growth and quality of Dioscorea fordii Prain
et Burk and Dioscorea alata plantlets using a
temporary immersion system
Huabing Yan1*, Litao Yang2
and Yangrui Li1
1Cash Crops Research
Institute, Guangxi Academy of Agricultural Sciences (GXAAS), 530007, Nanning,
Guangxi Province, People’s Republic of China.
Guangxi University, Nanning, Guangxi Province, 530007,
People’s Republic of China.
*Corresponding author. E-mail:
TIS, Temporary immersion system; SNCs, single
node leafy cuttings; NTPs, mean number of tubers per
Accepted 11 November, 2011
The effects of temporary immersion system (TIS)
culture on the growth and quality of Dioscorea fordii
Prain et Burk and Dioscorea alata plantlets were
investigated. Results indicate that TIS promoted the growth and
quality of D. fordii and D. alata plantlets.
Proliferation rate, shoot length, fresh weight (FW) and dry
weight (DW) of shoots, and total biomass production were
significantly (P≤0.05) higher in the TIS than in gelled and
liquid medium, respectively. The TIS also promoted tuberization
of D. fordii, and decreased vitrification of D. alata
significantly. The healthy plantlets of D. fordii and
D. alata obtained in the TIS would probably have positive
effects on transplanting in large-scale commercial production.
Dioscorea fordii Prain et Burk, Dioscorea alata,
culture system, TIS, micropropagation, tuberization.
is a well known edible and traditional medicinal plant. The
rhizomes of various species of Dioscorea had been used as
an important ingredient for invigorating the spleen and stomach,
promoting the body fluids, benefiting the lung and invigorating
the kidney, in addition to its used as a food crop (Wang et al.,
2006). Rhizome cuttings or aerial bulbils of Dioscorea
are normally used to initiate a crop, but these are sensitive to
viral disease infection, which finally degrade the cultivars and
dramatically decrease the yield. To overcome such problems,
in vitro propagation had been implemented for many
Dioscorea species, such as D. composita Hemsl. and
D. cayenensis Lam. (Viana and Mantell, 1989), D.
rotundata (Balogun et al., 2006), D. nipponica Makino
(Chen et al., 2007), D. cayenensis-D. rotundata
complex (Ovono et al., 2010) and D. prazeri (Thankappan
and Patell, 2011). In recent years, we have focused on the
micropropagation of two elite cultivars, viz.,
Dioscorea fordii Prain et Burk (“Guihuai No. 2”) and
Dioscorea alata (“Guihuai No. 6”) (Yan et al., 2010,
2011), which had been spread widely in Southwestern China owing
to their edible and medicinal dual functions.
The temporary immersion system (TIS), which was based on the
principle of temporary contact between plants and liquid medium,
had been extensively used for micropropagation (Alvard et al.,
1993; Cabasson et al., 1997; Martre et al., 2001; Wawrosch et
al., 2005; Alonso et al., 2009; Yan et al., 2010). Compared with
gelled and liquid culture, the TIS had been proven to have
quantitative advantages such as higher proliferation rate,
higher somatic embryogenesis, improved morphological
characteristics and reduced production cost (Etienne and
Berthouly, 2002). However, micropropagation of D. fordii
and D. alata using TIS had never been reported before.
Hence, this research was conducted to improve the growth and
quality of D. fordii and D. alata plantlets by
Materials And Methods
Single-node leafy cuttings (SNCs) obtained from in vitro
shoots of D. fordii and D. alata were used in all the
experiments. In vitro shoot regeneration methods had already
been established (Yan et al., 2011). Using SNCs as explants, the
effects of different culture systems (viz., gelled, liquid
and TIS) on shoot proliferation of both D. fordii and D.
alata were compared. As the gelled medium for proliferation,
Murashige and Skoog (1962) (MS) medium was used and supplemented
with 1.0 mg/L 6-benzylaminopurine (BA) (Sigma), 0.1 mg/L naphthalene
acetic acid (NAA) (Sigma), 3% (w/v) sucrose (Nanning, China),
1.5 g/L activated charcoal (AC) (Nanning, China) and 4.0 g/L agar
(Shanghai, China). The liquid and TIS medium for proliferation was
the same as that for gelled culture, but without the 4.0 g/L agar.
