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/ RESEARCH PAPERS BIOTECHNOLOGY OF SOMATIC POLYEMBRYOGENESIS AND PLANTLET REGENERATION IN LOBLOLLY PINE Praymod K. Gupta and Don J. Durzan* Department of Pomology, University of California, Davis, CA 95616. *Corresponding author. Plantlets of loblolly pine were regenerated and converted to soil by somatic polyem- bryogenesis (SPE). SPE was evoked in cells from an embryonal-suspensor mass whose nuclei were color-coded by a dou- ble-staining method. Color-coding with cell suspension cultures distinguished a free-nuclear stage, initial physiological states and the ultimate fate of each cell for estimates of yield based on the initial properties of embryogenic and nonem- bryogenic cell masses. Somatic embryos were encapsulated and stored in an algi- nate gel or in liquid nitrogen. omatic polyembryogenesis! can now be extended to loblolly pine (Pinus taeda L.), one of North America’s most valuable softwood and fiber spe- ies. We offer several important advances that distinguish’ our work from earlier reports on somatic embryogenesis, First we report a “true-to-conifer-type” SPE process that proceeds from a proliferating embryonal-suspensor cell mass as a “basal plan”. SPE compares favorably with zygotic embryogenesis, which develops in one of four ways characteristic of gymnosperms. Second, using a double- staining method, we report a previously unobserved free- nuclear stage in cell suspensions as found in the basal plan of zygotic embryo development’, Third, the control of, polyembryonic development in the basal plan is related to the initial physiological states of cells and nuclei in the proliferating embryonal-suspensor mass and to attributes Of calli derived from nonembryonic cells isolated from the same explant. Fourth, SPE was obtained repetitively (over 20 times) from a proliferating embryonal-suspensor mass. Calls of this mass were maintained under defined condi tions for over one year in suspension culture. Fifth, plantlets of loblolly pine are now in soil to complete a postulated cellular cloning cycle*, Sixth, developing em- bryos were encapsulated using an alginate gel and sev- enth, we report preliminary results on the storage and recovery of plants from low temperatures (4C and ""]96°C) in both encapsulated and free forms RESULTS Critical factors and step-wise observations leading to repetitive conifer-type SPE (Fig. 1) were as follows. Within 3 to 4 weeks after inoculation on a specially formulated medium (see Experimental Protocol), a white, mucila nous cellular mass was obtained in darkness from around the female gametophyte of seeds (4 to 5 weeks after fertilization). This embryonal-suspensor mass, similar to that described as embryogenic callus for sugar pine! and Norway spruce**, was recovered from a basal medium'® modified half-strength and supplemented with several factors (see Experimental Protocol). Mucilaginous embryonal-suspensor masses were found in 9 to 10% of the total explants cultured (Fig. 1). Light tom nk goth of enya maps a Teun tea panetpe yet aw SEDO Oe Spina RU aeEBRE ET oe Nar Yds RO ee aoe Er ee aa eee ea eee See ee Nat Peon trey Sarr PRT ATT Mf) alter 86 days. Bar represents 08 mm. 8. Develop: ment gui embiyron ean RAO 18s ee Fe icity Br a) CES Enea fens Sn enh rh pe ce eS SE ee ee te ae et ees ae ee te cnet Tam wna oe Seah ROO eae Sa a See oe ee ee. Sn ee Seed ca hh Coca Seen Ub arpa ernie Sec ES Wt Rape ee OS eee ga BOMECHNOLOGY VOLS FEBRUARY 1987 147 eves 2 Conifer-type somatic polyembryogeness in cel us- Pension cultures of loblolly pine as revesled by 2 double- Saining method and by fluorescence microscopy.’A. Control Tepresenting double tained cell from a nonembryonic callus ‘derived from the initial explant after 9 days in clture X25 B Elongnted cell from 2 double-stained proembryonal-au- pensor mass 1 day after subculture X50, Note acetoca Feacive protoplasmic strand and nuclei (arrows). ©. Free nuclear sage in embryonal-suspensor. mass showing four Giferentaly’ staining fed and ue nucle, x60. B, Sie diference bewween a fed (proembryonal) and blue nucleus at the late freemuclear stage: Note migration of nuceus to cell wal (arrow) where a proembryo wil emerge 90. E. Early division pattern of proembryo (red cells) and elongation of the suspensor@) (be) *19. F. Doablestained.proembyo Showing a red nucleus and a weakly staining cytoplasm. Note ue mucleus (arrow) astociated with the suspensor. 