IIfSURCII PAPER AlWYSIS

GYMNOSPERM TISSUE CULTURE: A RARE SUCCESSThe paper in this issue by PrarnodGupta and Don Durzan (Universityof California, Davis) on somatic poly-embryony in sugar pine describes oneof the few successes in generatingsomatic embryos, and in large num-ber, in gymnosperm tissue culture.Furthermore, the' team's suspensioncultures were derived from embryosexcised from mature seeds that hadbeen stored for nearly five years.Most successes in gymnosperm cul-

lure have been in the promotion ofshoot formation (organogenesis),usually on explanred cotyledons.Since these shoots typically form inbunches and without roots, eachshoot must be individually separatedand removed to a rooting medium.Many shoots do not root readily, andthose that do must then be movedthrough a series of repouings andchanges in environment until theycan be put out in the field. It is alabor-intensive process and thereforeexpensive.The forestry industry, always inter-

ested in breeding and propagatingelite species, has supported extensivetissue culture efforts for a good manyyears. Trial plantings that aim tospeed up propagation and reduceI COS.l are already in place:. .

Somatic embryogenesis If1 conifers(a in all plants) offers the potential oflarge numbers of embryos, all sepa-rate from each Other; roots alreadypart of the structure because there isa natural division of labor as embryosdifferentiate: and dormancy induc-tion in the embryos ami/or encap ula-tion for efficient: storage and deliveryto the fore. t. The elucidation of

Glasgow-A Human Leukaemia Vi-rus Centre, the first of its kind in theworld, is being established here at theUniversity of Clasgcw. Supported byfunds of almost £2 million over thenext five years, it will be located in thedepartment of veterinary pathology,where William Jarrett has carried outhis pioneering work over the past twodecades on feline leukemia.Following the demonstration by

Jarrett and his colleagues that leuke-mia in cats is usually associated with aretrovirus, the researchers found thatthe same virus is a major cause of animmunosuppressive AIDS-like syn-drome in cats. The center's formationcomes soon after the incrimination of

~

i;;;

~~L- ~ -"

Cytochemical attributes of somatic embryos (e) and suspensors (s) of sugar pine.Acetocarmine staining of callus mass reveals the origin of embryos, which containcells that have large nuclei and a strong affinity for the stain.

proper procedures for initiation andgrowth of the proernbryonic suspen-sions must go hand-in-hand with thedevelopment of the bioreactor anddelivery technologies.From a purely biological perspec-

tive, the white, mucilaginous callusthat ultimately produces the somaticembryos is also of interest. Similartissue has been observed in the earlypassages of cereal suspension cultures

t*

s

that form somatic embryos (such asmaize). Both conifers and cereals aredifficult to grow and regenerate inculture, and most successes have beenwith tissue derived from excised im-mature embryos. Determining thenature and possible role of these cellsand their mucilage may help eluci-date the factors that induce or pro-mote embryonic development.

-Philip V. Ammirato

IESUIOI fUNDING

GWGOW TO FORM A HUMAN LEUKEMIA CENTERa related agent in one form of humanleukemia, the pinpointing of HTLV-lUlL'\. V as the agent of humanAIDS, and suspicions that further re-troviruses may be responsible formultiple sclerosis and other condi-tions. Directed by David Onions, thecenter's research program will bringtogether epidemiology and molecularbiology. The Glasgow University Leu-kaemia Research Group, the largestin Britain working on oncogenic vi-ruses, has already produced a suc-cessful vaccine against feline leuke-mia virus (FeLV), and is collaboratingwith Robert Gallo of the U.S. Nation-al Cancer Institute (Bethesda, MD)on [he development of a vaccine

against human AIDS."It is marvelous to see the fruits of

the work of the past 20 years onanimal viruses combining with themodern technologies of molecular bi-ology to lead the search forward inthe field of human leukemia viruses,"says Jarrett, who has worked in Glas-gow throughout his career. Construc-tion to accommodate the new centershould be completed next spring, atwhich time a team of 10 people will berecruited. One of the first aims is toapply genetic engineering techniquesto investigate human leukemias thatoccur in clusters, the significance ofwhich is unknown to epidemiolo-gists. -Bernard Dixon

,IOffECHNOLOGY VOL.4 JULY1986 607

SOMATIC POLYEMBRYOGENESISFROM CALLUS OF MATURE SUGAR PINE EMBRYOSPramod K. Gupta and Don J. DurzanDepartment of Pomology, University of California, Davis 95616.

