Plant Biotechnol Rep (2008) 2:207–212
DOI 10.1007/s11816-008-0063-6
ORIGINAL ARTICLE
Effect of photoperiod and light intensity on in vitro propagation
of Alocasia amazonica
Eun-A. Jo Æ Rajesh Kumar Tewari Æ
Eun-Joo Hahn Æ Kee-Yoeup Paek
Received: 16 November 2007 / Accepted: 7 June 2008 / Published online: 12 July 2008
Ó Korean Society for Plant Biotechnology and Springer 2008
Abstract Plantlets of Alocasia amazonica regenerated Walters et al. 2003). The changes in chloroplast composi-
under a photon flux density (PFD) of 15 or 30 lmol tion that result from variation of the quantity of incident
m-2 s-1 showed better growth and development than those light have been well characterized. With increasing light
grown under higher PFDs. While chlorophyll a and chlo- availability, the requirement for light-harvesting complexes
rophyll b decreased, the number of stomata increased with (LHCs) is reduced in contrast with the demand for electron
increasing PFD. Photoperiods also affected plantlet growth transport and carbon assimilation components to support
and stomatal development. Highest growth was observed higher rates of photosynthesis (Anderson and Osmond
for the short photoperiod (8/16 h) and for equinoctial 1987; Anderson et al. 1995). For instance, in low light,
(12/12 h) light and dark periods. Very few stomata devel- levels of chlorophyll (Chl) a/b-binding LHCs are high,
oped in the leaves of plantlets grown under a short whereas in strong light, plants increase the levels of
photoperiod (8/16 h) and the number of stomata increased photosystems, the cytochrome b6/f complex, ATP synthase,
with increasing light period. In conclusion, both light and Calvin cycle enzymes—Rubisco (Walters et al. 2003).
intensity and photoperiod independently affect growth of Although light is the energy source for plant growth, excess
A. amazonica and development of stomata, depending on light can lead to depression of photosynthetic efficiency
the intensity and duration of light treatment. (photoinhibition) (Powles 1984). Plants grown in low light
have frequently been shown to be more susceptible to
Keywords In vitro culture Light intensity photoinhibition than those grown under high-intensity light
Photoperiod Stomatal development (Osmond 1994). Photon flux density (PFD) also plays a key
Relative water content role in plant acclimation and survival after transfer from in
vitro to ex vitro conditions (Björkman 1973). Apart from
the effect of PFD (Jeon et al. 2005; Ali et al. 2005), pho-
Introduction toperiod also affects various developmental processes, for
example rooting, elongation of stem (Ramanayake et al.
Plants are able to modify their growth, development, and 2006), tuberization (Omokolo et al. 2003), germination,
physiology in accordance with a variable environment; this induction of flowering (Taiz and Zeiger 2002; Heo et al.
plays a key role in determining their tolerance of stress, and 2003; Khokhar et al. 2007), and stomatal regulation
maintains efficient growth (Murchie and Horton 1997; (Stadler et al. 2003).
In micropropagation, plantlets should be maintained at
much lower levels of PFD than in the greenhouse or field,
E.-A. Jo R. K. Tewari E.-J. Hahn K.-Y. Paek (&)
Research Center for the Development of Advanced Horticultural because their responses to PFD are different from those
Technology, Chungbuk National University, grown in the greenhouse or field. Growth responses to
Cheongju 361-763, South Korea photoperiod could also be different depending on whether
e-mail: paekky@chungbuk.ac.kr
the plants are grown in vitro or ex vitro (Paek 2001).