25 G. Development of the embryonal-suspensor mass to pro- duce multiple embryos as part of a somatic polyembryonic Process. Embryos now atthe early embryonal sage sain red Sih acetocarmine. Nuceh in suapensor which Fetain their Minty for aectocarmine have the potential of producing tomatic embryos, Note next wave of embryonic development among double-stained cells (arrows). X80. H. Somatic poly tnbejonie masecs of lblelly pan a cell uapension camure Showing the spiraling and ‘ceavage of embryos asociated wrth the polyembrogens process” Note” the ignfcation (brown) astocted withthe surpensor. X15. I. Fluorescence Iniroscopy to reveal ignited ells in the embryona-susper tor mare 220. Ligne cels reveal a bright yellow Ruores ence (exc. 450-480 nm, em > 520 nm) under UV light BOTECHNOLOGY VOLS FEBRUARY 1987 TABU Affinity of organelles of cells in suspension cultures of loblolly pine atthe end of a 10-day subculture for stains in the double- Staining test: } very strong, 4 strong; 8 moderate; 2 weak: 1 very weak; Om. Nacleus Embryonal-suspensor Mass* _Acetocarmine & Feulgen Evan's Blue mbryonal cells 3 ° Epc 1 4 fs 2 2 freenuclear sager** 5 a4 Nonembryogen callus 2 2 Some transvacuolar strands stain strongly. ***Individual nuclei differ in their ability to accept stain. “Callus is not observed in the explant of the original embryonal suspensor mass. microscopic examination of masses revealed proem- bryonic stages and early embryonic stages (Fig. 2). Be- ‘cause of the origin, cellular composition and developmen- tal potential of this proliferating embryonal-suspensor mass, we hesitate to call it a callus. By contrast, cells derived from friable and nonfriable nonembryogenic callus under identical culture conditions are distinctively, different in shape and growth patern (Fig. 2A), ‘Two extreme major types of nuclei in the embryonal- suspensor mass are easily distinguished (Table 1, Fig. 2) by the double-staining method described in Experimental, Protocol. First are the large nuclei (ca > 10 p. dia.) of proembryonal cells that give rise to SPE (Fig. 2B, C, D). ‘These nuclei stain intensively with acetocarmine and Feulgen"’, Strands in the cytoplasm often show an affinity for acetocarmine and may represent proembryonal cyto- smic fibrils®* (Fig. 2B). Acetocarmine and Feulgen are stains normally used to, detect glycoproteins, chromatin and DNA in cytochemical studies". Second, smaller nu- 10%) in this mass do not fall into an’ easily distinguished category and may represent the tendency to, form callus ie. they do not stain intensely with either or both of the red and blue dyes (Table 1). This additional type reduces the efficiency of the SPE process. If and when callus develops, its contribution may be estimated by our color-coded double-staining assay. Somatic proembryogenesis begins with the migration and segregation of nuclei in proembryonal cells as in zygotic embryogenesis. While we have not actually ob- served the migration of nuclei, photographic evidence indicates that wherever a new somatic proembryo ‘emerges the red nuclei were always very closely associated ‘with the cell wall at one pole of the initially multinuclear cell (Fig. 2D). Mitosis splits off a proembryonal cell with a large nucleus that contributes to the vivid red color of the prospective embryo (Fig. 2E, F, G, H). The origin of the blue-staining nuclei was evident after freezing embryonal-suspensor masses in liquid nitrogen (196°C) for 30 min and recovering the mass by rapid thawing. The thawed embryonal-suspensor mass was placed on a modified half-strength Murashige-Skoo (MS) basal medium with 2,4-D (5 x 10° M) and KN an BAP (each 2 x 10° M). Upon recovery, nearly all of the suspensor cells with blue staining nuclei were killed leav- ing viable embryonal cells with large nuclei. After 