Plant Cell, Tissue and Organ Culture 77: 133–138, 2004.
© 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Plant regeneration from hypocotyl protoplasts of red cabbage
(Brassica oleracea) by using nurse cultures
Li-Ping Chen1,∗ , Ming-Fang Zhang1 , Qiu-Bing Xiao2 , Jian-Guo Wu3 & Yutaka Hirata2
1 Department
of Horticulture, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029,
P.R. China; 2 Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo
183-8509, Japan; 3 Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University,
Hangzhou 310029, P.R. China (∗ request for offprints: Fax: +86-571-86971431; E-mail: [email protected])
Received 22 November 2002; accepted in revised form 18 August 2003
Key words: chromosome observation, efficient plant regeneration, tuber mustard
Abstract
A protocol for rapid and efficient plant regeneration from protoplasts of red cabbage was developed by a novel nurse
culture method. When the protoplasts of red cabbage were cultured in modified MS medium containing various
combinations of BA, NAA and 2,4-D, they did not continue dividing due to browning. However, they successfully
divided and formed micro-calli at a high efficiency when they were mixed and co-cultured with those of tuber
mustard at a 1:1 ratio. The presence of tuber mustard protoplasts used as nurse cells was essential for sustainable
divisions and colony formation of red cabbage protoplasts. Red cabbage-like plantlets were regenerated from these
protoplast-derived calli at a frequency ranging from 33 to 56% in all the experiments where three cultivars of red
cabbage were tested. Over 120 protoplast-derived cabbage plants were transferred to the greenhouse, and they
showed no noticeable abnormalities in morphological features. Chromosome observation revealed that all of the
plants examined had the normal chromosome number of cabbage (2n = 18), suggesting that no spontaneous fusion
between the two species had occurred during protoplast culture.
Abbreviations: BA – benzyladenine; 2,4-D – 2,4-dichlorophenoxyacetic acid; MS – Murashige and Skoog (1962)
medium; NAA – α-naphthaleneacetic acid; CPW – cell and protoplast washing solution; MES – 2-(N-morpholino)ethane-sulfonic acid
Introduction
An efficient and reliable method of plant regeneration
from protoplasts provides an unique way for genetic
manipulation, for example, by gene transformation
and protoplast fusion. Previous studies on protoplast
regeneration have focused on seeking the optimal culture conditions (Glimelius, 1984; Fu et al., 1995;
Kunitake et al., 1995; Jiang et al., 1998). This strategy
has been effective for the successful culture of protoplasts of many species. However, it still has limitations
for practical applications because of the existence of
recalcitrant genotypes (Jourdan and Earle, 1989; Zhao
et al., 1995; Hansen et al., 1999) and a low plant
regeneration frequency.
Nurse culture has been practiced as a feasible way
for improving plating efficiency or for obtaining regenerants of some ‘recalcitrant’ genotypes that did
not respond to conventional protoplast culture methods (Walters and Earle, 1990; Matsuka and Sugimoto,
1997; Hu et al., 1999; Karamian and Ebrahimzadeh,
2001). However, nurse culture methods are not applicable to some species since liquid culture, which is
frequently employed in nurse culture (Kyozuka et al.,
1987; Liu, 1994), tends to result in aggregation and
necrosis of protoplasts of some genotypes.
There are a lot of economically important varieties in Brassica oleracea. Although plant regeneration
from protoplasts has been achieved in this species by
extensive screening of protoplast culture conditions
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(Glimelius, 1984; Fu et al., 1985; Hansen and Earle,
1994), several genotypes still cannot be recovered
from protoplasts (Jourdan and Earle, 1989; Kik and
Zaal, 1993; Zhao et al., 1995; Hansen et al., 1999). In
the present investigation, we have developed a simple
and novel nurse culture method, which allows plant regeneration at high frequency from protoplasts of three
red cabbage cultivars within 3 months.
with a 0.4 M mannitol solution containing CPW salts.
Then, the protoplasts were washed by resuspending
the pellet in modified MS liquid medium containing
2.5 mg l−1 2,4-D and 0.5 mg l−1 Kinetin and 0.4 M
mannitol, which was identical to the medium formula
for tuber mustard protoplasts (Chen et al., 2001). After
discarding the supernatant, the pellet was suspended in
liquid medium at a protoplast density of 1 × 105 ml−1
in Petri dishes (60 mm in diameter, 10 mm in depth,
IWAKI) and cultured at 25 ◦ C in the dark.
