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The Biotron Breeding System: A Rapid and Reliable Procedure for Genetic
Studies and Breeding in Rice

Article  in  Plant and Cell Physiology · May 2011

DOI: 10.1093/pcp/pcr066 · Source: PubMed

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The Biotron Breeding System: A Rapid and Reliable
Procedure for Genetic Studies and Breeding in Rice
Takayuki Ohnishi1, Mihoko Yoshino1, Hiromoto Yamakawa2 and Tetsu Kinoshita1,*
1
Plant Reproductive Genetics, GCOE Research Group, Graduate School of Biological Science, Nara Institute of Science and Technology,
Nara 630-0192, Japan
2
National Agricultural Research Center, Niigata 943-0193, Japan
*Corresponding author: E-mail, t-kinosita@bs.naist.jp; Fax, +81-0743-72-6210

Techniques
(Received April 7, 2011; Accepted May 18, 2011)

Oryza sativa is widely used as a model organism for many
aspects of research in monocots and cereals. However, it has
certain disadvantages as a model species compared with
Arabidopsis thaliana, the eudicot species most widely used
in plant sciences: first, it has a long cultivation time; and
second, it requires considerably more space for growth.
cereals. Resources for rice research have developed rapidly in
recent years, including a high-quality genome sequence
(Matsumoto et al. 2005), large-scale expression sequence tags
(ESTs), full-length cDNA collections (Kikuchi et al. 2003, Xie
et al. 2005, Lu et al. 2008), gene expression atlas (Li et al.
2007, Nobuta et al. 2007, Jiao et al. 2009, Wang et al. 2010,

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Here, we introduce a biotron breeding system, which
allows rapid and reliable rice cultivation using a well-
equipped artificial environmental chamber. This system in-
volves use of regulation of CO2 levels, removal of tillers and
embryo rescue to overcome the disadvantages of rice culti-
vation. The rice cultivars Nipponbare, Koshihikari, Taichung
65 and Kasalath all showed vigorous growth and sufficient
seed production in the biotron breeding system with accel-
erated flowering time. Nipponbare, which was the earliest
among these cultivars, flowered at about 50 d after sowing.
The life cycle of these plants could be further shortened
using an embryo rescue technique on immature seeds at
7 d after pollination, thereby avoiding the lengthy process
of seed maturation. Overall, it was possible to shorten the
life cycle of Nipponbare to about 2 months under the con-
trolled conditions. Furthermore, controlled crosses, which
can be difficult with conventional cultivation methods, were
easy to perform as we could control the exact timing of
anther dehiscence. Thus, our biotron breeding system offers
a valuable new approach to genetic and breeding studies
in rice.
Keywords: Controlled crosses  CO2 regulation  Embryo
rescue  Generation time  Oryza sativa  Rice biotron
breeding system.
Abbreviations: CO2, carbon dioxide; DAP, days after pollin-
ation; T65, Taichung 65.
Introduction
Rice is one of the most important food crops worldwide and is
also a significant model plant for research in monocots or
Fujita et al. 2011, Hamada et al. 2011, Ohnishi et al. 2011)
and mutant libraries (Jeon et al. 2000, Wu et al. 2003, Sallaud
et al. 2004, Miyao et al. 2007, Nakamura et al. 2007, Krishnan
et al. 2009, Satoh et al. 2010, Sakurai et al. 2011). These ad-
vances have added to the value of rice as a model plant species
for many types of research.
Despite these new resources in rice and the desire for re-
search into monocots and cereals, Arabidopsis is nevertheless
the most favored material for plant science studies such as
genetics, epigenetics, evolutionary biology and development.
Arabidopsis effectively plays the same role in plant sciences
as mice and fruit flies (Drosophila) in animal biology.
Although Arabidopsis has little direct significance for agricul-
ture, it does have a number of traits that are advantageous for
laboratory-based research and as a model for obtaining a basic
understanding of flowering plants in general. In particular, the
short life cycle and small size allow rapid culture in relatively
small spaces (Meyerowitz 1989). In comparison, research on
rice requires expensive facilities, such as large breeding fields
and well-equipped greenhouses. The mean generation time of
rice, depending on the particular cultivar, is much longer than
that of Arabidopsis. Moreover, because of the seasonality of rice
cultivation, many rice researchers are obliged to adapt their
research schedule to fit in with the annual life cycle of
field-grown rice. Research in rice is further complicated by Editor-in-Chief’s choice
the need to select a suitable cultivar for experimentation in
the particular location of the research station, i.e., differences
in day length and temperature at different locations affect the
growth and flowering of rice plants. Therefore the experimental
data obtained reflect particular environmental conditions. The
obvious alternative approach would be to develop an efficient
and reliable method of rice cultivation under artificial climate