/ RESEARCH NOTES

An embryogenic callus was isolated fromsuspensor cells of 5-year-old sugar pineseeds. With a newly formulated basal me-dium and by removal of 2,4-D followed byan addition of BAP, globular somatic em-bryos were induced having elongated sus-pensors. In darkness, embryos elongated

Somatic embryogenesis offers a technology aimed atclonally propagating elite trees for forests underintensive cultivation 1. However, since coniferoustrees are natural outbreeders, somatic embryogen-

esis does not offer a way to exactly duplicate the genotypeof the elite tree unless the breeding has been controlled toknow the genotype of the progeny. We report somaticpolyembryogenesis from callus of mature embryos ofsugar pine (Pinus lambertiana Lamb.) from controlledcrosses.Polyembryony refers to the fertilization of several eggs

(simple polyembryony) and the budding or cleavage ofzygotic embryos (cleavage polyembryony). Sporophyticpolyembryony occurs where adventitious embryos arise bybudding from the nucellus and integument and arepseudogamic and usually identical to each other and tothe mother plant. Our results with suspensor tissue areparallel to sporophytic polyembryony and much like thatfound in the polyembryonic nucellar tissue of Citrus»,However, Citrus embrvos are derived from the nucelluswhich are similar in genotype to the maternal parent. Inpine, the suspensor cells are basal cells of zygotic embryosand therefore contain the genotype of the new genera-_tion, not the mother plant. We prefer to use the termsomatic polyembryogenesis to emphasize the origin of theadventive embryos from suspensor tissue.This study differs from other recent results+" in four

other ways. First, somatic embryos are derived from anelite population of blister-rust resistant trees", Second, theprocess was observed in mature seeds stored for nearlyfive years as opposed to immature or freshly collectedseeds. Third, the process is associated with mucilaginouscallus initiated from radicles of embryos. Fourth, evidenceindicates that early stages of the process are repetitive,polyembryonic, and without overt signs of lethality asmight be expected from the earlier literature. Indeed,somatic polyembryogenesis in sugar pine is extremelytrue-to-type in temporal and developmental terms withthe production of large arrays of suspensor cells andembryos with multiple cotyledons. The control over thisprocessional process adds new insight to the concept andsimple, cleavage, and sporophytic embryogenesis+".

and formed numerous cotyledons in arepetitive and true-to-type polyembryonicprocess. When separated from one anoth-er, none of the embryos showed overtevidence of lethal or competitive effectsunder in vitro conditions.

643

",

i IIi!

I,i

I

I! ,!, 'I\ III I

I

Seeds from specific crosses in '1980 were provided by B.Kinloch, U.S. Forest Service, Berkeley, CA. They werecollected at the Institute of Forest Genetics, Placerville,CA, and stored at 20°C until use in this study. Seed coatswere removed, surface-sterilized, and embryos excisedaseptically before being placed on a range of modifica-tions for two culture media 10.11. Factors promoting em-bryogenesis were established using over 500 excised em-,bryos through ten-fold replicated treatments under two'different studies from June to September. Growth anddevelopment of cells were followed by the light micro-scope and histologically't-!". The critical factors, steps andobservations leading to embryogenesis were as follows.Excised embryos developed callus in all media 10,11 with-

