2. Materials and methods
2
.
1
. Plant material and establishment of cell suspension cultures
In vitro-grown plantlets of L. formosanum were maintained on half-strength MS medium [18] con-
taining 30 g l
− 1
sucrose and 2 g l
− 1
gellan gum at 25°C under continuous illumination 35 mmol m
− 2
s
− 1
with fluorescent lights. Bulb scales were ex- cised from the plantlets, cut into 5 – 10 mm trans-
verse segments, and inoculated for callus induction onto MS medium containing 5 mM 4-amino-3,5,6-
trichloropicolinic acid picloram PIC, 30 g l
− 1
sucrose and 2 g l
− 1
gellan gum. Cultures were maintained at 25°C in the dark.
Bulb scale-derived calli were transferred to 100 ml Erlenmeyer flasks each of which contained 40
ml liquid MS medium supplemented with 5 mM PIC and 30 g l
− 1
glucose to initiate cell suspension cultures. In L. formolongi, protoplasts isolated
from suspension cultures using a glucose-contain- ing medium showed a high plating efficiency as
compared with those using a sucrose-containing medium [15]. Suspension cultures were maintained
at 25°C in the dark on an orbital shaker 100 cycles min
− 1
. After stabilization of the growth rate, cell suspensions were subcultured every 2
weeks by transferring ca. 1 g fresh weight FW of cell clumps into 40 ml of the same fresh medium.
For measuring the growth rate of suspension cultures, samples 1 g FW were inoculated to a
100 ml Erlenmeyer flask containing 40 ml medium. Growth was monitored every other day from the
day of inoculation day 0 to day 20. To determine fresh mass, cells in each flask were collected on a
filter paper, drained under vacuum, transferred onto pre-weighed aluminum foil, and immediately
weighed again.
2
.
2
. Plant regeneration from cell suspension cultures
For inducing regeneration, cell clumps 1 – 3 mm in diameter were taken from suspension cultures 1
week after subculture and transferred onto MS media with or without plant growth regulators
a-naphthaleneacetic acid NAA, PIC, TIBA, 6- benzyladenine BA or N-1,2,3-thiadiazol-5-yl-
N-phenylurea thidiazuron TDZ. All media used for regeneration contained 30 g l
− 1
sucrose and 2 g l
− 1
gellan gum. Ten to 15 cell clumps were placed per plastic Petri dish 9 cm in diameter
containing 30 ml medium. Cultures were main- tained at 25°C under continuous illumination 35
m mol m
− 2
s
− 1
. After 2 – 3 months, regenerated shoot buds, so-
matic embryos and plantlets were transferred to 100 ml culture flasks, each of which contained 40
ml half-strength MS medium containing 30 g l
− 1
sucrose and 2 g l
− 1
gellan gum, and were cultured at 25°C under continuous illumination 35 mmol
m
− 2
s
− 1
with fluorescent lights. Plantlets with a well-developed bulblet were then subjected to a
cold treatment together with culture flasks at 4°C in the dark for 12 weeks. After that, scale-leaves
and roots were removed from the plantlets, and the remaining bulblets were then transferred to
pots containing vermiculite. They were incubated at 25°C under continuous illumination 35 mmol
m
− 2
s
− 1
. Following emergence of new scale- leaves, plants were transplanted to the greenhouse.
2
.
3
. Histological examination Cell clumps were sampled every 5 days after
their transfer to the regeneration medium without plant growth regulators. Cell clumps were fixed
with FAA 70 ethanol:formalin:acetic acid, 90:5:5, dehydrated in a butanol series, and em-
bedded in paraffin. Sections 10 mm thick were cut, double-stained with safranine and fast green
[19], and observed under a light microscope.
3. Results
3
.
1
. Establishment of cell suspension cultures Within 2 months after the initiation of culture,
creamy-white nodular calli formed mainly at the cut end of bulb-scale segments. Although the calli
initially contained many shoots and roots, callus lines without organized tissues were established
after two to three subcultures onto the same medium. When placed into a liquid medium, calli
easily broke apart and dispersed into cell clumps of 0.5 – 5 mm in diameter. Agitation further frag-
mented these clumps into small cell aggregates. About 1 month after the initiation of liquid cul-
tures, stably growing cell suspension cultures, which consisted of creamy-white cell clumps, were
established Fig. 1A. No changes in the appear- ance of cell clumps were observed during subcul-
tures for over 72 months.
Fig. 2 shows the growth FW of 12-, 36- and 72-month-old suspension cultures as a function of
time after subculture. In all cultures, the doubling time for FW between day 8 and 12 was about 3
days. Maximum FW was reached on day 16 – 18 and increased about 5- to 6-fold over the initial
FW. The rate of growth was stable for 72 months, but the rate gradually increased as the duration
after
the initiation
of suspension
cultures increased.
3
.
