CA2069964C - Method for reproducing conifers by somatic embryogenesis - Google Patents

Abstract

The invention is a method for reproducing coniferous trees by somatic embryogenesis using plant tissue culture. It comprises a multistage process in which a fertilized immature-embryo is excised and cultured to induce early state proembryos, which may be further cultured in liquid media without auxins or cytokinins but with the addition of abscisic acid, to develop late stage proembryos. In some species, it is desirable to significantly raise the osmotic potential of the late stage development medium. The medium may have added exogenous abscisic acid and must contain an absorbent material. After several weeks in culture the vigor and morphology of the embryos developed by the process are improved and the tendency of premature germination is reduced. The somatic embryos may be germinated before or after storage and transplanted to soil for further growth.

Description

~ -1- F ~ ~ 6 g g '~ 4 METHOD FOR REPROi~UClNG CONIFERS
- BY SOMATIC EMBRYOGENESIS
BACKGROUND OF THE INVENTION
The present ~nvention is a method for reproduclng coniferous 10 plants by somatic em~ g~ is using the techniques of plant tissue culture. It is especially suited for producing large clones of superior Douglas-fir selections useful for reEorestation.
Loblolly pine (Pinus taeda L.), its closely related southern pines, and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) are prob-15 ably the most important commercial species of temperate North Americantimber trees. Since the early 1940s, when serious privt~te reforestation - efforts began, literally billions of one and two year old nu- ,~r~ ~.own trees have been planted on cut-over or burned forest lands. For many years these seedling trees were grown using naturally produced seed from 20 cones collected as a part time effort of individuals seeking to supple-ment their incomes. As early as 1957 forest get~eticists began to plant seed orchards using either seed or gr~fted scions obtained from superior trees. These trees were selected for such inheritable characteristics as rapid growth, straightness of bole, wood density, etc. Now in both 25 th~ southern pine and Douglas-fir regions the bulk of the seed is pro-duced from selected trees grown in seed orchards, some of them now sec-ond and third generation orchards.
Despite the fact that the orchards were stocked with superior trees, pollination often cannot be carefully controlled and frequently 30 the seed trees are fertilized by wild pollen of unknown characteristics.
For this reason, the characteristics of the progeny produced by sexual reproduction have not been as predictable as hoped and genetic gain could not be attained as rapidly as desired.
Beginning about 1960, techniques were developed for reproduc-35 ing some species of plants by tissue culture. These were predominatelya~ 5 ~tnd usually ornamental house plants. The method employed use of a suitable explant or donor tissue from a desirable plant. This ;:
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wogl/05854 206996~ PCI/US90/06057 was placed on a series of culture media in which nutrients and growth hormones were carefully controlled from step to step. The usual pro-gression was growth froln the explunt lo a callus. The callus was placed on a budding medium where adventitious buds formed. These, in turn, 5 were separated, elongated, and rooted ~o ultimately form plantlets. A
plantlet has the nature of a seedling but is genetically identical to the explant donor plant.
Gymnosperms in general, and most forest tree species in par-ticular, proved to be much more dirficult to reproduce by tissue cul-10 ture. It was not until about 1975 that Douglas-fir was successrully reproduced by organogenesis. Loblolly pine was successfully reproduced about two years later.
Culture by organogenesis is tedious and expensive due to the large amount of delicate manual handling necessary. It was soon recog-15 nized that embryogenesis was potentially a much more desirable methodfrom the standpoints of quantity of plantlets produced, cost, and poten-tial genetic gain. Work on emt,~ of forest species began in the late 1970s. U.S. Patent 4,217,730 to El-Nil describes one early attempt at somatic embryogenesis of Douglas-fir. This approach was lAter set 20 aside because advanced stage embryos and plantlets could not be readily obtained. However, other workers entered the field in increasing num-bers and progress has been rapid even if it has not until the present time reached the commercial stage. A brief review of some of the most important work will follow. This is intended to be representative and 25 is not fully inclusive of all the work in the field. Literature cita-tions in the text are given in abbreviated form. Reference should be m~de to the bibliography at the end of the specification for full cita-tions of the literature noted herein.
The natural embryogeny of gymnosperms is described in great 30 detail by Singh (1978). Conifer-type embryogeny is one of four types noted for gymnosperms. This includes virtually all of the important forest species except Sequoia. Singh notes that the immature seeds typically contain more than one embryo. Most commonly this seems to occur when a single zygote forms multiple embryos, a phenomenon called 35 "cleavage polyembryony". As the seed matures one embryo becomes domin-ant while the others are suppressed. The ability to form multiple embryos from a single zygote forms the basis for most of the present embryog~nic processes for mulliplying conifers. However, Douglas-fir is 2~ PCI/US90/06057 -3-~ =.
arl exception. Most typically only a single embryo will be present throughout the formation and maturation of a seed. This may account for at least some of the difficulty ~,.~,.. ;~..c~ to date in multiplying Doug-las-fir by somatic eml,-yut~
Bourgkard and Favre ~1988) describe what is the apparently successful production of plantlets by somatic embryogenesis of Sequoia ~ senpervirens. As a historic note, this was one of the first forest tree species successfully r~ 1 by U.t;dl~Or,~
Hakman and her coworkers have concentrated on Norway spruce (Picea abies)t apparently with some success. In a paper by Hakmao, Fowke, von Arnold, and Eriksson (1985) the authors describe the produc-tion of "embryos" but not plantlets. Hakman und von Arnold (1985) do suggest that they have successfully obtained plantlets. This latter paper is interesting for its comments on the variability within the species and the poor success with many of the seed sources used for e~.plants. The authors suggest that this variability may be due to the pll~;olvt~l~dl condition of the source material. However, other workers have noted great differences in behavior between recogni~ed genotypes of the species.
l~agmani and Bonga (1985) describe cmb.yvg~ from megagam-etophytes or Larix decidua by tissue culture. The archegonia, pro-embryos, or embryos with their suspensors were removed prior to culture.
Some of the resulting embryos produced in culture were stated to have further advanced to become plantlets established in soil. The ploidy of these plants was not investigated.
Successful production of small quantities of plantlets has now been reported for loblolly pine. Teasdale, Dawson, and Woolhouse (1986) showed the criticality of proper mineral nutrients for cell suspension cultures of loblolly pine. The article by Becwar, Wann, nnd NflgmAni (1988) is enlightening for the differences shown in performance between different families (or genotypes). Three families out of the ten tried accsunted for most of their success. Even so, They appeared unable to grow ~vlyH~v~ embryos. A companion paper by Nagmani ~nd Becwar (1988) showed development of Pinus taeda to the precotyledonary stage.
35 In an earlier paper, Gupta and Durzan (1987) described their success in taking loblolly pine to the plantlet stage by embryogenesis. However, only one genotype was successfully taken to the plantlet stage and only one converted plant was produced. The authors note thr n~d for -~ ~ 206~6 4 ~improYed con~rersion r~tes~ as well ~s other informstion before the process can be considered commercially practical.
Sugar pine ~Pinas lambertiana) has also been cultured to the plantlet stage as reported bg Gupta and Durzan (1986). The authors note S a Yery low 1-2~i conYersion of embryos into piantlets.
- The ebove researchers appear to be the only ones who haYe f,reY;vu:,ly achieYed success in producing Douglas-fir plantlets (Durzan and Gupta 198~). Again, the success ratlo appears to be very low and they haYe obtained only two converted plsnts from a single genOtyi~e.
In ~nitev states Patent No. 4,957,866 i~;r,iued Septe~'ver ~.8,1990, we desc~ibed an ImproYed method for .~,.vviu~ g conlferous species by somatic embryo-genesis. An ~ntermediate high osmoticant culture medium wes used to genèrate strong iate stage p,o~ri.vl~v:" prior to the d.i~', I.~C~ of cot-15 yledonery embryos in a medium containing absc~sic acid. The methods disclosed were of particular effectiYeness in somatic polyembryogenesis of loblolly pine.

