401

25
Artificial Seeds: Encapsulation of Somatic
Emrrryos
Keitlr Redenbaugh, David Slade,
Peter Viss, and Mary Kossler

thln disk. Embryo survival after encapsulation was poor and sthgle embryo capsules were not produced. Thelr method deviates substantially from the concept of a true seed analog, which is a si-ngle somatic embryo encapsulated in an artificlal endosperm or seed coat matrix. Artificial seeds have the potential to serve as a rapid, inexpensive, and universal clonal delivery system to propagate elite and improved gerrnplasm of seed crops such as laborintensive, hand-pollinated hybrlds and genetlcally engineered plants where meLotic recomblnatlon (for seed pi'oduction 1s undesirable. elthough clonal propagatlon has been used for vegetatlvely propagated crops such as potatoes for millenla, this propagation has not been available for seed crops prior to the discovery of plant tissue culture technigues. Even then, typicat tissue culture propagation methods sueh as axlllary shoot production are expensive due to numerous, labor-ntensive steps which thereby lirnit commercializatlon to What 1s requlred is a bighly hlgh unit-value crops. greenhouse or fle)-d pl-anting systern that direct mechanlzed, productlon to be comparable to that of costs woul-d eoabl-e from seeds. Large scale derlved seed's or transplants produced from a few, tested production and encapsulation of SE potentXal of provldlng plant the materlal has hybrids or elite slmultaneously an inexpensive plant delivery system, whi.l-e providi-ng j.ncreased yields, harvest unlformlty, and disease resistance expressed. by the expl-ant parent In addition, it is anticipated that artlficlal seeds may prime melhod for flel-d delivery of genetically engineered be a particularly if a vector system such as caullflower plants, fiosaic vlrus ls used for transformation. The advantages of artlficial seedq, for genetic englneerlng are: 1) Rapld bulking-up of indivldual,
product j.on, engineered

INTRCDUCTION

'somatlc embryogenesls was first observed in r958 with carrot (steward et a1 19sg) and has been achieved since then in a Iarge number of species with subseguent recovery of compLete prants (Aunfrato 1983). rt was not untir, 20 years afte' steward's initlal discovery, however, that the concept of n artificial seed (i. e. encapsulated somatic embryo) appe,rred in prlnt (Murashige I97B), This and other subsequent appe.:rances of the artlflciaL seed concept contalned only a brlei mention of the ldea (Murashlge 1979; Annonymous IgeO; Durzirn 1980; Evans and Sharp 1982; and Krikorian l9B2). No resu.Lts appeared in the Literature prior to a report on encapsulatlon of alfalf and cerery somatlc embryos (sE) wtth natu.al hydrogel matrlees such as calcium alginate (Redenbaugh et aL 1984). Kitto and Janick (19S5, r983, rgg2, and rgBt) repor:ted coating clumps of.carrot SE, shoots, and callus wlth 2.51 polyoxyethylene (polyox WSR-N, Union Carbide) to form a

plants for

(
402

/
403

?\

Ellmlnatlon of re,qqlrement that inserted genes be stably lntegrated ln theii; genome, and Varlety protectlon lmaking the genetj.c rnodification unstabl-e through nelosls),

llowever, necessary for the introductlon of a new cultlvar. clonal propagatlon of alfal-fa 1s currently not avallable on a

varlety protectlon may likely be a major incentive for the .:ommercial sector to use artificial seeds. The great cost of g':netic engineering research makes it highly desirable that any Jeoe ldentlfled, cloned, and inserted into a host remain under proprietary protection. ln addlti.on, .the use of art1,'iclal seeds containlng mRioticarly-unstaure ioiiin genes as a dellvery system ,n.y ul#'uitfil some of the concern wlth relerse of geneti.carly englneered plants into the environment. Beca'se the artlficlal seeds are a substitute for crops whlch are l)ropagated almost exclusively by seeds, lt would be highJ_y unlli:ely that a foreign gene (which is lost upon seed set) woultl be maintalned after harvest or would ',escape,' into the envi.onment. In a sense, the foreign gene would be engineered to s,:lf-destruct after the growlng season.
SOMAI-']C EMBRYOGENESIS

basls, It is antlclpated that alfalfa artlflclal seeds r.ould ellmlnate the foundatlon and posslbly the reglstered seed generations,
conmercial-

l,t
1

lfaLfa has a well-developed, stage-speclflc somatlc embryogenesls systern (Stuart and Strlckland 1984). Plants were produced from SE uslng a flve stage process on agarsolldified medlum (TabLe 1).
TABLE

I.