Plant Cell Physiol. 52(7): 1249–1257 (2011) doi:10.1093/pcp/pcr066, available FREE online at www.pcp.oxfordjournals.org
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Plant Cell Physiol. 52(7): 1249–1257 (2011) doi:10.1093/pcp/pcr066 ! The Author 2011. 1249
 T. Ohnishi et al.

conditions, which would serve to remove one of the most im-

portant limiting factors in rice research.
The Japonica-type cultivar Nipponbare, whose genotype is
being sequenced (Matsumoto et al. 2005), was originally bred
for its ability to grow well in heavily manured fields (Koumura
1972a,b). In the process of producing this cultivar, Koumura
(1972a,b) was able to accelerate generation times by regulation
of temperature and photoperiod so that three generations
could be grown within 1 year. In this study, we sought to iden-
tify other environmental factors and techniques that could be
used to further shorten generation times and to reduce the
space required for cultivation of rice plants. It was also import-
ant that any of these changes did not adversely impair plant
growth, efficient seed production or the ability to perform ex-
perimental crosses. We found three parameters that fulfilled
these criteria for cv. Nipponbare: (i) regulation of CO2 level; (ii)
embryo rescue; and (iii) removal of tillers. Using our new culti-

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vation procedure, we were able to reduce generation times to
about 2 months in cv. Nipponbare and could grow the plants in
the reduced space offered by a biotron. Here, we describe the
details of our new rice biotron breeding system and demon-
strate its power and utility for laboratory experiments in many
aspects of research and for breeding.
Rice is very sensitive to environmental fluctuations during
reproductive development (Yamakawa et al. 2007, Endo et al.
2009, Hedhly et al. 2009, Yamakawa and Hakata 2010). One of
the benefits of the new system we describe here is the ability to
modify environmental conditions, which should be of particu-
lar value for investigating and understanding the effects of
environmental stress on rice physiology.
Results
Although rice plants normally have a growth habit that requires
a relatively large field space, we were able to modify the culti-
vation process to grow plants at a much higher density in a
biotron, a widely used growth chamber for laboratory research
on plants, in order to minimize their space requirements.
Individual plants were grown in disposable plastic pots, 6 cm
in diameter and 7 cm in height, comparable in size to those
used in Arabidopsis genetic research. Each pot was filled with
150 ml of rice nursery culture soil. In total, we could grow ap-
proximately 70 rice plants under programmed environmental
conditions in the biotron (Fig. 1A, see Materials and Methods).
The plant density in our procedure is comparable to that of
Arabidopsis in a growth chamber (Fig. 1B). In the closed and
limited environment of the biotron, air conditions, particularly
the partial pressure of carbon dioxide (CO2), can change rapidly
due to photosynthetic carbon assimilation by the plants. To
overcome this problem, we regulated CO2 levels in the biotron
using a CO2 cylinder so as to keep concentrations from falling
below that of the normal atmosphere (Supplementary
Fig. S1A). In agreement with previous studies showing that
elevated CO2 concentrations enhance rice growth and yield
Fig. 1 Rice plants and their population density in the controlled
biotron environment. (A) Rice plants growing in the biotron. (B) A
comparison of space requirements of biotron-grown rice and
Arabidopsis. The 20 rice plants (left) and 20 Arabidopsis plants
(right) are each grown in the same size disposable plastic pots
within the blue tray. (C) A panicle and seeds (D) of a rice plant
(cv. Nipponbare) grown in the biotron. In (D), the upper row shows
hulled seeds, the lower row shows unhulled seeds. Scale bar, 5 mm.
in a controlled environment (Kim et al. 2003, Sakai et al.
2006, Yang et al. 2006, Sasaki et al. 2007), we found that the
moderate supplementation of CO2 in the biotron increased
seed yield (Supplementary Fig. S1B). In addition to these
changes to growth and atmospheric conditions, we also
removed any tillers from the plants, except for the main
culm, in order to restrict plant growth and size to within the
limited nutrient sources and light conditions of the biotron. We
observed a lower fertility rate when tillers were not removed
(data not shown). The rice plants produced a single panicle and
normal-looking seeds when grown in the biotron under the
conditions described above (Fig. 1C, D). The other environ-
mental parameters, such as short-day condition, humidity
and temperature, were similar to those normally used for rice
cultivation in a greenhouse (see Materials and Methods).
We determined the number of florets and seeds, and the
flowering times for four cultivars [cv. Nipponbare, Koshihikari,
Taichung 65 (T65) and Kasalath] grown under the controlled-
environment conditions of the biotron. Although the number
of florets per plant was decreased in the biotron compared with
field-grown plants, all of the cultivars grew vigorously and had a
high fertility rate: >80% in Nipponbare, Koshihikari and T65,
and >60% in Kasalath (Fig. 2A). Seed numbers were highest in
Nipponbare, but >40 seeds were harvested on average from
each plant in the four cultivars (Fig. 2A). This seed yield is