Materials and methods
Protoplast nurse culture
Plant materials
Tuber mustard (Brassica juncea Coss. var. tumida
Tsen et lee) and three cultivars of red cabbage (cv.
Ruby ball, cv. Hongmu, cv. Zigan No. 1) were used
as the source of protoplasts. Seeds were surfacesterilized with 70% ethanol for 30 s, washed three
times with sterilized water, sterilized again with a
2% sodium hypochlorite solution supplemented with
a few drops of liquid detergent for 15 min, and
rinsed three times with sterilized water. Seedlings
were raised aseptically on half strength MS medium
(Murashige and Skoog, 1962) containing 0.1 mg l−1
6-BA with 0.8% agar under controlled conditions
(25 ◦ C, 16-h photoperiod, 84 µmol m−2 s−1 , fluorescent light).
Protoplast isolation
The protoplast isolation procedures were the same
for cabbage and tuber mustard. Protoplasts of tuber
mustard were isolated from cotyledons of 10-day-old
seedlings in an enzyme solution consisting of 0.5%
(w/v) Cellulase Onozuka RS, 0.1% (w/v) Pectolyase Y23, 2 mM MES, 3 mM CaCl2 and 0.4 M mannitol. Hypocotyls of red cabbages were excised from
aseptically raised seedlings, cut into 0.5–1 mm long
pieces in a 0.4 M mannitol solution containing CPW
salts (Frearson et al., 1973). After collecting the hypocotyl segments by filtering through a 108 µm mesh,
they were placed in Petri dishes (10 cm in diameter,
2 cm in depth FALCON, 3803) containing an enzyme
solution consisting of 0.2% (w/v) Cellulase Onozuka
RS, 0.1% (w/v) Pectolyase Y23, 2 mM MES, 3 mM
CaCl2 and 0.4 M mannitol. After 3–4 h incubation
with shaking at 50 rpm at 26 ◦ C in the dark, the mixture was sieved through a 50 µm mesh and the filtrate
was collected in a 10 ml tube and centrifuged at 50 × g
for 3 min. The supernatant was discarded and the pellet containing the protoplasts was washed three times
Two types of nurse culture were employed in the
present study by using protoplasts of tuber mustard as
the nurse cells. The protoplast culture system of tuber
mustard had been successfully established in our previous study (Chen et al., 2001). In the first method, the
agarose medium containing cabbage protoplasts was
cut into blocks that were then transferred onto a 6 cm
plate containing 3 ml agarose medium in which protoplasts of tuber mustard were embedded. In the second
method, protoplasts of cabbage and tuber mustard
were separately cultured in the modified MS liquid
medium mentioned above at a density of 1 × 105 ml−1
for 3 days. Then, the protoplasts of both species were
gently mixed, and the mixture was quickly mixed
with an equal volume of the modified MS agarose
medium to give evenly dispersed protoplasts. This
modified MS agarose medium contained 0.5 mg l−1
BA, 1 mg l−1 2,4-D, 0.5 mg l−1 NAA, 0.4 M mannitol
and 1.2% agarose, which was earlier found to be an
appropriate composition for protoplast culture of tuber
mustard (Chen et al., 2001). Finally, the protoplasts
were cultured at 25 ◦ C in the dark. In the latter method,
the frequency of division and plating efficiency of cabbage and tuber mustard were determined separately
with an inverted microscope based on the difference
in color and appearance of the protoplasts of the two
species.
Regeneration of plantlets
Upon micro-calli formation, the solidified agarose
medium with embedded colonies was cut into small
pieces and transferred to MS medium containing
0.25 mg l−1 NAA, 0.25 mg l−1 2,4-D, 1 mg l−1 BA
and 0.2 M mannitol for further proliferation under
dim fluorescent light (30 µmol m−2 s−1 at a 16-h photoperiod at 25 ◦ C). When the calli reached about 1 mm
in diameter, they were transferred to differentiation
MS medium containing 1 mg l−1 BA, 0.2 mg l−1 NAA
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and 0.8% agar. When red shoots appeared from the
calli after approximately 2–3 weeks in culture, the
frequency of shoot formation of cabbage was calculated as percentage of regenerative calli among total
cabbage-derived micro-calli. The shoots were cut and
transferred to 1/2 MS medium containing 0.1 mg l−1
NAA for regeneration of plantlets.
