Plant Science Letters, 2 (1974) 89--94

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

GROWTH,EMBRYOGENIC POTENTIAL AND STABILITY

OF A HAPLOID CELL CULTURE OF ATROPA BELLADONNA L.

A. RASHID* and H.E. STREET

Botanical Laboratories, University of Leicester (Great Britain)
(Received August 13th, 1973)

SUMMARY

Haploid cell suspension cultures of A tropa belladonna, initiated from a

plantlet of anther origin, grew well in a medium developed for diploid sus-
pensions and containing 0.1 mg/1 (ca. 5-10-7M)kinetin (K)and 1.5 mg/1
(ca. 10 -s M) naphthaleneacetic acid (NAA). Such suspensions remained pre-
dominantly haploid and highly enbryogenic during at least five successive
subcultures but ultimately showed a declining proportion of haploid cells and
a loss of embryogenic potential. When such suspensions were treated with
colchicine (1 g/l) for 24 h and subsequently transferred to auxin-free medium,
they grew and gave rise to a reduced number of embryoids, 70% of which
were diploid. Raising the level of K to 10 -s M cau, c~ poor separation of cells
in liquid medium and the rapid departure from haploidy.

INTRODUCTION

The technique of anther culture is an important source of haploid plantlets

and these in turn of haploid tissue and cell cultures. As a basis for the production
of homozygous mutants via such haploid cell cultures it is important to be able
(i) to achieve active growth of suspensions of haploid cells, (ii) to convert
mutant haploid cells into homozygous diploids, and (iii) to induce plantlet
formation from the cell cultures before and after diploidisation. We have pre-
viously described the control of embryogenesis and organogenesis in diploid
cultures of Atropa belladonna L. ~,2 and the production of haploid plants via
anther culture in this species 3. The present study has been concerned with
the growth and stability of a haploid cell line of Atropa belladonna, with
demonstration of its embryogenic potential and with its diploidisation.

* British Council Research Fellow from the University of Delhi.

Abbreviations: K, kinetin; NAA, naphthaleneacetic acid; SSM, standard synthetic medium.

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 bIATERIALS AND METHODS

Callus cultures were established from stem explants of haploid and diploid
plants on semi-solid (0.5% Difco bactoagar) SSM (as described by Thomas
and Street 4) supplemented with 2 mg/l NAA, 0.5 mg/l K, 100 mg/l m e s o -
inositol, 790 mg/l ammonium sulphate, vitamins after White s and 2% sucrose.
The haploid plants were obtained through anther culture and the present
study is based on clonal derivatives of a single such plant maintained by
meristem culture 3. Suspension cultures were initiated by transference of ca.
5.0 g callus to a 250-ml flask containing 60 ml liquid medium (differing from
the callus medium in containing 0.1 mg/1 K and 1.5 mg/l NAA) and agitation
during incubation on a horizontal platform shaker (120 rev./min horizontal,
throw 2 inch). Incubation was at 25 ° and at a light intensity of 1000 lux at
the platform surface. The suspensions were subcultured by transferring an
aliquot (0.1 culture volume) of stationary phase culture to new medium. Cell
number, packed cell volume and cell dry weight were determined on suspen-
sion cultures as described by Henshaw e t al. 6.
For chromosome counts aliquots of cells from suspension culture in their
exponential phase of growth were pretreated for 3 h with a saturated solution
of p-dichlorobenzene, fixed in acetic alcohol (1:3), hydrolyzed with 1 N HCI
for 10 min and stained with Feulgen reagent. The results are based on at least
100 metaphase counts from 5 or more slides. Cells in stationary phase were
used for DNA determinations. Cells were fixed overnight in 50% v/v formic
acid, washed with distilled water, hydrolyzed with 1 N HCI for 10--12 min
and stained with Feulgen reagent for 2 h, washed with SO2 water and mount-
ed in 25% aqueous acetic acid before squashing to prepare a monolayer. The
slides were made permanent and 100 nuclei were scored in the Vickers M85
scanning-microdensitometer. To obtain homozygous diploid plants the cells
in suspension were treated in their exponential phase of growth with filter-
sterilized colchicine solution in culture medium and chromosome counts were
made on root tips from the plantlets produced following transfer to auxin-
free medium ' .

RESULTS AND DISCUSSION

Growth of newly initiated suspensions of diploid and haploid cells was

studied over 28 days' incubation. Both sets M cultures were initiated at a
density of ca. 1.5-10 s cells/ml. Maximum cell numbers were reached by day
20 of culture at which time the diploid culture contained 2.7.106 cells/ml
and the haploid culture 5.8-106 cells/ml (Fig. 1). The biomass yields (from
28-day cultures in stationary phase) were equal in terms of packed cell volume
(ml packed cells per 10 ml culture) but the cell dry weight was higher in the
haploid culture. As would be expected from the above data, the cells of the
haploid suspension were smaller than those of the diploid (Fig. 2) and they
had thinner cell walls. Some cell debris was detected in the haploid suspension

90
 6-75
6-:30 E

-<..
O

"7"

~" 335-
~0,_270-
~2.25- o
= 180"
(J 1"35-
0-90-
045-
0.15~
i ! i i "t'' f | i i i I' i ! ' ~"" !
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time in days

Fig. 1. Growth of a haploid (H) and a diploid (D) cell suspension culture of Atropa bella-
donna. Packed cell volume (PCV) and cell dry weight measured after 28 days' culture.

