Karyomorphological studies in two species of Allium L.

Karyomorphological studies in two species of Allium L.
Keywords:
Allium wallichii Kunth., Allium hookeri Thw., karyomorphology, chromosome
classification, karyotype formula, metacentric.
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Journal of Research in
Plant Sciences
An International Scientific
Research Journal
Authors:
Harinita Toijam,
Borah SP, Bhaben Tanti
and Borthakur SK.*
Institution:
Department of Botany,
Gauhati University,
Guwahati-781014,
Assam, India
Corresponding author:
Borthakur SK.
Email:
Web Address:
http://plantsciences.info/
documents/PS0060.pdf.
Dates:
Received: 31 May 2013 Accepted: 03 July 2013 Published: 31 July 2013
Article Citation:
Harinita Toijam, Borah SP, Bhaben Tanti and Borthakur SK.
Karyomorphological studies in two species of Allium L.
Journal of Research in Plant Sciences (2013) 2(2): 213-221
An International Scientific Research Journal
Original Research
JournalofResearchinPlantSciences
Journal of Research in Plant Sciences
213-221 | JRPS | 2013 | Vol 2 | No 2
www.plantsciences.info
ABSTRACT:
A detail karyomorphological study was undertaken in Allium wallichii Kunth.
and Allium hookeri Thw. The study included determination of somatic chromosome
number, total chromosome length, volume, arm ratio and centromeric position. The
karyotypic study of Allium wallichii Kunth. and Allium hookeri Thw. showed that,
both of them have somatic chromosome number 2n=16 and 2n=22 respectively.
Chromosome classification was done on the basis of the position of the centromere.
In case of A. wallichii, the total chromosome length was found to be 150.94 µm while
the relative chromosome length varied in between 4.22 µm - 8.19 µm. The arm ratio
was varied from 1.00 - 3.06 .In A. wallichii, out of 16 somatic chromosomes, 9 were of
metacentric, 6 were of sub-metacentric and 1 was of sub-telocentric type. For
A.hookeri, the total chromosome length was found to be 164.05 µm and the relative
chromosome length was varied in between 2.59 µm- 6.95 µm; arm ratio also varied
from 1.00 - 4.36. Out of 22 somatic chromosomes, 2 chromosomes were of
metacentric, 13 were of sub-metacentric and 7 were of sub-telocentric type. No
satellite chromosomes were found in any one of the two species. The karyotype
formulae deduced for the two species of Allium may be represented as: A. wallichii
Kunth.: m9 +sm6 +st1 +t0 = 2n = 16 and A. hookeri Thw.: m2 +sm13 +st7 +t0 = 2n = 22.

INTRODUCTION
Allium wallichii Kunth. and Allium hookeri
Thw. belong to the family Liliaceae. Recent estimates
accept about 750 species in the genus Allium (Stearn,
1992), and 650 more synonymous species names exist
(Gregory et al., 1998). Alliums are perennial bulbous
plants that produce chemical compounds known as
cystein sulfoxide. And these sulfur containing
compounds give them a characteristics onion or garlic
taste and smell and many of them are used as food
plants. Many species of Allium are bulbous as well as
there are non bulbous species and variety also and many
of them have ornamental values. The young and tender
leaves of A. wallichii are used as vegetables and the dried
ones are used as spices (Manadhar, 1980; Dobremez,
1982). The bulbs are also boiled, fried with ghee and
used for the treatment of cholera and diarrhea (Coburn,
1984). A. hookeri also have some medicinal values. They
are used for treating cold and cough, for healing burn
injuries and wounds (Kala, 2005) and lowering the high
body temperature (Singh et al., 2003). The fresh leaf and
root extract is taken for anthelmintic and also used for
messaging (Sangtam et al., 2012).The bulbs and leaves
of A. cepa, A. sativum, A. cepa var. aggregatum,
A. fistulosum and A. chinense are used as vegetables and
as component of soup. Not only the members of this
family include many vegetables but also some of them
have great medicinal value, e.g. Allium sativum,
Allium hookeri, etc. The status of chromosome research
has undergone fundamental changes in recent years from
an emphasis on qualitative description of structure to an
interdisciplinary phase involving quantifications of
chemical components, identification of segments, even at
the molecular level, as well as in situ cytophotometry,
fluorometry and structural cytology. And also the
application of these techniques led to a more in depth
understanding of the structure, number and behavior of
chromosomes and such a useful study of chromosomes
serve as an aid for dealing with the systematic
relationships, phylogeny and evolution of related plant
groups and also helps to find out the possible modes of
karyotypic variation within and between species
(Mathew and Mathew, 1982 and Aswathanarayana,
2003). In fact, it cannot be denied that all these basic
aspects lay the foundation of the entire discipline of the
present day molecular genetics. So, the various aspects
of research, application of tissue culture, investigation on
molecular genetics and genetical engineering are based
on the basic chromosome research for better utilization
of plants towards commercial cultivation.
Chromosomal differences reflect the general
differences in genic content of the individuals. Thus,
chromosome morphology may be used to establish the
distinctiveness of individual plant species. Improving the
quality of this crop plant to meet the different needs
would involve hybridization programme. So in order to
improve these plants we need to know the thorough
knowledge of chromosome structure and number which
will facilitate the development of an appropriate
hybridization programme and thereby we can manipulate
both the chromosome structure and number for the
genetic improvement of such an important crop plant.
Despite the importance of these two species of
Allium in terms of their culinary as well as medicinal
values, they are poorly explored scientifically with
regard to survey, characterization and conservation.
Some cytological as well as taxonomic investigation of
certain species of Allium has been made but they are so
far quite inadequate. No systematic karyomorphological
classifications of chromosomes have been available for
them, except a few earlier reports (by Ved Brat, 1965).
Therefore, the aim of this study is to provide more
information on the mitotic chromosome in
Allium wallichii Kunth. and Allium hookeri Thw. and to
carry out the karyotypic analysis.
214 Journal of Research in Plant Sciences (2013) 2(2): 213-221
Toijam et al.,2013

