provide accessible surface targets for theAgrobacrerium-mediated or biolistic trans-formation of castor.

Materials and Methods

HORTSCIENCE 43(I):215-219. 2008.

In Vitro Regeneration of Castor(Ricinus Communis L.) UsingCotyledon ExplantsYeh-Jin Ahn' and Grace Qianhong Chen'United States Department of Agriculture, Agricultural Research Service,Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710Additional 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-derivedcotyledon explants produced adventitious shoots when placed on Murashige and Skoog(MS) medium containing thidiazuron (TDZ). The rate of shoot regeneration wasmaximal (25 shoots per explant) when explants were cultured on shoot inductionmedium supplemented with 5 lim TDZ and preincubated in the dark for the first 7 daysbefore transferring to the day/night cycle (16/8 h). Only the proximal ends of cotyledonexplants produced adventitious shoots, although green calli were observed in cotyledonveins. After 4 weeks in culture, explants with well-developed shoot buds were transferredto 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 toroot induction medium (MS medium supplemented with 5 ,ii indole-3-buty ric acid)where they developed roots in 4 to 6 weeks. Plantlets were transferred to soil andacclimatized to greenhouse conditions. Histological analysis showed the adventitiousinduction of the shoots originated from the cortical and epidermal cell layers of thecotyledon explants.

Castor (Ricinus communis L.), a semi-tropical perennial plant, is a valuable oilseedcrop. It is the only commercial source ofricinoleic acid that is used for numerousindustrial products (e.g., lubricants, paints,coatings, and plastics) (Caupin, 1997). How-ever, the seed contains the ricin toxin(reviewed in Hartley and Lord, 2004; Lordet al., 1994) and hyperallergenic storage pro-teins (2S albumins) (reviewed in Pastorelloet al., 2001; Shewry et al., 2002). Whenintroduced into cells, the ricin toxin inacti-vates ribosomes, resulting in inhibition ofprotein synthesis (Endo and Tsurugi, 1987).2S albumin was identified as the majorallergen of castor seeds, bringing about animmunoglobulin 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 forpublication 27 Sept. 2007.We thank Louisa yang for the assistance on thecastor tissue culture and medium preparation andChanman Ha (Plant Gene Expression center,USDA, Albany, CA) for the advice on the histo-logical analysis. Castor gerrnplasm was providedby the USDA-ARS National Genetic ResourcesPrograms, Southern Region Plant IntroductionStation.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;e-mail qhgc@pw.usda.gov

health concerns to farmers and oil plantworkers greatly limiting its cultivation.

These hazardous proteins could poten-tially be reduced through genetic engineer-ing. However, castor is highly recalcitrant toin 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 tipsas explants. However, the rates of putativetransformant recovery were very low (0.08%and 0.42%, respectively). Because a numberof independent transfonriants are typicallyrequired to identify a line with an appro-priate level of transgene expression, thecurrent transformation efficiency of castorneeds to be improved. Considering the highrates of plant regeneration using embryonictips (an average of 40 shoots per explant;Sujatha and Reddy, 1998), it is likely that themeristematic cells involved in plant regener-ation were difficult to transform using Agro-bacterium. To increase the transformationefficiency in castor, we are developing pro-tocols for plant regeneration through adven-titious shoot formation by testing varioustissues 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 fromcortex tissues at similar rates (24 and 25shoots per explant, respectively). Addition-ally, the cotyledon also used epidermis toproduce adventitious shoots, which would

Explant preparation and cultureiniliation. Castor seeds (Ricinus comniunisL. P1 P1215769) were obtained from theU.S. Department of Agriculture, GermplasmResources Information Network, SouthernRegional Plant Introduction Station (Griffin.GA). Seeds were decoated and surface-ster-ilized in a 5% (v/v) commercial bleachsolution (5.25% sodium hypochlorite) for15 min followed by five rinses in steriledeionized water. Cotyledon explants wereaseptically isolated from embryo and placedvertically (distal side in contact with me-dium) on the shoot induction media [MSmedium (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 ofthe explants were preincubated in the darkand the other half were incubated under theday/night cycle (16/8 Ii) at 100 pmolm<ssupplied by cool-white fluorescent lamps(Sylvania, Danvers, MA) at constant 26 °C.

