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25Artificial Seeds: Encapsulation of SomaticEmrrryosKeitlr Redenbaugh, David Slade,Peter Viss, and Mary Kossler

INTRCDUCTION'somatlc embryogenesls was first observed in r958 withcarrot (steward et a1 19sg) and has been achieved since thenin a Iarge number of species with subseguent recovery ofcompLete prants (Aunfrato 1983). rt was not untir, 20 yearsafte' steward's initlal discovery, however, that the conceptof n artificial seed (i. e. encapsulated somatic embryo)appe,rred in prlnt (Murashige I97B), This and other subsequentappe.:rances of the artlflciaL seed concept contalned only abrlei mention of the ldea (Murashlge 1979; Annonymous IgeO;Durzirn 1980; Evans and Sharp 1982; and Krikorian l9B2). Noresu.Lts appeared in the Literature prior to a report onencapsulatlon of alfalf and cerery somatlc embryos (sE) wtthnatu.al hydrogel matrlees such as calcium alginate (Redenbaughet aL 1984). Kitto and Janick (19S5, r983, rgg2, and rgBt)repor:ted coating clumps of.carrot SE, shoots, and callus wlth2.51 polyoxyethylene (polyox WSR-N, Union Carbide) to form a

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thln disk. Embryo survival after encapsulation was poor andsthgle embryo capsules were not produced. Thelr methoddeviates substantially from the concept of a true seed analog,which is a si-ngle somatic embryo encapsulated in an artificlalendosperm or seed coat matrix.Artificial seeds have the potential to serve as a rapid,inexpensive, and universal clonal delivery system to propagateelite and improved gerrnplasm of seed crops such as labor-intensive, hand-pollinated hybrlds and genetlcally engineeredplants where meLotic recomblnatlon (for seed pi'oduction 1sundesirable. elthough clonal propagatlon has been used forvegetatlvely propagated crops such as potatoes for millenla,this propagation has not been available for seed crops priorto the discovery of plant tissue culture technigues. Eventhen, typicat tissue culture propagation methods sueh asaxlllary shoot production are expensive due to numerous,

labor-ntensive steps which thereby lirnit commercializatlon tohlgh unit-value crops. What 1s requlred is a bighlymechanlzed, direct greenhouse or fle)-d pl-anting systern thatwoul-d eoabl-e productlon costs to be comparable to that ofseed's or transplants derlved from seeds. Large scaleproduction and encapsulation of SE produced from a few, testedhybrids or elite plant materlal has the potentXal of provldlngan inexpensive plant delivery system, whi.l-e slmultaneouslyprovidi-ng j.ncreased yields, harvest unlformlty, and diseaseresistance expressed. by the expl-ant parentIn addition, it is anticipated that artlficlal seeds may

be a prime melhod for flel-d delivery of genetically engineeredplants, particularly if a vector system such as caullflowerfiosaic vlrus ls used for transformation. The advantages ofartlficial seedq, for genetic englneerlng are:1) Rapld bulking-up of indivldual, engineered plants forproduct j.on,

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varlety protectlon may likely be a major incentive forthe .:ommercial sector to use artificial seeds. The great costof g':netic engineering research makes it highly desirable thatany Jeoe ldentlfled, cloned, and inserted into a host remainunder proprietary protection. ln addlti.on, .the use ofart1,'iclal seeds containlng mRioticarly-unstaure ioiiin genesas a dellvery system ,n.y ul#'uitfil some of the concern wlthrelerse of geneti.carly englneered plants into the environment.Beca'se the artlficlal seeds are a substitute for crops whlchare l)ropagated almost exclusively by seeds, lt would be highJ_yunlli:ely that a foreign gene (which is lost upon seed set)woultl be maintalned after harvest or would ',escape,' into theenvi.onment. In a sense, the foreign gene would be engineeredto s,:lf-destruct after the growlng season.SOMAI-']C EMBRYOGENESIS Of ALFALEA (nedrcago sailva L. )

