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,2-8050Ikarashi,Niigata950-2181,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 5mM 2,3,5-triiodobenzoic acid (TIBA) in combination with 0.5 or
5mM BA orN-(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.com/locate/plantsci
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 ofL. formosanumWallace, 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:[email protected] (M. Nakano).
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−1sucrose and 2 g l−1gellan gum at 25°C under continuous illumination (35mmol 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 5mM 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−1glucose to initiate cell suspension cultures. In L. formolongi, protoplasts isolated from suspension cultures using a glucose-contain-ing medium showed a high platglucose-contain-ing 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−1gellan 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 mmol 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−1gellan 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
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- and/or 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. 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 mean9S.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 mM 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 mM 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 and/or 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 meristeso-matic regions (arrowheads) (scale bar, 1 mm). (D) An elongated somatic embryo showing bipolarity of meristematic regions (arrow-heads) (scale bar, 500mm).
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.
Table 1
Effects of PIC and BA on the production of shoot buds and somatic embryos from 12-month-old suspension cultures of L.
formosanuma
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
0 0 100a 2.1c 15.2a
5 0 100a 0c 0c 0d 0c
100a 96.3a 26.5b
0 0.5 13.7a 1.3b
100a 100a 0c
5 8.6b
0 0c
27.0b 20.4b 0c 0.8c
0 50 0c
aData 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 and/or somatic embryos from suspension cell clumps ofL.formosanumat 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 mean9S.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 and/or 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 and/or somatic embryos 75 months
after the initiation of suspension cultures.
3.3. . Effect of TIBA on the restoration of regeneration potential of suspension cultures
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 5mM), the percentage was slightly higher on 0.5mM 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 formosanuma
TIBA (mM) BA (mM) TDZ (mM) % of viable % of cell clumps producing: Mean number per cell clump
cell clumps Shoot buds Somatic Shoot buds Somatic embryos embryos
0 0
0 100a 10.3c 1.1a 0.8c 0.1a
0.5 0 100a 12.4c 0a 0.8c 0a
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
In this study, fast-growing cell suspension cul-tures of L. formosanum were successfully estab-lished and could be maintained for over 6 years without an apparent change in the growth rate. This is the first report demonstrating the establish-ment and long-term maintenance of cell suspen-sion cultures in L. formosanum. Both shoot buds and somatic embryos were regenerated from sus-pension cell clumps in this study. In the early period after the establishment of suspension cul-tures (12-month-old culcul-tures), cell clumps prefer-entially produced somatic embryos on a plant growth regulator-free medium, while shoot buds were generally produced on BA-containing media. These results indicated that our suspension cul-tures were ‘embryogenic’ relatively soon after the initiation and, in addition, the morphogenic path-way of the cultures can be controlled by plant growth regulators.
In this study, regeneration potential decreased after 54 months, of culture and only few adventi-tious shoots and embryos were obtained from 75-month-old cultures. The decline with time of the regeneration potential of callus and suspension cultures has already been demonstrated for numer-ous plant species [20 – 22], and in some cases, the decline has been reported to be related to the increase of polyploid and/or aneuploid cells in the cultures [20,23 – 25]. In this study, however, no apparent differences in the ploidy level, as indi-cated by cytological observations and flow cyto-metric analyses of nuclear DNA content, were found in 12- and 72-month-old suspension cul-tures, and most cells in either culture had a diploid number of chromosomes (data not shown).
The decline in the regeneration potential of our suspension cultures was probably due to some physiological change of the cells since the potential could be restored using TIBA. Regeneration of shoot buds was stimulated on media containing only TIBA as a plant growth regulator, but the promoting effect of TIBA was further enhanced by combining with either cytokinin, BA or TDZ.
Beneficial effects of TIBA on the regeneration of shoot buds have so far been reported for several plant species [26 – 31]. Because TIBA specifically inhibits the polar transport of endogenous indole-3-acetic acid (IAA) [32], authors of the previous reports suggested that addition of TIBA to regeration media might inhibit the transport of en-dogenous IAA to the regeneration sites, so that an auxin/cytokinin balance become more favorable for the regeneration of shoot buds. A similar explanation may be possible for the promotive effect of TIBA observed in this study: endogenous and polar-transporting (or radial transporting in this case) IAA level in the cell and cell clumps might excessively increase during long-term sub-culture period, leading to the decline in the regen-eration potential of the cultures; and the balance between the level of auxin/cytokinin at the regen-eration sites might again become suitable for re-generation by treating old cultures with TIBA. In this study, suspension cell clumps produced only shoot buds, and no somatic embryos were regener-ated on TIBA-containing media, indicating that somatic embryogenesis may be inhibited through the inhibition of polar distribution of endogenous auxin in the cell clumps, which has been reported to be essential for the early stage of embryogenesis [33]. For clarifying the mechanism of the effects of TIBA, it is necessary to measure endogenous levels of IAA in cell clumps from both regenerable and nonregenerable old suspension cultures with and without TIBA treatments, and to treat the old cultures with the other auxin transport inhibitors, e.g. N-1-naphthylphtalamic acid.
In this study, plants could be regenerated from 72-month-old suspension cultures by TIBA treat-ments. Although neither apparent phenotypic vari-ations nor varivari-ations in chromosome number were detected in at least 30 plants regenerated from these old cultures, detailed examination on the genetic fidelity of the regenerated plants is needed using a larger number of regenerants, since long-term cultures are generally associated with high levels of somaclonal variation [34].
Acknowledgements
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