provide accessible surface targets for the
Agrobacrerium-mediated or biolistic trans-
formation of castor.
Materials and Methods
HORTSCIENCE 43(I):215-219. 2008.
In Vitro Regeneration of Castor
(Ricinus Communis L.) Using
Yeh-Jin Ahn' and Grace Qianhong Chen'
United States Department of Agriculture, Agricultural Research Service,
Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710
Additional index words, adventitious shoot, castor, dark preconditioning, regeneration,
oilseed, organogenesis, thidiazuron, tissue culture
Abstract. An efficient plant regeneration protocol using cotyledon explants was estab-
lished for castor (Ricinizs cwnmunis L.), an important oilseed crop. Mature seed-derived
cotyledon explants produced adventitious shoots when placed on Murashige and Skoog
(MS) medium containing thidiazuron (TDZ). The rate of shoot regeneration was
maximal (25 shoots per explant) when explants were cultured on shoot induction
medium supplemented with 5 lim TDZ and preincubated in the dark for the first 7 days
before transferring to the day/night cycle (16/8 h). Only the proximal ends of cotyledon
explants produced adventitious shoots, although green calli were observed in cotyledon
veins. After 4 weeks in culture, explants with well-developed shoot buds were transferred
to MS medium without plant growth regulators for the shoot elongation and develop-
ment. At 4 months after culture initiation, shoots (2 cm in length) were transferred to
root induction medium (MS medium supplemented with 5 ,ii indole-3-butyric acid)
where they developed roots in 4 to 6 weeks. Plantlets were transferred to soil and
acclimatized to greenhouse conditions. Histological analysis showed the adventitious
induction of the shoots originated from the cortical and epidermal cell layers of the
Castor (Ricinus communis L.), a semi-
tropical perennial plant, is a valuable oilseed
crop. It is the only commercial source of
ricinoleic acid that is used for numerous
industrial products (e.g., lubricants, paints,
coatings, and plastics) (Caupin, 1997). How-
ever, the seed contains the ricin toxin
(reviewed in Hartley and Lord, 2004; Lord
et al., 1994) and hyperallergenic storage pro-
teins (2S albumins) (reviewed in Pastorello
et al., 2001; Shewry et al., 2002). When
introduced into cells, the ricin toxin inacti-
vates ribosomes, resulting in inhibition of
protein synthesis (Endo and Tsurugi, 1987).
2S albumin was identified as the major
allergen of castor seeds, bringing about an
immunoglobulin E (IgE) response in 96%
of castor-sensitive patients (Thorpe et al.,
1988). These hazardous proteins pose serious
Received for publication 3 July 2007. Accepted for
publication 27 Sept. 2007.
We thank Louisa yang for the assistance on the
castor tissue culture and medium preparation and
Chanman Ha (Plant Gene Expression center,
USDA, Albany, CA) for the advice on the histo-
logical analysis. Castor gerrnplasm was provided
by the USDA-ARS National Genetic Resources
Programs, Southern Region Plant Introduction
Current address: Major in Life Science, College
of Natural Sciences, Sangmyung University,
7 Hongji-dong, Jongno-gu, Seoul 110-743, Korea.
'To whom reprint requests should be addressed;
health concerns to farmers and oil plant
workers greatly limiting its cultivation.
These hazardous proteins could poten-
tially be reduced through genetic engineer-
ing. However, castor is highly recalcitrant to
in vitro plant regeneration and genetic trans-
formation. Recently, Agrohacteriun-medi-
ated transformation was reported in castor
(Malathi et al., 2006; Sujatha and Sailaja,
2005) using meristems of embryonic tips
as explants. However, the rates of putative
transformant recovery were very low (0.08%
and 0.42%, respectively). Because a number
of independent transfonriants are typically
required to identify a line with an appro-
priate level of transgene expression, the
current transformation efficiency of castor
needs to be improved. Considering the high
rates of plant regeneration using embryonic
tips (an average of 40 shoots per explant;
Sujatha and Reddy, 1998), it is likely that the
meristematic cells involved in plant regener-
ation were difficult to transform using Agro-
bacterium. To increase the transformation
efficiency in castor, we are developing pro-
tocols for plant regeneration through adven-
titious shoot formation by testing various
tissues from mature seed, including endo-
sperm, embryo root, hypocotyl, and cotyle-
don. We found that hypocotyl (Alin et al.,
2007) and cotyledon (this report) were capa-
ble of producing adventitious shoots from
cortex tissues at similar rates (24 and 25
shoots per explant, respectively). Addition-
ally, the cotyledon also used epidermis to
produce adventitious shoots, which would
Explant preparation and culture
iniliation. Castor seeds (Ricinus comniunis
L. P1 P1215769) were obtained from the
U.S. Department of Agriculture, Germplasm
Resources Information Network, Southern
Regional Plant Introduction Station (Griffin.