The productlon of seed from elite, select germplasm foralfa"-ia ls a mul-ti-year process. rndividuar alfalfa parentsare :hosen, cloned (vla cuttlngs, and crossed to producebreeler seed. Breeder seed is then planted to producefounriation seed, whi-ch ln turn is grown to produce registeredseed Final1y, the reglstered seed is so$/n to produce thecert-.fied seed for sale to the growers. Di.sadvantages of thlsmethr,d are that the flna1, certified seed requires four ormore years for productlon and 1s often of l0wer quali.ty thanthe ,reeder seed due to loss of the initlal heterozygoslty,particularly if the selection index was hlgh, The foundationand r egistered seed generations coul-d be ornltted in productionuslng clona1 propagatlon, thereby reducing the time and cost

/( 403necessary for the introductlon of a new cultlvar. llowever,clonal propagatlon of alfal-fa 1s currently not avallable on aconmercial- basls, It is antlclpated that alfalfa artlflclalseeds r.ould ellmlnate the foundatlon and posslbly thereglstered seed generations,lfaLfa has a well-developed, stage-speclflc somatlcembryogenesls systern (Stuart and Strlckland 1984). Plantswere produced from SE uslng a flve stage process on agar-solldified medlum (TabLe 1).TABLE I. Procedure for alfalfa somatlc embryogenesls.

Maj.ntenance: SH + 25liM naphthaleneacetic acld + 10UM klnetinfnduction: SH + soltM 2,4-DLchlorophenoxyacetlc acld + 5pM

k inet lnRegeneratlon: SH + 3t sucrose + 30mM prollne + 10mM NH3+Maturatlon: 3 weeks ai oC wlth no medlum at 1o0t relatlvehunldltyConverslonI L/2 SH + 15t sucrose

Callus was lnitlated from leaf petloles and maintalnedwith a strict three-week subculture on Schenk and Hlldebrandt(sH) medium (1971.). The ca11us was passed through a three-dayinductlon phase wlth 2,-dichlorophenoxyacetlc acid (2,4-Dlprior to a regeneratlon phase to form SE on a hormone freemedium. Nurnerous sE formed after 3 weeks, but were stiIl

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Ellmlnatlon of re,qqlrement that inserted genes be stablylntegrated ln theii; genome, andVarlety protectlon lmaking the genetj.c rnodificationunstabl-e through nelosls),l,t1

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rather immature and woul_d not convert to plants at a very htghfrecuency. Consequently, the SE were matured for threeadd -t1ona1 weeks at 4oC without medium but under hlghhurnicity to prevent desiccation, The highest quality alfarfaSE vrere 5-8mm ln length, 3-4mm in diameter, with cotyledonsthat were weLl-developed and made up SOt of the entire SE. SEconverslon to plants was accompllshed on haLf-strength sH agarmedium wlthout hormones. Treatment effects could bestatistlcally assayed by counting the embryos produced and byscoring for embryo conversion.

Inltially, the conversion freguency of random_p1cked,alfarfa sE was less than rt. By optimlzing the amrnonlum andprol-ine levels, provldlng medlum replenlshment durlng theregeneration stage, and provldlng a post_regeneratlonmaturation stage of Iow temperature (40c) for 3 add1tl0na]weeks, the converslon frequency for random-plcked embryos /nvrtro was lncreased to 50-60t over a three_year period (Table2r.TABLE 2. tn vt tro conversion of alfalfa SE,

t Converslon of random SE

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embryo rather than j.n the endosperm, It 1s expected that theSE produced under appropriate condltlons can be lnduced toform seed-specific storage reserves. In fact, recently,storage protelns were detected ln alfalfa somatlc embryos(stuart et al 1985). Such storage reserves shoul"il be crltlcalfor artlflclal seed converslon uder greenhouse and fleldcondltlons,SOMATIC EMBRYOGENESIS O' gELERy lAplun gravcorsas L. )

Celery is transplanted lnto the fl.fA after a 6O-daygermlnation and growth perlod ln the greenhouse for most ofthe US productlon. Because of heterozygoslty 1n the seed,there ls a hlgh degree of varfabiltty Ln the transplants whlchresults 1n 5-20t off-types for the fLnal harvest-.. Inaddltlon, hybrtd celery ls not available on a cornmercfal baslsdue to the lack of good male sterlle and self-lncompatlbllltysystems. Celery can be vegetatlvely propagated, but thls hasTABLE 3. Procedure for celery somatlc embryogenesis.