Procedure

for alfalfa somatlc

embryogenesls.

Maj.ntenance: fnduction:

SH

+ 25liM naphthaleneacetic acld +

10UM

klnetin

SH + soltM 2,4-DLchlorophenoxyacetlc acld + 5pM

k

inet ln

Of ALFALEA (nedrcago sailva L.

Regeneratlon:
Maturatlon:

SH

+ 3t sucrose +

30mM

prollne +

10mM NH3+

The productlon of seed from elite, select germplasm for alfa"-ia ls a mul-ti-year process. rndividuar alfalfa parents are :hosen, cloned (vla cuttlngs, and crossed to produce breeler seed. Breeder seed is then planted to produce founriation seed, whi-ch ln turn is grown to produce registered seed Final1y, the reglstered seed is so$/n to produce the cert-.fied seed for sale to the growers. Di.sadvantages of thls methr,d are that the flna1, certified seed requires four or more years for productlon and 1s often of l0wer quali.ty than the ,reeder seed due to loss of the initlal heterozygoslty, particularly if the selection index was hlgh, The foundation and r egistered seed generations coul-d be ornltted in production uslng clona1 propagatlon, thereby reducing the time and cost

3 weeks ai oC wlth no medlum at 1o0t relatlve

hunldlty

ConverslonI

L/2

SH

+ 15t sucrose

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

404

405

rather immature and woul_d not convert to plants at a very htgh frecuency. Consequently, the SE were matured for three add -t1ona1 weeks at 4oC without medium but under hlgh hurnicity to prevent desiccation, The highest quality alfarfa SE vrere 5-8mm ln length, 3-4mm in diameter, with cotyledons that were weLl-developed and made up SOt of the entire SE. SE converslon to plants was accompllshed on haLf-strength sH agar medium wlthout hormones. Treatment effects could be statistlcally assayed by counting the embryos produced and by scoring for embryo conversion. Inltially, the conversion freguency of random_p1cked, alfarfa sE was less than rt. By optimlzing the amrnonlum and prol-ine levels, provldlng medlum replenlshment durlng the regeneration stage, and provldlng a post_regeneratlon maturation stage of Iow temperature (40c) for 3 add1tl0na] weeks, the converslon frequency for random-plcked embryos /n vrtro was lncreased to 50-60t over a three_year period (Table 2r.
TABLE

embryo

rather than j.n the endosperm, It 1s expected that the SE produced under appropriate condltlons can be lnduced to form 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 crltlcal seed converslon uder greenhouse and fleld for artlflclal
condltlons,
SOMATIC EMBRYOGENESIS O' gELERy

lAplun gravcorsas L.

Celery is transplanted lnto the fl.fA after a 6O-day germlnation and growth perlod ln the greenhouse for most of the US productlon. Because of heterozygoslty 1n the seed, there ls a hlgh degree of varfabiltty Ln the transplants whlch results 1n 5-20t off-types for the fLnal harvest-.. In addltlon, hybrtd celery ls not available on a cornmercfal basls due to the lack of good male sterlle and self-lncompatlblllty systems. Celery can be vegetatlvely propagated, but thls has
TABLE

2. tn vt tro conversion of alfalfa

SE,

3. Procedure for celery somatlc

embryogenesis.

t Converslon of
r.9

random

SE

Malntenance:

SH SH SH

+ 2.zSuM 2,4-D + 0.5uM klnetln + +

25mM

Preregeneratlon:
82

alanlne

(0

.5t
8*
30?

1983

Regeoeratlon Converslon:

8oM

alanlne and 0 tlH+

I984
l9 85

50-60r

t/2

sH

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

(
406 407

not bsen cost-effectlve. For celery production to be uniform and tc capture hybrld vigor, an artificial seed propagation systern is needed. {lelery SE were produced uslng a stage-specific procedure siml11r to that for alfafa (Tabl-e 3).
Sach stage $ras for tht."-t...k period, One major " diffgrence was that the celery callus was inltiated (from leaf petloles) and maintalned orl a 2,4-D medium. Tberefore, the calLus was ln a contlnual lnduced state. As with alfalfa, celery callus was removed from hormones to produce SE. A preregeneratlon subculture on SH medium with 25mM alanine produced hlgher quality SE. Removlng all ammonium from the medlurn during SE regeneratlon also increased SE guality. the SE were very thln (l-2mm ln dlameter) and lncreased tn length very rapldly. The cotyLedons, although well developed, did not
TABLE

make up a rnajority of the SE. SE conversion was hlghly correl-ated with SE size (Table {) and was completely dependent on nutrients provided 1n the conversion mediurn.