GA). Seeds were decoated and surface-ster-
ilized in a 5% (v/v) commercial bleach
solution (5.25% sodium hypochlorite) for
15 min followed by five rinses in sterile
deionized water. Cotyledon explants were
aseptically isolated from embryo and placed
vertically (distal side in contact with me-
dium) on the shoot induction media [MS
medium (Murashige and Skoog, 1962),
30 gL sucrose, 0.5 g .L' 2-(4-morpholino)
ethanesulfonic acid (MES). pH 5.7] supple-
mented with 1, 5, or 10 .tn thidiazuron
(TDZ). For the first week in culture, half of
the explants were preincubated in the dark
and the other half were incubated under the
day/night cycle (16/8 Ii) at 100 pmolm<s
supplied by cool-white fluorescent lamps
(Sylvania, Danvers, MA) at constant 26 °C.
Dcv weight of cotyledon explants. Light
grown (control) and dark preconditioned
cotyledon explants (40 each) on 5 pm TDZ
were harvested at the 7 d of culture initiation
and dried at 70 °C for 3 d to measure the dry
weight of the explants. The data were ana-
lyzed by the t test (P <0.01) using Sigmaplot
(Systat Software, Point Richmond, CA).
Adventitious shoot induction. After 7 d
of the dark preconditioning, explants were
transferred to the day/night cycle described
previously. After an additional 3 weeks on
shoot induction medium, explants with well-
developed adventitious shoot buds at the
proximal ends were placed on shoot develop-
ment medium [MS medium (pH 5.7, 30 gI..
sucrose, 0.5 gL' MES) without plant growth
regulators]. When shoots reached 5 mm in
length at c2 months, each adventitious shoot
mass was divided into three to four shoot
clumps. The number of shoots per cotyledon
explant was counted at 4 months, when
most shoots reached 2 cm in length. The
data sets for adventitious shoot formation
(Fig. I) were analyzed by the t test (P < 0.05)
using Sigmaplot (Systat Software). Cultures
were transferred to fresh medium every
2 weeks. All the plant growth regulators were
purchased from Phytotechnology (Shawnee
Mission, KS). They were filter-sterilized and
added to the autoclaved media. Experiments
were repeated two to three times with 30
to 40 explants per condition.
Rooting. Regenerated shoots that were
cm in length were transferred to root induc-
tion medium [MS medium (pH 5.7, 30 gL
sucrose, 0.5 gL MES) supplemented with
5 pm indole-3-butyric acid (IBA)]. After 4 to
6 weeks, rooted plantlets were transferred to a
peat—vermiculite growth mixture and cul-
tured in a greenhouse at temperatures ranging
HORTSCiENCE VOL. 43(1) FEBRUARY 2008?
from 18 to 28 °C (night/day) under a 15/9 h
(day/night) photoperiod with light supple-
mented by metal halide lighting at a photon
flux density of 1000 to 1250 FmoIm2s'.
