Plant Cell, Tissueand Organ Culture 36: 177-182, 1994.

(~) 1994KluwerAcademicPublishers. Printedin the Netherlands.

Induction and development of potato tubers in a jar fermentor

M o t o m u Akita 1 & Shinsaku T a k a y a m a 2

Tsukuba Research Laboratories, Kyowa Hakko Kogyo Co. Ltd. 2 Miyukigaoka, Tsukuba, lbaraki,
305, Japan (present addresses." 1Department of Biotechnological Science, Faculty of Biology-Oriented
Science and Technology, Kinki Univ., 930 Nishimitani, Uchita-cho, Naga-gun, Wakayama, 649-64,
Japan; 2Department of Biological Science and Technology, Tokai Univ. 317 Nishino, Numazu, Shizuoka,
410-03, Japan)

Received 11 February 1993; acceptedin revisedform 20 September 1993

Key words: culture temperature, in vitro tuberization, microtuber, Solanum tuberosum L., time course

Abstract

Potato (Solanum tuberosum L.) tubers were mass propagated in ajar fermentor using a two-step method
consisting of a shoot multiplication step (phase 1) followed by a tuber induction and development step
(phase 2). Tuberization was observed within 2 weeks of phase 2 and the number of tubers did not
increase after this culture period. In contrast, total tuber weight continuously increased for at least 10
weeks. Although the number of tubers and the total tuber weight clearly decreased under the lower
temperature (17°C), this weight decrease was partially prevented by changing the temperature from
17°C to 25°C after 2 weeks of phase 2. This result indicates that tuber development can be controlled
independently of induction. Lower temperatures influenced the localization and size distribution of tubers
in the jar fermentor.

Introduction Stacey 1984; Wang & Hu 1985). For example,

Since the potato (Solanum tuberosum L.) is one
of the most important crops in the world, numer-
ous researchers have been improving tissue cul-
ture techniques for the mass propagation of germ-
free and clonal plants (e.g. Miller & Lipschutz
1984; Wang & Hu 1985). In particular, in vitro-
propagated small tubers (microtubers) show many
advantages for supplying in vitro-derived plants,
because they are suitable for easy storage, trans-
fer and distribution. Moreover, microtubers can
be directly transplanted to fields without acclima-
tization. Thus, the conditions for the in vitro tuber-
ization of potatoes have been studies in depth
and the efficiency of their propagation has been
improved (e.g. Chandra et al. 1988; Hussey &
Wang & Hu (1982) reported that approximately
36,000 microtubers m -2 could be propagated in a
culture room using their static culture method.
Recently scale-up culture has been examined
with several plants because of possible econom-
ical advantages (Levin & Vasil 1989). In regard
to potato tuber propagation, Akita & Takayama
(1988a) reported a scale-up culture using jar fer-
mentor techniques (Fig. 1) by a method similar
to the liquid shaken culture method reported by
Estrada et al. (1986).
The responses concerning tuberization are dif-
ferent in the static culture and the jar fermentor
culture. For example, it was found that tuberiza-
tion is only stimulated at the liquid medium sur-
face area in the jar fermentor (Akita & Takayama
178
-W
STOCK PLANTS
JAR FERMENTOR CULTURE
SHOOT
MULTIPLI
P H A ,¢
Fig. i. Schematic diagram of in vitro mass propagation of
potato tubers using the jar fermentor technique.
1988a) and this localization of tuberization indi-
cates the existence of a peculiar factor regarding
tuberization in the jar fermentor culture. Thus,
the jar fermentor culture technique is also valu-
able for studying the physiology of tuberization
because the tuber-inducing tissues and the devel-
oping tubers can be mass propagated under an
experimental condition.
Thus, information about tuberization in a jar
fermentor, e.g. the time course of tuberization,
is indispensable for further study of tuberization
using this technique. In the present report, the
details of tuberization are described. The effects
of culture condition were also demonstrated by
changing the culture temperature.
Materials and methods
Plant material
In vitro potato stock plant (Solanum tuberosum cv.
Yukishiro) was established from an apical meris-
tern and was maintained by serial subculture on
the modified MS (Murashige & Skoog 1962) solid
medium as shown below.
Culture medium
Culture medium consisted of MS mineral salts
plus organic constituents including thiamine-
HC1 (0.4 mg l - l ) , myo-inositol (100 mg l - l ) ,
pyridoxine-HC1 (0.5 mg l - l ) , nicotinic acid (0.5
mg 1-1), glycine (2.0 mg 1-1) and sucrose. A
sucrose concentration of 30 g 1-~ was used for
shoot multiplication (phase 1) and of 90 g 1-1 for
the induction and development of tubers (phase
2). Solid medium that was used for maintenance
of the stock plants contained 30 g 1-1 sucrose
and gelrite (2 g 1- 1 ), Each medium was sterilized
by autoclaving (20 min, 121 ° C) after the pH was
adjusted to 6.2 with NaOH.
Culture conditions
Stock plants
Culture vessels were 25 mm (diameter) x 125
mm (length) test tubes containing 10 ml of the
solid medium for the culture of stock plants. Test
tubes were capped with silicone spongy plug T-
24 (Shin-Etu Silicon Co. Ltd., Tokyo). A sin-
gle nodal segment of a plant was transplanted
into a test tube and was incubated at 25°C under
continuous photosynthetic photon flux (PPF) of
approx. 9.4 ~tmol m -2 s -1 from fluorescent lamps
(Toshiba FL40 SW(100) V. (40 W)) for 4 weeks.
Whole plantlets were gently removed from the
solid medium and transferred to the jar fermen-
tors as described below.
Jar fermentor culture
An airlift type jar fermentor (vessel volume 8000
ml) was used. One hundred single nodal shoot
segments that were excised from the stock plants
were transplanted into a jar fermentor contain-
ing 2000 ml of MS medium that contained 30 g
1-1 sucrose and cultured for 4 weeks under 25°C,
continuous light (approx. 9.4 ~tmol m -2 s- 1 PPF).
Numerous shoots were induced and shoots were
elongated to 15-20 cm (approx.) in length. The
whole medium was then exchanged for 6000 ml