1250 Plant Cell Physiol. 52(7): 1249–1257 (2011) doi:10.1093/pcp/pcr066 ! The Author 2011.
 Enforced short generation times in rice

A
Florets number / plant

Seeds number / plant

80 80
100

60 60 80

Fertility [%]
60
40 40
40
20 20
20
0 0 0
re

ari

re

ari

re

ari

h
T6

lat

T6

lat

T6

lat
ba

ba

ba
ihik

ihik

ihik
sa

sa

sa
on

on

on
sh

sh

sh
Ka

Ka

Ka
p

p
Ko

Ko

Ko
Nip

Nip

Nip
B 110

100
Days after sowing [days]

90

80

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70

60

50

40
are

ari

h
T6

lat
ihik
nb

sa
po

sh

Ka
Ko
Nip

Fig. 2 Seed yield and flowering time of four rice cultivars grown in the biotron breeding system: Nipponbare (n = 10), Koshihikari (n = 20), T65
(n = 9) and Kasalath (n = 15). (A) The number of florets and seeds, and the proportion of fertile (%) seeds per plant. The data show the
mean ± SD. (B) The number of days to flowering from sowing. The colored symbols show the individual values in each cultivar. Black diamonds
represent the mean for each cultivar.

sufficient for research use. Flowering times were consistent and and good fertility. We examined biotron-grown plants to de-
highly reproducible in all four cultivars in the biotron breeding termine whether they were as useful for genetic crossing ex-
system. Differences in flowering times were present among the periments as those grown in the field during the summer.
cultivars (Fig. 2B), as has been reported previously (Yano et al. Several methods are commonly used for eliminating rice
2000, Nishida et al. 2002, Doi et al. 2004). The mean flowering pollen grains before flowering or fertilization, such as clipping
time for Nipponbare was about 11 d earlier than for Koshihikari, anthers, vacuuming of the anthers or dipping panicles into hot
and was almost half as long as the mean flowering times for T65 water for 7 min. We chose the hot water method because of its
and Kasalath (Fig. 2B). Therefore, the shortening of the gener- relative simplicity and effectiveness at preventing accidental
ation times was less effective for T65 or Kasalath; nevertheless, out-crossing in breeding experiments. Here, we dipped entire
the growth vigor, seed set and reproducible flowering times did panicles of plants grown in the biotron into hot water. After
offer some advantages even for these cultivars, and the use of treatment, 7–16 florets of the panicles opened soon after dip-
our biotron approach should also be of value to other cultivars ping. These plants were used as the female parents (Fig. 3,
and wild rice species. Table 1). The flowering plants to be used as the male parents
We also investigated the effect of CO2 regulation on flower- were taken from the biotron into the normal conditions of the
ing time in cv. Nipponbare. Our analysis showed that mean laboratory room at 5–6 h after the beginning of the light period;
flowering time was 6 d earlier under regulated CO2 conditions these plants started to flower within 15 min of being removed
compared with no CO2 regulation (Supplementary Fig. S1C). from the biotron (Video appendix, Supplementary data) pre-
In general, rice plants in a winter greenhouse show less vig- sumably in response to the environmental change. Immediately
orous growth and lower fertility compared with plants in the after dehiscence, anthers were grasped with tweezers and the
field in the summer. This may be due to pollen abnormalities pollen grains from the anther locules were scattered above the
under the unstable conditions in the greenhouse, such as tem- pistil of the female recipient. Using this procedure, we consist-
perature and humidity fluctuations. In the biotron, however, we ently obtained seeds from more than half of the pollinated
found that winter-grown plants still showed vigorous growth florets of every rice plant (Table 1). We confirmed that the