Dcv weight of cotyledon explants. Lightgrown (control) and dark preconditionedcotyledon explants (40 each) on 5 p m TDZwere harvested at the 7 d of culture initiationand dried at 70 °C for 3 d to measure the dryweight 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 dof the dark preconditioning, explants weretransferred to the day/night cycle describedpreviously. After an additional 3 weeks onshoot induction medium, explants with well-developed adventitious shoot buds at theproximal ends were placed on shoot develop-ment medium [MS medium (pH 5.7, 30 gI..sucrose, 0.5 gL' MES) without plant growthregulators]. When shoots reached 5 mm inlength at c2 months, each adventitious shootmass was divided into three to four shootclumps. The number of shoots per cotyledonexplant was counted at 4 months, whenmost shoots reached 2 cm in length. Thedata sets for adventitious shoot formation(Fig. I) were analyzed by the t test (P < 0.05)using Sigmaplot (Systat Software). Cultureswere transferred to fresh medium every2 weeks. All the plant growth regulators werepurchased from Phytotechnology (ShawneeMission, KS). They were filter-sterilized andadded to the autoclaved media. Experimentswere repeated two to three times with 30to 40 explants per condition.

Rooting. Regenerated shoots that werecm in length were transferred to root induc-tion medium [MS medium (pH 5.7, 30 gLsucrose, 0.5 gL MES) supplemented with5 pm indole-3-butyric acid (IBA)]. After 4 to6 weeks, rooted plantlets were transferred to apeat—vermiculite growth mixture and cul-tured in a greenhouse at temperatures ranging

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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 photonflux density of 1000 to 1250 FmoIm2s'.Transparent plastic covers were placed overthe plantlets for the first 2 weeks for accli-matization.

Histological analysis. Cotyledon explantswere dissected from dry seeds as describedpreviously and placed vertically (distal sidein contact with medium) on the shoot induc-tion medium (MS medium supplementedwith 5 IIM TDZ). They were preincubated inthe dark for 7 d and then placed under theday/night cycle (16/8 h) at constant 26 °C foranother 7 d. In some cases, cotyledon petiolesdeveloped from the cotyledon explants andreached 2 to 3 mm in length at the end of the7-d dark preincubation. The junction (5 mm)of cotyledon and cotyledon petiole with well-developed adventitious shoot buds wasobtained at 10 and 14 d after culture initia-tion. Histological analysis was performedas described previously (Ahn et al., 2007).Briefly, tissues were incubated in fixingsolution (formaldehyde, acetic acid, and eth-anol) overnight and dehydrated in a series ofethanol solutions with increasing concentra-tions. Embedding was performed with Tech-novit 7100 kit (Heraeus Kulzer, Wehrheim,Germany) according to the manufacturer'sinstructions. 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 asynthetic medium (Permount; Fisher Scien-tific, Fair Lawn, NJ). Sections were visual-ized using a Zeiss Axiophot microscope(Zeiss, Jena, Germany).

Results

Effect of dark preconditioning andthidiazuron on adventitious shoot induction.Our previous study showed that the darkpreconditioning of hypocotyl explants incastor increased the size of the explants andthe rate of plant regeneration, and TDZinduced more adventitious shoots than6-benzylaminopurine (Ahn et al., 2007).To test the effect of dark preconditioningand different TDZ concentrations on explantgrowth and subsequent adventitious shootinduction, cotyledon explants were culturedon shoot induction media containing TDZat 1, 5, or 10 .IM with or without 7 d darkpreconditioning. As shown in Figure 1, thelight-grown control explants producedadventitious shoots at an average numberof two, four, and seven, respectively. Whenexplants were dark preconditioned, the aver-age number of shoots regenerated increasedslightly at I FM TDZ (six shoots per explant).However, the increase was not statisticallysignificant based on the t test (P < 0.05). Adramatic enhancement in plant regenerationwas observed on 5 l.tM TDZ. Dark precondi-

tioned explants produced an average of 25shoots per explant, representing an v6-foldincrease compared with the light-grown ones.At 10 pm TDZ, dark preconditioning did notenhance plant regeneration efficiency. Thus,shoot production was maximal when cotyle-don explants were preincubated in the darkduring culture initiation and treated with5 F° TDZ.

