Experientia 36 (1980), Birkh~user Verlag, Basel (Schweiz) 305

Study of callus tissues from different parts of Nigella sativa (Ranunculaceae)

S. Chand and S. C. Roy 1

Chromosome Research Centre, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Calcutta-
700019 (India), 22 May 1979

Summary. Callus tissues were produced from leaf, stem and seed of Nigella sativa in modified Murashige and Skoog's
medium. Depending on the origin of explants, the time of callus initiation and also the properties of the callus tissue were
found to be different, especially regarding chromosome instability.

Plant tissue cultures have become important as an ex- slightly warmed in a 2% aceto-orcein-(N)HC1 mixture of
perimental tool to study many fundamental and applied 9:1 and kept for 1 h before squashing in 45% acetic acid.
problems in morphogenesis, cytology and genetics. Large Results and discussion: The 3 different types of explants
numbers of plant materials of both monocotyledons and were placed in the same medium to compare their growth
dicotyledons have been tried for the initiation of callus responses as well as their morphological and cytological
cultures in different media by different authors 2-4. The behaviour. Callus tissues were initiated from the leaf seg-
establishment of actively growing cultures depends mainly ments very quickly whereas the callus tissues from stem and
on the selection of the explants and on the culture medium. seed appeared more slowly (table 1). These leaf segments
It has also been found that the quantitative and qualitative and stern pieces were cultured from seedlings grown in the
nutritive requirements in vitro may vary with different agar medium, where the growth of the seedlings again took
plant materials and in some instances with different tissues at least 2 3 weeks. In the case of seed the total time of
of the same species. Another important point is that cul- growth for callus was less. The rate of growth in the case of
tured cells of both plant and animal tissues are charac- leaf segments was slightly better than that of seed callus.
terized by the instability of chromosome number and Callus proliferation in stem explants was very slow as
structure. According to Partanen 5'6 this chromosomal in- compared to other segments. Callus from leaf segments
stability in culture depends upon the type of explant from showed initiation of a vigorous, proliferating, soft and
which the callus originated. The present investigation was green-coloured tissue. A greenish-yellow and a yellow
undertaken to study the morphological and cytological colouration were characteristic of tissues grown from stem
behaviour of callus tissues of Nigella sativa derived from pieces and seeds respectively.
different explants. This species was chosen because of its During the proliferation of calluses from different parts of
low chromosome number (2n= 12) and large chromo- the plant, a number of changes in structure and number of
somes. chromosomes were noted. A high frequency of the normal
Seeds of Nigella sativa were surface sterilized in 0.1% number occurred mainly in calluses from leaf segments
mercuric chloride for 20 rain and rinsed several times in (table 2). The maximum chromosome number in leaf
sterile medium containing only 0.8% agar, and were kept in calluses was found to be 36, while up to 72 chromosomes
the dark. After 2-3 weeks seedlings were formed. Leaves were observed in stem calluses. The maximum frequency of
obtained from these seedlings were cultured in a medium tetraploid mitoses occurred in seed calluses. Fragmentation
N-1 which is a modification of Murashige and Skoog's 3 of chromosomes and micronuclei were observed in all
medium..Cultures were maintained in 16 h/8 h light-dark cases. Chromosomal instability thus appeared to be higher
photoperiod at 2 2 - 2 4 ~ Callus was als0 obtained directly in stem and seed calluses than in leaf calluses. From this
from seeds, whereby sterilized seeds were placed in N-1 point of view, il may be suggested that the leaf callus is
medium. These seeds were kept in complete darkness. better suited for tissue ctilture investigations than other
For cytological observations, callus tissues of stem, leaf and types of callus. Similar changes in ploidy level have been
seed were taken just after placing in light conditions for 2 h reported in differentiated tissues of many species 7"8.
and were fixed in Carnoy's fluid overnight. They were then

Table 1. Growth pattern and nature of callus in Nigella sativa

Plant parts used for initiation of callus: Leaf Stem Seed
Time taken for initiation of callus (days): 5-6 8-10 22-30
Time taken for full development of callus (days): 22-28 30-35 35-60
Colour of callus: Green Yellowish-green Yellow
Growth rate of callus after 3 weeks: +++ + ++
Nature of the callus: Soft Soft More compact

Table 2. Percentage of metaphases with various chromosome numbers (2 n = 12) in initial passage of leaf, stem and seed callus tissue from
Nigella sativa
Origin Chromosome numbers
of callus 12 24 36 48 72 aneuploids
Leaf 55.56 38189 5.56
From 1 to 6 micronuclei in many cells; fragments present.
Stem 33.33 29.16 25.00 8.33 4.16 -
From 1 to 5 fragments in some polyploid ceils.
Seed 24.49 51.02 18.37 4.08 2.04
From 1 to 6 fragments in many polyploid cells.
306 Experientia 36 (1980), Birkh~iuser Verlag, Basel (Schweiz)

The differential response of the different explants in the investigation tetraploid cells occur at m a x i m u m frequency
same m e d i u m may be due to changes in the physiological in seed calluses, while a tendency toward diploidy was
conditions of the material. Although there is evidence that found in leaf calluses. Although Partanen 5'6 reported that
polyploid cells may arise from endoreduplicated nuclei in chromosomal instability in culture depends upon the type
the original explant, the range of c h r o m o s o m e n u m b e r and of plant parts from which the callus originated, the present
structure observed in established cultures strongly points to investigations show variation in c h r o m o s o m e n u m b e r in all
the origin of these changes during culture. In the present the 3 different explants cultured in the same medium.