Transparent plastic covers were placed over
the plantlets for the first 2 weeks for accli-
Histological analysis. Cotyledon explants
were dissected from dry seeds as described
previously and placed vertically (distal side
in contact with medium) on the shoot induc-
tion medium (MS medium supplemented
with 5 IIM TDZ). They were preincubated in
the dark for 7 d and then placed under the
day/night cycle (16/8 h) at constant 26 °C for
another 7 d. In some cases, cotyledon petioles
developed from the cotyledon explants and
reached 2 to 3 mm in length at the end of the
7-d dark preincubation. The junction (5 mm)
of cotyledon and cotyledon petiole with well-
developed adventitious shoot buds was
obtained at 10 and 14 d after culture initia-
tion. Histological analysis was performed
as described previously (Ahn et al., 2007).
Briefly, tissues were incubated in fixing
solution (formaldehyde, acetic acid, and eth-
anol) overnight and dehydrated in a series of
ethanol solutions with increasing concentra-
tions. Embedding was performed with Tech-
novit 7100 kit (Heraeus Kulzer, Wehrheim,
Germany) according to the manufacturer's
instructions. Cross-sections (3 l.Im) were ob-
tained using a manual microtome and ad-
hered to glass slides using water. Tissues
were stained with 0.1% toluidine blue (Serva,
Heidelberg, Germany) and mounted using a
synthetic medium (Permount; Fisher Scien-
tific, Fair Lawn, NJ). Sections were visual-
ized using a Zeiss Axiophot microscope
(Zeiss, Jena, Germany).
Effect of dark preconditioning and
thidiazuron on adventitious shoot induction.
Our previous study showed that the dark
preconditioning of hypocotyl explants in
castor increased the size of the explants and
the rate of plant regeneration, and TDZ
induced more adventitious shoots than
6-benzylaminopurine (Ahn et al., 2007).
To test the effect of dark preconditioning
and different TDZ concentrations on explant
growth and subsequent adventitious shoot
induction, cotyledon explants were cultured
on shoot induction media containing TDZ
at 1, 5, or 10 .IM with or without 7 d dark
preconditioning. As shown in Figure 1, the
light-grown control explants produced
adventitious shoots at an average number
of two, four, and seven, respectively. When
explants were dark preconditioned, the aver-
age number of shoots regenerated increased
slightly at I FM TDZ (six shoots per explant).
However, the increase was not statistically
significant based on the t test (P < 0.05). A
dramatic enhancement in plant regeneration
was observed on 5 l.tM TDZ. Dark precondi-
tioned explants produced an average of 25
shoots per explant, representing an v6-fold
increase compared with the light-grown ones.
At 10 pm TDZ, dark preconditioning did not
enhance plant regeneration efficiency. Thus,
shoot production was maximal when cotyle-
don explants were preincubated in the dark
during culture initiation and treated with
5 F° TDZ.
Effect of dark preconditioning on explant
development. During the initial 7 d in culture,
the light-grown (control) explants did not
enlarge much and accumulated chlorophyll
slowly (Fig. 2A). Approximately 10% of the
light-grown explants failed to produce chlo-
rophyll until 4 weeks after culture initiation
(data not shown). On the other hand, the dark
preconditioned explants were much bigger
than the light-grown ones (Fig. 213). When
their dry weights were compared, the former
was 6 times heavier than the latter (Fig. 3).
Although these data were shown for cotyle-
don cultures with 5 FM TDZ, similar results
were obtained for the cultures with 1 or 10 FM
TDZ (data not shown).
.4 dventitious s/toot induction and plant/ct
formation. When the dark preconditioned
cotyledon explants were transferred to the
day/night cycle, they rapidly accumulated
chlorophyll within a couple of days (data
not shown) and gradually developed adven-
titious shoot buds at their proximal ends.