2
. Plant regeneration from cell suspension cultures and changes in the regeneration ability
during long-term culture Creamy-white cell clumps that had been grown
as cell suspension cultures in the dark were trans- ferred to regeneration media, incubated under
continuous illumination, and they rapidly turned dark purple within 1 week. These cell clumps
started to produce bud- andor embryo-like struc- tures about 2 weeks after transfer to regeneration
media. Bud-like structures were tightly attached to the original cell clumps Fig. 1B. Histological
Fig. 1. Plant regeneration from cell suspension cultures of L. formosanum. A Cell clumps 0.5 – 5 mm in diameter in 12-month-old suspension cultures scale bar, 5 mm. B – D Two weeks after transfer of cell clumps from 12-month-old
suspension cultures onto the regeneration medium without plant growth regulators scale bars, 1 mm: B production of shoot buds; C formation of an oval-shaped somatic embryo arrowhead; D an elongated somatic embryo possessing a cotyledon
arrow and a root arrowhead. E Regenerated plants from 72-month-old suspension cultures growing in the greenhouse scale bar, 2 cm.
Fig. 2. Growth curve of 12-month-old, 36-month-old and
72-month-old cell suspension cultures of L. for-
mosanum. One gram FW of cell clumps was inoculated into a 100 ml Erlenmeyer flask containing 40 ml liquid medium.
Values represent the mean 9 S.E. of five independent experi- ments.
and 26.5 of the cell clumps also produced a few somatic embryos on the medium containing 0.5
m M BA. Although shoot bud formation was ob-
tained in all of the cell clumps, production of somatic embryos was never observed on the
medium containing 5 mM BA. On the medium containing 50 mM BA, cell clumps frequently
browned and 20.4 of the cell clumps produced a few shoot buds. Significantly greater numbers of
shoot buds 15.2 and somatic embryos 13.7 per cell clump developed on the media containing 0.5
m M BA and without plant growth regulators,
respectively. Using the regeneration medium without plant
growth regulators, changes in the regeneration potential of suspension cultures, which were evalu-
ated with the percentage of cell clump produced shoot buds andor somatic embryos and by the
Fig. 3. Histological examinations of the regeneration from 12-month-old suspension cultures of L. formosanum 2 weeks
after transfer of cell clumps onto the regeneration medium without plant growth regulators. A A shoot bud consisted
of an apparent shoot meristem arrowhead and several leaf primordia arrows scale bar, 500 mm. B An oval-shaped
somatic embryo scale bar, 1 mm. C An oval-shaped so- matic embryo showing bipolarity of meristematic regions
arrowheads scale bar, 1 mm. D An elongated somatic embryo showing bipolarity of meristematic regions arrow-
heads scale bar, 500 mm.
observation revealed that these structures con- sisted of an apparent shoot meristem and several
leaf primordia Fig. 3A, and, therefore, they could be called ‘shoot buds’. These shoot buds
thereafter developed into shoots with a small bul- blet at the basal region. In comparison, embryo-
like structures were oval to club-shaped, easily detached from the original cell clumps Fig.
1C,D, and they had a shoot and root meristem Fig. 3B – D, indicating that they were ‘somatic
embryos’. These embryos simultaneously devel- oped a shoot and a root, and plantlets with a
small bulblet were obtained from them.
Effects of PIC and BA on the formation of shoot buds and somatic embryos were examined
using cell clumps taken from 12-month-old sus- pension cultures Table 1. On the medium con-
taining 5 mM PIC, only callus growth was observed, and neither shoot buds nor somatic
embryos were produced. All of the cell clumps produced numerous somatic embryos, and 36.5
of the cell clumps also produced shoot buds on the plant growth regulator-free medium. With the in-
crease in BA concentration, the formation of so- matic embryos was inhibited, whereas shoot bud
formation was promoted. Almost all of the cell clumps 96.3 vigorously produced shoot buds
Table 1 Effects of PIC and BA on the production of shoot buds and somatic embryos from 12-month-old suspension cultures of L.
formosanum
a
PIC mM BA mM
of viable cell clumps of cell clumps producing:
Mean number per cell clump Shoot buds
Somatic embryos Shoot buds
Somatic embryos 100a
36.5b 100a
2.1c 15.2a
5 100a
0c 0c
0d 0c
100a 96.3a
26.5b 13.7a
0.5 1.3b
100a 100a
0c 5
8.6b 0c
27.0b 20.4b
0c 0.8c
0c 50
a
Data were recorded 3 months after transfer of suspension cell clumps 1–3 mm in diameter to the regeneration media without TIBA. Values represent the mean of at least three independent experiments, each of which consisted of at least ten cell clumps.
Means in the same column followed by the same letter are not significantly different PB0.05; LSD test.