Chromosome observation
For chromosome observation, the seeds of mother
plant were put in water for several hours, and transferred to wet filter paper for germination at 25 ◦ C.
When the roots reached 1–1.5 cm in length, seeds
were treated with 2 mM 8-hydroxyquinoline for 3 h
at 22 ◦ C. The seeds were then fixed in Carnoy’s solution for 24 h, and stored in 70% ethanol at 4 ◦ C. The
meristematic tissues of root tips, stem tips and young
leaves were cut from plantlets growing on the culture medium and were treated like the seed root tips.
Chromosome preparations were made according to the
method of Wu et al. (1997).
Results
Protoplast isolation of cabbage
Hypocotyls from one partner are commonly used as
protoplast donors for protoplast fusion because its
chloroplasts are less visible, which is a useful characteristic for hybrid selection. However, in our previous
study, the yield of protoplasts isolated from hypocotyl
was extremely low (Chen et al., 2001) and a large
number of seeds were needed to obtain a sufficient
number of protoplasts. Therefore, we first examined
the yield of protoplasts from hypocotyls of three cabbage cultivars. The results showed that the hypocotyls
of 3–5-day-old seedlings offered enough good quality protoplasts for culture, that is, around 1.8 × 104
per seedling. Protoplasts isolated from 3 to 5-day-old
seedlings also showed a high division frequency in
culture (Figure 1).
Protoplast culture
When the protoplasts from hypocotyls of red cabbage
were cultured in MS agarose medium containing various combinations of BA, NAA and 2,4-D, they divided
(Figure 2a, b) but failed to reach beyond a 6–10 cellcolony stage. Then they gradually turned brown and
died. From our observation, protoplasts of red cabbage
Figure 1. Effect of seedling age on hypocotyl protoplast division of
red cabbage (cv. Ruby ball). Each value is a mean ± S.D. for three
independent experiments.
often gave rise to necrosis after several divisions. Although we had succeeded in preventing aggregation
and necrosis by using agarose for embedding protoplasts of B. juncea (Chen et al., 2001), agarose had no
beneficial effect on protoplasts of B. oleracea in the
present experiment. The same phenomenon was observed when cotyledons and mature leaves were used
as the source for protoplast culture of cabbage (results
not shown).
To induce sustainable cell divisions and prevent
necrosis, the effect of two different nurse culture methods on colony formation was investigated. The first
method, in which cabbage and tuber mustard protoplasts were cultured separated from each other, had
no positive effect on protoplast culture of cabbage. In
the second method, the effect of mixing of protoplasts
of cabbage and tuber mustard in different proportions
on micro-callus formation was tested. Table 1 shows
that the presence of nurse cells (tuber mustard) in the
medium was essential to keep protoplasts of cabbage
in a healthy condition, and to obtain colony formation.
After about 4 weeks of culture, numerous micro-calli
became visible (Figure 2d). When the protoplasts of
cabbage (cv. Ruby ball) were mixed with those of
tuber mustard at a 1:1 or 1:2 ratios, the plating efficiency of micro-callus formation reached 18.3 and
19.9%, respectively, whereas the control culture of
cabbage showed no callus formation. Similar results
were obtained when the protoplasts of cabbage cv.
Hongmu and cv. Zigan No. 1 were nursed by tuber
mustard protoplasts (data not shown). These microcalli continued to grow after cutting of the agarose
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Figure 2. Plant regeneration from hypocotyl protoplasts of red cabbage (cv. Ruby ball) by nurse culture. (a) First division after 3 days of
culture; (b) second division after 6 days of culture; (c) colony formation after 3 weeks of culture; (d) callus formation after 5 weeks of culture;
(e) red shoot initiation; (f) plantlet transplanted.