ActiYated charcoal has been widely used before In tissue cul- s ture media where it is believed to function as an adsorbent for tox~c 25 r~etabolic products and undesirable amounts of residual hormones.
Abscisic ac~d has also been recognized as being a useful plant hormone in cultures inducing conlfer embryogenis; e.g., Boulay, Gupta, i~rog-strup, and Durzen (1988). The combination of these two materiels hes been used by e number of workers, generelly with indifferent or negstiYe 30 results. Johansson~ Andersson, and Ericksson (1982) cultured anthers ol several ornsmentel plant species using a two phese liquid oYer solid medium in which the agsrified solid phase contained actiYated chercoel.
The chercosl eppeered to be useful for ebsorbing small umounts endoge-nous abscisic acid. In a related paper Johansson (1983), tested the 35 e~fects of charcoel es sn edsorbent of meteriels inhib~ting the initie-tion of emb. ~o~.r..,is. In a test intended as a model, he added exo-genous ABA in emounts Yerying by orders of magnitude between 10 9 ?~i and 10~3 ~ to medis with end without sctiYsled charcoel in the solid portion ;

20-6~996:4 i WO91/058~4 ; ~ 3~ PCltUS90/06057 _5_ of 8 two phase medium. His conclusion was that initiation was complet-ely inhibited for all of the test species at Af~A concentrstions above 10-6 M, when no charcoal was used, and 10 4 ~1 when charcoal was present.
Thus, charcoal was seen as an effective mAterial for removing inhibitory 5 amounts of ABA and other undesirable materials such as phenolics.
~ ;iY and Gadasi (1986) studied embryogenesis in several geno-types of cucumber (Cucumis sativus L.). They us~d liquid cultures as well as the two layer technique with activated charcoal in the solid layer of the medium and low (0.4 ~uM) levels of abscisic acid in the 10 liquid layer. In the liquid cultures abscisic acid by itself only slightly improved embryo formation and was significantly more effective th~n the combination of abscisic acid with activated charcoal. Plantlet development in the liquid over solid cultures was slightly improved by th~ combination of the two Materials.
Buchheim, Colburn, and Ranch (1989) suggest that exogenous abscisic acid and activated charcoal would probably not be a very useful combination of ingredients in a culture medium because of adsorption of the abscisic acid by the charcoal with subsequent loss of its biological effectiveness.
Since the importance of the osmotic environment within a developing seed is known (Yeung and 8rown 1982), it has been assumed by others that the osmotic potential of the media during a culturing pro-cess could have an important effect (e.g., Raghavan 1987). Lu and Thorpe (1987), using white spruce (Picea glauca), noted that increasing the osmolarity of a medium and reducing the auxin concentration enhanced development and maturation of somatic embryos. They observed that more embryos developed on media containing 6% than on those with 9% sucrcse and that similar results were obtained when sorbitol replaced 3 % of the sucrose in the medium. Sorbitol is known to be only poorly metabolized SO presumably its effect was osmotic rather than as a carbon source for the developing embryos. Quite in contrast to their findings, HakmAn snd von Arnold (1988), using the same species and a combination of abscisic acid and sucrose in a development medium, found a very sharp falloff in success in going from 3% to 4% sucrose.
Becwar and Feirer (1989) note work involving the trsnsfer of a loblolly pine embryonal ...u~ mass to development media containin~
10 uM abscisic acid with 3-6% sucrose. Ilow~ver, they reported no details of their experimental protocol and only tha~ th~ media "promot~d 4 ~ r~ ~ PCI/US90/06057 embryo developmen-t". While a report (Becwar et si.) is noted as being in press it hss spparently not yet been published.
Finer, Kreibel and Becwsr (1989), studying eastern white pine (Pinus strobus L.), inttiated snd mslntsined cultures on medla wlth a 3%
S sucrose level. Further embryo developmment W85 then attempted on 8 medium with 1-12~i sucrose combined with a high concentrstion of sbscisic scid snd varging amounts of glutsmine. Best results were found with 50 mM glutsmine, 38 uM abscisic acid, and 6% sucrose. However, the number of embryos formed under any of the conditions wss not high And, ss of 10 the time of reporting, none hsd been successfully germinsted snd con-verted into plsnts.
Schuller and Reuther (1989), in the sbstrsct of a psper, describe the study of seversl sugsrs snd soluble stsrch ss csrbohydrste sources for the culture of Abies slbs. They note thst develoement wss 1~ obtsined only on 8 medium using soluble stsrch snd lactose. No detsils were given snd appsrently no somstic embryos were developed to the cot-yledonsry stsge.
Von Arnoid (1987) investigsted carbohydrste level of the init-istion medium for Norwsy spruce. Sucrose wss vsried between about 1-396 20 with successful initistion being obtsined st the higher level on hslf strength medium. By replscing 8 portion of the sucrose with sorbitol she showed thst the poorer results on full strength medium were not due to incressed osmotic pressure.
Von Arnold snd Hskman (198a) took 8 Norwsy spruce embryogenic 25 csllus and trsnsferred it to 8 modified~ intermedi~t~ medium prior to full embryo development. The intermediate medium contsined sbscisic ~cid snd from 1-3X sucrose. The higher sucrose levels, slong with the sbscisic scid, resulted in incressed freqùency of sdvsnced stage pro-embryo development.
The potentisl for schievlng genetic gsin using somstic embryo-genesis is recognized ss being very grest. However, the problems to dste hsve been so overwhelming thst commercisl spplicQtion has seemed ressonsbly close st=hsnd only for Norwsy spruce and, to 8 lesser e~tent, loblolly pine using the methods described in our psrent applicstions.
3~ Successful embryogenesis of Douglss-fir hss been much more elusive.
Until the present time, while some converted trees hsve been obtsined, the percentsge of success hss been fsr below thst of the two previously nsmed species. Possible commercisl production ot Douglas-fir replsnting ~ 9 g ~ 4 stock by embryogenesis has I~ inPd no more than a fond hope in the minds of the people working in the f ield.
BRIEF DESCRIPTION OF THE DRAWINGS
The figures show various stages of plant embryog-enesis in which:
Figure 1 shows early stage proembryos.
Figure 2 shows late stage proembryos.
Figure 3 depicts cotyledonary stage embryos.
Figure 4 shows a plantlet ready for transfer to soil .
Figure 5 shows the variation in behaviour in tissue culture of various genotypes of a single coniferous species.
Figure 6 is a photomicrograph of a clump of Douglas-f ir early proembryos .
Figure 7 is a photomicrograph of embryos after singulation .
Figure 8 is a photomicrograph of a clump of unsingulated Douglas-f ir cotyledonary embryos . -Figure 9 is a photomicrograph of high quality Douglas-f ir cotyledonary embryos .
Figure 10 is a graph showing typical levels of osmotic potential and abscisic acid concentration during the culture of Douglas-f ir .
SUM~RY OF THE INVENTION
The present invention is a method of reproducing selected plants by somatic embryogenesis using tissue culture techniques. The method is particularly suitable for reproducing woody gymnosperms of the order Coniferales.
It is PCrP~iAlly well suited for generating large clones of superior forest trees for reforestation, including, species - within the families Pinaceae, Cupressaceae, and Taxodia-F 201;
-7a-ceae. Mo6t or all species within the genera Abies, Pinus, Plcea~ ~suqa, Pseudotsuqa, Thuia, Juniperis, Larix, and Sequoia are believed to be well suited for multiplication by the present method. The present method is most es-5 pecially useful for reproducing Douglas-fir (Pseudotsuqa menziesii (Mirb. Franco. ) The method is particularly advantageous in that it enables greater quantities and more robust somatic 10 embryos to be produced. This results in higher numbers of embryos that can be successfully converted into plants growing in soil. Costs per plant can be significantly reduced over prior known tissue culture methods. In addition, use of the method generates embryos that can be 15 retained for extended periods of time in cold storage without transferring them from a development medium.
A number of terms are known to have differing r--nin~s when used in the literature. The following 20 definitions are believed to be the ones most generally used in the field of botany and are consistent with the usage of the terms in the present specif ication .
"Auxins" are plant growth hormones that promote 25 cell division and growth.
"Cytokin;ns" are plant growth hormones that affect the organization of dividing cells.
"Callus" is generally considered to be a growth of unorganized and either unconnected or loosely connected plant cells generally produced from culturing an explant.
"Embryogenic callus" is a translucent white mucilagenous mass that contains early stage proembryos attached to suspensors. This is also referred to as an ,'~s "embryonal-suspensor mass" or "ESM" by some investigators.