3 weeks, these acetocarmine-reactive cells divided with the produc- tion of suspensors with nuclei having the typical affinity for Evan's blue, We have now recovered several rooted plantlets from this study®. Early embryogeny was produced repetitively over one year by subculture on agar every 10 to 12 days. Delaying the rate of subculture caused browning of cells, the loss of embryogenic potential, the proliferation of nonembryo- genic callus and death. Further growth and development of all cells were arrested on the initial medium, and further embryonic development as illustrated in Figure 1 was evoked by sequential subculturing, Longitudinal-sections of somatic ‘embryos “revealed shoot and root apices (Fig. 1 [5)). Within 9 to 10 weeks, complete plantlets developed. A plantlet grown in a mixture containing peat moss, vermiculite and perlite (1:2:1) is shown in Figure 18). ‘The above sequence was repeated up to the globular stage with cell suspensions derived from embryonal sus- pensor masses. Growth and development of homoge- neous cell suspensions was encouraged by subculturing in growth-regulator free medium. After 30 days, globular embryos (0.43 + .02 mm dia.) with suspensors (4.25 = 0.23 mm length) were recovered (Fig. 1 {9) at a level of 1040 + 200 embryos per 100 ml medium. In somatic and zygotic embryogenesis, while subsequent divisions lead to the formation of massive suspensors with blue- and red- staining nuclei, many cells (45 + 20%) in the embryonal- suspensor mass retained nuclei with dominant red-stain- ing properties (Fig. 2G, H). The division of the later contribute to the efficiency of conifer-type somatic po- Iyembryogenesis (Fig. 2H). From 0 to 10 days, the osmo- lality of the medium increases from 190 to over 220 mOsm as the pH at 23 + 1°C drops from 5.7 to 4.6. At lower pH’s the cell suspensions deteriorate rapidly. Histological inspection of suspension cultured embryos revealed shoot and root apical meristems. This contrasted sharply to the unorganized growth and histological pat- terns obtained from nonembryogenic callus and suspen- sion culture under the same treatments. By coupling the double-staining procedure with fluorescence microscopy we have observed the lignification of suspensors as late embryogeny begins (Fig. 2H, 1) Results with callus and cell suspensions with and with- ‘out 2,4-D indicate that 2,4-D is essential for the induction and maintenance of a proliferating embryonal-suspensor mass. After removal of 2,4-D, the addition of NAA (0.5 X 10-®M) improved the growth and development of somatic embryos. White light (50, 20, 05 wW em™* nm” in blue, BOMECHNOLOGY VOLS FEBRUARY 1987 red and far-red spectrum respectively) was inhibitory to the early stages of SPE but stimulated late embryogeny and plantlet formation. ‘Somatic embryos with cotyledons were separated and encapsulated in an alginate gel. Over 50 encapsulated somatic embryos were stored in darkness at 4° + 2°C for 4 ‘months (Fig. 1 (7)). All encapsulated embryos returned to 20°C produced chlorophyll upon transfer to light. While survival was not affected, their reliable conversion to plantlets in soil has not yet been obtained, DISCUSSION Currently, several methods of micropropagation are employed in conifer tree improvement programs®' Compared to micropropagation, repetitive conifer-type somatic polyembryogenesis (SPE) provides an alternative and appropriate biotechnology involving a cellular clon- ing cycle® with advantages for true-to-type mass propaga- tion. However, the conversion of embryos to plantlets in soil needs to be improved substantially, In conifer-type zygotic embryogenesis, the free-nuclear stage is initiated by the rst zygote division to produce the proembryonal tir of ces. This first division results in two and then four free nuclei. The nuclei migrate or are pulled to the base of the archegonia where they partially wall off and initiate the proembryo. In somatic embryo- genesis, repetitive divisions also give the initial free- nuclear stage. Mitosis in the embryonal-suspensor mass somehow contributes to the multiplicity of embryos in the polyembryogenie