Factors Influencing In Vitro Flowering from Styles of Saffron

Z. Jun, C. Xiaobin, C. Fang

Life and Environment Science College
Shanghai Normal University
Shanghai 200234
PR China
Keywords: Crocus sativus, explants age, growth regulator, style, temperature
Abstract
This paper refers to the factors affecting in vitro flowering as follows in turn:
the important factor was explants age according to style length, the second was
culture condition containing temperature and light, the third was growth regulator.
Style incised from 55-70 mm length floral buds with light blue perianths as explants
cultured on media regulated with 26.8 M 1-naphthaleneacetic acid and 31.1 M 6Benzylaminopurine in the dark at 20 was optimal condition for in vitro flowering
induced, the induction ratio was 37.5-40.0 %. As for growth regulator only, the higher
6-BA/NAA in the dark at 20 was beneficial to in vitro flowering induced.
INTRODUCTION
Reasons for studying flower formation in vitro can be summarized as (1) to
provide a model system for studying flower induction and development, (2) to
provide a means for conducting microbreeding, and (3) to provide a source of
biochemicals and pharmaceuticals (Tisserat et al., 1990). Saffron plant (Crocus
sativus L.) is the worlds most expensive and precious, not only because it is used as a
source of food additives but also as a component of traditional medicine. During the
last few years the antitumoral properties of crude saffron stigma extracts, both in vitro
and in vivo, have been demonstrated (Salomi et al., 1991; Abdullaev et al., 1992;
Escribano et al., 1996). As a geophyte, saffron crocus grows slowly and propagates
only by vegetative production through daughter corm, and only its stigmas can be
used for medicine and spice, therefore its supply cant meet the demand.
Biotechnology provides a possible means for reducing the time needed for
establishing an alternative, more effective system for secondary metabolite production
and the accumulation of medicinally important substances. Attempts have been made
to induce saffron in tissue cultures from stigmas (Hori et al., 1988; Sarma et al., 1990)
and ovaries (Himeno et al., 1987; Fakhrai et al., 1990), and stigmalike structures had
been successfully induced from different floral explants of Crocus sativus. However,
there arent any reports on in vitro flowering of Crocus sativus so far. So, it is
important for inducing in vitro flowering. Here, the preliminary result of in vitro
flowering induced from styles of Crocus sativus is described.
MATERIALS AND METHODS
Saffron Crocus (Crocus sativus L.) corms came from local plantation of
ChengduSichuan ProvincePR China. According to the length of top floral buds,
sprouted corms were divided into four development stages which amounted to 40
days (Table 1). In each stage, 30 sprouted corms containing about 60-64 top floral
buds were taken as explants for four medium. That means 15-16 floral buds were
taken as explants for each media (Table 2).

