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http://www.sciencedirect.com/science/article/pii/S1871678411001300
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In this study, morphogenesis from explants of Vietnamese
ginseng (Panax vietnamensis) main roots, including
somatic embryogenesis, callus, shoot, and root regeneration,
were first established using thin cell layer (TCL)
techniques. The optimal conditions for somatic embryogenesis
were Murashige and Skoog (MS) medium containing 0.05 mg/l
thidiazuron (TDZ). MS medium supplemented with 1.0 mg/l
benzyladenine (BA) and 0.1 mg/l 2,4–dichlorophenoxyacetic
acid (2,4–d)
efficiently induced shoot regeneration under 16 h
photoperiod while MS medium with 2.0 mg/l
α–napththaleneacetic
acid (NAA) in dark was optimal for root regeneration. Calli
were regenerated from root tTCL in MS medium containing 0.2
mg/l BA and 1.0 mg/l 2,4–d
under 16 h photoperiod or MS medium supplemented with 1.0
mg/l 2,4–d and
0.1 mg/l TDZ in dark. This is the first time developmental
programs for direct shoot, root, and somatic embryo
formation from explants without going through the callus
stage were established for Vietnamese ginseng.
Key words:
Panax vietnamensis, root regeneration, somatic
embryogenesis, thin cell layer, Vietnamese ginseng.
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Thin
cell layer (TCL) technique was first introduced to plant organogenesis
studies by Tran Thanh Van in 1970’s. Since then, TCL has been
successfully applied on different species, including tobacco, lily, and
chrysanthemum. Tobacco is the most well-studied species, with four
established organogenesis programs, including direct flower, shoot, root
and callus regeneration. Other than tobacco, this technique has been
proven successful in many other species. Ohki (1994) was able to
generate more than 70,000 African violet plantlets within only three to
four months. Nhut et al. (2001) effectively applied TCL to in vitro
culture of lily. Ahn et al. (1996) also applied this technique on
ginseng for somatic embryogenesis directly from cotyledons. Similarly,
Whei–Lan et al. (2002) reported embryogenesis, root and callus formation
from Korean and American ginsengs. TCL system, with its advantage of
being thin, is very suitable for rapid and uniformed development of
explants. Also, it is very applicable for valuable species, especially
ginseng. In addition, utilization of TCL also provide various
advantages, such as: direct contact of explants to culture media, hence,
better nutrient and hormone uptake, leading to the formation of
different structures including primary shoot/roots, and embryos. In this
study, we report the application of TCL in studying morphogenesis of
Vietnamese ginseng (P. vietnamensis). Somatic embryogenesis and
callus, shoot and root formation were induced from main root
transverse–TCLs (tTCLs) in different culture conditions. Of these,
direct embryogenesis has particularly significant impacts in propagation
for the ability of somatic embryos to develop completely into mature
plants with shoots, leaves, and roots. This study proved the importance
of somatic embryogenesis through TCL technology in Vietnamese ginseng
propagation and preservation.
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Explant source
Vietnamese
ginseng plants grown for three months on Schenk and Hildebrandt (SH)
medium supplemented with 30 g/l sucrose, 0.5 g/l activated charcoal, and
9 g/l agar were used as the source of explants. Selected plants were
vitrification–free, equally well–growing and healthy with leaves,
shoots, and main and fiber roots. tTCLs with 1–mm thickness were cut
from in vitro main roots as initial explants.
Culture media
The
basic medium for all experiments was MS medium (Murashige and Skoog,
1962) supplemented with 30 g/l sucrose and 8 g/l agar. Plant growth
regulators (PGRs) including α–napththaleneacetic acid (NAA), 2,4–dichlorophenoxyacetic acid (2,4–d),
and thidiazuron (TDZ) were added separately and in combination into
culture medium for different experiments. All culture media were
adjusted to pH 5.7 to 5.8 before autoclaving.
Experimental design
Callus formation, direct embryogenesis, and root and shoot formation of tTCL explants from in vitro
Vietnamese ginseng main roots was investigated. The appropriate medium
for each morphogenesis process was determined based on evaluating the
individual and combinatorial effects of TDZ (0.01, 0.05, 0.1, 0.2, 0.5,
and 1 mg/l), benzyladenine (BA) (0.1, 0.2, 0.5, 1, 2 mg/l), NAA (0.1,
0.2, 0.5, 1, 2 mg/l), and 2,4–d (0.1, 0.2, 0.5, 1, 2 mg/l) after eight weeks of culture.