Figure 2C shows a dark preconditioned
explant with initial shoot buds (3 weeks after
culture initiation, 5 FM TDZ). In some cases,
cotyledon petioles also developed and adven-
titious shoot buds were formed at the junc-
tion of cotyledon and petiole. The shoot buds
developed more vigorously on the adaxial
surface of cotyledon explants. More than
90% of the dark preconditioned explants
were responsive and produced adventitious
shoot buds (data not shown). Small shoots
started to be visible at 4 weeks after culture
initiation (Fig. 2D). Then, explants were
transferred to MS medium without plant
growth regulators for shoot elongation. The
adventitious shoots reached 5 mm in length
at 2 months after culture initiation (Fig. 2E)
and 1 cm at 3 months (Fig. 2F). When shoots
reached 2 cm in length, at rv4 months,
individual shoots were transferred to root
induction medium (MS medium supple-
mented with 5 FM IBA) and developed the
root system in 4 to 6 weeks (Fig. 2G). The
rooted plantlets were transplanted to a com-
mercial peat—vermiculite growth mixture and
successfully acclimatized in the greenhouse
Histological survey. Transverse sections
of the cotyledon explants developing adven-
titious shoot buds were examined to learn the
origin of the regenerated shoots. In some
cases, petioles developed from the cotyledon
explants, and adventitious shoot buds were
formed at the junction of cotyledon and
petiole. Figure 4 shows the histological char-
acteristics of the junction tissue producing
adventitious shoot buds during the first 2
weeks of culture initiation. At 10 d, the
cross-section of the junction tissue retained
Fig. I. Optimization of adventitious shoot production. Cotyledon explants dissected from mature seeds
were placed on shoot induction media containing 1,5, or 10 gu thidiazuron (TDZ). For the first week of
culture initiation, cotyledon explants were cultured under the day/night (16/8 Ii) cycle (light) or in the
dark (dark) at constant 26 °C. At 8 d, dark preconditioned explants were also transferred to the day/
night cycle. After 4 weeks on the shoot induction media, explants with adventitious shoot buds were
transferred to shoot development medium (Murashige and Skoog medium without plant growth
regulators). The number of shoots regenerated per explant was calculated at 4 months when most of the
shoots reached m2 cm in length. Values followed by the same letter are not significantly different at the
level of 5% according to the t test (Sigmaploo Systat Software, Point Richmond, CA). TI, I? TDZ
T5, 5 1M TDZ Tb, 10 pm TDZ.
HORTSCIENCE VOL. 43(1) FEBRUARY 2008
Fig. 2. Adventitious shoot induction and plantlet establishment in castor. Cotyledon explants dissected
from mature seeds were placed on Murashige and Skoog medium supplemented with 5 p'a thidiazuron
(TDZ). For the first week of culture initiation, (A) half of the explants were placed directly under the
day/night cycle (16/8 h) and (B) the other half were preconditioned in the dark at constant 26 °C.
Pictures were taken at 7 d. From the second week, the dark preconditioned explants were also cultured
under the day/night cycle. Adventitious shoot buds were induced at the proximal ends of cotyledon
explants [(C), 3 weeks after culture initiation; (D). 4 weeks] and developed into multiple shoots [(E),
2 months; (F), 3 months] without morphological defects. (C) Regenerated shoots developed root
system in 4 to 6 weeks when treated with 5 .1M indole-3-butyric acid. (H) Plantlets were successfully
acclimatized in the soil. Bars - I cm.
a typical structure of petiole where a con-
caved adaxial surface was still distinguish-
able (Fig. 4A). Active cell division was
observed in the epidermal and cortical cell
HORTSCtENCE VOL. 43(1) FEBRUARY 2008?
layers of the adaxial side of the tissue. At
14 d, the concaved surface expanded outward
and developed protrusions (Fig. 413). When
examined closely, leafpriniordium-like struc-
lures were observed in the protrusions (Fig.
4C). Our results indicate that the adventitious
shoot buds originate from the cortical and
epidennal cell layers of the adaxial side of the
cotyledon —petiole junction.
This report describes the adventitious
shoot formation and plant regeneration from
cotyledon explants in castor. Plant regenera-
tion has been achieved by using cotyledons as
explants in a number of plant species. In most
cases, cotyledon tissues were obtained from
Young seedlings such as in squash (Cucurhita
pepo; Ananthakrishnan ci al., 2003), bottle
gourd (Lagenaria siceraria Standl.; Han
et al., 2004), and an oilseed crop, niger
[Guizotia abyssinica (L. f.) (ass.; Murthy
et al., 2003]. In castor, we used cotyledon
explants obtained directly from mature seeds,
like in black locust (Robin/a pseudoacacia
L.; Zarago7b et al., 2004) and radiate pine
(Pious radiata D. Don; Grant et a)., 2004).
