全 文 ：植物学通报 2004, 21 (4): 455~460
Chinese Bulletin of Botany
①Foundation item: 863 Program.
②Author for correspondence. E-mail: email@example.com
Received: 2003-04-01 Accepted: 2003-04-08 Managing editor: SUN Dong-Hua
陈书安 王晓东 赵 兵 王玉春②
(中国科学院过程工程研究所生化工程国家重点实验室 北京 100080)
摘要 对藏红花(Crocus sativus L.) 愈伤组织的诱导条件进行了优化。结果表明：MS 是藏红花芽愈伤
组织的最佳诱导培养基，而 B5是 叶子和花愈伤组织的最佳培养基。藏红花芽、叶和花愈伤组织的最佳
诱导。1.5～2.0 mg.L-1 NAA和 0.25 mg.L-1 6-BA是愈伤组织诱导的最佳激素组合。通过目视法和HPLC
方法, 从229株细胞系中筛选出细胞系Corm1，其藏红花素1 的含量是1 677 mg.g-1，生长较快，且不易
关键词 藏红花, 藏红花素1, 愈伤组织，筛选
Screening of Crocus sativus L. Callus Lines for Crocin Production
CHEN Shu-An WANG Xiao-Dong ZHAO Bing WANG Yu-Chun②
(State Key Laboratory of Biochemical Engineering, Institute of Process Engineering,
the Chinese Academy of Sciences, Beijing 100080)
Abstract Induction conditions of Crocus sativus L. callus were optimized. MS medium was optimal
for shoot callus induction while B5 medium was optimal for leaf and flower callus induction. 18℃, 25
℃ and 21℃ were the optimal temperature for callus induction of shoot, leaf and flower, respectively.
Light was positive factor for leaf callus induction but negative for shoot and flower induction.
Naphthaleneacetic acid (NAA, 1.5-2.0 mg.L-1) and 6-benzyladenine (6-BA, 0.25 mg.L-1) were the
optimal hormones for callus induction. Under the optimized conditions, the induction rate reached
60% for shoot callus, 80% for leaf callus, and 35% for flower callus. Using eyesight and HPLC
methods, one callus line which gave rise to 1 677 mg.g-1 crocin and would not brown was obtained
from 229 callus lines.
Key words Crocus sativus L., Crocin, Callus, Screening
Crocin, the main pigment of saffron (Crocus sativus L.) stigma, is soluble in water and exhibits a
high coloring capacity. A powerful quencher of free radicals and endowed with anticancer activity,
研 究 简 报
crocin could be a first choice of hydrosoluble natural yellow food colorant (Escribano and Alonso,
1996; Dufrene et al., 1999). Commercial utilization of saffron pigment is restricted by a prohibitive price
and limited availability. As a geophyte, saffron grows slowly and propagates only by vegetative
production through daughter corm. It takes 150 000-200 000 flowers and over 400 h of hand-labor to
produce 1 kg of saffron stigma (Plessner et al., 1989). Plant tissue culture methods offer a great
potential for crocin production.
One approach is to produce crocin by in vitro stigma-like-structure (SLS) culture; another is by
the induction of crocin in callus tissues. Problems encountered in these approaches are the low
frequency of SLS regeneration, slow growth, low crocin content and the browning of callus tissues
(Loskutov et al., 1999).
In order to get the quick growth, high crocin content and not easily browning callus tissues,
induction and screening of saffron callus lines were systematically studied.
1 Materials and Methods
1.1 Plant material
Crocus sativus L. was collected from Zhejiang province of China.
1.2 Shoot, leaf and flower callus induction
Shoot, leaf and flower explants were rinsed by tap water for 1-2 h, washed with distilled water,
dipped in 70% alcohol for 20 s, surface sterilized for 20 min in 1% sodium hypochlorite solution, and
then rinsed five times with sterile distilled water. Afterward, explants were dissected as sided pieces of
0.5-1 cm and inoculated on MS medium (Murashige and Skoog, 1962), B5 medium (Gamborg et al.,
1968), White medium (White, 1963) and N6 medium (Nitsch and Nitsch, 1969), respectively.
