Plant Cell, Tissue and Organ Culture 55: 15–22, 1999.

© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

15

In vitro culture of Safflower L. cv. Bhima: initiation, growth optimization

and organogenesis

T. D. Nikam1,∗ & M. G. Shitole2

1 Post-grad.
Research Centre, Dept. of Botany, Modern College, Shivajinagar, Pune - 411 005, India; 2 Dept. of
Botany, University of Pune, Pune - 411 007, India (∗ requests for offprints)

Received 13 November 1996; accepted in revised form 7 January 1999

Key words: Carthamus tinctorius L. organogenesis, prolong callus culture

Abstract
Callus induction and in vitro plantlet regeneration systems for safflower (Carthamus tinctorius L.) cv. Bhima using
root, hypocotyl, cotyledon and leaf explants were optimized by studying the influence on organogenesis of seedling
age, media factors, growth regulators and excision orientation. Supplementation of the medium with an auxin:
cytokinin ratio > 1 enhanced the growth rate of callus cultures; however, for 2,4-D the ratio was < 1.34–11.41 µM
concentrations of growth regulators (IAA, NAA, BA and Kinetin) in the medium were found effective for callus
induction and regeneration in all explants. The calli could be maintained over 32 months. BA (4.43 µM) combined
with casein hydrolysate (10 mg l−1 ) yielded the highest rate of shoot production on hypocotyl (3–6) and cotyledon
(5–7) explants and cotyledonary derived callus (4–8). More shoots were produced on explants cut from the most
basal region of cotyledons from 5 to 7-day-old seedlings than from older seedlings or more distal cut sites. Apolar
placement of explants, inhibited shoot regeneration. The shoot regeneration potential remained upto 7 months in
calli developed on NAA + BA. Of three media tested, MS was superior to SH-M and B5 . Rooting of shoots was not
efficient; 42% of the shoots were rooted on MS medium containing sucrose (7–8%) + IAA (2.8–5.7 µM). Capitula
induction was observed in both callus mediated shoots on cotyledons and shoots on rooting medium with sucrose,
IAA, NAA and IBA. Well developed plantlets were transferred to the field with a 34% success rate.

Abbreviations: IAA – indoleacetic acid; IBA – indolebutyric acid; NAA – alpha-naphthaleneacetic acid; 2,4-D –
2,4-dichlorophenoxyacetic acid; BA – 6-benzylaminopurine; MS – Murashige & Skoog’s; SH-M – Mitchell &
Gildow; B5 – Gamborg