For the gelled and liquid culture, about 35 ml medium was dispensed
into glass vessels (90 mm height and 64 mm diameter).
TIS used Plantima containers (A-Tech Bioscientific Co., Ltd.,
Taipei, Taiwan) with 250 ml medium in each container. The container
comprised two compartments, an upper one with the plantlets and a
lower one with the medium. The application of pressure in the lower
compartment pushed the medium into the upper one. Plantlets were
immersed as long as forced pressure was applied. During the
immersion period, air was bubbled through the medium, gently
agitating the tissues and renewing the air in the head space inside
the culture container, with the forced pressure escaping through
outlets in the upper part of the container. The explants were
immersed for 3 min every 4 h by forced pressure, which propelled the
liquid towards the plant material. The pH of all media used was
adjusted to 5.8 ± 0.1 before autoclaving at 121°C for 20 min. All
cultures were incubated at 25 ± 1°C under a 12/12 h (day/night)
photoperiod with light supplied by white fluorescent tubes (25 μmol
m-2s-1). After in vitro culture for six
weeks, shoot length, proliferation rate, fresh weight (FW) and dry
weight (DW) of shoot, frequency of tuberization and mean numbers of
tubers per plantlet (NTPs) were scored. Shoot length was measured
from the apical shoot tip to the base of stem. DW of shoots was
determined after drying at 80°C for 72 h.
the experiments were carried out in a completely randomized design
with three replicates for each treatment, and using 18 SNCs in each
replicate. The statistical analyses were performed using statistical
analysis package (SAS version 8.01). Data were presented as mean ±
standard error. When necessary, statistical significance was
determined by using analysis of variance (ANOVA), the t- test
and extreme deviation analysis.
Results and Discussion
The TIS clearly promoted shoot formation (Figure 2A and B).
Proliferation rate and shoot length of D. fordii plantlet
in the TIS were significantly (P≤0.05) higher than those in
gelled and liquid medium, respectively as shown by proliferation
rate (2.1 and 1.1 times greater than those in gelled and liquid
medium, respectively) and shoot length (2.9 and 1.3 times
greater than those in gelled and liquid medium, respectively)
(Table 1). Similar results
shown in Figure 1. FW (423.3 mg) and DW (39.4 mg) of D.
fordii plantlet in the TIS were also significantly higher
than those in gelled medium (72.2 and 6.3 mg,
respectively) and those in liquid medium (306.8 and 28.2 mg,
respectively) (Table 1). The positive effects of the TIS on
shoot growth had been demonstrated by many authors in earlier
studies (Alvard et al., 1993; Lorenzo et al., 1998; Etienne and
Berthouly, 2002; Escalona et al., 2003; Yan et al., 2010). Under
the same growth conditions, compared to gelled and liquid
culture, plantlets in the TIS showed a significant (P≤0.05)
increase in total biomass production
expressed as DW and FW per plantlet (Table 1 and Figure 1).
Alonso et al. (2009) confirmed that TIS was a promising method
for biomass production of Digitalis purpurea by
in vitro shoot multiplication. The total biomass of
Siraitia grosvenorii plantlet cultured in TIS increased
significantly, compared to that in gelled and liquid culture (Yan
et al., 2010). The most important reason for the efficiency of
the TIS was that it combined the advantages of both gelled
culture (gas exchange) and liquid culture (increa- sed nutrient
uptake), which improved the growth of the plantlets (Etienne and
Compared to gelled culture, the liquid and TIS culture
significantly (P≤0.05) promoted in vitro tuberization,
indicated as frequency of tuberization
and NTPs (Table 1). TIS could increase the tuber size and
numbers of tubers per plantlet by extending the culture time
without renewing the culture medium, compared to the liquid
culture. After three months of culture, various sizes of
microtubers of D. fordii (Figure 2C) were obtained, 70%
of which were able to sprout in the seed bed (data not shown).