The proximal end of the cotyledon explant
(the Junction of cotyledon and cotyledon
petiole) was proven to be highly regenerative
in castor. Calli were often produced along the
cotyledon vein but did not regenerate into
shoots (data not shown). Similarly, the prox-
imal tissues of cotyledon explants were much
more responsive than the distal ones in
soybean (GIt'cine mar; Mante et al., 1989).
squash (Ananthakrishnan et al.. 2003), and
bottle gourd (Han et al.. 2004).
We have shown here that the dark pre-
conditioning of cotyledon explants increased
the size of explants and the number of ad-
ventitious shoots regenerated. The enlarge-
ment of dark preconditioned explants was
observed at all the TDZ concentrations tested
(1, 5, and 10 .txt). However, the number of
shoots regenerated increased significantly
(6-fold) only when adventitious shoots
were induced on 5 l.tM TDZ. When treated
with I lt\1 TDZ, the dark preconditioned
explants did not induce shoot buds at a
significantly higher level than the light-
grown control ones. On 10 1.IM TDZ, numer-
ous shoot buds were initially induced from
the dark preconditioned explants. However,
most of the shoots remained small in elonga-
tion medium free of TDZ and did not elon-
gate further to develop into plantlets. Thus,
the maximal plant regeneration efficiency
was achieved by using the dark precondition-
ing of explants and optimizing the TDZ
concentration for the adventitious shoot in-
duction and subsequent development. Pre-
viously, the dark preconditioning of explants
also enhanced the plant regeneration in castor
when hypocotyl tissues were used as explants
(Ahn et al., 2007).
In other plant species, there are a number
of studies reporting the beneficial effect of
the dark preconditioning of explants on the
subsequent plant regeneration through organ-
ogenesis such as in watermelon (Citrullus
lanatus; cotyledon explants; Compton, 1999)
and Chinese plant jujube (Zizvphus jujube
Mill.; leaf explants; Gu and Zhang, 2005).
Fig. 3. Dry weight of cotyledon explants. Explants dissected from mature seeds were cultured under the
day night (16/8 h) cycle (light) or in the (lark (dark) for 7 d. Then, 40 explants per condition were
harvested and dried at 70 °C. After 3 d, the dry weight of each explant was measured. The har,
of the moans. Val tics foil (O\ nd b\ the sailic letter are not 1 on ficantl\ di fThrcnt at ii
lIo,:rc. f'i:t? ']?
I lie \,tCt iitncli:titi'.iit of 111C cithiticod 11()o(
bud produetiott by US111, 1 dark preconditioning
of explants is not clearly understood. As we
discussed previously (Ahn et al., 2007),
studies suggested that explants experience
changes during the dark preconditioning on
a molecular or cellular level, resulting in the
enhancement of plant regeneration. Several
characteristics of dark-grown tissues such
as 1) preservation of light-sensitive endoge-
nous or exogenous plant growth regulators
(Hutchinson et al., 2000), 2) a higher level of
undifferentiated cells (Herman and Hess,
1963), and 3) reduced cell wall thickness or
cell wall deposits (Herman and Hess, 1963)
may confer the regenerative capacity on the
TDZ has been found to be the most
effective plant growth regulator to induce
shoots from various explants in castor (Ahn
et al., 2007: Sujatha and Reddy, 1998). Our
previous study using hypocotyl explants
revealed that TDZ induced adventitious
shoots from the cortex (Ahn et al., 2007),
similar to what we found in this study using
cotyledon explants in which the surface
tissues, including the cortex and epidermis,
were involved in the formation of adventi-
tious shoots. The adventitious shoot forma-
tion at the outer cell layers of cotyledon
explants could be beneficial for the .4gro-
bacterium-mediated or biolistic transforma-
tion of castor when applied, because those
cells are readily accessible to the Agrohacte-
rium or particles bombarded. We believe that
this highly efficient and simple plant regen-
eration protocol will be useful for the devel-
opment of efficient systems for genetic
transformation in castor.
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