Fig. 4. Production of shoot buds andor somatic embryos from suspension cell clumps of L. formosanum at different months after the initiation of suspension cultures. Data were recorded 3 months following transfer of cell clumps 1 – 3 mm in diameter to the
regeneration medium without plant growth regulators. Values represent the mean 9 S.E. of at least three independent experiments, each of which consisted of at least 20 cell clumps.
total number of shoot buds and somatic embryos per cell clump, were examined over 72 months of
subculture Fig. 4. Suspension cultures main- tained a high regeneration ability for up to 54
months. During this period, almost all of the cell clumps produced shoot buds andor somatic em-
bryos, and the total number of these differentiated tissues per cell clump was routinely over 30. The
percentage gradually decreased thereafter, and only about 10 of the cell clumps produced a few
shoot buds andor somatic embryos 75 months after the initiation of suspension cultures.
3
.
3
. . Effect of TIBA on the restoration of regeneration potential of suspension cultures
In order to induce an efficient regeneration from 75-month-old suspension cultures, cell clumps
were transferred onto medium containing 0.5 or 5 m
M plant growth regulators, including NAA, PIC, TIBA, BA and TDZ. An improvement in regener-
ation was observed only when cell clumps were
cultured on TIBA-containing media data not shown; therefore, further experiments on the ef-
fects of TIBA were conducted Table 2. On medium without TIBA, only about 10 of cell
clumps produced shoot buds, and less than one shoot bud was obtained per cell clump. On the
other hand, some improvements in regeneration were observed on medium containing only TIBA:
the percentage of cell clumps forming shoot buds increased to 36.4 and 25.7 on 0.5 and 5 mM
TIBA-containing media, respectively, and over three shoot buds were obtained with both concen-
trations. Regeneration of shoot buds was further stimulated by combining TIBA with a cytokinin:
65 – 80 of cell clumps produced shoot buds, and eight to ten shoot buds were obtained per cell
clump on media containing TIBA and either BA or TDZ. Although no significant differences both
in the percentage of cell clumps producing shoot buds and the mean number of shoot buds per cell
clump were observed between different concentra- tions of TIBA 0.5 and 5 mM, the percentage was
slightly higher on 0.5 mM TIBA-containing media. TIBA did not affect viability and appearance of
the cell clumps, but only shoot buds were regener- ated and no somatic embryos were produced on
TIBA-containing media. On media containing TIBA and 5 mM TDZ, the production of multiple
shoot buds was occasionally observed.
3
.
4
. Plant regeneration Shoot buds regenerated from 12- or 72-month-
old suspension cultures were excised from the cell clumps and transferred to a plant growth regula-
tor-free medium on which they formed roots and developed into plantlets. Although some shoot
buds regenerated on TIBA-containing medium did not readily form roots, complete plantlets were
obtained after one or two subcultures onto the same plant growth regulator-free medium. Most of
the somatic embryos could develop into plantlets on the regeneration media, but their growth was
promoted by transferring them to the fresh plant growth regulator-free medium. Shoot bud- and
somatic embryo-derived plantlets formed a small bulblet at the basal region.
After cold treatment, regenerated plantlets were transferred to pots, in which over 90 of them
developed new scaly leaves within 2 weeks and successfully transferred to the greenhouse Fig.
1E. The first flowers opened 5 – 6 months after transfer to the greenhouse. Among 30 plants,
which had been derived from 72-month-old sus-
Table 2 Effects of TIBA, BA and TDZ on the production of shoot buds and somatic embryos from 72-month-old suspension cultures of
Lilium formosanum
a
TIBA mM TDZ mM
BA mM Mean number per cell clump
of cell clumps producing: of viable
cell clumps Shoot buds
Somatic Somatic embryos
Shoot buds embryos
0.1a 100a
0.8c 1.1a
10.3c 0.5
100a 12.4c
0a 0.8c
0a 5
100a 9.6c
0a 0.6c
0a 0a
0.9c 0a
9.3c 100a
0.5 5
85.3b 10.8c
0a 0.6c
0a 100a
0.5 36.4b
0a 3.3b
0a 0a
7.9a 0a
75.3a 0.5
100a 0.5
5 100a
0.5 80.2a
0a 8.5a
0a 0.5
100a 78.9a
0a 8.3a
0a 0.5
5 85.7b
0.5 73.5a
0a 9.1a
0a 5
96.3a 25.7bc
0a 3.1b
0a 77.4a
100a 0.5
5 0a
9.4a 0a
5 5
0a 100a
8.3a 0a
68.3a 5
88.2b 65.2a
0a 9.8a
0a 0.5
5 88.8b
67.7a 0a
8.8a 0a
5
a
Data were recorded 3 months after transfer of suspension cell clumps 1–3 mm in diameter to the regeneration media. Values represent the mean of at least three independent experiments, each of which consisted of at least ten cell clumps. Means in the
same column followed by the same letter are not significantly different PB0.05; LSD test.
pension cultures, no apparent variant phenotype was detected. In addition, all of them had a
diploid number of chromosomes 2n = 2x = 24 data not shown.
4. Discussion
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