following the chromic acid treatment involved in the cell count so that there
may have occurred some cell destruction; if so, the cell numbers fo~ the
haploid culture in Fig. 1 may be underestimates.
When the stationary phase suspensions were subcultured to medium de-
void of auxin they gave a high yield of embryoids {Fig. 2) and the embryoids
forming in the haploid cultures frequently showed superficial adventive em.
bryoids {Fig. 2E).
The suspensions were serially subcultured in the standard liquid medium
containing NAA and then subcultured at the end of each passage to auxin-
free medium to test their embryogenic potential. The diploid cultures showed
a decline of potential to a very low level by the end of the second passage
and when tested after further passages showed no embryogenic potential and
variable but low rhizogenic potential 1. By contrast, the haploid suspensions
retained a high embryogenic potential up to the 5th passage but this declined
steadily in subsequent passages; some rhizogenic potential was retained up to
the end of the experiment (10 passages) and low embryogenic potential was
observed up to the 8th passage. Chromosome counts were made on the haploid
suspension cultures over the 10 passages and on the roots of embryoids prod-
uced during the first 5 passages. Chromosome counts in the range 33--36 were
scored as haploid. These counts revealed that the cultures remained 85--95%
haploid up to passage 7 and then the proportion of haploid cells declined
to reach 50% by passage 10 (Fig. 3). In addition to haploid cells, the cultures
contained cells approximating to diploids and aneuploids between 36 and 72.
The aneuploid cells were abnormally elongated. Observations on mean cell

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Fig. 2. Cellular composition ~nd embryogenic potential of a haploid and a diploid cell sus-
pension culture of Atropa beiladom~a. A, B, Stationary phase cell suspensions. A, diploid;
B, haploid. C, D, Yield of embryoids and roots from a 30-ml culture in auxin-free medium
initiated from 1st passage suspensions. C, diploid; D, haploid. E, A haploid plantlet devel-
oped from the suspension culture and showing adventive embryoids developing from its axis.

size of the cultures agreed with e x p e c t a t i o n from the c h r o m o s o m e counts.

The e m b r y o i d s e x a m i n e d from the first 5 passages were haploid a l t h o u g h
occasional cells in the r o o t s were diploid.
5 ml of the 4 t h passage haploid suspension (3- 106 cells/ml) was t r e a t e d
with colchicine (1 g/l) for 24 h, washed with m e d i u m devoid of colchicine
and auxin and the cells used to initiate a culture in auxin-free m e d i u m . After
a d e t e c t a b l e lag phase this culture showed satisfactory growth and y i e l d e d
a reduced n u m b e r of embryoids, 70% of which were diploid (the r e m a i n d e r
were haploid }.

92
 ~oo]
U

E
8

10-'
h
4 5 6 "'7 I~ § {0
Number of passages

Fig. 3. P e r c e n t a g e h a p l o i d c e l l s p r e s e n t in s u c c e s s i v e p a s s a g e s o f a s e r i a l l y s u b c u l t u r e d cell
s u s p e n s i o n c u l t u r e i n i t i a t e d f r o m t h e h a p l o i d p l a n t l e t o f Atropa belladonna via a c a l l u s
c u l t u r e passage.
40~
A

30-

K 5 x 1£~7M
t-20.

---•
NAA 1()5 M
Passage VIII
8 T -
CL I
I
10,

I~ , f. . . . . . .

..... L . . -. . . . . . -4 . . . . . . . .
2 3 4 5 6 7 8 11 1~3
Arbitrary units of ~NA

20- K ~O'~M B
NAA 10-5 M
Passage T_I
"9 15.
r

~10-

2 3 4 5 6 7 8 9 10 1! 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Arb,trary units of DNA
Fig. 4. M i c r o d e n s i t o m e t e r p r o f i l e s f o r t h e cells o f s u s p e n s i o n c u l t u r e s d e r i v e d f r o m a
h a p l o i d Atropa belladonna p l a n t l e t . A, a c u l t u r e o b t a i n e d b y t h e s t a n d a r d p r o c e d u r e a n d
a n a l y s e d a t t h e e n d o f t h e 8 t h p a s s a g e as a s u s p e n s i o n . B, a c u l t u r e o b t a i n e d b y i n i t i a t i n g
t h e c a l l u s a n d g r o w i n g t h e s u s p e n s i o n in a m e d i u m c o n t a i n i n g a h i g h e r level o f K; s u s p e n -
s i o n a n a l y s e d a t t h e e n d o f its 2 n d p a s s a g e .

93
 The low level of K used for callus initiation (0.5 mg/l) and for suspension
cultures (0.1 mg/1; ca. 5-10-7 M) was important for good cell dispersion and
the maintenance of the haploidy of the suspensions. When the K level was
raised to 2.0 mg/l (ca. 10 -s M) in both the callus initiation and suspension
culture media, a very coarse suspension was obtained. This grew slowly, con-
tained enlarged and irregular cells and when examined by microdensitometry
in the 2nd suspension passage showed a wide range of DNA levels suggestive
of aneuploidy (Fig. 4B). The corresponding profile for passage 8 suspension
in the standard medium is shown in Fig. 4A. This profile shows three distinct
peaks which can be interpreted as 2C haploid nuclei (3--4 arbitrary units),
4C haploid plus 2C diploid (6--7 units) and 4C diploid (10--13 units). This
correlates well with the % of haploid cells at passage 8 as estimated by chro-
mosome counts (Fig. 3).

REFERENCES

1 E. Thomas and H.E. Street, Ann. Botany, 36 (1972) 239.

2 R.N. Konar, E. Thomas and H.E. Street, Ann. Botany, 36 (1972) 249.
3 A. Rashid and H.E. Street, Planta, 113 (1973)263.
4 E. Thomas and H.E. Street, Ann. Botany, 34 (1970) 656.
5 P.R. White, The Cultivation of Animal and Plant Cells, Thomas and Hudson, London,
1954.
6 G.G. Henshaw, K.K. Jha, A.R. Mehta, D.J. Shakeshaft and H.E. Street, J. Exptl. Botany,
17 (1966) 362.

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