Dichlrophenoxyacetic acid (2,4-D, 2.5 mg.L-1) and 6-BA (0.25 mg.L-1) were added to all of the above
mediums. The explants were cultured at 20℃ under darkness.
1.3 Optimization of the cultural conditions for induction of shoot, leaf and flower callus
The culture medium, light or temperature optimized in the last experiment was used to the subse-
quent experiments instead of basal condition.
1.4 Corm callus induction
Callus was developed from corm mainly as described by Piqueras et al. (1996). Briefly, surface
sterilized and dissected corms were cultured on MS medium supplemented with 2,4-D (2.0 mg.L-1) and
6-BA (0.25 mg.L-1) at 21± 0.3℃ in dark. After 14 days, calli could be observed on the top of the
1.5 Crocin extract
Calli were dried in an oven at 60℃ until their weight did not change and the dry weight was
recorded. Each sample was ground, resuspended in ethanol:water (50:50, V/V), stirred for 1 h at 25℃ in
dark. Later, the suspension was centrifuged at 3 500 r.min-1 for 20 min to separate callus residue and
the supernatant was collected. The residue was further extracted by ethanol:water (50:50, V/V) and
centrifuged again under the same conditions above. The residue was discarded and the combined
4572004 陈书安等：产藏红花素 1(crocin)愈伤组织的诱导及其细胞系的筛选
extracts were passed through a filter (0.22 mm) for high performance liquid chromatograph (HPLC)
1.6 Crocin detection
HPLC was performed with a GB11A quad pump and with LCQ DECA XP Photodiode Array
Detector (a UV-visible spectrophotometer: 190-800 nm). Reverse phase HPLC was done using a
Nucleosil C18 column (3.9 mm×150 mm, 5 mm particle size). Samples were eluted over 20 min linear of
ethanol:water (53:47, V/V). Other conditions were as follows: detection meter, 440 nm; flow rate, 0.7 mL.
min-1; injection volume, 10 mL.
1.7 Standard crocin
Standard crocin was obtained from natural stigmas of Crocus sativus L. by thin layer chromatog-
raphy (TLC) and purified three times through silica gel plate. Crocin showed a chromatographic (HPLC)
purity of 97%, and its molecular weight was identified as 977.36 by Mass Spectrometry test (van
Calsteren et al., 1997).
1.8 The screening method of callus lines
Callus lines were firstly screened according to their color, growth, and browning characters.
Either the yellow, red, quickly growing, or not easily browning lines were selected by eyesight method.
The lines selected above were further selected according to crocin content analyzed by HPLC method.
2 Results and Discussion
2.1 Crocus sativus L. callus induction
Callus initiation time, induction rate and growth character varied with kind of medium, culture
temperature, light and growth hormone used (Table 1-3). MS medium was the optimal for shoot callus
induction while B5 was optimal for leaf and flower.
Temperature had a significant effect on callus initiation time, induction rate and growth character.
18℃, 25℃ and 21℃ were the optimal temperature for shoot, leaf and flower callus induction,
respectively. Light was positive factor for leaf callus induction but was negative for shoot and flower
Naphthaleneacetic acid (NAA, 1.5-2.0 mg.L-1) and 6-benzyladenine (6-BA, 0.25 mg.L-1) were
the optimized hormones for callus induction. Under these optimized conditions, the induction rate
reached 60% for shoot callus, 80% for leaf callus, and 35% for flower callus. 2, 4-D has been reported
to be effective for callus induction in many monocotyledonous plant, including Crocus sativus L.
(Vasil and Vasil, 1984), but it was not effective in this study. The discrepancy between this report and
others may be due to different kind of explants used.
2.2 Crocus sativus L. callus lines screening by eyesight method
Table 4 indicated that the morphology, browning and growth characters of four kinds of callus
lines were different, and the differences might be due to the different explants and mediums used.