MATERIALS AND METHODS
The species of plant Allium: Allium wallichii
Kunth. and Allium hookeri Thw. were collected from
Darjeeling and Manipur (India) respectively.
Methods for cytological study
For detail karyotypic studies, karyotypes were
prepared from the somatic chromosomes. For cytological
studies, root tips were collected from the plants between
7.30a.m to 8.30a.m. and washed them thoroughly with
distilled water and then they were pretreated with para-
dichlorobenzene for 3 hours at 4° C. Then, fixation was
done in Carnoy’s fluid (1:3 glacial acetic acid and
ethanol) for 24 hours at room temperature. After fixation
the root tips were washed with 70% ethanol and finally
stored in 70% ethanol.
Hydrolysis and staining of chromosomes
For preparation of slides, root tips were first
hydrolyzed in 0.1 N HCl at 60° C for 10 -12 mins and
then washed with double distilled water and then they
were kept in 45% acetic acid for 5 – 7 mins and then
transferred to 2% aceto orcein solution, warmed over a
flame for 15 mins and kept for 2 -3 hours at room
temperature (Sharma and Sharma, 1980).
Squash preparation
For preparation of squash the meristematic parts
of root tips were cut out and transferred to a small drop
of 45% acetic acid on a grease free clean slide; then
covered with a cover slip and squashed continuously
without heating and finally sealed with paraffin. Then
temporary slides were prepared and observed under a
compound microscope at a magnification of 10X, 45X
and 100X (using oil immersion). Well scattered
metaphase stages were selected for karyomorphological
analysis of the chromosomes by using camera lucida
apparatus. Drawings of the chromosomes were also
made. The following parameters were considered for the
karyomorphological analysis:-
(i) Total chromosome length (ii) Arm ratio =
Length of long arm/Length of short arm (iii) Volume of
the chromosome (πr2
h) (iv) Relative length of the
chromosome = (Length of the individual chromosome/
Total chromatin length of the diploid set) X 100. (v) On
the basis of length, chromosomes will be categorized as:
Type A, Type B, Type C, Type D, Type E and Type F
(vi) F% = (Length of the short arm / Total length of
individual chromosome) X 100. (vii) Total form percent
or TF % = (Total sum of short arm length / Total sum of
chromosome length) X 100. (viii) On the basis of the
centromeric position, the chromosomes may be classified
into metacentric, sub - metacentric, sub - telocentric, and
telocentric (Levan et al., 1964; Tanti et al., 2009; Tanti
et al., 2012).
The chromosomes were grouped into different
categories depending upon their length. These were Type
A= 11.00µm and above, Type B = 10.00µm – 10.99µm,
Type C = 9.00µm – 9.99µm, Type D = 8.00µm –
8.99µm, Type E = 7.00µm – 7.99µm, Type F = 6.99µm
and below.
Finally the chromosomes were classified on the
basis of their centromeric position by following Levan
et al., (1964).
RESULTS
The detail karyomorphological analysis of the
two species of Allium L. is given below:
Allium wallichii Kunth.:
The chromosome no. of Allium wallichii Kunth
was found to be 2n=16 in the somatic cells. The
chromosome length varied from 6.38µm to 12.36µm
while their volumes ranged from 5.00µm3
to 14.96µm3
.
The relative length of the chromosomes varied from
4.22µm to 8.19µm. On the basis of the length, the
chromosomes were classified into Type A, Type B,
Type C and Type D (Table 1). The total genomic
chromosome length was found to be 150.94 µm. The
different types of chromosomes categorized on the basis
of the length are represented as:
Journal of Research in Plant Sciences (2013) 2(2): 213-221 215
Toijam et al.,2013
A6 +B2 +C0 +D2 +E3 +F3 = 2n= 16