1 The authors are grateful to Prof. A.K. Sharma for his advice 6 C.R. Partanen, in: Proceedings of International Conference on
and suggestions during the course of work. Plant Tissue Culture. Ed. P.R. White and A.R. Groove.
2 P.R. White, The Cultivation of Animal and Plant cells. Ronald McCutchan, Berkeley, Ca. 1965.
Press, New York 1954. 7 N. Sunderland, in: Plant Tissue and Cell Culture, p. 177. Ed.
3 T. Murashige and F. Skoog, Physiol. P1. 15, 473 (1962). H. E. Street, Blackwell, Oxford 1977.
4 H.E. Street, Plant Tissue and Cell Culture. Blackwell, Oxford 8 W.F. Sheridan, in: Genetic Manipulation with Plant Materials,
1977. p.263. Ed. L. Ledoux, Plenum Press, New York 1975.
5 C.R. Partanen, Int. Rev. Cytol. 15, 215 (1963).

P r e s e r v a t i o n o f Bordetella pertussis

Sarita Sood Angra, G. Saran and P. G u p t a 1

Triple Vaccine Division, Central Research Institute, Kasauli, H.P. 173205 (India), 20 June 1979

Summary. Bordetella pertussis has been shown to remain viable for a period of 13 years on freeze-drying. The revived
cultures had unaltered antigenic composition and biological activity.

Frequent subculture of a microbial species is liable to result effect of an inert gas on the viability o f cultures, gaseous
in change of antigenic and other characters. Therefore, nitrogen was passed into some o f the tubes before sealing.
maintaining viability of an organism for longer periods All tubes containing freeze-dried ceils were stored at
without resorting to repeated subcultures is highly desir- 4-10 ~ until used. The viability o f the cultures was tested
able. Freezing at - 7 0 ~ with 15% glycerol was found to be on duplicate tubes of B o r d e t - G e n g o u (BG) medium. Tubes
a satisfactory means of preservation o f Treponema pallidum were incubated at 35 ~ for a m a x i m u m of 96 h. Antigenic
and other bacteria 2. The factors affecting the death rate of composition of B G - g r o w n cultures was checked with B. per-
Escherichia coli at temperatures between - 1 . 5 ~ and tussis monospecific factor 1, 2, and 3 sera by s i d e aggluti-
- 1 9 5 C have been studied 3. Reports of the preservation o f nation method 6. Test for dermonecrotic activity was per-
B.pertussis for 45 months in 15% glycerol at - 7 0 ~ and of formed in rabbits. 0.1 ml of a suspension containing
freeze-dried cultures for 10 years are available 4,5. The pre- 1.0• 109 organisms/ml was inoculated intradermally. Re-
sent communication reports the preservation of B. pertussis suits were recorded at 48 and 72 h. The m a x i m u m reaction
for a period of 13 years. obtained has been reported.
Materials and methods. Strains 134 and 509 routinely used Result and discussion. The findings have been summarized
by us for the manufacture of pertussis vaccine were sus- in the table. A freeze-dried culture under v a c u u m showed
pended separately either in L o m o d e x (10% dextran in 5% viability upto 13 years - the m a x i m u m period of observa-
dextrose, Rallis India Ltd) or aqueous gum acacia solution. tion in this study. Revived cultures of strain 134 agglutinat-
0.2 ml o f the culture suspension containing approximately ed with factors 1 and 3 and those of strain 509 with factor
200x 109 organisms/ml were freeze-dried in 100x 8 m m 1, 2, and 3 sera. This is in agreement with the k n o w n anti-
sterile glass tubes and sealed under vacuum. To study the genic composition of the 2 strains. The cultures from the

Viability and other characteristics of B.pertussis cultures after various periods of freeze-drying
Strain Date of Conditions of freeze-drying Growth up to 96 h Agglutination with factor sera Dermone-
freeze- at 35 ~ crotic
drying Suspending Gaseous BG BG reaction
fluid condition Tube I Tube II 1 2 3 (mm)
509 March, 66 Gum acacia Vacuum + + + + + 10• 10
509 July, 72 Gum acacia Vacuum + + + + + 15• l0
509 July, 72 Lomodex Nitrogen - -
509 April, 76 Lomodex Vacuum + + + + + 10x 10
509 April, 76 Lomodex Nitrogen - -
509 April, 79 Lomodex Vacuum + + + + + 15 • 10
134 July, 66 Gum acacia Vacuum + + + - + 15• 10
134 July, 72 Gum acacia Vacuum + + + - + 15• 10
134 July, 72 Lomodex Nitrogen - -
134 April, 76 Lomodex Vacuum + + + - + 20x 10
134 July, 76 Lomodex Nitrogen - -
134 April, 79 Lomodex Vacuum + + + - + 15x 10
+ = Growth, agglutination; - = no growth, no agglutination.