R. K. Tewari Despite the enormous knowledge accumulated on the
e-mail: rktewari_bot@yahoo.com photoregulation of plant development under different light
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208 Plant Biotechnol Rep (2008) 2:207–212
regimes, no report is available on micro-propagation of chlorophyll content, relative water content (RWC), size of
A. amazonica under different light intensity and photope- leaf, and size and number of stomata were estimated.
riod conditions. Thus the objectives of this work were to
establish whether the quantity of light and photoperiod PFD experiment
affect micro-propagation, growth of plantlets, and devel-
opment of stomata in A. amazonica foliage. Light treatment was varied by placing culture vessels at
different PFD (15, 30, 60, and 90 lmol m-2 s-1) supplied
by fluorescent lamps fitted with a timer set at 16/8 h light/
Materials and methods dark photoperiod. PFD was measured (on top of the vessel)
with a data logger (LI-1400; LI-COR, Lincoln, NE, USA).
Plant material and maintenance of culture Thirty days after initiating treatment, shoot length, root
length, fresh weight, dry weight, leaf area, chlorophyll
Rhizomes with nodal buds of A. amazonica were selected content, RWC, and number and the size of stomata were
and washed with running tap water. The explants were estimated.
surface disinfected with 70% ethanol for 10 s followed by
surface sterilization with 3% sodium hypochlorite solution Plantlet growth, photosynthetic pigments, and relative
for 30 s then thorough rinsing with sterilized distilled water content
water. The nodal explants were transferred to 100 ml
Erlenmeyer flasks containing 20 ml MS (Murashige and To determine dry weight, plantlets were oven-dried at 70°C
Skoog 1962) solid medium supplemented with 2.0 mg l-1 until they reached constant mass. Leaf area was measured
benzyladenine and 3% sucrose 7.5 g l-1 agar (Duchefa, with a leaf-area meter (LI-3100; LI-COR). Chlorophyll and
Haarlem, the Netherlands) to induce adventitious shoots. carotenoid content were measured according to Lichtent-
The pH of the medium was adjusted to 5.7 before auto- haler (1987). RWC was determined by measuring fresh,
claving. After 5 weeks of culture, the adventitious shoots hydrated, or saturated (for 3 h at 4°C in the dark on glass-
were cut into 1.0 cm lengths and cultured in polypropylene distilled water), and oven-dry weights of 45 leaf discs
growth vessels (107 9 107 9 97 mm; Osmotek, Israel) (three replicates of 15 discs each; Weatherley 1950).
containing 50 ml MS basal medium supplemented with
30 g l-1 sucrose. Cultures were maintained at 25 and 18°C Stomatal observations
during day and night, respectively, with 30 lmol m-2 s-1
PFD (using cool white fluorescent lamps, 40 W) with a For stomata observation, segments of the fully developed
16 h photoperiod. leaves were cut into 3 9 3 mm pieces and fixed in a for-
malin–acetic acid–alcohol solution for 24 h. This segments
Culture conditions and treatments were washed three times with distilled water, stained for
15 min in 0.01% acridine orange, then stained for 15 min
Five apical bud explants (1 cm) were placed in a 900-ml in a 0.01% rhodamine. Finally they were washed well with
square-type transparent glass vessel containing 200 ml distilled water before observation. Stomata size and fre-
solid MS medium as described above. To provide air quency were determined by microscopic examination of
exchange (0.1 vvm), gas-permeable micro-porous filters five leaf segments with a laser scanning confocal micro-
(Mill-Seal, Millipore, Tokyo; pore size 0.5 lm) were scope (1024 ES; Bio-Rad MRC, UK) equipped with a
attached to the tops of the culture vessels. Cultures were Kr/Ar mixed gas laser (Bio-Rad MRC). Samples were
maintained for 30 days in a growth chamber at 25°C air observed with a 209 dry objective lens (0.6–1.0 NA) by
temperature and 70 ± 5% relative humidity. the method of Gray et al. (1999). The length and width of
stomata were measured and compared with the mean by
Photoperiod experiment using the standard error of the mean.
In the photoperiod experiment, the light period was regu- Statistical analysis
lated by timers attached to cool white fluorescent lamps
(40 W) and set at 8/16, 12/12, 16/8, and 24/0 h day/night There were ten replicates for each treatment and data were
cycles. Illumination was from above the vessels and the tested using the statistical analysis system (SAS 1989).
