Meiotic behavior in tetraploid populations of Pfaffia tuberosa (Amaranthaceae)

Abstract

Four populations of Pfaffia tuberosa, the Brazilian ginseng, collected in the central region of the state of Rio Grande do Sul (Brazil) were cytologically analyzed for meiotic behavior. All were tetraploid (2n = 4x = 68) and this condition could be observed through multiple chromosome pairing in pachytene and quadrivalent formation at diakinesis. Irregular chromosome segregation in both divisions led to the formation of a wide diversity of meiotic products. Chromosome stickiness was also frequently found among microsporocytes. Pollen grains of different sizes were observed, while pollen sterility ranged from 49.56 to 64.20% among analyzed populations. This is the first cytological report involving chromosome number determination and meiotic behavior for the species.

Full text

Page 1

ActaBot.Croat.63(1),17–24,2004CODEN:ABCRA25
ISSN0365–0588
Meiotic behavior in tetraploid populations of
Pfaffia tuberosa (Amaranthaceae)
ONILDES MARIA TASCHETTO1, MARIA SUELY PAGLIARINI2*
1State University of Western Paraná (UNIOESTE), Department of Biology,
85.814-110, Cascavel PR, Brazil.
2State University of Maringá, Department of Cell Biology and Genetics, 87.020-900,
Maringá PR, Brazil.
FourpopulationsofPfaffiatuberosa,theBrazilianginseng,collectedinthecentralregion
of the state of Rio Grande do Sul (Brazil) were cytologically analyzed for meiotic behav-
ior.Allweretetraploid(2n = 4x = 68)andthisconditioncouldbeobservedthroughmulti-
ple chromosome pairing in pachytene and quadrivalent formation at diakinesis. Irregular
chromosomesegregationinbothdivisionsledtotheformationofawidediversityofmei-
oticproducts.Chromosomestickinesswasalsofrequentlyfoundamongmicrosporocytes.
Pollen grains of different sizes were observed, while pollen sterility ranged from 49.56 to
64.20% among analyzed populations. This is the first cytological report involving chro-
mosome number determination and meiotic behavior for the species.
Key words: Pfaffia tuberosa, chromosome, polyploidy, meiosis, pollen, sterility
Introduction
PfaffiatuberosaisanativespeciesfromthestateofRioGrandedoSul,southernBrazil,
with wide occurrence in the Santa Maria municipality. It is an herbaceous plant with a sub-
terranean ligneous system primarily composed of roots. Roots show alternate tuberose and
non-tuberose zones. Root tuberosity is caused by the growth in the number of parenchy-
maticcellsbetweentheconductingelementsinthecenteroftheorganandbythefibro-vas-
cular groups (SIQUEIRA et al. 1993).
Due to its wide use, Pfaffia tuberosa, like P. glomerata, is also known as the »Brazilian
ginseng«. In folk medicine, it is used in the treatment of rheumatic and vascular diseases
andasanaphrodisiactonic(TERÀN1990,SIQUEIRAetal.1993,TANIGUCHIetal.1997).ARENAS
andAZORERO(1977)reportedtheuseofthisspeciesinthetreatmentoffemalesterility.Ac-
cordingtoNISHIMOTOetal.(1986),theecdysteronecontentinP.tuberosaislowerthanthat
in P. iresinoides.
ACTA BOT. CROAT. 63 (1), 200417
* Corresponding address: e-mail: mspagliarini@uem.br