WO 91/05854 2 ~ gl~9~ PCI/US90/06057 A "proembryo" is 8 cell or group of cells having the potential to become a plant but lacking defined mer~stematic organ primordia.
An "eQrly stage proembryo" is u mass generally of 1 - 10 cells with dense cytoplasm and Idrge nuclei that have the potential of forming 5 a plant. The early stage proembryo is normally found as a head assoc-iated at the end of a long thin-walled suspensor cell (FIG. 1).
A "late stage proembryo" is a proembryo with a smooth embry-oral head of at least about 100 cells associated with multiple suspensor cells. The late stage proembryo is a very robust advanced proembryo 10 (FIG. 2).
A "cotyledonary embryo", sometimes simply referred to as an "embryo", has a well defined elongated bipolar structure with latent meristem with cotyledonQry primordia at one end and a potential radicle at the opposite end. The cotyledonary structure frequently appears as a 15 small "crown" at one end of the embryo (FIGS. 3 and 9). A cotyledonary somatic embryo is analogous to a developed zygotic embryo.
An "explant" is a piece of tissue taken from a donor plant for culturing.
A "meristem" or "meristematic center" is a group of tissue forming cells capable of further development into plant organsj e.g., shoots and roots.
An "osmoticant" or "osmoticum" is a chemical material used for controlling the osmotic potential of a solution. In the present context the solution would be a culture medium.
A "plantlet" is a plant asexually reproduced by tissue culture (FIG. 4).
A "converted embryo" is an embryo that has germinated and been established as a plant growing in soil "Somatic embryogenesis" is the process using tissue culture techniques for generating multiple embryos from an explant. The embryos from a given tissue source are presumed to be genetically identical.
The present method comprises a multistage culturing process.
A suitable explant is first placed on an induction or initiation culture medium. This usually will contain relatively high qusntities of growth 35 hormones including at least one auxin and frequently one or more cyto-kinins. However, growth hormones at this initial stage are not alwsys necessary or desirable for induction of early stage proembryos. A num-ber of sour~es of explants may ultimately prov~ to be satisfsctory for WO91/05854 2069~ PCltUS90~06057 -9~
culturing. These include, but are not limited to, tissue froln cotyle-dons, hypocotyls, epicotyls, buds, meristematic centers for buds or roots, and seed embryos. Zygotic embryos remoYed from seeds are pr~-sently preferred. In particular, for species which in the past hav~
5 proved to be very difficult or impossible to propagate by somatic emb.~yv~ the embryos from immature seeds may be preferred. In the case of Douglas-fir, an embryo selected between the time that an apical dome begins to form but before the first appearance of cotyledon pri-mordia appears to be optimum.
lD The first stage or induction medium will normally be one of those well known from past work which contain a balanced concentration of inorganic salts and organic nutrient materials, with plant growth hormones included as noted above. Auxins are normally present in con-centrations which may initially be as high as about 600 ~UMtL, more typi-15 cally not exceeding about 500 ,uM/L. Cytokinins, if present, may initi-ally be as high as 500 uM/L. The plant growth hormones may include at least one auxin and one cytokinin in a combined initial concentrrtion not exceeding about 1100 uM/L, more typically not exceeding about 900 uM/L The particular auxins and cytokinins used and their exact 20 concentrations, or whether they are used at all, will depend somewhat oIl the species being cultured and even on the particular genotype within that species. This is something that cannot be easily predicted but can be readily determined experimentally. These very high levels of growth hormones assume the presence in the medium of an adsorbent material, 25 such as activated charcoal. Where charcoal is not present the levels of growth hormones would normally be much lower than those just noted.
Culturing during this stage may be carried out in the dark, under very low light conditions, or in full light until an embryogenic mass forms. Lighting conditions will depend in large part on the compo-30 sition of the particular medium selected. This embryogenic mass hasbeen described by various other names by r.~e~ . who have reported it in the past; e.g., embryogenic callus (Hakman and von Arnold 1985) or embryonal-suspensor mass (Durzan and Gupta 198~). It has the appearance of a whitish, translucent, mucilagenous mass containing early stage pro-35 embryos which are readily apparent by low power light microscopy. Intl~e case of Douglas-fir the presence of activated charcoal or a similar adsorbent in the initiation medium appears to be quite advantageous. It was noted earlier that Douglas-fir does not experience polyemb~yony ss -WO 91/0~854 2 ~ PCT/US90/06057 do most other coniferous species. The reasons for this are not well understood but one hypothesis suggests that Douglas-fir seeds contsin a high endogenous level of abscisic acid which prevents polyembryony.
Activated charcoal in the initiation medium may remove this endogenous 5 A~3A, as well as other undesirable metabolic byproducts, to allow poly-embryony to occur in vitro. Because the charcoal will also gradually remove growth hormones over time the initial concentrations of these materials are necessarily higher than might otherwise be the case. The preferred induction medium for Douglas-fir will preferably contain an 10 auxin or auxins in amounts of about 400-600 uM/L and a cytokinin or cytokinins in the a~nount of about 240-500 ~uM/L.
Early stage proembryos from the first culture may be directly transferred to a late proembryo development culture medium having s~g-nificantly reduced plant growth hormones and, ~or some species, Q higher 15 concentration of osmoticunts. However, they are preferably first sub-cultured in a maintenance medium of similar or slightly higher osmotic potential than the induction medium for multiplication. This multiplication med~um will also usually have the concentration of plant hormones significanfry reduced below that of the induction medium. 13y Z0 "significantly reduced" is meant lowered by a factor which may typically be one whole order of magnitude. In the case of Douglas-fir it may be two full orders of msgnitude. No hormone ~dsorbent is usually necessary or desirable at this time. The osmotic pOtentiQI of the induction ~nd maintensnce medium will most often not exceed ~bout 160 mM/kg.
Z5 The composition and use of the late proembryo development culture medium is Important to the success of the present process. It differs from the induction medium by hAving a similar level of plant growth hormones to those present in the maintenance and multiplication medium However, ~or many species such as Pinus taeda and Pseudotsuga menziesii, the late proembryo development media should have a concentra-tion of osmoticants that is significantly raised above that of the induction or multiplication media. The optimum osmoticant levels at each stage will usually differ for each species and often for individual genotypes within a species ~or loblolly pine the osmotic level should 35 typically be of the magnitude of at least 200 mM/kg and preferably about 240 m M/kg or even higher. ~owever, lower levels of about 1~0 m M/kg mintmum will suffice for most genotypes of Douglas-fir. The key advan-tage of this osmotic "pulse" is th~t proembryo quality and/or si~e can WO 9~/05854 ~ PCr/US90/06057 be significantly improYed. Some species such as Picea abi~s, which are relatively easy eO reproduce, may not generally require this raised osmotic level, or it may only be necessary for some genotypes. In these cases late proembryo deYelopment may usually be achieved without a 5 change in medium composition from the maintenance and multiplication medium.
Incubation at this stage is usually carried out in the dark or in greatly reduced light until robust late stage proembryos have formed.
These may then be l~<,..ar~.,v~ to an embryo development medium which 10 preferably lacks auxins and cytokinins entirely.
Many investigators refer to cotyledonary embryo development simply as a "development" stage and that usage will be understood herein unless the word "development" is otherwise qualified.
Douglas-fir requires an intermediate step between the late lS proembryo development stage and cotyledonary embryo development stage wllich is not necessary for other species. The proembryos tend to form in tight clumps or clusters (FIG. 6) which must first be singulated - before going to the development stage. This singulation is carried out in a liquid shake culture which lacks auxins and cytokinins but has 20 exogenous abscisic acid as a necessary new hormone. The level of osmotic potential is also reduced from that of the late stage proembryo development medium. ABA will typically be within the range of S-15 ppln (20-60 uM/L) with osmotic potentia~ levels in the range of 130-140 mM/kg It is most desirable when transfers to fresh media are made that 25 the initial ABA level of the fresh medium should not be higher than the final level of the medium at the end of the preceeding culture period.
Tilis will ensure a continuously dropping level of ABA during the singul-ation period. The singulated late stage proembryos (FIG. 7) can then be 1- r~ to a cotyledonary embryo development medium. If the embryos 30 are not singulated they will develop into a tight clump of cotyledonary embryos which cannot be readily separated and are useless for furth~r germination (FIG 8.).
Especially when Douglas-fir is being cultured, the osmotic potential of the development medium should be shflrply raised above that 35 of any of the preceeding media. Initial levels may be in th~ 300-350 mM/kg range but these should be increased to levels of at least about 4~0 mM/kg as development proceeds. If development is stsrted at levels around 300-350 mM/kg, the osmotic level may be increased during d~v~lop--.