Celery ls an albuminous specles wlth an endoslerm that ls very much larger than the embryo. Almost all of tbe storage reserves for the germination of a zygotic embryo ls provlded by the endosperm. It is expected that for celery artlficial seeds, the capsule wllL have to provlde a major portlon of the needed nutrients.

CONVERSION

4. ln V rtro converslon of celery

SE.

SE

size

(run)

t Conversion at 2 weeks
r00
76

The term "conversj-on" eras chosen to define the productlon of a high-quallty, seedilng-like plant from a somatlc embryo. Thls process 1s not clearly dellneated by the speclflc developmental stages such as those that occur durlng the embryogeny and germination of true seeds. In general, SE do not undergo desiccation, developmental arrest, and tnhibltlon. Rather, as the SE continue to mature, the radlcl-e gradually elongates and a plant growth axls forms (processes that are slmilar to zygotlc embryo precocious germinatlon). There is no cl-ear demarcation between the end of somatic embryogeny and the beginning of plant formation.

10

7-10 5-7 3-5 1-3

72 64
39

The

correlatlon coefficient for

SE

size

and

converslon was 0.95

The ability of encapsulated SE to convert into complete plants is directly proportlonal to the quallty of the SE. Blpolar alfalfa and celery SE that are s-8rrun ln length and have well-developed cotyl-edons $rl11 convert to plants at a much higher frequency than SE lacking these characterlstlcs. SE quaLity ls influenced by the condj.tions durlng each stage t, of somatic embryogeny: callus lnitlatlon, callus lnductlon, regeneration (embryogenesis ), and maturatlon. The flnal assessment of .SE qualj.ty- ls whether conversion to plants occurs . Unfortunately, literature regardlng somatic

408

409

ogenesis contalns 1tttle information on conversion frequ:ncies, usually because the research has necessarily dealt with the in1t1a1 events of SE formatlon and the demon:;tratlon that at teast a few plants can be produced, In cases where ,'germloatlon' data are given, the term "germioatlon'r ls usually not deflned as to whether lt ls radlc.r.e elongatlon, full or partlal development of elther a root or shoot, or productlon of a compl"ete, hlgh-qual1ty plant Emphasis must now be dlrected to improving embryo quali -y. Converslon frequencles must approach the germination frequr:ncj,es of true seeds for artlficlal seeds to be used on a
embr: comnei:clal basls.

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

SEAWEED EXTRACTS

Agar (Ge t tdtun gract tartal carrageenan (chrondrus crrspus and Glgartlna nant / losa) F\rrcellaran \Furce ! I ar t a fast tg ! atal AIglnate (Phaeophycae specles)
PLANT EXUDATES

ENCAPIJULATION OF SOMATIC EMBRYOS

'--'he concept of SE encapsulation to produce an analog to true seeds ls based on the slmllarity of SE wlth zygotlc embryi)s ln terms of morphology. physiology, and blochemistry. SE pr oceed through slmllar developrnental stages to form struc'--ures with cotyledons and both shoot and root aplcal meris :ems connected by a common vascular system. The gross morph)logy of SE 1s very slmilar in ppearance to zygotic embry os. Under certalc condltions, SE tolerate severe desict:ation, such as a water loss to 2.251 fresh welght (Kltto and Jrnlck L9821 , an ablllty shared only wlth zygotlc embryos. Final--y, in some specles, such as Brass/ca napus atd Medlcago satrv.r, SE produce the same embryo-specific protelns as do zygot-.c embryos (Crouch 1980; Stuart et al" 1995),

Arabic (Acacta senega! | Ghatti (Anogene /ssus /at lfo! lal Karaya (Stercu/i a specles) Tragacanth (Astraga !us gunnt ferll
SEED GUMS

Guar (Cyamos/s tetragrono lobl aJ Locust Bean Cum (Ceratonla st t lgtua, Tamarind (Tanar tndus I ndlcal
MICROBIAL PRODUCTS

'-'he dellvery of SE directly . to the greenhouse or field requi: es an encapsulatlon matrlx pl1able enough to cushion and protect the embryos and a1low germination, yet sufficiently rlgid to all"ow for rough handJ.ing of the capsules durlng manuf,cture, transportatlon, and planting. The matrix should

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

4r0

be aLIe to contaln and deliver sufficient nutrients, growth and (levelopmental control agents, and other chemical or bloJ-o,;lcal components necessary for embryo-to-plant conversion and plant establishnent. Ideally, the capsules could contain plani: growth prornotlng microorganisms and agricultural cheml:als speclfically chosen f,or cultlvar and envlronmental condiblons. The encapsulatlon,process should also a1low for the 1'ornatlon of slngfe-embryo capsules. Furthermore, the encapsulated SE shoulil be handled and planted uslng existing seed plantlng equlpment to facllitate acceptance at the farm
leve1.