in 4-5 weeks. By 10 to 12 weeks (5-year-old seeds) and 3-4 weeks (immature seeds) an unusually white, mucilagi-nous callus was obtained from explants around the radicleon DCR basal medium 10 containing 3.0, 50 and 500 mg 1-1of 2,4-dichlorophenoxyacetic acid (2,4-D), L-glutamineand casein hydrolysate, respectively [Fig. 1 (1) and (2)].The clear mucilage surrounding the white callus retainedat 23°C the, same mas molality of the medium, ca., 125mOsgl. The mucilaginous callus was found in only 4-5percent of the total of 200 embryos cultured under theseconditions. Microscopic examination of the white callusrevealed globular embryos at various stages of develop-ment with large suspensors protruding from the callus[Fig. 1 (3-7)]. Cross-sections revealed that the somaticembryos contained shoot and root apices. Cells at theembryonal end were densely cytoplasmic with large nuclei[Fig. 1 (8)]. Development time and morphology weremuch like early stages of zygotic embryogenesis in coni-fers/-". Observations on free nuclear stages arecurrently in progress. Other types of callus did not revealsigns of organized growth on the same medium.Embryogenic callus has been maintained so far for up

to 5-6 months on a 2,4-D supplemented medium. Howev-er, the development of the globular somatic embryos didnot proceed beyond 12 weeks! unless they were trans-ferred to a medium lacking 2,4-D and containing 0.1 mg1-1N6-benzyladenine (BAP). Transfer of embryos encour-aged elongation of the embryonic axis and the true-to-

I

BIOIfECHNOLOGYVOL4 JULY1986

Ii'I

III

644

;

~~.. .~.,;.- 4i

FIGURE I (I) Somatic polyembryogenesis in sugar pine. Callusderived from 5-year-old seeds showing somatic embryos (e)embedded in mucilaginous matrix after 90 days on DCR. (2)Callus from immature seeds (3-4 weeks) after fertilizationshowing embryonic cells (e) in a network of suspensor cells (s).Embryos and totipotent cells are stained with 0.1 % acetocar-mine. Callus remains clear and poorly stained. Moribund cellsshow strong fluorescence (10). (3) Embryo (e) with small cellsand dense nuclei emerging from suspensor callus mass (s)after 60 days on DCR. (4) Rapidly growing multiple somaticembryos (e) after 120 days. (5) Globular stage attached to

suspensors (s) after 140 days. (6) Torpedo stage in advance offormation of cotyledons after 170 days. (7) Family of somaticembryos derived from suspensor cultures. Large embryo hasmultiple cotyledons, x 10. (8) Longitudinal section of somaucembryo showing cotyledons and apices, x 30. (9) Cytochemicalattributes of somatic embryos (e) and suspensors (s) of sugarpine. A. Acetocarmine staining of callus mass reveals origin ofembryos with cells having large nuclei and a strong affinity ~orstam. (10) Fluorescence (exc. 365 nm em > 418 nm) dlstlll-guishes somatic embryos from suspensor cells and moribundcells (m) filled with tannins and pigments, x20.

BIOfrECHNOLOGY VOL. 4 JULY1986

i

~~'

_ ~-pe development of 6-8 cotyledons in all cases [Fig. 1 (6-I 8)]. Cross sections revealed that the somatic embryoscontained shoot and root apices.Embryogenic callus originated from suspensor cells

which remained attached to the radicle of zygotic embry-os. Embryos induced in the callus mass of suspensor cellsstained bright red with 0.10% (w/v) acetocarmine [Fig 1(2), (9)]. When unstained callus was viewed under UVlight, embryonic cells exhibited a characteristic greenfluorescence. Moribund cells gave a bright yellow fluores-cence. Suspensor cells revealed a weak fluorescence. Thisdisplay enabled us to distinguish among the developmen-tal fates in the callus [Fig. 1 (l0)].The acetocarmine staining and fluorescence of cells

revealed that numerous embryonic cells are present ini-tially in the mucilaginous matrix of elongated suspensorcells. This is the natural result of simple and cleavagepolyembryony. In seeds of our seed lots only one embryodevelops completely in the mature seed.The recovery of healthy and totipotent suspensor tissue