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wo g~o~ 2 0 6 9 9 ~ ~ PCI/US90/060~7 -12~
ment by a complete medium change, ~ partial change in which some old medium is replaced, or by adding an appropriate form, such as a solu-tion, of osmoticants to the medium without replacement of any of the original medium. Any of these changes msy be considered a transfer to a "new" medium. It is preferred that the osmotic leveis at the end of the development period should be at least about 450 mM/kg although with some genotypes lower levels are acceptable. These higher levels tend to prevent deterioration and callusing of the embryos.
Osmotic potential is best controlled by a combinDtion of osmoticants. One of these should be a readily metabolized carbohydrate energy source, preferably a sugar such as sucrose, glucose, fructose, maltose, or galactose. Sucrose is a preferred ingredient but should be present in amounts only in the range of 2-3%. The other is a poorly metabolized osmotlcant of which sorbitol, lactose, or a polyalkylene glycol would be examples. In a solid development medium, a combination of sorbitol, lactose and polyethylene glycol has proved very eEfective.
Polyethylene glycol alone, in concentrations of 20-3096 of the medium, - has worked very well in liquid development media. While the salts andorganic components of the medium make a small contribution to the osmol-ality, the osmotic potential is primarily controlled by the energy-providing sugar an=d the other osmoticants. It is within the scope of the invention to use one combination of osmoticants at the beginning of development and transfer to a medium having a different combination at some point during the development stage.
For ~any species a supply of exogenous abscisic acid is a desirable component in the development medium. This is always used in combination with an ~dsorbent, such as activ~ted charcoal. The adsor-bent should be present in a sufficlent amount and form to slowly reduc~
the abscisic acid and remove metabolic waste products. It shouid not be present in such a high concentration as to deplete the abscisic acid in a very short time; e.g., in a matter of days. The combination of abscisic acid with the adsorbent will usually require a higher initial concentration of abscisic acid than would be the case if no adsorbent was present in the medium. In the particular case of Douglas-fir, and perhaps other species as well, the level of exogenous abscisic acid should be generally continuously lowered over time from the 5-15 ppm normally found necessary at the beginning of the singulation step to a level ~erhaps of about 1-2 ppm, or even to ~ero, at the end of th~

WO 91/05854 2 0 6 9 ~ 6 ~ i PCr/US90/060~7 development stage. Accurate measurements of abscisic acid presenf in the development system have not yet been made due to the extreme diffi-culties of analyzing the medium.
In some cases when Douglas-fir is being cultured, sufflcient ~ S abscisic acid will be carried oYer wfth the medium associated with the embryos from the singulation step so that no additional ABA is needed in the development medium. In other cases, the level of endogenous ABA
after singulation is sufficiently high so that no exogenous ABA need be present at all. The terms "sufficient" or "having an adequ~te supply 10 o~" should be considered broad enough to encompass all of these situa-tions. A small amount of activated charcoal, usually in the range of about 0.02-0.04% still appears to be necessary in the development medium to effect the continuing reduction in ABA that began with the singula-tion treatment. Reduction of ABA to low levels at the end of the devel-15 opment stage seems to help continue late embryo development andmaturation and also reduces the tendency of precocious germination of the embryos.
Following embryo development the embryos (FIG.9) may be placed directly on a germination medium for conversion into plantlets. Alter-20 natively, they may be converted into artificial seeds by any of a numberof published processes.
An advantage of the present process was the discovery that the more robust somatic embryos produced by the use of the abscisic acid-adsorbent combination could be readily stored for extended periods of 2s tillle. ''~everal genotypes of at least two coniferous species (Pinus taeda and Picea abies) have now been stored without loss of vitality for three months at 4~-5~C without removing them from the development medium.
P~eudotsu~a menziesii has been stored for oYer one month. This has not been believed possible with any degree of success before the present 30 invention The germination medium has no hormones, a lowered organic nitrogen content, and a reduced level of osmoticants. After a suffic-ient time in darkness followed by light, or a 16 hour light and 8 hour dark photoperiod, the cotyledonary embryos will have developed into 35 RIQntlets Douglas-fir does not require an initial dark period although a one week dark period is useful for Norway spruce. The time period for germination will be about 1-2 months. The resulting plantlets will have a well developed radicle and cotyledonary structure with a growing ~pi-.-.