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

This technlque provided an extremely useful method for encapsulating SE. lfalfa and celery SE were very tolerant to various encapsulation parameters such as 'sodlum alglnate and calcium chloride sources and concentratlons, and length of
complexation time.

of naturally occurring hydrogels (Table 5) have the 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 SE encapsulatlon: lt solublllzes at room temperature, lt does not reguire heat to produce a ge}, and lt gels upon contact wlth a non-toxlc, divaLent rnetal salt such as calclum chloride. The dlvalent salt causes complexation by forming lonlc bonds between carboxyllc acld groups on the guluroni.c acld inolecules of the alginate to form calcium alginate gels. AlgIrlate produced from brown algae fronds has a high mannuronlc acld:guluronic acld ratio (M:c), whereas alglnate from stems has a 1ow ratio. Harder gels are produced from brown algae specles and plant parts that have'a low M:G ratlo. Capsule hardness 1s also control-led by the concentratj.on of dlvalent salts used and the complexatlon time. As ttre salt concentratlon and complexatlon time lncrease, harder capsules are produced. Capsule slze ls controlled by varylng the lnslde dlameber of the nozzLe used for forming the alginate beads. Other useful dlvalent metal salts incl"ude Ianthanum chlorlde, cobaltous chlorlde, ferrous chloride, calcium hydroxide, and calcjum nitrate. Algloate has previously been used for encapsulatlon of microorganlsms, plant cells, organelles, and enzymes (Table 6), but not SE or organlzed plant tissue.
-q.

number

Sonr

c frvo

Pnoclrv Porvtn
Covrre

FIGURE

1, Construct,lon of an artlficlal

seed.

Alginate-encapsulated alfalfa and celery sE converted to complete planLs ln vltro oq ag.ar-solldlfied, half-strength SH medlum at frequencies equal to those of non-encapsulated

4r2

6-. M-lcroorganlsms, plant ce}1s, organelles and enzymes encapsulated uslng sodlum alginate. Most capsules were folmed by complexatlon wlth calclum and were l_ess than I mm ln dlaneter. The overall purpose of such encapsulation $ras to produce s_peclflc compounds from the microrganisms, plant cel1s,. and_ enzym-es. Organelles were encapsulatd generaliy to study blochemical events.
TABLE

A. Ml(:roorganlsms Artnrobacter slnplex Baci I lus stearothernophl lus Can<l lda t rop lca I I s chlorel la pyranatdosa Clostrldlun specLes Corlolus varslcolor Curvularla lunata Ervlnla rhapontlcl
GI

Reference

Larsson et al L976 Hackel et al L975 Aldercreutz et aL 1982 Dallyn et al l97z Llvernoche et a1 tggl
Ohl-son
Cheetham

Dallyn et al

1977

Gluconobacter oxydans Kluyveronyces specles Lactobacl rrus Specles

ornus nosseae

lasc/goc I aduS I anl nosus llet/-ranosarc lna barker I Pachyso I en tannoph I lus Rhl roblun Japonlcun
S

Zynononas nobl I ls

Spl ru I I na nax lna Str3ptorryce s ph aeoch r onogenes Tr l;hoderna specles

ac,i h a r ony

ces specles

et al I9B0 et aL IgAz canry et aL t9B2 Aldercreutz et a1 19g2 Klerstan and Bucke l97z Llnko I980 Sawa et al 1980 Scherer et al- 19Bf MaLeszka et aI l98t Jung et aI 1982 Klerstan and Bucke I927 Grlsby and Hall tgBr vleth et aL 1976 Matteau and SaddLer 1982 Grote et al l"9go
Jones and Vellky IgBl Brodellus et al 1979 Jones and vellky 198f

embryos. Encapsulated al-fal-fa and celery SE also converted to plants under I00t rel"atlve humldlty in sand trays, wlthout SH medium, or in transplant plugs (Techniculture, Inc,, Sa1lnas, CA) sratered wlth half-strength SH llguld medlum, respectlvely. Conversion of encapsulated alfalfa SE was lmproved from Ot to 8t by improving the overall guallty of the SE. For celery, conversj-on was improved from an lnltlal 1t to lot by uslng 810nun SE and adding 3t sucrose to the capsules, These converslon data supported the hypothesls that exalbumlnous species such as alfalfa wlll produce SE tht contaln necessary storage reserves (providing that the somatlc embryogenesls system is optlmized) and that albuminous specles such as celery require addltional nutrlents for converslon.
REQUIREMENTS FOR COMMERCIAL PRODUCTION OF