from 5-year-old seeds is difficult and unpredictable. Thesuccess often depends upon how seeds were stored andtheir origin (viz. seed lot number). To overcome thisproblem we found that a much higher percentage ofembryonic cells is obtained by culturing suspensor tissuefrom immature seeds (2-4 weeks after fertilization). Thisobservation is supported by the increased number of j 8.cytoplasmically dense cells with large nuclei (embryoniccells!") that stain with acetocarmine. The totipotent em-bryonic cells are strikingly similar under the light micro- ., 9.scopic to polvembryonic nuclear tissue in Citrus and to""~ 10.zygotic embryos in vitrol5,]6True-to-type developmental stages of embryogenesis II.

have been recapitulated within 6 weeks of culturingsuspensor cells from immature seeds. The temporal pro- 12.cessional stages of development occurred on the DCR 13.basal medium supplemented with casein hydrolysate(500mg l-I),L-glutami.ne \200 mg 1-1), ~-indoleace~ic acid(IAA) (0.2 mg 1"\ kinetin (0.1 mg 1"), and myo-mosltol 14.500 mg 1"1) at pH 5.9 (23°C).In ex lants from immature seeds, the resence of the

female ameto h te attac ed to sus ensor cells aids the 16.establishmen ~,s and in.Jl1s:deve!Q.Qment of somatic£~I~!Ebr:gg,e sis. Llll: e~ ill!, ro'Ve~e t in the er- 17.fQ.L.f!1~I1S$.•~LE.l!e_emb2J:ogen!.u,!rocess IS s$!s:D..E.r~mov~L,of the dommant embr 0 [e.g., Fig. 1 (7)]. RemovaTfdeasest e growth of smaller embryos. We do not know if thisdominance involves inhibitor(s), the depletion of sur-rounding nutrients, or some other factor. Limited experI-ence reveals that somatic polyembryogenesis remains re-petitive as long as the callus can be maintained on the 2,4-D medium.Transfer of embryos to a filter paper support in liquid

medium lacking growth regulators promoted embryoelongation and the greening of cotyledons over 30 days. ,t this stage transfer of embryos to a basal mediumithout supplements and with 0.25% (w/v) activated char-oal under continuous light (2.8. 2.0, 0.5 j.LWcm2 nm") inhe blue, red, and far-red produced complete plantletsithin 40 days at a low (1-2%) conversion (unpublishe~observations).Results support the view!" that the original initial phys-

iological state of the explant, nutritional factors in themedium, sequential relationships among growth regula-tors as applied exogenously, and competitive influencesamong embryos, all contribute to the overall control of thedevelopmental process.The next step involves improvement in the germination

or conversion of somatic embryos into plantlets and thebehavior of somatic embryos in coating and encapsulating

gels aimed at the development of artificial seeds.

AcknowledgmentThis work was supported in part by Cooperative Agreements

(PSW-83-0038CA and PSW-84-0011CA) with the Institute ofForest Genetics, Pacific Southwest Forest and Range ExperimentStation, USDA Forest Service, Berkeley, California. We thankTom Ledig for his interest and help in obtaining access to foresttrees at Placerville, California.

Received 13 January 1986; accepted 2 April 1986.

ReferencesI. Farnum, P., Timmis, R., and Kulp, ]. L. 1983. Biotechnology andforest yield. Science 219:694-702.

2. Spiegel-Roy, P. and Vardi, A. 1984. Tropical and subtropical fruitscitrus. p. 355-372. In: Handbook of Plant Cell Culture, Vo!. III. P. V.Ammirato, D. A. Evans, W. R. Sharp, and Y. Yamada (eds.). McMillanPub!. Co., NY.

3. Hackman, I., Fowke, L. C., von Arnold, S. and Ericksson, T. 1985.The development of somatic embryos in tissue culture initiated fromimmature embryos of Picea abies (Norway spruce). Plant Sci. Lett.38:53-63.