r.9 821983I984l9 85

(0 .5t8*30?50-60r

Malntenance:Preregeneratlon:RegeoeratlonConverslon:

SH + 2.zSuM 2,4-D + 0.5uM klnetlnSH + 25mM alanlneSH + 8oM alanlne and 0 tlH+t/2 sH

Al-falfa ls an exalbumlnous species;seed storage reserves are located in therefore, most ofthe cotyledons and

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(406

not bsen cost-effectlve. For celery production to be uniformand tc capture hybrld vigor, an artificial seed propagationsystern is needed.{lelery SE were produced uslng a stage-specific proceduresiml11r to that for alfafa (Tabl-e 3).Sach stage $ras for " tht."-t...k period, One majordiffgrence was that the celery callus was inltiated (from leafpetloles) and maintalned orl a 2,4-D medium. Tberefore, thecalLus was ln a contlnual lnduced state. As with alfalfa,celery callus was removed from hormones to produce SE. Apreregeneratlon subculture on SH medium with 25mM alanineproduced hlgher quality SE. Removlng all ammonium from themedlurn during SE regeneratlon also increased SE guality. theSE were very thln (l-2mm ln dlameter) and lncreased tn length

very rapldly. The cotyLedons, although well developed, did notTABLE 4. ln V rtro converslon of celery SE.

SE size (run) t Conversion at 2 weeks

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make up a rnajority of the SE. SE conversion was hlghlycorrel-ated with SE size (Table {) and was completely dependenton nutrients provided 1n the conversion mediurn.Celery ls an albuminous specles wlth an endoslerm that lsvery much larger than the embryo. Almost all of tbe storagereserves for the germination of a zygotic embryo ls provldedby the endosperm. It is expected that for celery artlficialseeds, the capsule wllL have to provlde a major portlon of theneeded nutrients.

CONVERSIONThe term "conversj-on" eras chosen to define the productlonof a high-quallty, seedilng-like plant from a somatlc embryo.Thls process 1s not clearly dellneated by the speclflcdevelopmental stages such as those that occur durlng theembryogeny and germination of true seeds. In general, SE donot undergo desiccation, developmental arrest, and tnhibltlon.Rather, as the SE continue to mature, the radlcl-e graduallyelongates and a plant growth axls forms (processes that areslmilar to zygotlc embryo precocious germinatlon). There isno cl-ear demarcation between the end of somatic embryogeny andthe beginning of plant formation.The ability of encapsulated SE to convert into completeplants is directly proportlonal to the quallty of the SE.Blpolar alfalfa and celery SE that are s-8rrun ln length andhave well-developed cotyl-edons $rl11 convert to plants at amuch higher frequency than SE lacking these characterlstlcs.SE quaLity ls influenced by the condj.tions durlng each staget,of somatic embryogeny: callus lnitlatlon, callus lnductlon,regeneration (embryogenesis ), and maturatlon. The flnalassessment of .SE qualj.ty- ls whether conversion to plantsoccurs . Unfortunately, literature regardlng somatic

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The correlatlon coefficient for SE size andconverslon was 0.95

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embr: ogenesis contalns 1tttle information on conversionfrequ:ncies, usually because the research has necessarilydealt with the in1t1a1 events of SE formatlon and thedemon:;tratlon that at teast a few plants can be produced, Incases where ,'germloatlon' data are given, the term"germioatlon'r ls usually not deflned as to whether lt lsradlc.r.e elongatlon, full or partlal development of elther aroot or shoot, or productlon of a compl"ete, hlgh-qual1typlant Emphasis must now be dlrected to improving embryoquali -y. Converslon frequencles must approach the germinationfrequr:ncj,es of true seeds for artlficlal seeds to be used on acomnei:clal basls.ENCAPIJULATION OF SOMATIC EMBRYOS