There were three tubers produced on one plantlet occasionally
(Figure 2D). Furthermore, vitrification often happened in
the liquid culture of D. alata (Figure 2E), but not in
TIS. Leaf malfunction in the liquid culture partly attributed to
high relative humidity in the culture container (Ziv, 1991). By
the combination of adequate culture ventilation and intermittent
contact between shoots and the liquid medium, the
microenvironment inside the temporary immersion container
probably was improved, which contributed to improved shoot and
root formation of D. alata. More also, Calathea
plants from TIS presented more functional photosynthetic and
respiratory apparatus, and could adapt more successfully to the
environmental changes during ex vitro acclimatization
(Yang and Yeh, 2008).
It is worth mentioning that the healthy plantlet obtained in TIS
had well-developed roots, which probably shortened the time to
transplant. The renewal of the head space in the TIS with every
immersion led to the higher oxygen concentration (Roels et al.,
2006), which probably contributed to the well-developed root
formation of D. fordii. and D. alata plantlets.
low dissolved oxygen concentration around the shoot base in the
agar medium partially resulted in the poor rooting of sweet
potato in vitro shoots (Zobayed et al., 1999). Similarly
in the liquid medium, the base of shoots was totally immersed in
the liquid medium during the whole culture period and so the
concentration of oxygen around the root system was also limited.
Comparative effects of
gelled, liquid and temporary immersion system (TIS) on
D. fordii shoot
proliferation with six weeks of culture.
Shoot length (cm)
Fresh weight (FW) (mg)
Dry weight (DW) (mg)
The ratio of
FW to DW
Frequency of tuberization (%)
Mean number of tubers per plantlet
2.4 ± 0.4b*
2.0 ± 0.2c
72.2 ± 22.4c
6.3 ± 1.1c
11.3 ± 2.2a
8.3 ± 14.4b
0.1 ± 0.1b
4.8 ± 0.8a
4.5 ± 0.6b
306.8 ± 57.6b
28.2 ± 6.5b
11.1 ± 1.8a
83.3 ± 8.3a
0.9 ± 0.1a
5.0 ± 0.1a
5.7 ± 0.3a
423.3 ± 54.6a
39.4 ± 4.8a
10.7 ± 0.3a
73.8 ± 2.1a
0.8 ± 0.1a
*Values with the different alphabets within the same column are
significantly different (P≤0.05), according to the t
This is the first report on the micropropagation of two
Chinese yams, D. fordii. and D. alata, using
TIS. Our results show that TIS could be used for shoot
multiplication of these two yams, thereby providing a
valuable way for propagation and seedling production.
Meanwhile, TIS also provided an alternative valuable option
for microtuber production of D. fordii. Therefore,
with the availability of reliable microtuber production, the
germplasm propagation, conservation and exchange of in
vitro propagated, pathogen-tested elite clones would be
facilitated (Balogun, 2009).
This research was financially supported by Guangxi Natural
Science Foundation (2010GXNSFB013032). Also, the comments
and suggestions on the manuscript from the editor and two
anonymous reviewers are gratefully acknowledged.
Alonso NP, Wilken D, Gerth A, Jahn A, Michael H, Kerns G, Perez
AC, Jimenez E (2009). Cardiotonic glycosides from biomass of
Digitalis purpurea L. cultured in temporary immersion
systems. Plant Cell Tissue Organ Cult. 99: 151-156
Alvard D, Cote F, Teisson C (1993). Comparison of methods of
liquid medium culture for banana micropropagation. Plant Cell
Tissue Organ Cult. 32: 55-60
Balogun MO (2009). Microtubers in yam permplasm conservation and
propagation: the status, the prospects and the constraints.