~O91/05854 ~ 9~ -i t PCI/US90/06057 cotyl and are ready for planting in soil.
The present invention is most particul~rly concerned w~th thecomposition of the cotyledonary embryo development medi~ snd method of its use. For Douglas-fir, it has been found that a very high osmotic 5 level in combination with a diminishing level of exogenous abscisic acid is essential. This combin~tion gives greatly improved numbers and qual-ity of somatic embryos that are not subject to precocious germinstion.
It is an object of the present invention to produce coniferous plantlets by somstic emtJIy~l6...~Ji~
It is another object to produce a large clone of a genetically selected forest specles for reforest~tion using the methods of somatic emt,.~y~,6..~ , and plant tissue culture.
It is a further object to provide a method of somatic embryo-genesis that will dependably and consistently provide conlferous plant-lets in large quantities.
It is yet another object to provide a method of somatic embryogenesis that can dependably snd consistently reproduce large clones of selected individuals of forest species that heretofore have not been successfully reproduced by this method.
zo rt is still s further object to provide a method whereby superior genotypes of coniferous trees can be multiplied by tissue cul-ture in the large quantities needed for reforestation.
It is also an object to provide a method that will produce somatic embryos in large quantities with improved robust morphology for converston into plantlets.
It is a particular object to provide a method and suitsble culture media for somatic embryogenesis of Douglas-fir that produces robust somatic embryos with ~ high percentage of conversion to plants growing in soil.
It still ~nother object to provide a method that generates robust somatic embryos capable of withstanding extended periods of cold storage.
These and many other objects will become readily apparent to those skilled in the art by resding the following detailed description, t~ken in conjunction with the drawings.
-~ -Is- ~2~g6 4 .
s .

20 DETAILED DESCRIPTION OF THE PREFERRED EM~ODIMENTS
The process of the present invent~on is not limited to sny single culture medium or to the use of spec~fic growth hormones. Any of a ~lumber of well known media, such as that of Murashige and Skoog (1962), may be used. Howeverl the present inventors have found the 2~ basal medium described in Table 1 to give excellent results, partic-ularly when used for culturing loblolly pine ~Pinus taeda). The basal medium is modified for each of the various culturing stages as shown in Table 2. Simiiar media ~.li~ preferred for Norway spruce (Picea abies) are g5ven in Tables 4 and 5.
.. :
.

WO91/058~4 20~64 '~ PCI/US9O/06057 Ta Pinus Taeda B~s~l Medium (Modified 1/2 P6 Bssal S~lts ) Constituent Concentr~tion, mg/L
NH4NO3 603.8 KNO3 909.9 KH2PO4 136.1 Ca(NO3)2 4H2O 236.2 MgSO 7H O 246.5 Mg(NO3)2 6H2O 256.5 : . MgC12'6H20 50.0 1~1 4.15 H3BO3 15.5 MnS04'H2o 10.5 ZnSO4 7H2O 14.4 NaMoO4 2H2O 0.125 CuSO4 5H2O 0.125 CoCI 6H O 0.125 FeSO 7H O 6.95 Na2EDTA 9.33 Sucrose 30,000.
myo-lnositol 1,000.
Casamino acids 500.0 L-Glutamine 1000.0 Thiamine HCI 1.00 Pyridoxine HCI 0.50 Nicotinic acid 0.50 Glycine 2 00 Agar+ 6,000.
pH adjusted to 5.7 According to Teasdale, Dawson, and Woolhouse (1986) as modified + Used if a solid medium is desired WO91/0'~54 2d69gl6~; Pcr/US90/06057 Tsble 2 Composition of Media for Different St~ge Treatments BMl -- Induction Medium BM + 2,4-D (50 uM) + KIN (20 ~uM) + BAP (20 uM) BM2 -- Maintenance and Multiplication Medium BM + 2,4-D (5 uM) + KIN (2 uM) + BAP (2 yM) BM3 -- Late Proembryo Development Medium BM2 + 9000 mg/L myo-inositol B~4 -- Embryo DeYelopment Medium 8M + 4.0 to 8.0 mg/L abscisic acid 15 . B.\q5 --Germination Medium BM modified by reducing sucrose to 20,000 mg/L, myo-inositol to 100.0 mg/L, glutamine to 200.0 mg/L, and cusamino acids to 0.0 mg/L
A number of abbreviAtions are used in the following text.
These ~re in common use in the field of tissue culture.
BAP-- N6-benzylamir ;~e (or N6~ .yL~d~ e)~ a cytokinin KIN -- klnetin (6-furfurylaminopurine), also a cytokinin 2,4-D -- 2,4-dichlol ~ ~ yllcetic acid, an auxin.
NAA -- 2-Naphthylacetic acid (Naphthalene-2-acetic acid) ABA -- Abscisic acid It will be understood by those skilled in the art that other pl~nt growth hormones can be substituted for those just not~d. As examples, IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), and 30 NAA (naphthalene-2-acetic acid) are effective auxins and 2-lP (N6-iso-pentenylaminopurine) Qnd zeatin are frequently used as cytokinins.
As an aId in comparing the present work with other published data, the following table of conversions from weight to molar concentra-tions might be useful.
1 uM/L_ 1 mg/L
BAP 0.225 rnglL 4.44 ~uM/L
KIN 0.215 4.65 2,4-D 0.221 4.52 NAA 0.816 5.38 40 ABA 0.264 3.78 , ..

-18~ 1 6 g g ~ i~
~ United st~tes Patent No. 4,957, 866 1ssued ~ f' ' lr~, 1990, we pointed out the importance Or the control of osmotic poten-tial of the media used in the various cu~uring stQges. A l~rge group of chemicQI msterials are suiteble Qs osmoticQntS. In generQI these are highly water soluble polyhydroxylated molecules thQt include either s~mple or complex sugsrs, hexitols, and cyclitols. The cyclitols are normally six carbon ring compounds thQt are h~dliyd~ yl6ted~ The most readily Qvailable cyclitol is _yo-inositol but any of the other eight stereoisomeric forms, such as scyllo-inositol are believed to be quite 10 suitQble. Among the sugars, sucrose and glucose are known to be very effective but many others should prove to be equQlly useful. Sorbitol (3-glucitol), D-mannitol, and galactitol (dulcitol) are straight chsin sugQr alcohols suitQble as osmoticants. Lactose is e SUgQr effective as an osmoticant. Other materials suitable Qs osmoticQnts mQy include 15 glycol ethers such Qs poly(ethylene glycol) Qnd poly(propylene glycol) and their respective monomers.
LOBLOLLY PINE CULTURE
Exa m ple The following schedule of treQtments hQs been very success-fully used for the growth of plQntlets by somQtic embr~og~r.csia of loblolly pine (Pinus tQedQ). ExplQnts were immQtUre embryos dissected from seeds 4 to 5 weeks after fertilizQtion. Seeds were obtained from t cones supplied by a W~J_.hC~ CompQny seed orchard locQted at WQsh-25 ington, North Carolina. The cones were stored Qt 4~C until used.
ImmediQtely before remoYdl of the immQture embryos the seeds were steri-lized using Q modified method of GuptQ Qnd DurzQn (1885). Briefly, this involves Qn initial washing and detergent treatment followed by Q first sterilizQtion in 30~ H2O2 Qnd a second in diluted 10% v/v household 30 bleQch. The QdditionQI HgCl2 treQtment used by GuptQ and Durzan WQS not found to be necessary to ensure sterility. The eXplQntS were thoroughly wQshed with sterile distilled water after each treatment.
StaFe I - Induction Sterile dissected embryos were plQced on Q solid BM1 culture medium Qnd held in Qn environment Qt 22~-25~C with Q
35 24 hour dQrk photoperiod for Q time of 3-5 weeks. The length of time depended on the pQrtiCUlQr genotype being cultured. At the end o~ this time Q white mucilQgenous mass hQd formed in associQtion with the orig-inQI explQnts. This appears to be identicQI with thQt described by 2~ ~9 9 ~