'--'he concept of SE encapsulation to produce an analog totrue seeds ls based on the slmllarity of SE wlth zygotlcembryi)s ln terms of morphology. physiology, and blochemistry.SE pr oceed through slmllar developrnental stages to formstruc'--ures with cotyledons and both shoot and root aplcalmeris :ems connected by a common vascular system. The grossmorph)logy of SE 1s very slmilar in ppearance to zygoticembry os. Under certalc condltions, SE tolerate severedesict:ation, such as a water loss to 2.251 fresh welght (Klttoand Jrnlck L9821 , an ablllty shared only wlth zygotlc embryos.Final--y, in some specles, such as Brass/ca napus atd Medlcagosatrv.r, SE produce the same embryo-specific protelns as dozygot-.c embryos (Crouch 1980; Stuart et al" 1995),'-'he dellvery of SE directly . to the greenhouse or fieldrequi: es an encapsulatlon matrlx pl1able enough to cushion andprotect the embryos and a1low germination, yet sufficientlyrlgid to all"ow for rough handJ.ing of the capsules durlngmanuf,cture, transportatlon, and planting. The matrix should

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TABL8 5. Classlfication and sources of nabural hydrogels.These r:ompounds will gel when 'mixed with or dropped intoapproprl-ate electrolytes. Ionlc bonds are formed to producestable complexes.

SEAWEED EXTRACTSAgar (Ge t tdtun gract tartalcarrageenan (chrondrus crrspus and Glgartlna nant / losa)F\rrcellaran \Furce ! I ar t a fast tg ! atalAIglnate (Phaeophycae specles)

PLANT EXUDATES

Arabic (Acacta senega! |Ghatti (Anogene /ssus /at lfo! lalKaraya (Stercu/i a specles)Tragacanth (Astraga !us gunnt ferllSEED GUMS

Guar (Cyamos/s tetragrono lobl aJLocust Bean Cum (Ceratonla st t lgtua,Tamarind (Tanar tndus I ndlcalMICROBIAL PRODUCTS

Dextran ( Leuconosstoc /Desenterordes )Xanthan Gun (Xanthononas canpestrls)Gel-lan Cum (eseudononas etadea')

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be aLIe to contaln and deliver sufficient nutrients, growthand (levelopmental control agents, and other chemical orbloJ-o,;lcal components necessary for embryo-to-plant conversionand plant establishnent. Ideally, the capsules could containplani: growth prornotlng microorganisms and agriculturalcheml:als speclfically chosen f,or cultlvar and envlronmentalcondiblons. The encapsulatlon,process should also a1low forthe 1'ornatlon of slngfe-embryo capsules. Furthermore, theencapsulated SE shoulil be handled and planted uslng existingseed plantlng equlpment to facllitate acceptance at the farmleve1.

-q. number of naturally occurring hydrogels (Table 5) havethe aove matrix qualltles necessary for embryo encapsulation,One cf the most useful of these hydrogels, sodium alginate,has i varlety of propertles that make 1t amenable to SEencapsulatlon: lt solublllzes at room temperature, lt doesnot reguire heat to produce a ge}, and lt gels upon contactwlth a non-toxlc, divaLent rnetal salt such as calclumchloride. The dlvalent salt causes complexation by forminglonlc bonds between carboxyllc acld groups on the guluroni.cacld inolecules of the alginate to form calcium alginate gels.AlgIrlate produced from brown algae fronds has a highmannuronlc acld:guluronic acld ratio (M:c), whereas alglnatefrom stems has a 1ow ratio. Harder gels are produced frombrown algae specles and plant parts that have'a low M:G ratlo.Capsule hardness 1s also control-led by the concentratj.on ofdlvalent salts used and the complexatlon time. As ttre saltconcentratlon and complexatlon time lncrease, harder capsulesare produced. Capsule slze ls controlled by varylng the lnsldedlameber of the nozzLe used for forming the alginate beads.Other useful dlvalent metal salts incl"ude Ianthanum chlorlde,cobaltous chlorlde, ferrous chloride, calcium hydroxide, andcalcjum nitrate. Algloate has previously been used forencapsulatlon of microorganlsms, plant cells, organelles, andenzymes (Table 6), but not SE or organlzed plant tissue.