Toijam et al.,2013
Chr.
Type
Chr.
No.
ChromosomeLengthTotal
Length
(l+s)µm
RelativeChr.
Length(µm)
Arm
Ratio(l/s)
ChromosomeCentromeric
Index(F%)
Positionof
Centro-mere
Nomenclature
ofChromosome
LongArm
(l)µm
ShortArm
(s)µm
Radius
(r)µm
Vol.(πr2
h)
µm3
A18.164.2012.368.191.940.6013.9733.98smSubmetacentric
B76.443.8110.256.791.690.559.7337.17mMetacentric
B86.403.8010.206.751.680.558.9937.25mMetacentric
D95.443.408.845.861.600.537.5038.46mMetacentric
D105.443.408.845.861.600.527.5038.46mMetacentric
E116.001.967.965.273.060.526.7524.62stSubtelocentric
E124.713.127.835.191.510.526.6539.84mMetacentric
E133.513.507.014.641.000.525.5049.92mMetacentric
F143.502.966.464.271.180.505.0745.82mMetacentric
F153.482.906.384.221.200.505.0045.45mMetacentric
F163.482.906.384.221.200.505.0045.45mMetacentric
Table1.AlliumwallichiiKunth.

These 16 somatic chromosomes comprised of 9
metacentric chromosomes, 6 submetacentric
chromosomes and 1 subtelocentric chromosome. The
karyotypic formula for Allium wallichii Kunth. is
represented as:
The microphotograph of the chromosomes are
represented in Fig. 1. The idiotype and camera lucida
diagram are shown in Fig. 3 and 5A respectively.
Allium hookeri Thw.:
The chromosome no. of Allium hookeri Thw.
was found to be 2n=22 in the somatic cells. The
chromosome length varied from 4.25µm to 11.41µm
while their volumes ranged from 3.34µm3
to 12.89µm3
.
The relative length of the chromosomes varied from
2.59µm to 6.95µm. On the basis of the length, the
chromosomes were classified into Type A, Type B, Type
C and Type D (Table No.2). The total genomic
chromosome length was found to be 164.05 µm. The
different types of chromosomes categorized on the basis
of the length are represented as:
These 22 somatic chromosomes comprised of 2
metacentric chromosomes, 13 submetacentric
chromosomes and 7 subtelocentric chromosomes. The
karyotypic formula is represented as:
The microphotograph of the chromosomes is
represented in Fig. 2. The idiotype and camera lucida
diagram are shown in Fig. 4 and 5B respectively.
DISCUSSION
Karyomorphology and chromosome number of a
variety or species are useful in its identification. Each
plant species is characterized by its karyotype. The
importance of karyotype analysis in different plant
species is well known. Karyomorphology and
chromosome number of a variety or species are useful in
its identification and also in establishing the relationships
among related species .The study of variation in
chromosome morphology indicates the progress of
microevolution and to some phenomenon of speciation.
Chromosomal differences are the sources of
genetic variation. Karyomorphology and chromosome
number of a variety or species are useful in its
Toijam et al.,2013
M9 +sm6 +st1 +t0 = 2n = 16.
A2 +B2 +C0 +D4 +E4 +F10 = 2n= 22
m2 +sm13 +st7 +t0 = 2n = 22.
Figure 1. Microphotograph of chromosomes of
Allium wallichii Kunth. 2n=16
Figure 2. Microphotograph of chromosomes of
Allium hookeri Thw. 2n=22
Figure 3. Idiotype of Allium wallichii Kunth. (2n=16)