Gupta and Durzan (1987). Microscopic examination revealed numerous early stage proembryos associated with the mass. These are generally characterized as having a long thin-walled suspensor associated with a small head generally hsving less than 10 individual cells, each with ~ 5 dense cytoplasm and large nuclei. Early proembryos are illustrated in FIG. 1.
~ Osmolality oE the induction medium may in some instances be as hiFh as 200 mM/kg. Normally it will be below 1~5 mM/kg and, more typi-c~lly, about 160mM/kg or even lower. The osmolality of the medium 10 described above was 158 mM/kg.
Stage 11 - Maintenance and Multiplication Early stage pro-embryos removed from the masses generated in the induction stage were pl~ced on a BM2 medium. This differs from the induction medium in that the growth hormones (both auxins and cytokinins) were reduced by a full 15 order of magnitude. The temperature and photoperiod were again 22~-25~C
with 24 hours in the dark. Osmolality of this medium will typically be similar or identical to that of the induction medium. In the present example it was identical. Proembryos developed in this stage were similar in appearance to those from Stage 1 and were subcultured every 20 12-15 days on BM2 medium.
Stage 111 - Late Staf~e Proembryo Development Early stage proembryos from either Stage I or Stage ll,-preferably the latter, were placed on a BM3 solid medium. This medium has the same growth hormone concentration as BM2, however, the osmoticant was raised to e much 25 higher concentration. In this case the osmoticant, myo-inositol, was at a concentration of 10,000 mg/L or 1% on a w/v basis. Osmotic potenti~l was measured as 240 mM/kg. Temperature and photoperiod were the same as for Stages I and 11. After 3 or 4 subcultures of about 12-lS days each, very robust late stage proembryos hsd formed. These ~ure characterized 30 by smooth eribryonal heads generally having in the r.~:bl-L~ od of over lQ0 individusl cells with multiple suspensors, as exemplified in FIG. 2.
Osmotic potential of the l~te proembryo development medium should usu-ally fall within the range of about 200-400 m M/kg for Pinus tueda. Most typically it should be in the neighborhood of about 1.5 times higher 35 than that of the induction or multipliction media. As was noted ear-lier, the requirements for elevation of osmotic potential at this stage will vary for different species.
:

2 0 6~
WO91/05854 ~ PCI/US90/060~i7 AlternatiYely, the Stage 11 and/or Stage 111 proembryos could be cultured for late proembryo development in suspension in a liquid medium of similar composition to BM3 but Iscking the agar. In this case subcultures could be msde every 7-8 days.
It is preterred that early stsge proembryos brought into Stage Ill culture should have a Stage 11 subculturing for rapid multiplication of the particular clone. However, on occasions where time may be of greater importance than quantity, early stage proembryos from Stage I
may be taken directly into Stage 111.
Stage IV - Embryo Development The late stage proembryos from Stage Ill culture were transferred to a solid BM4 medium. This medium either lacks growth hormones entirely or has them present only at very low levels and has the same lower level of osmoticants as Stages I and II. However, abscisic acid (5-(1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclo-15 hexen-1-yl)-3-methyl-2,4-pentadienoic acid) had been included here as a necessary material for further development. A critical aspect of the present invention is the further inclusion of an adsorbent material in this medium. The adsorbent may be chosen from a number of chemical materials having extremely high surface area and/or controlled pore size Zo such as activated charcoal, soluble and insoluble forms of poly(vinyl pyrrolidone), activated alumina, silica gel, molecular sieves, etc. The adsorbent will normally be present in a concentration of about 0.1-5 g/L, more generally about 0.25-2.5 g/L. The contribution of the adsorb-ent appears to be complex and is not well ~ r~ o~1 Adsorbent mater-Z5 ials, especially activated charcoal, haYe been widely used in the pastin various culture media. However, the particular combination of activa-ted charcoal with relatively large amounts of abscisic acid in a late stage somatic embryo development medium is believed to be entirely new.
The prevalling wisdom found in the literature clearly teaches away from 30 use of this combination, especially at this point in the process.
The osmotic potential of this medium will generally be no greater than about 175 mM/kg. In the present case it was measured as 168 mM/kg. As before, development was carried out in complete darkness at a temperature of 22~-25~C. Development time was 4-6 weeks after 35 which elongated cotyledonary embryos 4-5 mm long were present. These appeared as represented in FIG. 3.

WO 91/05854 2 û 6 9~8~ p~/US90/06057 -21 - ~ =
Stage V - Germination Cotyledonary embryos from Stage IV were pl,lced on solid BM5 medium for germination. This is a basal medium lacking growth hormones which has been modified by reducing sucrose, m o-inositol and organic nitrogen. After about 6-8 weeks under environ-5 mental conditions of 23~-25~C and a 16 hour light/8 hour dark photo-period the resulting plantlets were Approximately 20 mm in length and had a well deYeloped radicle and hypocotyl and green cotyledonary struc-tu~e and epicotyl. The young plsntlets are shown in PIG. 4.
Because of the reduced carbohydrate concentration, the osmotic 10 potential of the germination medium is further reduced below that of the development medium. It will normally be below about lS0 mM/kg and was, in the present example, about 100 mM/kg.
Stage Vl - Plant Frowth Plantlets from Stage V were removed from the culture medium and planted in a soil comprising equal parts of 15 peat and fine perlite.
To the present time, three distinct genotypes of Pinus taeda ha~e been successfully cultured through Stage V. Some of the plantlets ha ~e already been successfully transferred to soil and these are growing with good vigor. Two additional genotypes are being multiplied in Stage 20 ll prior to Stage Ill treatment. In work that preceeded that just described, all five genotypes when cultured without the Stage 111 high osnnoticant treatment ultimately browned and died in Stage IV. Stated differently, the method failed completely when early stage Pinus taeda _ =
proembryos from Stage Il were taken directly into Stage IV, as is taught 25 in the prior art.
While inorganic salts and pure simple organic chemicals gener-ally behave similsrly in culture media regardless of suppiier, there are occasions when this is not the case for the more complex materials.
Without intending endorsement of any product over available alterna-30 tives, chemicals from the following suppliers were used throughout theexperiments to be described in the examples. Agar was obtained from Di~co Laboratories, Detroit Michigan. Where specified as "tissue cul-tuI~e agar" the supplier was Hazleton Biologics, Inc., Lcnexa, iiansas.
Casamino acids, a casein hydrolysate, was also supplied by Difco i abor-35 atories. Activated charcoal was obtained from Sigma Chemical Company,St. Louis Missouri, as their grade NuC-4386.