process. For loblolly pine, somatic po- Iyembryogenesis has now been repeated for 20 months in our laboratory. So far, developmental patterns have been consistent with zygotic pine polyembryony in vitro™, In the initial explant, some nuclei may represent “relict” nuclei occasionally associated with zygotic proembry- ‘ogeny’*, Relict nuclei are found in the upper portion of zygotic cells. Nuclei vary in number and size even within the same species and represent several origins. Since we cannot yet identify relict nuclei specifically we are unsure of their staining properties. Somatic embryos generated from cells bearing relict nuclei could be designated as “somatic phenocopies” or “phenoclonal variants”. Staining with Evan's blue indicates that organelles are leaky or at least permeable to the dye'*, Alteration in the cell wall and/or in plasmalemma and nuclear membrane organization facilitates rapid and intense staining behav- ior. This was demonstrated by enzymic removal of the cell wall to prepare protoplasts (P. K, Gupta, D. J. Durzan, unpublished), Protoplasts of highly actocarmine-eactive embryonal cells stained strongly with Evan's blue. Aceto- carmine staining indicates a concentration and enrich- ment of yet unidentified materials. ‘The origin of the repetitive or cleavage phenomenon was related to the division (mitosis) and fate of acetocar- mine and Feulgen-positive nuclei in somatic proem- bryonal cells in cell suspension cultures. Based on these observations we are forced to consider the embryonal- suspensor mass not as a callus but as a cellular array of significant developmental potential provided that cultural conditions resembling the true environment of the egg, are maintained. Nonembryogenic callus shows none of the above staining or developmental properties under the same conditions. Observations with nuclei have now been repeated with Douglas-fir, Norway spruce and sugar pine. ‘When red and blue stains are combined sequentially we have a useful nuclear-cytoplasmic diagnostic method that provides new insight into the sequential stages of conifer- type somatic and, zygotic polyembryogencss, The end result is a diagnostic method that identifies a proliferating proembryonal cells from all stages of the life cycle ofa tree BIOTECHNOLOGY VOLS FEBRUARY 1987 that are suitable for mass true-to-type clonal propagation. Double-staining can be followed through artificial seed formation and associated with low-temperature storage for assessment of embryonic potential until the field- testing of progeny can be completed. The emergence of embryos (Fig. 1 [4]) pushed into air from embryonal-suspensor mass supports the notion that the suspensor provides the connection, and much of the nourishment for the developing embryo. The develop- ment of only 1 to 5 plantlets per gram of fresh cell mass on paper bridges is due presumably to competition i.e., the depletion of nutrients in the callus and possibly to the formation of substances by the suspensors that inhibit the development of other somatic embryos. By contrast, with cell suspensions, we have recovered (from 100 ml) over 1,000 acetocarmine staining proem- bryos. Lignification provides structural stability to the developing embryonal-suspensor mass, and lignification of suspensor cells has been recorded earlier'*. This prop- erty should facilitate the hydrodynamic handling of em- bryos in bioreactors. If polyembryonic masses are not kept separate, a fabric is quickly formed by the proliferating and interweaving suspensions that makes subsequent me- chanical separations very difficult. However, the recovery of fully developed somatic embryos from cell suspension cultures again falls off dramatically even though the early sage of polyembryogeness can be maintained indefinite. ly. SPE we cannot escape the notion that by its seperate, it reflects the cleavage process in zygotic lyembryogenesis. Cleavage polyembryony is usually de- Eidi'es the production of ore than one cays by division of the zygote into two or more embryos. The resultant embryos represent the new generation, ie., they are monozygotic in origin and products