Plant Cell Physiol. 52(7): 1249–1257 (2011) doi:10.1093/pcp/pcr066 ! The Author 2011. 1251
 T. Ohnishi et al.

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Fig. 3 Images of panicles selected for use in controlled crosses; the insets show florets at each step of the process. (A) Panicle selected for hot
water emasculation. (B) Same panicle 15 min after hot water emasculation. Eleven florets have opened (white triangles). (C) Unopened florets are
clipped off, and the top one-third of each open floret is cut for pollination (magnified image of a single floret is shown in the inset). (D) Seed
growth (white solid triangles) at 7 DAP.

Table 1 Results of controlled crosses between female cv. harvested seeds were hybrid using PCR with appropriate mark-
Nipponbare and male cv. Kasalath in the biotron breeding system ers (data not shown). Plants from all four cultivars were suc-
Plant number Number of Number of Success cessfully used as the pollen parent, and both Nipponbare and
florets opened hybrid seeds rate (%) Koshihikari were used as the female parent in the crosses car-
#1a 11 6 55 ried out. Although the numbers of F1 seeds were not large, the
#2 16 11 69 success rate in these crosses was similar to that obtained in the
#3 7 6 86 summer field.
#4 10 8 80 In our biotron breeding system, Nipponbare had the short-
#5 10 7 70 est flowering time of the cultivars tested (Fig. 2B). Nipponbare
Meanb 10.8 ± 3.3 7.6 ± 2.1 72 ± 11.9 took about 50 d from sowing to flowering and then required
a
This plant is shown in Fig. 3. about another 30 d for seed maturation, a total generation time
b
Data are mean ± SD. of approximately 80 d. To further shorten the generation time,

1252 Plant Cell Physiol. 52(7): 1249–1257 (2011) doi:10.1093/pcp/pcr066 ! The Author 2011.
 Enforced short generation times in rice

we applied an embryo rescue technique to immature seeds of
Nipponbare in order to avoid the long period necessary for seed
maturation and dormancy breaking. Although growth regula-
tors such as auxin, cytokinin and gibberellin are widely used for
embryo rescue techniques in other plant species, their effects
are complicated (Gaspar et al. 1996). It is possible to perform
embryo rescue in rice without recourse to plant hormones.
Here, we used a 0.5 concentration of Murashige and Skoog
(MS) agar medium supplemented with 2% sugar without any
growth regulators. In general, the success rate of embryo rescue
varies with the stage of embryonic growth. At earlier stages, it
can be difficult to dissect the embryo from the immature seed,
and the embryo may not have the ability to sustain autono-
mous growth. However, use of later stages somewhat
undermines the rationale of embryo rescue as a shortcut for
the long seed maturation process. We therefore chose to carry
out the embryo rescue at 7 days after pollination (DAP), when
the embryos have already formed root and shoot primordia in
the immature seeds. Preliminary experiments confirmed that 7
DAP was suitable for our purpose. We surface sterilized 7 DAP
seeds, and surgically separated the embryo and transferred it to
the medium under a binocular microscope (Fig. 4A–C). These
dissections were performed under aseptic conditions.
We monitored the daily growth of the rescued embryos on
the medium (Fig. 4D–G). The embryo is essentially a bipolar
structure with root and shoot primordia; the shoot and root
tissues are derived from each apical meristem. None of the
rescued embryos showed any morphological abnormalities or

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Fig. 4 Embryo rescue technique. (A) Surface sterilized 7 DAP immature seed. (B) A seed after removal of the seed coat. White endosperm and
yellowish embryo (bottom right) can be distinguished. (C) A seed after isolation of the embryo. (D) Embryo at 24 h after transfer to embryo
rescue medium. (E) Germinating embryo at 48 h after embryo rescue. (F) Seedling at 72 h after embryo rescue. (G) Seedling at 96 h after embryo
rescue. Scale bars, 1 mm.