PFD was adjusted to 30 ± 2 lmol m-2 s-1 (on top of the Where a significant difference (P B 0.05) was observed for
culture vessel). Thirty days after initiating treatment, shoot a measured value, means were separated using Duncan’s
length, root length, fresh weight, dry weight, leaf area, multiple range test (DMRT) at the 5% level.
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Plant Biotechnol Rep (2008) 2:207–212 209
Fig. 1 Effect of photoperiod (a–d) or light intensity (e–h), on the phenotypic appearance of plantlets of Alocasia amazonica regenerated in
day/night cycles of 8/16 (a), 12/12 (b), 16/8 (c), and 24/0 (d) h or in a PFD of 15 (e), 30 (f), 60 (g), 90 (h) lmol m-2 s-1
Results and discussions Table 1 Effects of photoperiod on growth, chloroplast pigments,
relative water content (RWC), and stomatal attributes in the leaves of
A. amazonica plantlets
Effects of photoperiods
Measured characteristic Photoperiod (light/dark h)
Among the environmental factors affecting plant activity, 8/16 12/12 16/8 24/0
photoperiod is one of the most important factors regulating
plant development by maintaining a specific ratio of photo- Shoot length (cm) 3.9a 3.6a 3.1b 2.9b
receptors during specific photoperiods (Taiz and Zeiger Root length (cm) 4.4a 2.7b 3.3b 3.1b
2002; Heo et al. 2003; Stadler et al. 2003). Significant Number of root (explant-1) 5.7a 6.0a 5.0a 4.3a
2 -1
differences were observed regarding the growth and devel- Leaf area (cm leaf ) 3.21a 3.68a 3.82a 2.00b
opment of plantlets in our study also. The length of Leaf emergence (%) 100c 100c 91.6b 83.3a
photoperiod affected growth and morphology of A. amazo- Bulb size (cm) 0.66a 0.67a 0.63a 0.64a
nica differently (Fig. 1a–d). Shoot length, root length, Fresh weight (g plant-1) 0.44a 0.44a 0.39ab 0.36b
number of roots, leaf area, fresh weight, dry weight, and Dry weight (g plant-1) 0.040a 0.041a 0.039a 0.039a
-1
chlorophyll and carotenoids content (Table 1) increased Chl a (mg g fresh wt) 1.64a 1.33b 1.38b 0.97c
under shorter day (8/16 h) and equinoctial (12/12 h) (light/ Chl b (mg g-1 fresh wt) 0.60a 0.51b 0.50b 0.34c
dark cycle) photoperiods. Similarly, short photoperiods Carotenoids (mg g-1 fresh wt) 0.52a 0.40b 0.39b 0.28c
resulted in a higher rate of tuberization of Habenaria mac- Car/chl ratio 0.231a 0.217b 0.207b 0.215b
roceratitis (Omokolo et al. 2003; Stewart and Kane 2006). RWC (%) 92a 96a 91a 81b
However, in plum, maximum shoot proliferation was recor- Number stomata (mm-2) 59.50c 81.75b 83.75b 102.50a
ded for 12/12 and 16/8 h light/dark periods, but chloroplast Length of stomata (lm) 27.94a 27.65a 27.05a 26.52a
pigments and shoot quality were not affected by light period Width of stomata (lm) 23.84a 22.16b 22.14b 18.60c
(Morini et al. 1991). Increased chloroplast pigments at low
Data are means from duplicate experiments (n = 10 per experiment).