are genetically identical. SPE is distinct from somatic embryogenesis derived from nucellar tissues of Citrus where embryos are similar in genotype to the mother plant”. Our use of “repetitive” and “‘conifer-type” is based on comparisons with true-to-type zygotic embryogenesis, on specific stain- ing properties of cells and the origin and fate of nuclei in the free-nuclear stage of SPE. ‘Singulation of embryos by encapsulation and the low temperature storage of somatic embryos representing elite genotypes (Fig. 1 [7]) may someday reduce our dependence on unpredictable seed years and on insect- infested seeds, Refinements in the encapsulating gels will allow us to study the process of seed development and ‘maturation in the laboratory. Through SPE we can cap- ture elite gains, amplify these gains and reduce risks associated with current breeding practices. But before all of this is possible and before these cells can be used for genetic engineering, we need improved conversion rates and more information as to the behavior of these plantlets under nursery and field conditions. EXPERIMENTAL PROTOCOL source ant improved teed of loblolly pine (Ph toda 2) were collec in June 1985 and Soained 4 is of Weyerhaeuser’s forest seed orchard (Lyons, GA). Seed cones were stored at 4°C until used for this study. Seeds from cones were caved and surface terized Female gametophytes with attached suspensors and proembryos were excised from seeds every wesk Jus afer ferization June 10-18) untl seeds PE amt ne ay week tisues were inoculated on two defined eaiture media Fac evoking SPE and associated wih thee media, were established from over 1000 explants using 5-fold replica nine es mle pean Shc tum** with ‘modification of NH,NO3 (550 mgt), KNOs (4674 ‘gi and thiamine HCl (1.0 mg/l). Halfatrength modifed MS ‘meditim was supplemented with myo-inositol (1000 mg), sucrose (8%), teglutamine (gin) (450 mg, cascin hydrolysate CH (500 (28) alanine 450 m,n ype CH 00 BAP) Gach at 2X 10M at an inal prt of 27 before Stoclaving, Cultures were maintained on 0.6% agar (Difco Bacto aaa) pate in darkness at 29-2 2 “To complete early embryony the proiferaing embryonal. supenor as rng the ty sage oF SPE seed to bye sane medium & desenbed above except for 3D (9 TOr*M) RN and BAP (each at 2 10"MD. after 9 or 4 subcultures, dhe globular stage of embryogenesis was Fully evr dem (Fig: |S). Embryos eongucd and developed coyledons Sih S15 foes a 1S PE when wanted tos were tered liquid medium with fier pape support witout grow Fefglaton and under continuous white light (6.0, 20, 63 :W Gait nm" in blue, red and farred,respecvely) (Pg. 1 omplte plants were developed ina 0.3 basal medion contain: fog 65% wv actvntcd chafcoal (E- Mere), menos! (100 Imo) and sucrone 5), Carin hydrolysate and glutamine were ‘elnoved, ‘This subeulture sequence competed the recovery of Blantca from embryonal sorpewser tees on agar pstee ate uproar macs in 0 ml) weve placed in shaking (20 rp) E00 mi Erlenmeyer fifsks with fated bases. The cultuge medium was 0.5 strength MS Gee ove) with 24¢b (6° 10™°M), KN and BAP (ath 2 656M) Cel suspensions formed, rapidly im darkness when toainted and plcoutured every 5108 days Repeated subeal thre produced welldispersed suspensions of single cll, aggre fates of to 3 cell and larger emiryonal-suspensor mass Fasked‘cal volume was meatured after centrifugation of ell spenon of ech aa 380% fr 10 cultures = gals nee ated follows. Samples sre cll volume of 5 0 10) were suspended in ‘Resta to which 2% acetocarmin (1:1) wavadded. Cultares were heated slighty for 13 see and fiteed to remove exces Dain: 03% Evat’s Bue (Jel ys) yas added to an acctocarmine Saiped cc surpetson washed wi medi wo ranove exons of iin ad lefed» Afr dutletaing cures eve seu med in 100% giyceral to improve te optical dary of cll on Bile for merecpic mspecion and the datouton of dyes {ollwed mcropborograpizealy. The proces was repeated wi alge "and oa el capsulation and freeze of somatic embryos, Somatic embryos were dpoed 18 wiv sodium alginate coming from a separatory funnel. After exposure to alginate the coa embryos were Gropped