The optirnum encapsulation procedure was to mix alfalfaand celery embryos (5-8 un in leogth) with sodlurn alglnate,(3.2t BDH Gallard-schlesinger and drop them lndividually intoa calcium chLoride solution (5omM) for 30 mlnutes. Single-embryo beads approximately four millimeters in dlameter r,rereproduced (r19. 1).

This technlque provided an extremely useful method forencapsulating SE. lfalfa and celery SE were very tolerant tovarious encapsulation parameters such as 'sodlum alglnate andcalcium chloride sources and concentratlons, and length ofcomplexation time.

Sonr t c frvo

FIGURE 1, Construct,lon of an artlficlal seed.Alginate-encapsulated alfalfa and celery sE converted tocomplete planLs ln vltro oq ag.ar-solldlfied, half-strength SHmedlum at frequencies equal to those of non-encapsulated

Pnoclrv PorvtnCovrre

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4r2TABLE 6-. M-lcroorganlsms, plant ce}1s, organelles and enzymesencapsulated uslng sodlum alginate. Most capsules were folmedby complexatlon wlth calclum and were l_ess than I mm lndlaneter. The overall purpose of such encapsulation $ras toproduce s_peclflc compounds from the microrganisms, plantcel1s,. and_ enzym-es. Organelles were encapsulatd generaliy tostudy blochemical events.A. Ml(:roorganlsmsArtnrobacter slnplexBaci I lus stearothernophl lusCan

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PRODUCTION QUALITY CONTROL

Biochemical Markers andProduction of Storage Protein

Conversion ofSE to Plants

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sion fr.e(luency and rate in the greenhouse or 1n the field.Holrever, the SE of most lf not all crops lack the vigor andstorage reserves needed lo convert under greenhouse or fieldconditloos, Flnally, the varlatlon of the pl-ants producedfrom sE must be controlled so that stable and uniform plantswj-th consisteot yields are achleved.REFERENCESAl,dercreutz, P,, O, Holst, and B. Mattlasson. f982. Oxygensuppl"y to immobilized cells. Enzyme Microb Technol 4:395-400.Anmirato, P. L983. Ernbryogenesls. In: Handbook of Plant Cel Icutture vol 1. (eds) D. Evans et al' Macmlllan PubJ.lshlngCo, pp 82-L23.Anonymous. f980. tnpacts of Appl ted Genettcs. Washlngton,DC: U.S, Government Printlng Offlce' *81-60046.---. 1982. world crops, Jan/Feb pp 30*3r.Brodelius, P. , B. Deus, K. Mosbach, and M. zenk. I979.Immobi liz ed plant cell"s for the production andtransformabion of natural products. FEBS Lett 103:.93-97.Cheetham, P., C. Imber, and J, Isherwood. 1982. The formatlonof isomaltulose by immobilized Ertvtnla rhaponttcl. Nature(tondon) 299:628-631.Crouch, M. 198o. Storage Proteins as Einbryo-speclflcDevelopmental Markers ln zygotlc, Microsporlc, and SomatlcEmbryos of Erasslca napus L. Untverslty of Mlchlgan, Ann

Arbor: University Microfj.lms.Dal1yn, H,, w. Falloon, and P. Bean. L977. Methocl for thelmmobj-Iizaton of bacterla.l- sPores in alglnate ge1. LabPract 26 :77 l-775 .Durzan, D. 1980. Progress and promise ln forest genetlcs. In:Proc SOth Anntversary Conf 'Paper Sc/ance and Technology -The Cutttng Edge,- epplton, wisc: The Institute of PaperChemistry. pp 3r-60.ABLE 7. Comnerclal productlon of artlficial- seeds.

Select Plants, Hybrids orGenetically Engineered CellsSomatic Embryogenesis

ScaIe-up r^,ith Bioreactofor 5E Production

Viability ofArtificial SeedsStorage ofArtificial Seeds

Transplant Productionin GreenhougeUniformity ofCrop and Yield

P,rckaging of SEirr Gel CapsuleC(,ating Capsuleswith Hard Shell

Freld Production