Zhaonan recently have achieved very good results on studies on
tetraploid rice and tetraploid barley [2, 12]. This clearly shows that it is still effective to
induce autopolyploid into cultivated crops.
4. TetrapJoid tartary buckwheat has superior characteristics. Its protein, fat, vitamins as well as
amino acid content are all higher than those ofdiploid tartary buckwheat, in which vitamin P
increases the most. Therefore, it is quite evident that tetraploid tartary buckwheat is one
variety of health food which is high in quality and rich in nutrition. It is worthy of further
study and development.

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2. Seeds ofEluowuqie Diploid
3. Seeds ofShichee Tetraploid
4. Seeds ofShichee Diploid
5. Seeds ofKuciqiao Tetraploid
6. Seeds ofKuciqiao Diploid

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