Effect of dark preconditioning on explantdevelopment. During the initial 7 d in culture,the light-grown (control) explants did notenlarge much and accumulated chlorophyllslowly (Fig. 2A). Approximately 10% of thelight-grown explants failed to produce chlo-rophyll until 4 weeks after culture initiation(data not shown). On the other hand, the darkpreconditioned explants were much biggerthan the light-grown ones (Fig. 213). Whentheir dry weights were compared, the formerwas 6 times heavier than the latter (Fig. 3).Although these data were shown for cotyle-don cultures with 5 FM TDZ, similar resultswere obtained for the cultures with 1 or 10 FMTDZ (data not shown).

.4 dventitious s/toot induction and plant/ctformation. When the dark preconditionedcotyledon explants were transferred to theday/night cycle, they rapidly accumulatedchlorophyll within a couple of days (datanot shown) and gradually developed adven-titious shoot buds at their proximal ends.Figure 2C shows a dark preconditionedexplant with initial shoot buds (3 weeks afterculture 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 budsdeveloped more vigorously on the adaxialsurface of cotyledon explants. More than90% of the dark preconditioned explantswere responsive and produced adventitiousshoot buds (data not shown). Small shootsstarted to be visible at 4 weeks after cultureinitiation (Fig. 2D). Then, explants weretransferred to MS medium without plantgrowth regulators for shoot elongation. Theadventitious shoots reached 5 mm in lengthat 2 months after culture initiation (Fig. 2E)and 1 cm at 3 months (Fig. 2F). When shootsreached 2 cm in length, at rv4 months,individual shoots were transferred to rootinduction medium (MS medium supple-mented with 5 FM IBA) and developed theroot system in 4 to 6 weeks (Fig. 2G). Therooted plantlets were transplanted to a com-mercial peat—vermiculite growth mixture andsuccessfully acclimatized in the greenhouse(Fig. 21-I).

Histological survey. Transverse sectionsof the cotyledon explants developing adven-titious shoot buds were examined to learn theorigin of the regenerated shoots. In somecases, petioles developed from the cotyledonexplants, and adventitious shoot buds wereformed at the junction of cotyledon andpetiole. Figure 4 shows the histological char-acteristics of the junction tissue producingadventitious shoot buds during the first 2weeks of culture initiation. At 10 d, thecross-section of the junction tissue retained

Fig. I. Optimization of adventitious shoot production. Cotyledon explants dissected from mature seedswere placed on shoot induction media containing 1,5, or 10 gu thidiazuron (TDZ). For the first week ofculture initiation, cotyledon explants were cultured under the day/night (16/8 Ii) cycle (light) or in thedark (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 weretransferred to shoot development medium (Murashige and Skoog medium without plant growthregulators). The number of shoots regenerated per explant was calculated at 4 months when most of theshoots reached m2 cm in length. Values followed by the same letter are not significantly different at thelevel of 5% according to the t test (Sigmaploo Systat Software, Point Richmond, CA). TI, I TDZ

T5, 5 1M TDZ Tb, 10 pm TDZ.

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Fig. 2. Adventitious shoot induction and plantlet establishment in castor. Cotyledon explants dissectedfrom 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 theday/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 culturedunder the day/night cycle. Adventitious shoot buds were induced at the proximal ends of cotyledonexplants [(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 rootsystem in 4 to 6 weeks when treated with 5 .1M indole-3-butyric acid. (H) Plantlets were successfullyacclimatized in the soil. Bars - I cm.

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a typical structure of petiole where a con-caved adaxial surface was still distinguish-able (Fig. 4A). Active cell division wasobserved in the epidermal and cortical cell

layers of the adaxial side of the tissue. At14 d, the concaved surface expanded outwardand developed protrusions (Fig. 413). Whenexamined closely, leafpriniordium-like struc-

lures were observed in the protrusions (Fig.4C). Our results indicate that the adventitiousshoot buds originate from the cortical andepidennal cell layers of the adaxial side of thecotyledon —petiole junction.