WO91/05854 20~ PCr/US90/06057 --22--~
Example 2 The combination of ABA and activated charcoAI in the Embryo Development Medium has proved to be very effective not only with Pinus taeda but with other important conifer species such~ as Picea abies and Pseudotsuga menziesii. Tn the following experiments the~ Loblolly Pine Basal Media of Tables 1 and 2 were used~ .In the Embryo Development Medium the ABA was adjusted as described in Table 3 and Activated char-coal was included in a concentration of 2.0 g/L. All of the ingredients except the abscisic~ acid were combined, autoclaved, and cooled to 50~-60~C. A filter sterilized solution Pf ABA was then added and mixed.
After 10 minutes the medium was poured into petri dishes.
Late stage proembryo cells of two loblolly pine genotypes, grown as described in the first example, were settled from a suspension culture, the supernatant liquid poured off, and 1 - 1.5 mL of the set-tled cells were plated on the solid Embryo Development Medium in S cm dishes. These cultures were incubated in the dark at about 22~C for six weeks. Control cultures having 2 and 4 mg/L ABA without activated - charcoal were also prepared. The following results were obtained.
Table 3 Medium Composition Embryos Produced ABA, mg/L Activated Charcoal, g/L Genotype A Genotype B
25 2.0 0.0 2.5 4.0 0.0 5.5 --20.0 2.0 0 0 40.0 2.0 2 2 60~0 2.0 4 3 30 80.0 2.0 10 4.5 100.0 2.0 8.5 2 The embryos produced on the charcoal containing media were of better morphology with a well developed cotyledonary structure but 35 without evidence oE germinating precociously when compared to those grown without activated charcoal in the medium. The media described here are not represented as being optimized for the species or any genotype.

WO 91/05354 2 ~ 6 ~ ~ 6 ~'t~ PCI~US90/06057 ~ -23- = ~ = ~
~ORl''AY SPRUCE CULTURE ~ _ Exa m ple 3 Some coniferous species sre relatiYely easier to propagate by somatic embryogenesis than others. Coastal redwood, Sequoia semper-S virens, is considered be be a relatively easy species while Norway spruce, Picea abies, is usually thought to be of only moderate diffi-culty. Most members of the genus Pinus as well as Douglas-fir, Pseudo-tsuga menziesii, are regarded as very difficult. This has posed a major challenge to researchers since the latter two genera include a major 10 percentage of the worlds most economically import~nt timber species.
Even though past ~ rcl~ have reported success with somatic embryo-genesis of several pines and of Douglas-fir, others in the field have frequently not been able to duplicate the work of these competent inves-tigators. There are probably several reasons for this. Most certainly, 15 one of them is over optimism on the part of researchers who have achieved and reported early stage embryogenesis or embryo-like struc-tures but who later have not been able to succeed in producing signifi-cant numbers of cotyledonary embryos or plantlets. Another is the great differences in performance between different genotypes within a given 20 species. Picea abies is a case in point. As noted earlier it is usu-nlly regarded as a species of only moderate difficulty to reproduce by somatic emvlyv~ wia using present state-of-the-art technology. ~low-ever, there are some genotypes of Picea abies that haven proven intract-able to all previous efforts. Most researchers have limited themselves 25 to working with only one or two genotypes that are known from past experience to give good results.
Our method has resulted in successful production of late stage proembryos and cotyledonary embryos on 23 of the 26 genotyp~s of Picea abies that have been investigated to date. This sample includes a 30 considerable number of previously intractable genotypes. FIG. 5 shows the maximum yield of embryos per culture plate for 19 genotypes grown on the same nonoptimized culture (Medium No. 2 as describ~d in Exampl~ 6).
Seven of these are from non-select wild seed and twelve are select seed from known half-sib orchard fr~milies. The enormous differences in 3s bellaYior constituting two full orders of magnitude, especially within the non-select seed, are immediately apparent. As has been noted ~ eallier, similar results have be~n obtain~d with Pinus taeda, although nol all genotypes h~ve b~n proc~ss~d to the later stages of treatment ! ' WO 91/05854 2 0 6 ~ 9 6 ~-24- PCI/US90/060~7 a[ the present time.
While the plsnt growth hormone usages noted in Table 2 are near optimum for loblolly pine, different concentrations and mixtures may prove more suitable for other species. It is fairly well estab-5 lished that growth hormones are usually necessary in Stages 1-111, although some workers have apparently achieved early stage proembryos using growth hormone-free media. However, even when initially cultured on hormone-free media, these early stage proembryos were then transfer-red to cultures having the usual growth hormones. These hormones may in 10 some instances be a single auxin or a mixture of auxins with or without one or more cytokinins. As a general rule the total concentration of all growth hormones should be below about 250 ,uMIL, preferably below about 100 uM/L in the St~ge I medium. These cQncentrations should be reduced about tenfold in the Stage 11 and Stage 111 media.
The folrowing tables show preferred media for culture of Norway spruce by somatic embryogenesis.
.

2~6~9~
WO 91/058~4 r ~ ~ ~ 3 ~; PCr/usgo/o6057 Table 4 Picea Abies Basic Culture Media __ Constituent Concentration, mg/L
- ~ A(l) B(2) NH4NO3 -- 206.3 KCI 372.5 RNO3 50.0 2340.0 KH2P~4 85.0 85.0 MgS04 ?H2~ 160.0 185.0 CaC12 6H2~ 220.0 220.0 -~1 0.415 0.415 H3BO3 3.10 3.10 MnSO4-H2O 8.45 8.45 ZnSO4-7H2O 4.30 4.30 NaMoO4 2H2~ 0.125 0.125 CuSO ~5H O 0.0125 0.0125 CoC12-6H O 0.0125 0.0125 FeSO ~7H O 13.90 13.93 Na2EDTA 18.65 18.63 ORGANIC A~DITIVES
Sucrose 10,000. 30,000.
myo-lnositol 50.0 1000.0 Casamino acids -- 500.0 z5 I~Glutamine 750.0 450.0 Thiamine-HCI 0.05 1.00 Pyridoxine-HCI 0.05 0.50 Nicotinic acid 0.25 0.50 Glycine -- = 2.00 L-Asparagine 50.0 pH 5.8 5.7 (1) Institute of Paper Chemistry medium (~erhagen and Wann 1989) (2) Gupta and '~urzan medium BM3 (1986b).
, Tabie 5 Composltion of Picee Abies Media for Different StaFe Treatments BMI -- Induction~Medium BMA~1) + NAA(3) (10.8uM) + BAP(4) (4.4uM) + 7.0g/L Difco agar.
BMM -- Mainte~nce and (M5)ultiplication Medium BMB + 2,4-D (5 uM) + BAP (2uM) + KIN(6) (2~uM).