ARTIFICIAL

SEEDS

B. Plant Cells Cannabl s satrv

Catharanthus roseus Oaucus carota Dlgltalls lanata Hunu lus I apu lus
I

Norlnda cltrlfolla Vlcta faba protoplasts

ponoea specLes

Brodellus et al 1979 Klrsup 1982 Jones and vellky IgBl BrodeLius et aI tgBI Scheurlch et al" 1980
Rhodes and

C. Organelles and Enzjmes

Chloroplasts Mltochondrla Inulase Glucose oxldase

rogenase Glucoamylase
Hyd

cLucosldase Uricase
Est 3rase
Any

Loglucosldase

Anonymous L982

Klerstan and Bucke l97z Klerstan and Bucke 1977 Klerstan and Bucke I977 Klerstan and Bucke 1972 crlsby and HalI IggO Svensson and Ottesen lggI Salleh and Nohamed I9B2 Salleh and Nohamed I9g2 Kumaraswamy et a1 I98l

The various steps for commercial productlon of artiflclal seeds for a gj.ven crop are outllned 1n Table 7. After chooslng the specles desired, a somatlc embryogenesls system must be devised. FortunateLy, for many crops, SE can be produced. However, maturatlon research has not progressed far enough for any specles to date to produce SE that are of comparable quality to zygotlc embryos. Higher conversion frequencies are needed and more research on blochemlcal markers to assess guality 1s requlred. Slngulatlon of SE 1s not a maJor difflculty when l1guld culture methods lflasks or bloreactors are used. The calclum alglnate encapsulatlon system is non-damaging to the embryo, allows handllng of the capsule wlthout breakage, and allows for embryo converslon. Furthermore, alglnate can serve as an artlflclal endosperm/seed eoat to hold and del-iver nutrfents and carbon sources to the developlng ernbryo, Because the alglnate capsule ls hydrated, an outer, water-retalnlng coatlng is necessary for storage and planting the capsules. A further requirement is for the encapsulated SE to have a hlgh conver-

415

PRODUCTION

QUALITY CONTROL

Select Plants, Hybrids or Genetically Engineered Cells

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 and storage reserves needed lo convert under greenhouse or field Flnally, the varlatlon of the pl-ants produced from sE must be controlled so that stable and uniform plants wj-th consisteot yields are achleved. conditloos,
REFERENCES

Somatic Embryogenesis

Biochemical Markers and Production of Storage Protein

Al,dercreutz, P,, O, Holst, and B. Mattlasson. f982. Oxygen Enzyme Microb Technol 4:395suppl"y to immobilized cells.
400.

ScaIe-up r^,ith Bioreacto for 5E Production

Conversion of SE to Plants

P,rckaging of SE irr Gel Capsule

C(,ating Capsules with Hard Shell

Viability of Artificial Seeds

Artificial

Storage of
Seeds

Transplant Production

in

Greenhouge

Freld Production

Uniformity of Crop and Yield

TABLE

7.

Comnerclal productlon

of artlficial-

seeds.

Anmirato, P. L983. Ernbryogenesls. In: Handbook of Plant Cel I cutture vol 1. (eds) D. Evans et al' Macmlllan PubJ.lshlng Co, 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 and transformabion of natural products. FEBS Lett 103:.93-97. Cheetham, P., C. Imber, and J, Isherwood. 1982. The formatlon of isomaltulose by immobilized Ertvtnla rhaponttcl. Nature (tondon) 299:628-631. Crouch, M. 198o. Storage Proteins as Einbryo-speclflc Developmental Markers ln zygotlc, Microsporlc, and Somatlc Embryos of Erasslca napus L. Untverslty of Mlchlgan, Ann Arbor: University Microfj.lms. Dal1yn, H,, w. Falloon, and P. Bean. L977. Methocl for the lmmobj-Iizaton of bacterla.l- sPores in alglnate ge1. Lab Pract 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 Paper Chemistry. pp 3r-60.