In order to get appropriate callus lines for crocin production, calli were firstly screened by eye-
sight according to their color, growth rate and browning characters. Yellow or red callus lines were
selected since they might contain high concentration of crocin, and the quickly growing or not easily
browning lines were selected as well. Thus, fifteen callus lines induced from corm, three from shoot, six
from leaf and four from flower were obtained and used in the following experiments.
2.3 Screening of Crocus sativus L. callus lines by HPLC method
Table 5 shows the red, yellow and white callus lines contained a certain quantity of crocin.
Table 1 Effects of different mediums on callus induction of Crocus sativus L. shoot, leaf and flower
Medium Initiation time (d) Induction rate (%) Growth
Shoot Leaf Flower Shoot Leaf Flower Shoot Leaf Flower
B5 21 18 35 30 40 10 Slow; some brown Quick; some brown Slow; easily brown
MS 15 22 40 40 25 5 Quick; some brown Slow; some brown Slow; brown
N6 22 - - 15 0 0 Slow; some brown
White 16 25 - 35 10 0 Slow; brown Slow; brown
“-” indicates having no results and the callus induction rates were calculated within 60 d
Table 2 Effects of temperature and light on callus induction of Crocus sativus L. shoot, leaf and flower
Temperature (℃) 18±0.3 18±0.3 21±0.3 21±0.3 25±1 25±1
Light or dark Dark Light Dark Light Dark Light
Initiation Shoot 15 20 15 20 16 21
time (d) Leaf 25 19 26 20 23 18
Flower 42 - 35 - 38 -
Induction rate Shoot 45 30 40 35 40 40
(%) Leaf 30 40 25 40 35 50
Flower 5 0 20 0 10 0
“-” indicates having no results and the callus induction rates were calculated within 60 d. Light conditions were under
16 h cool-white fluorescent
Table 3 The effect of plant hormones on callus induction of Crocus sativus L. shoot, leaf and flower
Growth hormone 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D NAA NAA NAA NAA NAA
(mg.L-1) 0.5 1.0 1.5 2.0 3.0 0.5 1.0 1.5 2.0 3.0
Initiation Shoot 15 15 15 16 15 15 15 15 15 15
time (d) Leaf 18 20 18 18 18 18 18 18 18 18
Flower - - 45 - - 35 40 40 35 38
Induction rate Shoot 35 35 45 40 35 45 50 60 25 35
(%) Leaf 40 40 40 35 35 40 70 80 50 50
Flower 0 0 10 0 0 5 25 20 35 15
“-” indicates having no results. The callus induction rates of corm, shoot and leaf were calculated within 30 d while
that of flower was within 45 d
Table 4 Screening of Crocus sativus L. calli by eyesight method
Corm callus Shoot callus Leaf callus Flower callus
Callus Granule or clump; Slice or clump; loose Granule or clump; Clump; loose; red
morphology loose or compact; or compact; white loose or compact; white or yellow;
white, yellow or red; or yellow; opacity or yellow; opacity opacity or
opacity or translucence or translucence or translucence translucence
Brown Some easily brown Some easily brown Some easily brown Easily brown
Growth A few grow rapidly A few grow rapidly A few grow rapidly Grow slowly
Callus lines 102 55 50 22
4592004 陈书安等：产藏红花素 1(crocin)愈伤组织的诱导及其细胞系的筛选
However, Visanath et al. (1990) reported that the combination of NAA plus kinetin or 2,4-D plus kinetin
gave rise to yellow callus, but crocin was not detected in these yellow callus lines and was only
detected in the red callus lines by TLC method. The differences may be due to the different mediums,
plant hormones or crocin detection methods used as well as genotypic factors.
As shown in table 5, the colors of saffron callus were correlative to the crocin content and callus
browning. Red and most of yellow callus lines had high crocin content (which confirms the applicabil-
ity of eyesight method in screening lines of high crocin content), but most of them grew slowly and
browned easily. On the contrary, white lines grew quickly and did not brown easily, but the crocin
content was very low. In order to obtain a callus line with higher capability for crocin production, the
crocin content and growth ability of lines should be considered in general. Therefore, Corm1 line was
selected for further studies.