in beaker containing 100 mM calcium nitrate and stirred for 8-10 minutes” Encapsulated embryos were then whe wih sere water to rove exe cau ‘ate, Embryonal-uspensor masses were freee preserved an recovered by the procedure described by Ulrich tal” Results of this work are in preparation®= Acknowledgments ‘We thank B. Finkle, D. Parfitt (National Germplasm Re} tory, Davis, CA) and K, Redenbaugh (Plant Genetics, Davis, CA) for access to facilities and assistance in cryopreservation and encapsulation of somatic embryos. The U.S. Forest Service con- tract, PSW-84-0011CA provided postdoctoral salary for P. K. Gupta. W. Carlton, Weyerhaeuser, Hot Springs, Arkansas, pro- vided the experimental material, P. Krogstrup contributed to the initiation of callus and in identification of embryogenic explants. Don Edwards provided color photographic plates. Received 4 September 1986; accepted 27 October 1986, Ratrnen Tape PK. and Durzan, D. J. 1986. Soma frm eas of maie vga poe embry 8 oa" 2. Butan, DJ and Seard, FC 170 Morphogenssin cel cares alin some rts tera fC Fores es Og Selon 3 worsahop May So une, Het Pinan. 20 pp. Abi or Fe. 7401617). Fk i oy, 1; von Amol and iso, 18, The sevsopiet soma eyes pine care ated rom irae cinryr of Paar ale Rory pce) Pane Sete sae as 4 Ha, and von Awol S. 1985, Pant regenera though tema iyopeent in Peach Norway sprees Part Pye secs 5. Ragman By apd Bong J.M: 196. Et Cal ot tr dead Est Forex 151000081 6. Gap FR rsa} 1980 Pate repeneratin va oma neat from nies ele of mae cy of ace thes Ntrway pe) Rapid Comm Cel il 85-288 bryogencss echnology 42043 sin subcultured 48 16, i 18. 3. Gi Doyle, J. 196. Progmbrogeny in Pinu in raion to that in other fou ecg a aC Eo ae See Rea oes i a a Bape TR role ps7, ie gs ay CS ec ci Murashige, T and Skoog, F. 1962. A revised medium for the rapid fiesta se ay oa eee Py Pa isca73-07 Sharma, A. K.and Sharma, A. 1980. Chromosome techniques, theory andi, pal. abcham Pre Norio Gan ol Otang O-Ogaa 0.1L The ue of nonpermeting cna for texing th mrt of cl J Exp. Boe. 48790-758 {Eilan 8860 Beer and reveal agg npn el of diferent age, p. 185-162 fot Cel Aging and Call Deu. Dave, Sige, D.C feat), Cambridge Univ. Nese ‘Amerson HV. Frampton, jr L- fu MeKeand, 8. Mot, R. Land ‘Weir, he} 1085: Lobloy pie sult culture: Laboratory, Greene dna ei suey p28 Tue Clr in Foren aod ‘Agrcuture R-. Sienke, XW. Hughes, NM.) Consantn and A. NSizender (eis) Pcmun Prem, NewYork. Farum, P. Timms, and Kulp, J-L. 1989, Biotechnology and forest yl Science 10:004-702 Radforth, N; We and Bonga, JM, 1960. Nature 185982 angan, i. 1969. Ovary ovules mucelis clr, p. 105-125. fn Experimental embryology of vascular plants Johrt B.M. (ed). Springer-Verlag, New York Cpu Pe thd Dursan, D. J. 1985. Shoot mulptiction from mature trees of Doughscir (Plewdange menses) and ugar pine (Gina tombersiona). Plane Cll Reps. 4197-179, Redenbaugh, M. K. 1986, Analogs of botanic seed, US, Patent No 503608 inch, 'M., Mickler, RA, Finkle, B.]. and Karnosky, DF. 1984. Survival and regeneration of American ei callus cultures after being frozen in guid nityogen. Can. J. For. Res, 1€780-758, Beri, GP and Pao, PC. 1985. Cytoplasni bien proembryo formation in Pins, Can J: Bot a8:175-176. Barly C.F, 1062, Devdlopmenal paverns in pine polyembryon ‘An foi d0gar-aae hha Pater in poe poem. rks Duran, ,. and Pale, BJ. Recovery of somatic fmbryon of Plea abe and Pinu taeda from Ronen eultred ce. in preparation) THE NEW MULTI METER Why have 4 separate meters cluttering up bench space when you can have the compact 4 in 4 pH/pOa! Tempim¥ meter for much less. - Bright easy fo ready LED digital readout. + Simple push button sequencing from one mode to the ‘other. +4-20 mA output for pH and POe (polarographic) Automatic temperature compensation for pH ond Oe. -Metars can operate Independently while operating with & control system. Add on our compact modules with 4-20 mA input to ‘expand this meter into a control system. Pegasus® industrial Speciatties Ltd. P.O. Box 349, Agincourt, Ont. MAS 389 Phone: (416) 298-3144. Telex: 065-26447 Write in No. 19 on Reader Service Card BOMECHNOLOGY VOLS FEBRUARY 1987