4. Krogstrup, P. 1985. Embryoid-like structure from cotyledons and ripeembryos of Norway spruce (Picea ames (L.) Karst.). Can.]. For. Res.(submitted).

5. Nagmani, R. and Bonga, ].]. 1985. Embryogenesis in immaturesubcultured callus of Larix decidua. Can. J. For. Res. 15:1088-1091.

6. Kinloch, B. B., Parks. Jr., G. K. and Fowler. C. W. 1970. White pineblister rust; simply inherited resistance in sugar pine. Science167:193-195.

7. Dogra, P. D. 1967. Seed sterility and disturbances in embryogeny inconifers with particular reference to seed testing and tree breeding inPmaceae, Studia Forestalia Suecica. 45: 1-95.lorstog, K. 1982. Experimental embryology of gymnosperms, P: 25-51. In: Experimental Embryology of Vascular Plants. B. M. Johri (ed.).Springer Verlag, NY.Thomson. R. B. 1945, "Polyembryony" sexual and asexual embryoinitiation and food supply. Trans. Roy. Soc. Can. 34: 143-169.Gupta. P. K. and D. J. Durzan. 1985. Shoot multiplication frommature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine(Pinus lambertiana). Plant Cell Repts. 4: 177-179.Murashige and Skoog. 1962. A revised medium for rapid growth ofbioassay with tobacco tissue culture. Physio!. Plant. 15:473-493.Feder, N. and O'Brien, T. P. 1968. Plant microtechnique. Someprinciples and new methods. Am. J. Bot. 55: 123-142.Warrnke, H. E. and Lee, S. L. J. 1976. Improved staining proceduresfor semi-thin epoxy sections of plant tissues. Stain Techno!' 51: 179-185.Berlyn, G. P. and P. C. Passoff. 1965. Cytoplasmic fibrils in proembryoformation in Pinus. Can.]. Bot. 43:175-176.

15. Rad forth , N. W. and Pegoraro, L. C. 1955. Assembly of early differ-entiation in pine proembryos transplanted in vitro conditions. Trans.Royal Soc. Can. 49:69-82.Berlyn, G. P. 1962. Developmental patterns in pine polyembryony.Am.]. Bot. 49:327-333.Durzan, D. J. 1984. Special problems: Adult vs. juvenile ex plants. P:471-503. In: Handbook of Plant Cell Culture, Vol, 2. W. R. Sharp,D. A. Evans, P. V. Ammirato, and Y. Yamada (eds.). MacMillan Pub!.CO.,1\Y.

GUIDE FOR AUTHORSBio/Technology publishes original papers on applied research in, and onbasic research with practical implications for, biotechnology. The disci-plines we cover include molecular biology, microbiology, biochemistry,plant and animal biology, and chemical engineering, among others.Because of a substantial increase in the volume of high-quality manu-scripts now regularly submitted to Bio/Technology, and in order to sus-tain our policy of rapid publication (often three to four months afteracceptance), we must ask that research papers be no more than 3,000words long. with a maximum of five illustrations (figures or tables).Bio/Technology also welcomes submission of shorter, single-topic re-search notes of no more than 1,500 words. with at most three illustra-tions. Only original research will be published; material submittedelsewhere will not be considered.

Manuscripts should be addressed to the journal's research editor.Please include a covering letter describing the originality and potentialapplication of research. All manuscripts selected for possible publica-tion will undergo peer review by two or more referees. Authors areencouraged to suggest two referees, but the final selection of reviewersis the editor's. Bio/Technology customarily publishes articles withoutpage charges. In the case of articles that are exceptionally long orcontain color illustrations. however; the authors may be asked to helpdefray publication costs. '

Reviews and book reviews are commissioned by the editor; proposalsare most welcome. -The Editors!.

BIOITECHNOLOGY VOL 4 JULY1986

!i

II:I

645