Page 2

Despite its importance for medicinal purposes, few cytological studies have been de-
votedtoP.tuberosaorthegenusPfaffia(TASCHETTOandPAGLIARINI2003a,b).Thecurrent
cytological study reports, for the first time, the meiotic behavior of four Brazilian popula-
tions of P. tuberosa.
Materials and methods
Cytogenetic studies were carried out on four populations of P. tuberosa collected in the
central region of the state of Rio Grande do Sul: the Santa Maria, São Vicente do Sul,
Jaguari and Santiago municipalities where they widespread (Fig. 1).
For meiotic studies, floral buds in the proper stage were collected and fixed in acetic
ethanol (3:1) fixative for 24 hr, transferred to 70% ethanol, and stored under refrigeration
until use. Microsporocytes were prepared by squashing in 0.5% propionic carmine. More
18ACTA BOT. CROAT. 63 (1), 2004
TASCHETTO O. M., PAGLIARINI M. S.
Fig. 1.Morphology of Pfaffia tuberosa (Scale bar denotes 30 cm)

Page 3

than 5.000 cells, including microsporocytes in meiosis I and II, and meiotic products, were
analyzed in each population. Pollen fertility was evaluated also by using 0.5% propionic
carmine. More than 3.200 pollen grains were analyzed in each population. Photomicro-
graphs were taken with Kodak Imagelink-HQ, ISO 25, black and white film.
Results and discussion
The four populations of P. tuberosa collected in the state of Rio Grande do Sul presen-
ted 2n = 68 chromosomes and reported tetraploid in relation to other species of the genus,
P. glomerata, previously analyzed by the same authors (TASCHETTO and PAGLIARINI2003 a),
ACTA BOT. CROAT. 63 (1), 200419
MEIOTIC BEHAVIOR IN TETRAPLOID POPULATIONS OF PFAFFIA TUBEROSA
Fig. 2.Microsporogenesis in Pfaffia tuberosa. a – microsporocyte in pachytene showing multiple
chromosome association. b – diakinesis with a tetravalent. c – metaphase I with precocious
chromosome migration to the poles. d – anaphase I with laggards. e – telophase I with sev-
eral micronuclei. f – telophase I with two nuclei of different sizes. g – telophase I with three
nuclei of different sizes. h, i – metaphases II with a big micronucleous (h) and precocious
chromosome migration to the poles (i). j – anaphase II showing stickiness. l – asynchronous
division metaphase II / telophase II. m – telophase II with some micronuclei.

Page 4

with chromosome number scoring 2n = 34 in nine populations. The polyploid condition of
the species under analysis could be inferred also by the pattern of multiple chromosome
pairing visualized at pachytene (Fig. 2a), and multivalent chromosome association at
diakinesis (Fig. 2b). Quadrivalents were frequently observed.
Consequently to polyploid condition, many abnormalities related to irregular chromo-
some segregation were observed from the metaphase I to the tetrad stage. The percentage
of meiotic abnormalities for each meiotic phase was similar among populations (Tab. 1).
Precocious chromosome migration to the poles at metaphase I and II (Figs. 2c and i), lag-
gardchromosomesatanaphaseIandII(Figs.2dandl)andmicronucleiattelophaseIandII
(Figs. 2e and m) were observed. Micronuclei gave rise to microcytes at tetrad stage (Figs.
3a to h) which, in turn, gave rise to small and sterile pollen grains (Fig. 3i). Pollen sterility
among populations ranged from 49.56 to 64.20%.
Meiotic abnormalities related to irregular chromosome segregation leading to pollen
sterility are a common feature in polyploids. However, chromosomes with precocious mi-
gration to the poles or laggard chromosomes generally remain isolated or in small groups
sothatsmallmicronucleiareformed.Besidesthistypicalbehavior,adistinctivefeaturehas
also been reported in the four polyploid populations of P. tuberosa. Sometimes the amount
of chromosomes irregularly segregated to the poles was so different that two telophase nu-
cleiofextremelydifferentsizeswereobserved(Fig.2f).Thisbehaviorledtotheformation
of a tetrad with two big microspores and two small ones (Fig. 3g). In other cases, chromo-
somes formed two telophase nuclei of different sizes at the same pole (Fig. 2g), giving rise
to hexads with microspores of three different sizes. The amount of different kinds of
segregationalabnormalitieswassogreatthataninfinitediversityofmeioticproductscould
be observed. Figure 3 illustrates only a small parcel of this diversity. In spite of representa-
tive aspects of irregular meiosis shown in Figure 2, certain meiotic products, such as those
represented in Fig. 3a, 3b and 3d, and many others not given in this plate, could not be ex-
plained merely by irregular chromosome segregation. Monads (Fig. 3a), dyads (Fig. 3b),
triads with three microspores of different sizes (Fig. 3d) suggest that restitutional nuclei
may also occur. Another cause of triad formation could be asynchrony in the second divi-
sion, as observed in Figure 3l, where one cell is in metaphase and the other is entering
telophase. If the former fails to enter the cycle, in due time a triad with one 2n microspore
and two n microspores will be formed. The causes and consequences of 2n and jumbo pol-
len formation in P. tuberosa and P. glomerata are better discussed by TASCHETTO and
PAGLIARINI (2003 b).
Another segregational meiotic abnormality observed in a small percentage of cells in
all populations was related to the inability of some bivalents to reach the metaphase plate,
and thus remaining non-congressed. Non-oriented bivalents are very common among
plants. They were reported in Chlorophytum comosum (PAGLIARINI et al. 1993), in some
Paspalumspecies(PAGLIARINIetal.2001)andinsoybean(BIONEetal.2000).Theinability
of chromosomes to congregate on the equatorial plate may be related to the kinetochore.
NICKLAS and WARD (1994) enumerated some factors that might impair the attachment of
kinetochores to the spindle fibers.
Chromosome stickiness, frequently reported among microsporocytes of all popula-
tions, was another important cytological feature of P. tuberosa. Sticky chromosomes are
seen as intense chromatin clustering affecting chromosome segregation. The phenomenon
20ACTA BOT. CROAT. 63 (1), 2004
TASCHETTO O. M., PAGLIARINI M. S.