Table 1. Effect of nurse culture on division and plating efficiency of red cabbage protoplasts
Protoplast source
Cabbage (CK)
Cabbage + tuber mustard
Cabbage + tuber mustard
Cabbage + tuber mustard
Tuber mustard
Mixing ratio of protoplasts
(cabbage:tuber mustard)
5:1
1:1
1:2
% of protoplasts with first
division in cabbage
(in tuber mustard)∗
Micro-callus formation in cabbage
(in tuber mustard)∗∗
31.0%
28.2% (23.1%)
29.6% (26.7%)
29.3% (26.5%)
27.7%
0
0
18.3% (19.1%)
19.9% (18.9%)
16.0%
∗ Number of protoplasts with first division/total number of protoplasts of cabbage (or tuber mustard) × 100.
∗∗ Number of formed micro-callus/total number of protoplasts from cabbage (or tuber mustard) × 100.
medium into small pieces and transfer onto propagation medium.
Plant regeneration
After reaching 1 mm in diameter on propagation medium, individual calli from cabbage or tuber mustard
were placed randomly on differentiation medium.
They grew further, and some of them developed red
spots on the surface that grew into visible shoots in
2–3 weeks. Shoots were obtained with a frequency
of 33% (cv. Zigan No. 1), 47% (cv. Hongmu) and
56% (cv. Ruby ball). The calli (of cv. Ruby ball)
with red spots produced 13 shoots per callus on average. When differentiated red shoots were transferred
onto rooting medium, they readily rooted and over
120 plantlets of red cabbage were transferred to pots
in the greenhouse (Figure 2f). To date, apparent
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Figure 3. The chromosome observation of red cabbage at metaphase (cv. Ruby ball). (a) Root tip cell of mother plant (2n = 18); (b) shoot tip
cell of the red regenerant (2n = 18). Bar = 5 µm.
morphological variants have not been observed among
regenerants.
Chromosome observation
In order to clarify the origin of the cabbage plantlets, the chromosome numbers were examined of the
mother red cabbage cultivars and their regenerants.
The results showed that the chromosome number of
the original red cabbage was eighteen (Figure 3a). The
red regenerants were also 2n = 18 (Figure 3b). Consequently, no variation in the chromosome number
was observed in the red cabbage-type regenerants.
Discussion
Although plantlets were successfully regenerated
from protoplasts of B. oleracea in previous studies
(Glimelius, 1984; Fu, 1985; Hansen and Earle, 1994),
their protocols varied greatly in either culture parameters or genotypes and we failed to obtain plantlets from
red cabbage protoplasts with their protocols. Thus,
it is very imperative to establish a standard culture
system that would work within species, genus or family as much as possible. In the present investigation,
plant regeneration from three cultivars of cabbage protoplasts was successfully and reproducibly achieved
with identical medium formula and culture conditions
by a nurse culture method.
In the first method, where agarose-blocks containing protoplasts were transferred onto a feeder layer
of nurse cells, the protoplasts failed to form colonies, suggesting that the growth factors released by
the nurse cells did not accumulate sufficiently in the
agarose blocks to stimulate proliferation of cabbage
cells. The presence of nurse cells of tuber mustard was
necessary to induce sustainable division of protoplasts
of red cabbage.
It is necessary to exclude the possibility of the genomic contamination with the nurse protoplasts due
to the spontaneous fusion between nurse and nursed
protoplasts. To exclude this possibility, we employed
the following precautionary measures:
– separate pre-cultures of protoplasts of cabbage
and tuber mustard for 3 days to allow cell wall
formation;
– well-dispersed low density (0.5 × 105 ml−1 ) culture of mixed protoplasts to avoid the fusion or
aggregation of the protoplast of the two species.
Meanwhile, picking callus pieces with red pigmentation is a simple and practical way to ensure that only
B. oleracea are being regenerated. The chromosome
number of the regenerants was 18, which is the proper
number for red cabbage, while that of tuber mustard
is 2n = 36. The regenerants were uniform and genetically stable in phenotype in the vegetative growth
stage. To date, no evidence was found for spontaneous
fusion. Therefore, the nurse culture method employed
138
in the present study was effective for protoplast culture
of red cabbage. Experiments aimed at producing regenerants from other recalcitrant plants are in progress
by using the present nurse culture system.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (30270851). The first author
would like to thank Prof. Masahiro Mii of the Laboratory of Plant Cell Technology, Faculty of Horticulture,
Chiba University, for comments and critical reading of
the manuscript.
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