light periods seem to be an acclimatory mechanism to Values in the same row with different letters are significantly different
increase quantum absorption under conditions of limited at (P B 0.05)
photoperiod. The increases in the amounts of chloroplast
pigments (Table 1) in plantlets grown in shorter day pho- et al. 2003). Increased carotenoids-to-chlorophyll ratio fur-
toperiods might have maximized the antenna molecules as a ther substantiates this suggestion. Higher fresh and dry mass,
compensatory mechanism for stationary-state photosynthe- with other growth attributes, indicate A. amozonica requires a
sis, similar to plants growing in low light intensity (Walters shorter photoperiod for its best vegetative growth. Decline in
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Fig. 2 Effect of photoperiod (a–d), or light intensity (e–h), on (c), and 24/0 (d) h or in a PFD of 15 (e), 30 (f), 60 (g), and 90 (h)
the development of stomata in the leaves of Alocasia amazonica lmol m-2 s-1. Scale bar = 25 lm
plantlets regenerated in day/night cycles of 8/16 (a), 12/12 (b), 16/8
water status of leaf tissue, as reflected by decreased RWC at Table 2 Effects of PFD on growth, concentration of chloroplast
higher photoperiods (16/8, and 24/0 h), suggests increased pigments, relative water content (RWC), and stomatal attributes in the
leaves of A. amazonica plantlets
stomatal transpiration. Because numbers of stomata
increased with increasing photoperiod (Table 1, Fig. 2a–d), Measured characteristic PFD (lmolm-2 s-1)
they might have resulted in greater loss of water from the leaf 15 30 60 90
surface, reflected as decreased RWC of plantlets grown under
-1
16/8 or 24/0 h photoperiods. Length of stomata was, how- Number of shoots (explant ) 3.67b 6.33a 5.00ab 4.33b
ever, not affected by photoperiod, but width of stomata Shoot length (cm) 5.73a 5.37ab 4.93ab 4.17b
declined with increasing photoperiod (Table 1, Fig. 2a–d). Rooting (%) 80b 90a 70c 75c
These observations indicate that light period affects the dif- Leaf area (cm2 leaf-1) 4.14a 4.60a 2.50b 1.88b
ferentiation of leaf epidermal tissues of A. amazonica. Bulb size (cm) 1.03a 0.93a 0.94a 0.89a
Fresh weight (g plant-1) 0.37a 0.38a 0.28b 0.23c
-1
Effects of PFD Dry weight (g plant ) 0.019bc 0.022a 0.020ab 0.018c
Chl a (mg g-1 fresh wt) 1.20b 2.10a 1.10b 0.44c
PFD affected plant growth and morphological features of Chl b (mg g-1 fresh wt) 0.47b 0.85a 0.47b 0.21c
A. amazonica (Fig. 1). Plantlet growth was largest in a PFD Carotenoids (mg g-1 fresh wt) 0.28b 0.53a 0.32b 0.13c
of 30 lmol m-2 s-1. Shoot length, bulb size, leaf area, and Car/chl ratio 0.167a 0.179a 0.204b 0.200b
fresh and dry matter yields (Table 2) did not change RWC (%) 96.7a 94.7a 87.3a 83.3a
between PFD of 15 and 30 lmol m-2 s-1 and appeared to Number stomata (mm-2) 29.01c 39.99c 77.01b 96.01a
be largest at these PFDs. However rooting rate (%) Length of stomata (lm) 27.44b 33.05a 25.70b 22.20c
was highest at a PFD of 30 lmol m-2 s-1 (Table 2). Width of stomata (lm) 17.45c 23.39a 20.51b 21.76ab
For plantlets cultured at higher PFDs, viz. 60 or
Data are means from duplicate experiments (n = 10 per experiment).
90 lmol m-2 s-1, these values decreased. These results for Values in the same row with different letters are significantly different
growth and morphological features show that A. amazonica at (P B 0.05)
can perform well in low rather than high light intensity
(Fig. 1e–h). Apart from the effect on growth and mor- observed at a PFD level of 30 lmol m-2 s-1 (Table 2) and
phology, PFD levels also had effects on chlorophylls and declined at PFDs of 60 and 90 lmol m-2 s-1. It has been
carotenoids contents (Table 2). Maximum concentrations reported that leaves of plants growing at low PFD had
of chloroplast pigments (chl a, chl b, and carotenoids) were relatively higher contents of chloroplast pigments and
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Plant Biotechnol Rep (2008) 2:207–212 211
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