Plant Cell Physiol. 52(7): 1249–1257 (2011) doi:10.1093/pcp/pcr066 ! The Author 2011. 1253
 T. Ohnishi et al.

defects. We achieved a 94.9% (131/138) success rate in our Selective crossing is an inherent part of plant genetics and
embryo rescue experiments, a rate that is comparable to the breeding new varieties. The naturally long life cycle of rice and
germination rate of mature seeds. To check that embryo rescue the seasonal limitations for cultivation are disadvantageous for
did not have an adverse effect on later plant growth, we these purposes. Even under generally suitable conditions in the
analyzed the numbers of florets and seeds, and the flowering field during the summer, unsettled weather can greatly affect
times of rice plants that originated from rescued embryos (Fig. the timing of flowering in the daytime. After dehiscence, rice
5A, B). We found that the rescued rice plants had a slightly pollen grains are viable for only a short period (Khatun and
lower numbers of seeds, and their flowering time was 4 d longer Flowers 1995), and thus uncontrolled timing of flowering can
on average. This effect may be a consequence of an impose limits on experimental design. Inevitably, the uncon-
observed delay in early growth by the rescued embryos after trollable effects of the environment cause experimental over-
transplantation to the artificial medium (Fig. 5C). We also per- runs in the time necessary to complete research. In contrast, the
formed repeated embryo rescues over five generations and did biotron breeding system we describe here provides the possi-
not observe any morphological abnormalities or growth bility of a well-scheduled experimental design. Flowering of the
defects. rice plants in the biotron is largely dependent on programmed
lighting and temperature. Furthermore, in mature plants, the
flowers open shortly after the plants are taken from the biotron,
Discussion
presumably in response to changes in temperature, brightness

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In this study, we developed a procedure for growing and breed- and humidity. Therefore, the researcher can precisely control
ing rice in a biotron that has three principal elements: (i) regu- the flowering of the rice plants regardless of external weather
lation of CO2 level; (ii) embryo rescue; and (iii) removal of tillers. conditions and seasons. Improvements in artificial cultivation
Through this procedure, we have been able to substantially conditions will also be of use for growing transgenic rice in
shorten the generation time of cv. Nipponbare plants to ap- countries that have strict regulations regarding growth of gen-
proximately 2 months, thereby allowing a potential six gener- etically modified plants. The biotron breeding system could be
ations per year (Fig. 6). In addition, our procedure permits applied to analyses of transgenic rice plants since the rate of
controlled crosses under laboratory conditions. fertility is high enough to study reproductive processes and/or

A
100 70 100
Seeds number / plant
Florets number / plant

60
80 80
Fertility [%]

50
60 40 60

40 30 40
20
20 20
10
0 0 0
Nipponbare Nipponbare Nipponbare Nipponbare Nipponbare Nipponbare
from seeds from embryo rescue from seeds from embryo rescue from seeds from embryo rescue

B C
60 16
Days after sowing [days]

14
Seedling length [cm]

55 12
10 Nipponbare
50 8 from seeds
6
Nipponbare
45 4 from embryo rescue
2
40 0
Nipponbare Nipponbare 5 6 7 8 9
from seeds from embryo rescue Days after transplantation [days]

Fig. 5 Comparison of floret numbers, seed numbers, fertility and early growth in plants derived from normally matured seeds (n = 10) and 7 DAP
embryo rescue (n = 20) in cv. Nipponbare. (A) The derived plants of both groups were grown in the biotron system. Values show the mean ± SD.
(B) The number of days to flowering from sowing seeds or transplantation of 7 DAP embryos. Colored symbols indicate individual plants, black
diamonds are the means. (C) Seedling lengths from mature Nipponbare seeds or from rescued Nipponbare embryos at different times after
transplantation to medium.