All
treatments were triplicated, each with 15 explants in five culture
vessels. Morphogenesis was allowed at 25 ± 2°C, 80% relative humidity,
under regular lighting conditions (2,000 to 2,500 lux) or darkness.
Histological studies
Histological
analysis was performed, according to Gonzalez and Cristóbal (1997), for
explants at 15 days after culture initiation. Samples of cultured
explants were fixed in formaline, acetic acid, 70% ethanol – 5:5:90
(FAA), dehydrated with Deshidratante histológico BIOPUR®, then embedded in paraffin wax as described by Johansen (1940), and sectioned into 8 to 10 μm
thick serial section with a rotary microtome. Section were mounted on
glass slides and stained with safranin-Astra blue (Luque et al., 1996),
and observed under a light microscope.
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Effect of separately–supplemented PGRs on the morphogenesis of main root tTCLs
The effect of PGRs on
the explant morphogenesis was clearly visible after eight weeks of
culture. Explants cultured on control medium (without PGRs) under both
16 h photoperiod and darkness died off. This suggests that the
endogenous PGRs of the tTCLs were not sufficient for any morphogenesis
processes (Tables 1 and 2).
Although
browning of the explants were observed on medium supplemented with TDZ
or BA separately, the formation of embryos and shoots still occurred
(Figure 1) at the edge of the tTCLs.
The
highest rate of embryogenesis (60%) (Table 1) was recorded from tTCLs
cultured on medium containing 0.05 mg/l TDZ under 16 h photoperiod with
milk–white or yellowish globular embryos and some embryos were observed
under a light microscope (Figure 3b). When embryogenic tissue sections
containing groups of 10 to 20 somatic embryos (Figure 3a) were
transferred to the free-PGRs MS medium, some embryos germinated within a
few days (Figures 3c and d) and complete plantlets were obtained
(Figures 3e and f).
Medium
supplemented with 0.05 mg/l TDZ also allowed shoot formation (26.7%)
with large green shoot clusters (Figure 1). Interestingly, some shoots
were formed directly from tTCLs without going through callus formation.
Under the dark condition, embryogenesis was reduced with the highest
rate at 26.7% on medium with 0.2 mg/l TDZ. This medium, however, gave the highest rate of shoot formation (Table 2).
On
culture medium that had BA as the sole exogenous PGR, embryogenesis
rate increased with increased BA concentration, reaching 36.7% with 2
mg/l BA under both regular light and dark conditions. All developmental
stages of embryogenesis including globular, heart– and torpedo–shaped,
and cotyledonary embryos were observed (Tables 1 and 2). Under regular
lighting conditions, BA induced the formation of shoots with the highest
rate (23.3%) at 2 mg/l BA (Table 1). No shoot formation was seen in
dark condition (Table 2, Figure 1).
Only callus formation without embryogenesis, shoot and root formation occurred when 2,4–d was supplemented into the medium. More callus formation was recorded as 2,4–d concentration increased, reaching the highest rates of 60% at 1 mg/l 2,4–d and 66.7% at 2 mg/l 2,4–d
under regular lighting and dark conditions, respectively. Three types
of callus appearance were observed, including white and friable, yellow,
and green and hard (Tables 1 and 2, Figure 2).
Small
and long adventitious hairy roots with milk white or transparent white
colours (Figure 2) were induced on medium supplemented with NAA. The
highest rate of root formation was recorded at 2 mg/l NAA in dark and
was higher than that under regular lighting condition. Low concentration
of NAA under light induced embryogenesis and shoot formation at low
rates (Table 1) while only embryogenesis was seen at high NAA
concentration in dark (Table 2).
In
general, different PGRs induced different morphogenesis processes from
Vietnamese ginseng main root tTCLs. Optimal conditions for embryogenesis
and shoot, callus, and adventitious root formation were 0.05 mg/l TDZ
under light, 2 mg/l 2,4–d in dark, and 2 mg/l NAA in dark, respectively (Table 2, Figure 4).
Table 1. Effect of separately–supplemented PGRs on the morphogenesis of (P. vietnamensis) main root tTCLs after eight weeks of culture under 16 h photoperiod.
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