Introduction vitro plant regeneration system is a basic necessity for

Carthamus tinctorius L. (safflower), Asteracere, is an
important oilseed crop of semi-arid regions. It occu-
pies a unique position among oil seed crops due to
the high linoleic content of its seed oil which has
therapeutic value. The seed oil is used for edible and
industrial purposes. The seeds are diuretic and used as
a tonic. The young plants are used as leaf vegetable
(Anonymous, 1950).
Gene transfer techniques are currently being used
for genetic modification of agronomically important
characters and also the structure and composition of
fatty acids in oil seed yielding crop. An efficient in
such approaches. In recent years, direct somatic em-
bryogenesis from cotyledon explants (Mandal et al.,
1995) and in vitro shoot regeneration has been repor-
ted in safflower (George and Rao, 1982; Tejovathi and
Anwar, 1987; Orlikowska and Dyer, 1993; Rani et
al., 1996; Baker and Dyer, 1997). However, the re-
sponse varies with the cultivar and regeneration and
frequency of the shoot rooting was low. Attempts have
been made for capitula induction (Tejovathi and An-
war, 1984), androgenic plant production (Prasad et al.,
1991) and host-parasite interaction (Tietjen and Ma-
tern, 1984). As an alternative, the culture of callus
tissue provides an important technique that can be pre-
16
liminary to the regeneration of whole plants. Because
of the potential genetic variability associated with this
system, regenerated plants may assume importance
for genetic improvement and selection strategies when
evaluated for somaclonal variation.
In addition, the establishment of cell culture has
considerable potential in the future as an alternative
to traditional agriculture for the production of known
and new secondary metabolites (Stockight et al., 1995)
the production is released from all the disadvantages
of whole plant field cultivation. This technology can
provide a continuous and constant year-round sup-
ply of natural plant products, which may be more
easily purified. Methods for the production of import-
ant components from plant cells have been developed
in safflower, such as o-tocopherol (Gan, 1991) and
red pigment (Hanagata et al., 1993), although fur-
ther research is necessary to improve their economic
efficiency.
In this paper, we report the optimization of a mi-
cropropagation and callus culture protocol in safflower
cv. Bhima (an high yielding Indian cultivar).
Materials and methods
Plant material
Certified seeds of cultivar (cv.) Bhima of safflower
(Carthamus tinctorius L.) were obtained from the
Nimkar seed Research Centre (NSRC), Phaltan, MS,
India. Seeds were surface sterilized with 0.1% mer-
curic chloride for 7 min followed by three washes for
5 min each in sterile distilled water. Seeds were then
germinated and grown on a sucrose (1%)–agar (0.8%)
medium under a 9-h photo period of fluorescent light
(19.75 µmol m−2 s−1 ). Root (8–10 mm), hypocotyl
(8–10 mm), cotyledon (15–18 mm2), and entire coty-
ledon (58–62 mm2 ) explants were isolated from 3 to
15-days-old seedlings. Leaf explants (15–18 mm2 )
were isolated from the shoots obtained in vitro from
the cotyledon explants on medium supplemented with
4.43 µM BA. Explants were transferred onto callus
induction medium (see below).
Induction of callus and regenerants
Callus induction was carried out on MS SH-M and B5
medium supplemented with BA, Kinetin, NAA, IAA
and 2,4-D (0.44–85.62 µM) either alone or in com-
binations. Samples of 250 ± 10 mg of fresh calli of
root, hypocotyl, cotyledon and leaf explants were sub-
cultured onto fresh optimum callus induction media
every 30 days (total of 32 subcultures). The average
dryweights of the calli were determined at each sub-
culture after 30-days growth. To obtain dry weights,
weighed fresh calli were dried at 60 ◦ C in a hot air
oven until constant weight (for 48 h).
Shoot induction from explants and calli (250–
300 mg/culture) was carried out on MS, SH-M and
B5 medium containing Kinetin, BA (0.44–85.62 µM
g) plus IAA, NAA (0.53–5.71 µM) plus caseinhydro-
lysate (1–30 mg l−1 ). When regenerated shoots were
about 1 cm long, they were separated from the explant
and callus and counted.
Rooting of resulting shoots (1–1.5 cm long) from
explants and calli was attempted on MS, SH-M, and
B5 media without growth regulators and with sucrose
(1–9%), IAA, NAA and IBA (0.49–28.54 µM) either
alone or in combination. Addition of BA or Kinetin
(0.44–4.64 µM) was also tested with rooting medium.
Culture conditions
The basal media MS, SH-M and B5 consisted of salts
and vitamins, 3% sucrose and gelled with 0.8% agar.
The growth regulators BA, Kinetin, NAA and 2,4-D
added in the medium before autoclaving while IAA
and IBA were filter sterilized with 0.22 µM filter and
added under aseptic conditions to the autoclaved me-
dium, after cooling to 60 ± 4 ◦ C. Basal media salts,
sucrose and agar (extra pure grade) were obtained
from Glaxo India Ltd. Vitamins and growth regulators
were from Sigma Chem. USA. The pH of the media
was adjusted to 5.7 with 0.5 N NaOH before autoclav-
ing at 121 ◦ C for 15 min. A volume of 15 ml nutrient
medium was dispensed into 150×25 mm corning glass
test tubes that were plugged with non-absorbent cot-
ton. In vitro cultures were incubated at 25 ± 2 ◦ C
in a temperature-controlled growth room under a 9-
h photoperiod (cool white fluorescent Philips lamps,
19.75 µm m−2 s−1 ).
Hardening
Rooted plantlets were removed from the culture vi-
als. After the agar had been removed by washing
with sterile water, the plantlets were planted in a pots
containing a 1:1 sterilized potting mixture soil and
washed sand (with a pebblesize of 0.5–1.2 mm). The
plants were placed outside in the shade (light max-
imum 83.46 µm m−2 s−1 , temperature 25 ± 4 ◦ C)
and irrigated at 3-day intervals with tap water. For the