1254 Plant Cell Physiol. 52(7): 1249–1257 (2011) doi:10.1093/pcp/pcr066 ! The Author 2011.

Fig. 6 Schematic summary of the schedule of the biotron breeding
system. Biotron breeding allows a dramatic shortening of the gener-
ation time of the rice plants (cv. Nipponbare). Cultivation of cv.
Nipponbare plants started from either mature seeds or embryo
rescue from 7 DAP immature seeds. Fertilization can be by
self-pollination or a controlled cross at 49 d when using mature
seeds as the starting point, or 53 d when using embryo rescue.
Harvesting of dry seeds requires a further 30 d from pollination to
maturation of the seeds. Alternatively, the maturation process can be
abbreviated by the use of the embryo rescue technique with 7 DAP
immature seeds. In the latter case, the Nipponbare life cycle can be
shortened to about 2 months. Thus, it would be theoretically feasible
to perform six backcrosses per annum using the biotron breeding
system.
seed maturation. This would also provide opportunities for rice
research using transgenic plants with green fluorescent protein
(GFP) reporters, T-DNA insertion mutations and targeted gene
mutations from homologous recombination (Terada et al.
2002).
The set-up described here includes specific parameters for
growing rice in the biotron breeding system; however, these
parameters could be modified depending on experimental re-
quirements. For example, we observed an inverse correlation
between the number of seeds and the flowering time (Fig. 2A),
possibly due to depletion of nutrients in the soil in the relatively
small pot. Therefore, it might be feasible to add additional fer-
tilizer or use larger pots according to the experimental design.
Hence, higher plant densities could be used where a low seed
yield is acceptable, as in repeated inbreeding, or vice versa.
Plant Cell Physiol. 52(7): 1249–1257 (2011) doi:10.1093/pcp/pcr066 ! The Author 2011.
Enforced short generation times in rice
The biotron used here had an inside height of 110 cm; when
necessary, we could split this space to grow adult rice plants on
a bottom shelf and young rice plants on an upper shelf. If a
dwarf gene such as Hosetu-dwarf (Kurita et al. 2002) was intro-
gressed to cv. Nipponbare, then the space requirements for rice
cultivation would be similar to those of Arabidopsis ecotypes
that have the erecta allele. The embryo rescue technique can
also be modified according to the experimental materials. The
success of culturing plants from immature embryos largely de-
pends upon the stage of maturity of the embryos, which differs
between genotypes and with the environmental factors set in
the biotron.
As mentioned earlier, day length and environmental condi-
tions, which vary considerably with latitude, greatly affect the
flowering time of many cultivars in the field. The ability to
control environmental variables to ensure vigorous plant
growth, as in a biotron or closed greenhouse, is an important
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prerequisite for the use of plants as laboratory model organ-
isms. Since the biotron breeding system is unaffected by season
or locality, widespread uptake of this procedure would encour-
age the development of rice resources for genetic studies, and
provide a more rapid and reliable means of implementing
breeding programs. The six generations of rice plants (via five
rounds of embryo rescue) within a year in the biotron is com-
parable to what is possible in Arabidopsis genetic studies.
Materials and Methods
Plant materials and growth conditions
Rice (Oryza sativa cv. Nipponbare, Koshihikari, T65 and
Kasalath) was grown at 30 C/25 C (11 h day/13 h night) in a
biotron (NC350HC; Nippon Medical & Chemical Instruments
Co. Ltd, Osaka, Japan), which provided illumination at an in-
tensity of 350 mmol photons m 2 s 1. Relative humidity was
regulated at 70%; a CO2 level of 20% was supplied from a CO2
gas cylinder and regulated by a biotron setting of 475 ppm (see
Supplementary Fig. S1). Mature seeds were dehusked with a
rice husker and were surface sterilized in 2.5% sodium hypo-
chlorite for 30 min with gentle mixing. The seeds were washed
five times in water and then incubated overnight in water at
