Plant Science 158 2000 129 – 137
Decrease in the regeneration potential of long-term cell suspension cultures of Lilium formosanum Wallace and its restoration by the
auxin transport inhibitor, 2,3,5-triiodobenzoic acid
Masaru Nakano, Toshiaki Sakakibara, Sakae Suzuki, Hiroyuki Saito
Faculty of Agriculture, Niigata Uni6ersity,
2
-
8050
Ikarashi, Niigata
950
-
2181
, Japan Received 8 February 2000; received in revised form 5 June 2000; accepted 6 June 2000
Abstract
Cell suspension cultures of Lilium formosanum Wallace were initiated from bulb scale-derived calli and subcultured every 2 weeks using a medium containing 5 mM 4-amino-3,5,6-trichloropicolinic acid picloram. Almost all cell clumps from the
suspension cultures developed numerous somatic embryos following their transfer onto a plant growth regulator-free medium, while they vigorously produced shoot buds on media containing 0.5 or 5 mM 6-benzyladenine BA. The high regeneration
potential on a plant growth regulator-free medium was maintained for up to 54 months, but it gradually decreased thereafter, and only a few adventitious shoots and embryos were obtained from 75-month-old cultures. For restoring the regeneration potential
of these cultures, various treatments with plant growth regulators were applied, among which about 10-fold increases in the number of regenerated shoot buds were obtained with 0.5 or 5 mM 2,3,5-triiodobenzoic acid TIBA in combination with 0.5 or
5 mM BA or N-1,2,3-thiadiazol-5-yl-N-phenylurea thidiazuron. Only shoot buds were produced from the cell clumps cultured on TIBA-containing media, and these shoot buds developed into complete plantlets after they were excised from the calli and
transferred to a plant growth regulator-free medium. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Lilium formosanum; Wallace; Long-term culture; Regeneration ability; Shoot bud; Somatic embryo; 2,3,5-Triiodobenzoic acid www.elsevier.comlocateplantsci
1. Introduction
Fast-growing, highly-regenerable callus and cell suspension cultures have been used as a suitable
source of regenerable protoplasts and as a target material for producing transgenic plants in many
monocotyledonous plant species [1 – 7]. In the genus Lilium, callus and cell suspension cultures
have already been established in several species and cultivars [8 – 13]. In addition, plant regenera-
tion from suspension culture-derived protoplasts of Lilium formolongi [14,15] and production of
transgenic plants via microprojectile bombard- ment of callus cultures of Lilium longiflorum [16]
have recently been reported. However, only few studies have demonstrated the establishment and
characterization of long-term callus and cell sus- pension cultures in this genus [8,9,13].
In this study, we examined the establishment and long-term maintenance of cell suspension cul-
tures of L. formosanum Wallace, which has several advantageous characteristics including self-com-
patibility, taking only less than 1 year from seedling to flowering stages, and vigorous growth
[17], and thus this species is suitable as a target for basic research using cell suspension cultures. Char-
acterization of the suspension cultures was made with special reference to their regeneration ability
for over 72 months. In addition, we demonstrated the restoration of regeneration ability of the cul-
tures by treating with 2,3,5-triiodobenzoic acid TIBA, the auxin transport inhibitor.
Corresponding author. Tel.: + 81-25-2626858; fax: + 81-25- 2626858.
E-mail address
:
mnakanoagr.niigata-u.ac.jp M. Nakano. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 1 3 - 7
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