Out surface of floral buds were removed thoroughly for taking styles and perianths as
explants. Then they were asepticized with 70 % ethyl alcohol for 1min, followed by
mercuric chloride at 0.1 % (w/v) for 8min and rinsed 3 times with sterile distilled
water. Styles and perianths were aseptically dissected from others of floral buds parts
and cut into 1020 mm single node sections, cultured on Murashige and Skoog (1962)
medium containing 3 % (w/v) sucrose, supplied with different combinations of 2,4Dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA), and 6Benzylaminopurine (6-BA). Per stage floral buds(60-64) were averaged into four
groups (15-16 floral buds) and cut into 1020 mm single node sections for four
growth regulator combinations medium maintained in the dark or light at 20 or 25 C.
The schemes of inducing in vitro flowering were show as table 2, 3.
RESULTS AND DISCUSSION
The perianths obtained from different stages couldnt be induced in vitro
flowering under any condition (see Table 2). Only multiperianths or stigma-like
structures could be induced from perianths. Detailed results about in vitro flowering
induced from styles of Crocus sativus were reported as the following.
The Effects of Explants Age on In Vitro Flowering
After the material was cultured for four months, the in vitro flowerings were
induced from styles of 55-70 mm floral buds (stage 3). 7 in vitro flowerings were
induced out in the stage3. No in vitro flowering was induced from styles before (less
than 40 mm) or after (70-85 mm) the stage 3 in our experiment schemes (Table 2, 3).
So the age of material used is important to inducing saffron flowers. The same results
were found in induction of stigma-like structures from perianth (Wenliang, 1992) and
stigmas especially for stigma. With different age of saffron stigmas as explants to
induce stigma-like structures, Smarma (1990) found that orange yellow stigmas could
get higher induction rate than light yellow stigmas, and white stigmas could get
lowest induction. The induction rate of stigma-like structures was higher with older
explants, whereas pink stigmas couldnt be initiated, which could be easily
understood as their inability to differentiate. Juvenile plants did not flower because of
inability to produce flowering factor(s) or inability of meristems to respond to a
flowering factor (Lang, 1965; Hackett, 1985).
Physiological studies have sought for many years about what is florigen and
have shown that flowering time control are influenced by environmental factors and
endogenous cues. Plants can integrate these signals, such as day length, vernalization,
ambient temperature, irradiance, water/mineral availability and presence/absence of
neighbors, to relate flowering time. One of these factors, Size and age of plants, were
demonstrated to be particularly important. Day length and light quality are usually
believed to be essentially perceived by expanded leaves, then florigen(sucrose and
isopentenyladenine) will be produced and moved directly or indirectly to shoot apical
meristem (SAM) to guide flowering determination (Bernier, 2005). These may be
why in vitro flowering can only be induced from 55-70 mm floral buds in our
experiment, which is the optimal age and have prepared ample accumulated florigen
from leaves in this stage.
Floral gradient in vitro has been reported with several species and explant
sources (Scorza, 1982). Because in our experiment, all in vitro flowering originated
close to style apex, floral gradient maybe also existed. The floral gradient may be
regarded as a result of changes in the capacity for development, changes in the
capacity of particular cells to produce or react to a floral stimulus, a gradient of a

floral promoter or inhibitor at the time of explant excision, or a combined promoterinhibitor gradient (Wardell, 1969a, b; Tran Thanh Van, 1973; Scorza et al., 1980;
Lang., 1987). Therefore, a further study on floral gradient will increase inducing rate
of Crocus sativus.
Effects of Culture Temperature on In Vitro Flowering
The induction results of in vitro flowering in different combinations of
temperature and light were shown in Table 3. Only one was induced in the dark at
251 C (in lower ratio of 6-BA/NAA). There wasnt in vitro flowering in the light at
251 C with higher 6-BA/NAA or lower 6-BA/NAA. As for in vitro culture of
Crocus sativus, there is a common finding that the culture condition was in the dark at
201 C. Isa and Ogasawara reported that 15-20 C is the best temperature range of
inducing callus of Crocus sativus. Sarma (1990) reported that stigma-like structures
could be induced at a higher rate at 20 C than at 25 C. It was also proved in our
experiment that the best environment was in the dark at 201 C (higher ratio of 6BA/NAA), in which six of seven in vitro flowering were induced. As for basal media,
exogenous hormone and culture temperature, culture temperature is the most crucial
for the regeneration of stigma-like structures (Wenliang et al., 1990). Anyhow high
temperature and/or light are unfavorable for inducing stigma-like structures, spice
accumulation and in vitro flowering. This is in accordance with nature cultivation in a
certain extent. In nature cultivation, proper environment for cultivation and flowering
of saffron is 15 -20 C.
In some plants vernalization alone suffices for flowering evocation, but others
require subsequent exposure to inductive photoperiods (Usually long days), and in
them the changes at the apex wrought by vernalization and the photoperiodic stimulus
are presumably different, possibly complementary (Evans, 1971). Saffron as short-day
plant and enjoying gloomy and cold situation in nature, vernalization instead of the
photoperiods is more important for in vitro flowering initiation. In response to
vernalization, chilling occur at low temperature during the period for saffron, which is
referred to as a direct effect of chilling rather than an inductive effect.
Effects of Growth Regulator on In Vitro Flowering
6 of 7 in vitro flowerings were obtained from one media (Table 2), which was
regulated with 26.8 M NAA and 31.1M 6-BA. The induction ratio was 37.5-40.0 %
(6/16-6/15), whereas, 1 of 7 in vitro flowerings was induced from another media
regulated with 21.4 M NAA and 22.2 M 6-BA. From this result, we know that the
higher ratio of 6-BA/NAA is beneficial to in vitro flowering induced from saffron
styles. Induced flowers were cultured for 3 months on higher 6-BA/NAA and then
planted on media with lower concentrated growth regulator, one anther was detected
quickly, but it was not clear whether the induced anther was initiated before or after
planting from higher 6-BA/NAA. Nevertheless the perianths grew long only on lower
concentration of hormone, on which flowers couldnt be induced from styles. There is
no obvious response for perianths on higher growth regulator media.
Auxins have frequently been reported to inhibit the formation of flowering
buds in vitro in both long-day plants and short-day plants; low concentrations,
however, may promote flowering even when higher ones are inhibitory (Evans, 1971).
Chrungoo (1984) reported that, in saffron plants, NAA had an inhibitory effect on
sprouting, vegetative growth, and flowering. This is same with our result--the higher
ratio of 6-BA/NAA is beneficial to in vitro flowering induced from saffron styles. At
the stage of flower initiation and differentiation of Crocus sativus, Ebrahimzadeh