room temperature. About 30 mature seeds were placed on
filter papers in a Petri dish and soaked with 10 ml of water
and the plate was then sealed with surgical tape (Micropore
Surgical Tape; 3 M, Germany). After germination, the rice seed-
lings were incubated in the biotron for 7 d. The 7-day-old rice
seedlings were transferred to individual disposable plastic pots
(TO polipot diameter 6 cm, height 7 cm; Tokaikasei Co. Ltd,
Gifu, Japan) filled with 150 ml of rice nursery culture soil (con-
taining 0.8 g each of nitrogen, phosphate and potassium per
3 kg of soil). Each plant was restricted to the main culm by the
removal of tillers. Twenty plant pots were put in a plastic tray
(31  22  9 cm) and placed in the biotron. Water was supplied
to the plant until the soil was covered. Additional fertilizer was
not supplied.
1255
 T. Ohnishi et al.
Controlled crosses
Female parents were prepared by selecting flowering stage
plants for hot water emasculation. The entire panicle was
soaked in water at 42 C for 7 min at 3–4 h after the beginning
of the light period. After soaking the panicle, unopened spike-
lets were clipped off and the top part of opened florets and
of the anthers were removed (see Fig. 3C); the panicle was
then enclosed in a paraffin-paper bag to prevent accidental
pollination until used for the cross. Male parents were prepared
by removing rice plants at the flowering stage from the biotron
at 5–6 h after the beginning of the light period. The florets
normally opened within 15 min of the plant being transferred
to laboratory conditions (Video appendix, Supplementary
data). The female parents prepared above were
pollinated with fresh pollen grains from the male parents. In
detail, we selected fresh anthers from a caryopsis just after
flowering, and then placed the fresh pollen grains or fresh an-
thers into each treated female floret. Each panicle was enclosed
in a paraffin-paper bag, and the plants transferred to the
biotron.
Embryo rescue
Immature seeds at 7 DAP were dehusked using a pair of twee-
zers and sterilized in 2.5% sodium hypochlorite for 30 min with
gentle mixing. They were then washed five times in water. Up to
30 seeds were placed on filter papers in a Petri dish and, under a
binocular microscope, their seed coats were removed using a
surgical knife and a pair of tweezers. Each dissected embryo
was transferred to embryo rescue agar medium. The embryo
rescue medium contained 0.5 MS salts (Murashige and Skoog
Plant Salt Mixture; Wako Pure Chemical Industries, Ltd), 0.8%
agar and 2% sucrose, pH adjusted to 5.8 with potassium hy-
drate, and was prepared in a 50 mm Petri dish (Bibby Sterilin
Ltd, Staffordshire, UK). Dishes with rescued embryos were
sealed with surgical tape and transferred to a growth cabinet
(Growth Cabinet MLR-350; SANYO Electric Co., Ltd, Osaka,
Japan, illumination: 225 mmol photons m 2 s 1) with continu-
ous light conditions at 28 C for 11 d. Growing seedlings were
transferred to plastic pots filled with soil, and placed in the
biotron.
Measuring seedling length and flowering times
The length of seedlings in mature seeds and of rescued embryos
were measured as the length of the aerial part from dehusked
seeds or of the rescued embryos when cultured in a cylindrical
plastic container (Agripot; Kirin Brewery Co., Tokyo, Japan) on
embryo rescue agar medium. Flowering time was defined as the
number of days from sowing day to opening of the first floret
on each panicle.
Supplementary data
Supplementary data are available at PCP online.
1256 Plant Cell Physiol. 52(7): 1249–1257 (2011) doi:10.1093/pcp/pcr066 ! The Author 2011.
Funding
This work was supported by the Ministry of Agriculture,
Forestry and Fisheries of Japan (Genomics for Agricultural
Innovation, Molecular cloning and characterization of agrono-
mically important genes of rice, IPG-0017, IPG-0020); National
Agriculture and Food Research Organization (Development of
innovative crops through the molecular analysis of useful genes,
No. 1211).
Acknowledgments
We thank Drs Takeshi Izawa and Amane Makino for discussion
and advice on the biotron breeding system.
Downloaded from http://pcp.oxfordjournals.org/ by guest on December 24, 2015
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