Toijam et al.,2013
Chr.
Type
Chr.
No.
ChromosomeLengthTotal
Length
(l+s)µm
Relative
Chr.Length
(µm)
Arm
Ratio(l/s)
ChromosomeCentromeric
Index(F%)
Positionof
centro-mere
Nomenclatureof
Chromosome
LongArm
(l)µm
Short
Arm(s)
µm
Radius
(r)µm
Vol.(πr2
h)
µm3
A19.262.1511.416.954.300.6012.8918.84stSubtelocentric
A29.262.1511.416.954.300.6012.8918.84stSubtelocentric
B38.512.2410.756.553.790.6012.1520.83stSubtelocentric
B48.402.1410.546.423.920.6011.9120.30stSubtelocentric
D56.452.518.965.462.560.558.5128.01smSubmetacentric
E94.003.927.924.821.020.557.5249.49mMetacentric
E103.923.907.824.761.000.557.4250.00MMetacentric
E115.102.147.244.412.380.556.8729.55smSubmetacentric
E125.022.147.164.362.340.505.6229.88smSubmetacentric
F134.981.416.393.894.360.505.0122.06stSubtelocentric
F144.381.776.153.742.470.555.8428.78smSubmetacentric
Table2.AlliumhookeriThw.

identification and also in establishing the relationships
among related species (Lavania and Srivastava, 1999;
Liu et al., 2000; Frame, 2001; Sharma and Sen, 2002;
Salimuddin and Ramesh, 2005; Ying et al., 2006). In the
present investigation, karyotype analysis of A. wallichii
Kunth. showed that the species contain diploid
chromosome number, 2n=16 and A. hookeri Thw.
contain chromosome number, 2n=22. The
karyomorphological studies in both the species of
Allium L. revealed a number of features. The cytological
study reveals that the length of the chromosomes and
gross appearance of the karyotype show a general
resemblance in both the species investigated but, upon
closer examination the karyotypes however showed
variations in the type of chromosomes, absolute
chromosome size, variation in the position of centromere
and variation in relative chromosome size. According to
Ved Brat (1965), most Allium species possess
metacentric to submetacentric types of chromosomes,
but some subtelocentric and telocentric chromosomes are
also present in few species. With respect to chromosome
morphology the karyotype of A. wallichii have a majority
of metacentric and submetacentric types of chromosomes
with the former being more frequent. This shows the
symmetry in morphology of the chromosomes which is a
reflection of relatively primitive karyotypes of the
members of this genus (Stebbins, 1971). In case of
A. hookeri, the chromosomes mostly consist of
submetacentric and subtelocentric chromosomes. It
consists of 13 submetacentric, 7 subtelocentric and only
2 metacentric chromosomes. So it may be described as
asymmetrical, since the karyotype consisting of more
numbers of submetacentric and subtelocentric
chromosomes may be termed as asymmetrical (Stebbins,
1971). The evolution of karyotype is a continuous
process as Jones (1978) has pointed out that the
karyotype alterations during course of evolution are
brought about by various structural alterations of
chromosomes. This implies that the more number of
submetacentric and subtelocentric chromosomes in
A. hookeri Thw. has shown that the genome of this
species has undergone some amount of structural
modifications. This increase in the number of
submetacentric and subtelocentric chromosomes of this
species may be attributed to the phylogenetic changes
occurring in the length of the chromosomes that causes
shortening in the size of the arms leading to the shifting
of the centromeric position as well as reduction in the
absolute length of the chromosomes (Delaunay, 1926).
No secondary constriction could be detected in
any one of the chromosomes of the two species under
investigation.
Toijam et al.,2013
Figure 4: Idiotype of Allium hookeri Thw. (2n=22). Bar indicates 10µm
Figure 5: Camera lucida diagram of Allium
wallichii Kunth. and Allium hookeri Thw.

CONCLUSION
It is an established fact that karyomorphological
analysis forms a prerequisite for the genetic
improvement of any plant species. Therefore, the
detailed cytological investigations undertaken in these
two species A. wallichii. and A. hookeri will contribute
significantly towards their genetical improvement.
Realizing their importance and utility, ex-situ
conservation measures may be initiated for protection,
preservation and regeneration of such a remarkable plant
species. It may further help in research such as breeding
programme, gene exploitation as well as germplasm
conservation.
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