Acknowledgement This research was sponsored by 863 Program (the National High Technology
Research and Development Program of China).
Dufrene C, Cormier F, Dorion S, Niggli U A, 1999. Glycoslation of encapusulated crocetin by a Crocus sativus L. cell
culture. Enzyme Microb Tech, 24:453~462
Escribano J, Alonso G L, 1996. Crocin, saffronal and picrocrocin from saffron (Crocus sativus L.) inhibit the growth
of human cancer cell in vitro. Cancer Lett, 100:23~30
Gamborg O L, Miller R A, Ojima K, 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell
Res, 50: 151~158
Loskutov A V, Benginger C W, Ball T M, 1999. Optimization of in vitro conditions for stigma-like-structure produc-
Table 5 Screening of Crocus sativus L. callus lines by HPLC method
Callus Color Brown Growth Crocin (mg.g-1) Callus Color Brown Growth Crocin (mg.g-1)
Corm1 Yellow Not Rapidly 1667±58 Corm15 White Slowly Slowly 333±23
Corm2 White Slowly Rapidly 647±52 Shoot1 Yellow Slowly Slowly 1105±67
Corm3 Yellow Easily Slowly 2171±101 Shoot2 Yellow Slowly Rapidly 771±54
Corm4 Yellow Easily Slowly 1086±46 Shoot3 Yellow Slowly Slowly 1095±48
Corm5 White Slowly Slowly 419±28 Leaf1 Yellow Easily Slowly 2714±103
Corm6 White Slowly Slowly 187±16 Leaf2 Yellow Easily Slowly 2771±132
Corm7 Yellow Slowly Slowly 1229±72 Leaf3 White Not Rapidly 457±35
Corm8 Red Easily Slowly 6376±158 Leaf4 White Slowly Rapidly 495±38
Corm9 White Slowly Slowly 487±41 Leaf5 White Not Rapidly 343±42
Corm10 White Not Rapidly 343±25 Leaf6 Yellow Slowly Rapidly 695±50
Corm11 White Not Rapidly 209±38 Flower1 Yellow Easily Slowly 3171±153
Corm12 White Not Rapidly 505±40 Flower2 Yellow Slowly Slowly 1201±122
Corm13 Yellow Slowly Slowly 1124±72 Flower3 Red Easily Slowly 5213±184
Corm14 Yellow Slowly Slowly 1943±95 Flower4 Yellow Slowly Slowly 2281±102
The values are means of three separate experiments ± standard deviation
tion from half-ovary explants of Crocus sativus L. In Vitro Cell Dev Biol (Plant), 35: 200~205
Murashige T, Skoog F A, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol
Plant, 15: 473~479
Nitsch J P, Nitsch C, 1969. Haploid plants from pollen grains. Science, 163: 85~87
Piqueras A, Han B H, Gomez R, Debergh P C, 1996. Development of cromogenic corms nodules and microcorms: two
alternatives for saffron micropropagation. In Vitro Cell Dev Biol (Plant), 32: 314
Plessner D, Neghbi M, Ziv M, 1989. Effect of temperature on the flowering of saffron (Crocus sativus L.): inducing
of hysteranthy. Israel J Botany, 38: 1~7
van Calsteren M R, Bissionnette M C, Cormier F, 1997. Spectroscopic characterization on crocetin derivatives from
Crocus sativus and Gardenia jasminoides. J Agr Food Chem, 45:1055~1061
Vasil V, Vasil L K, 1984. Cell Culture and Somatic Cell Genetics of Plant. Orlando: Academic Press, 36~42
Visanath S, Ravishankar G A, Venkataraman L V, 1990. Induction of crocin, crocetin, picrocrocin and safranal
synthesis in callus cultures of saffron— Crocus sativus L. Biotechnol Appl Bioc, 12: 336~340
White P R, 1963. The Cultivation of Animal and Plant Cell. New York: Ronald Press, 226~229