(2001) reported that the IAA level in the bud was the lowest and increased sharply
during the elongation of leaves and floral organs in the perianth tube. The IAA levels
play an important role for the initiation and differentiation of floral organs of Crocus
sativus (Ebrahimzadeh et al., 2001). So, from what have been done implied us that it
should provide more satisfying results to do detailed growth regulator research about
in vitro flowering of saffron.
In conclusion, our work has laid a preliminary foundation for a further
research of in vitro flowering of Crocus staivus. The description of flower
morphology in the dark at 201 C was shown as table 4. Two of in vitro flowerings
were formed from one style, with one containing 6 perianths and 3 stigmas, while the
other containing 8 perianths and 5 stigmas. The rest were 2 stigmas or multistigmas. It
is significant that one anther was induced in vitro when planted on medium containing
1.34 M NAA, 1.13 M 2,4-D and 2.22 M 6-BA. The related results have been
reported: the occurrence of pollen fertility under in vitro conditions may also be due
to the action of some of the media components, or the increased humidity in the
culture vessel (Rajani et al., 1997). In terms of these results, we can do more working
about in vitro induced anther.
ACKNOWLEDGEMENTS
This work was supported by Shanghai Municipal Education Commission
(NO.04DB10).
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Table 1. Four development stages of explants

Len. of
Len. of
Stages
floral buds (mm) perianths (mm)
(1)
(2)
(3)
(4)

20-30
35-40
55-70
70-85

<5
5-6
5-9
<12

Colour of
stigma

Colour of
perianths

Light yellow
Yellow
Red
Purplish red

White
White
Light blue
Blue

Table 2. Effects of different growth regulator on in vitro flowering

NAA 2,4-D 6-BA
(M)
21.4
26.8

0
0

Perianths
Planted
Induced
No./per stage
No.

22.2
31.1

90-95
90-95

Styles
Planted
Induced No.
No./per stage

0
0

15- 16
15- 16

1
6

1.34 1.13 2.22

90-95
0
15- 16
1.34 0.90 11.1
90-95
0
15- 16
Note: The planted NO.was from 60 to 64 in the 4 stages

0
0

Table 3. Numbers of in vitro flowering in different culture conditions

201 C
251 C
Growth regulators (M)
in the dark
in the light
NAA
2,4-D 6-BA
styles
perianths
styles
perianths
21.4
26.8
1.34
1.34

0
0
1.13
0.90

22.2
31.1
2.22
11.1

0
6
0
0

0
0
0
0

0
0
0
0

251 C
in the dark
styles
perianths

0
0
0
0

Table 4. Morphologic of in vitro flowering in the dark at 201 C

Color, len.(mm)
No. of
Color, len.(mm)
No. of Perianths
of perianths
stigmas
of stigmas
6
8
7
Multi-perianths

White 5-7
White 5-7
Light blue 2
White 5-7

3
5
2
15

Yellow 1-1.2
Yellow 1-1.2
Red 1.5
Red 1.0

1
0
0
0

0
0
0
0

No. of
anthers
0
0
1
0