Biotechnol. Mol. Biol. Rev. 4(1): 001-010
Balogun MO, Fawole I, Ng SYC, Ng NQ, Shiwachi H, Kikuno H
(2006). Interaction among cultural factors in microtuberization
of white yam (Dioscorea rotundata). Trop. Sci. 46: 55-59
Cabasson C, Alvard D, Dambier D, Ollitrault P, Teisson C (1997).
Improvement of citrus somatic embryo development by temporary
immersion. Plant Cell Tissue Organ Cult. 50: 33-37.
Chen FQ, Fu Y, Wang DL, Gao X, Wang L (2007). The effect of
plant growth regulators and sucrose on the micropropagation and
microtuberization of Dioscorea nipponica Makino. J. Plant
Growth Regul. 26: 38-45.
Etienne H, Berthouly M (2002) Temporary immersion systems in
plant micropropagation. Plant Cell Tiss. Organ Cult. 69:
Martre P, Lacan D, Just D, Teisson C (2001). Physiological
effects of temporary immersion on Hevea brasiliensis
callus. Plant Cell Tissue Organ Cult. 67: 25-35.
Murashige T, Skoog F (1962). A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol. Plant, 15:
Ovono PO, Kevers C, Dommes J (2010). Tuber formation and
development of Dioscorea cayenensis-D. rotundata
complex In vitro effect of polyamines. In Vitro
Cell Dev. Biol. Plant 46: 81-88.
Roels S, Noceda C, Escalona M, Sandoval J, Canal MJ, Rodriguez
R, Debergh P (2006). The effect of headspace renewal in a
temporary immersion bioreactor on plantain (Musa AAB)
shoot proliferation and quality. Plant Cell Tissue Organ Cult.
Thankappan SS, Patell VM (2011). In vitro propagation
studies and genetic fidelity assessment of endangered medicinal
wild yam-Dioscorea prazeri. Plant Omics J. 4(4): 177-189.
Viana AM, Mantell SH (1989). Callus induction and plant
regeneration from excised zygotic embryos of the seed-propagated
yams Dioscorea composita Hemsl. and D. cayenensis
Lam. Plant Cell Tissue Organ Cult. 16: 113-122
Wang SJ, Gao WY, Liu HY, Chen HX, Yu JG, Xiao PG (2006). Studies
on the physicochemical, morphological, thermal and crystalline
properties of starches separated from different Dioscorea
opposita cultivars. Food Chem. 99: 38-44.
Wawrosch C, Kongbangkerd A, Kopf A, Kopp B (2005). Shoot
regeneration from nodules of Charybdis sp.: a comparison
of gelled liquid and temporary immersion culture systems. Plant
Cell Tissue Organ Cult. 81: 319-322.
Yan HB, Fang F, Dong WQ, Bu ZY, Bi ZQ, Li YR (2010). In vitro
and ex vitro rooting culture of Dioscorea fordii
Prain et Burk plantlets. Guangxi Agric. Sci. [in Chinese],
Yan HB, Liang CX, Li YR (2010). Improved growth and quality of
Siraitia grosvenorii plantlets using a temporary
immersion system. Plant Cell Tissue Organ Cult. 103: 131-135.
Yan HB, Yang LT, Li YR
Axillary proliferation and tuberization of Dioscorea fordii
Prain et Burk. Plant Cell Tissue Organ Cult. 104: 193-198.
Yang SH, Yeh DM (2008) In vitro leaf anatomy, ex vitro
photosynthetic behaviors and growth of Calathea orbifolia
(Linden) Kennedy plants obtained from semi-solid medium and
temporary immersion systems. Plant Cell Tissue Organ Cult. 93:
Ziv M (1991). Quality of micropropagated plants-vitrification.
In Vitro Cell Dev. Biol. Plant, 27: 64-69.
Zobayed FA, Zobayed SMA, Kubota C, Kozai T, Hasegawa O (1999).
Supporting material affects the growth and development of in