Page 5

ACTA BOT. CROAT. 63 (1), 2004 21
MEIOTIC BEHAVIOR IN TETRAPLOID POPULATIONS OF PFAFFIA TUBEROSA
Tab. 1. Number of analyzed cells and percentage of meiotic abnormalities in tetraploid populations of P. tuberosa and pollen sterility.
Phases
Abnormalities
No of cells analyzed and percentage of abnormalities/populations
Santa Maria
São Vicente do Sul
Jaguari
Santiago
Metaphase I
Precocious migration to poles
(1545)
23.30
(1525)
19.15
(1691)
10.88
(1515)
18.28
Non-congressed bivalents
5.70
3.02
1.01
3.49
Chromosome stickiness
27.90
19.15
20.05
37.82
Anaphase I
Laggard chromosomes
(201)
7.46
(198)
6.06
(167)
10.18
(212)
4.72
Chromosome stickiness
25.87
18.69
17.56
21.23
Telophase I
Chromosome bridges
(810)
6.92
(795)
7.04
(760)
2.50
(855)
1.52
Micronuclei
39.14
23.43
25.66
27.37
Trinucleate cells
2.60
1.00
0.0
0.94
Prophase II
Micronuclei
(425)
17.65
(830)
2.89
(388)
9.02
(440)
8.18
Chromosome bridges
6.82
0.72
1.29
1.81
Trinucleate cells
2.82
0.48
1.55
1.59
Metaphase II
Precocious migration to poles
(68)
67.64
(152)
27.73
(162)
28.57
(185)
30.28
Anaphase II
Laggard chromosomes
(126)
16.66
(136)
16.24
(131)
20.18
(128)
26.74
Telophase II
Micronuclei
(935)
3.42
(893)
2.64
(901)
1.87
(907)
2.75
Polynucleate cells
2.24
2.27
2.77
2.20
Tetrad
Microcytes
(1095)
41.83
(920)
41.42
(1024)
30.08
1034)
40.62
Polyads
7.30
4.35
2.73
3.48
Pollen sterility
3390
(58.84)
3420
(49.56)
3280
(61.28)
3380
(64.20)