Discussion

This report describes the adventitiousshoot formation and plant regeneration fromcotyledon explants in castor. Plant regenera-tion has been achieved by using cotyledons asexplants in a number of plant species. In mostcases, cotyledon tissues were obtained fromYoung seedlings such as in squash (Cucurhitapepo; Ananthakrishnan ci al., 2003), bottlegourd (Lagenaria siceraria Standl.; Hanet al., 2004), and an oilseed crop, niger[Guizotia abyssinica (L. f.) (ass.; Murthyet al., 2003]. In castor, we used cotyledonexplants obtained directly from mature seeds,like in black locust (Robin/a pseudoacaciaL.; 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 cotyledonpetiole) was proven to be highly regenerativein castor. Calli were often produced along thecotyledon vein but did not regenerate intoshoots (data not shown). Similarly, the prox-imal tissues of cotyledon explants were muchmore responsive than the distal ones insoybean (GIt'cine mar; Mante et al., 1989).squash (Ananthakrishnan et al.. 2003), andbottle gourd (Han et al.. 2004).

We have shown here that the dark pre-conditioning of cotyledon explants increasedthe size of explants and the number of ad-ventitious shoots regenerated. The enlarge-ment of dark preconditioned explants wasobserved at all the TDZ concentrations tested(1, 5, and 10 .txt). However, the number ofshoots regenerated increased significantly(6-fold) only when adventitious shootswere induced on 5 l.t M TDZ. When treatedwith I lt \1 TDZ, the dark preconditionedexplants did not induce shoot buds at asignificantly higher level than the light-grown control ones. On 10 1.IM TDZ, numer-ous shoot buds were initially induced fromthe 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 efficiencywas achieved by using the dark precondition-ing of explants and optimizing the TDZconcentration for the adventitious shoot in-duction and subsequent development. Pre-viously, the dark preconditioning of explantsalso enhanced the plant regeneration in castorwhen hypocotyl tissues were used as explants(Ahn et al., 2007).

In other plant species, there are a numberof studies reporting the beneficial effect ofthe dark preconditioning of explants on thesubsequent plant regeneration through organ-ogenesis such as in watermelon (Citrulluslanatus; cotyledon explants; Compton, 1999)and Chinese plant jujube (Zizvphus jujubeMill.; leaf explants; Gu and Zhang, 2005).

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Fig. 3. Dry weight of cotyledon explants. Explants dissected from mature seeds were cultured under theday night (16/8 h) cycle (light) or in the (lark (dark) for 7 d. Then, 40 explants per condition wereharvested and dried at 70 °C. After 3 d, the dry weight of each explant was measured. The har,rprent theof the moans. Val tics foil (O\ nd b\ the sailic letter are not 1 on ficantl\ di fThrcnt at iiLo'a ail1In i.lIo,:rc. f'i:t'](hi

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bud produetiott by US111,1 dark preconditioningof explants is not clearly understood. As wediscussed previously (Ahn et al., 2007),studies suggested that explants experiencechanges during the dark preconditioning ona molecular or cellular level, resulting in theenhancement of plant regeneration. Severalcharacteristics of dark-grown tissues suchas 1) preservation of light-sensitive endoge-nous or exogenous plant growth regulators(Hutchinson et al., 2000), 2) a higher level ofundifferentiated cells (Herman and Hess,1963), and 3) reduced cell wall thickness orcell wall deposits (Herman and Hess, 1963)may confer the regenerative capacity on thedark-grown cells.

TDZ has been found to be the mosteffective plant growth regulator to induceshoots from various explants in castor (Ahnet al., 2007: Sujatha and Reddy, 1998). Ourprevious study using hypocotyl explantsrevealed that TDZ induced adventitiousshoots from the cortex (Ahn et al., 2007),similar to what we found in this study usingcotyledon explants in which the surfacetissues, 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 cotyledonexplants could be beneficial for the .4gro-bacterium-mediated or biolistic transforma-tion of castor when applied, because thosecells are readily accessible to the Agrohacte-rium or particles bombarded. We believe thatthis highly efficient and simple plant regen-eration protocol will be useful for the devel-opment of efficient systems for genetictransformation in castor.

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