6.0 g/L Difco agar added if solid medium is desired.
10 BMD --Colyle~ y Embryo Development Medium BMB + 40.0 mg/L Arginine + 100 mg/L Asparagine + 6.0 g/L
Tissue Culture Agar + Abscisic acid (as specified) + Adsorbent (e.g., activated charcoal) (as specified). KNO3 is reduced to 1170mg/L in basal salts.
BMG --Germination Medium BMB with KNO3 reduced to 1170 mg/L, myo-lnositol reduced to 100 mg/L, Sucrose reduced to 20.0 g/L, and L-Glutamine and Casamino acids removed. 2.5 g/L of Adsorbent and 6.0 g/L of Tissue Culture Agar are added.
(1)Basic medium A from Table 4 (2)Busic medium B from Table 4 (3)2-Naphthylacetic acid (NaphthAl~ne-2-acetic acid) 25 (4)N6-8en~ylaminopurine (5)2,4-Di ~ ~,L,h~ .y~lcetic acid (6)Kinetin Example 4 3Q The following screening experiment was made as a comparison between embryo development stage cultures contAining only abscisic acid as a hormone additive with cultures containing a mixture of abscisic ac~d and activated charcoal. Mature Picea abies seed embryo explants were cultured on an Initiation Medium and Maintenance Medium as 35 described in Tables 4 and 5 . Explants were incubated in light of an intensity approximately 50 ~uEm 2sec 1 In this case the Induction Medium BMI had a relatively low carbohydrate content with a resulting low osmolarity of about 62 mM/kg. After an early stage embryogenic mass had developed, it was transferred to a solid and later to A liquid 40 liquid Maintenance and Multiplication Medium BMU hQving a higher osmol-ar~ty of about 158 mM/kg. In this case the proembryos had attained a sufficiently late stage of development without the need for further culturing on a very high osmotic potential Late Proembryo Development Medium. These proembryos were settled and washed twice with liquid ~ _z7_ r 2 0 6 9 g ~ 4 Embryo Development Mediurn BMD of Table S to which 10 mg/L of abscisic acid had been sdded. The washed cells were then drained on polyester pads. Approximately 2 ml: of the wsshed cells were transferred to solid Embryo Oevelopment Medium B'.~D on 50 mm petri dishes. The growing celis S were transferred twice at two week intervals to fresh medis of the same composition. Culture room conditions were ebout 23-24~C in dqrkness throughout the experiment. The following table shows the compositions of the media used and the results obtained.
Table 6 Medium Comr~qi~ion BMD Cotyledoifary Embryos, ABA, m~/L Activated Charcoal, ~/L Average Yield/2 mL
2.0 0 18;6 15 5.0 0 20.0 7.5 0 13.0 10.0 0 12.9 25.0 0 14.7 25.0 2.S 2.0 20150.0 2.5 4.1 _ 100.0 2.5 16.1 250.0 2.5 0.1 The ABAlcharcoal media did not in general produce as many 25 cotyledonQry embryos in this experiment as the media with ABA alone.
However, it was noted that the embryos produced on the media containin~
charcoal were frequently larger and of superior morphology to those cultured on the media containing only ABA. This experiment showed that emt)ryos could be successfully cultured on media hav~ng relatiYely high 30 concentrations of ABA and activated charcoal. Further, there was an indication that these embryos would have superior strength to those cultured on media containing only ABA.
It was noted during the experiment that development and mass growth on the charcoal containing media was so rapid that 10-14 days after 35 starting the experiment most growth had stopped and the mass containing the embryos appeared dry. This suggested that aQailable liquid was absorbed during early growth and may have become limiting.
The above experimenl was carried out on three additional genotypes of Plcea abies. In these tests no preliminary washes with ABA-40 containing medium were given. Results were variable. One genotypeproduced no cotyledonary embryos under any cond~tions. Another produced an average of 10.2 embryos on th( m~dia without charcoal and only 0.4 , .
f*
.

wo gl/05854 ~ A ?~ " PCr/US90/06057 ~ 2~69964 -28- ~
eMbryos on the charcoal containing media. The third genotype produced an average of only 1.7 embryos on the ABA only media and 2.0 embryos on the charcoal containing media.
It should be noted that the above reported experiments were of 5 a preiiminary screening nature only and do not represent optimi~ed conditions. They were primarily made to see if the use of activated charcoal in the cotyledonary embryo development stage would be advuntag-eous.
Example 5 In a followup experiment to that just described, five geno-types of Picea abies were cultured as described above using a solid co~yledonary development medium containing 100 mg/L ABA and 2.5 g/L
activated charcoal. This time the liquid BM~q medium containing the 15 proembryos was simply settled and the supernatant liquid poured off.
Then 2 mL of the settled cells were pipetted onto the surface of the solid medium without washing or further draining in order to provide additional water for the system.
Embryos were visible within two weeks and harvestable by 3 1/2 20 weeks. One genotype produced 34 robust cotyledonary embryos per mL of settled cells. Another produced 5.3 embryos per mL. The other geno-types produced few or no embryos.
160 cotyledonary embryos of the best performing genotype were transferred to a germination medium, incubated three weeks in the dark, 25 ~ sf~ d to fresh media and moved to the light. About 25 % of these embryos began epicotyl development after five weeks.
Again, it should be noted that no optimization of the ABA-charcoal ratio had been attempted.
Example 6 Another set of experiments was made under conditions similsr to the previous eYample in which activated ch~rcoal in the cotyledonary embryo development medium was varied between 0 and 2.5 g/L and ABA
varied between 5 and 10D mg/L. Four genotypes of Picea abies was used 35 in the eYperiment. At least five replicates were made at each test condition.
Media compositions are given in the following table.

-WO 91/~;854 - 2 ~ 6 9 g 6 ~ ~ PCr/US90/06057 -zq- \ ~
Table 7 _ _ _ Medium ABA, Charcosl, AverAFe Embryos/mL, Genotype No. m~/L i~/L _ B _ _ D
100 2.5 46 0 0.5 13.2 2 50 1.25 102 0 1.8 9.5 3 10 0.25 76 0 0.6 20.4 4 10 0.10 18 0 0.2 5.4 10 5 5 0,0 0 0 0 0 Medium No. 2 contAining 50 mg/L ABA and 1.25 g/L activated cllarcoal appeared to be significantly better than the others from the standpoints of number and vigor of embryos formed.
In a further modification of this experiment the best perform-ing genotype above (Genotype A) WAS trensferred onto the medium sup-ported on laborAtory filter paper. The experiment was repiicated using the same culture media as Above. As is seen in the following table, the results of cultures grown on agar medium alone and on filter paper 20 supported on Agar media are generslly compArable. This opens up the pQssibility of direct mAss trAnsfer to germinAtion or other media with a consfderable savings in hAndling required. It further opens the possib-ility of a system in which either the charcoal or ABA is locat~d on the filter pAper with the other component being in the medium.
2~
TAble 8 ~ . =
Medium ABA, Charcoal, Avera~e Embryos/mL, 30 No. ~ mg/L g/L Directly on Agar On i'ilter Paper 100 2.5 46 43 2 50 1.25 ~ 102 93 3 10 0.25 75 55 4 10 0.10 17 30 35 5 5 oo 0 0 Example 7 A comparison was mAde using four methods of culturing PiceA Abies at the embryo development stage. The first method used Embryo Develop-40 ment Media containing both ABA and activated charcoal. One subset was a - replication of the Medium 2 composition from the previous example thAt gaYe the best results. Another subset used reduced amounts Or both ABA
and Ac~ivsled ch~rco~i. in th~ second method the charcoAI WAS omitted from the culture medium but th~ lAte stAge proembryos were first coAted WO91/05854 2~6~a~ PCI/US90/060~7 -30- ,~
with Qctiv~ted charco~l by rinsing them with a chsrcosl-containing liquid medium. The third method ~irst cultured the late stflge pro-embryos on 8 medium contsining only sctivsted charcosl followed by cul-turing on a medium containing only ABA. ~inslly, cultures were made on 5 ABA-containing medium without any charcosl. In one subset of this method the growing embryos were transferred three times to fresh medium.
In the other subset the embryos remsined on the original medium the entire culturing period.
Two new genotypes of Picea abies not tested in the eQrlier 10 examples were used for the present set of experiments. Results are given below. The compositions of all the medis used are given in the Table 9. Late stage proembryos, cultured ss in the l~st three examples, were used for all trials.
Table 9 15 Medium No. _ _ Composition BMD (Tnble ) + 15 mg/L ABA + û.75 g/L activated charcoal 2 BMD + 50 mg/L ABA + 1.25 g/L activated charcoal 3 BMM without hormones or agar + ~.5 g/L activated charcoAI
4 BMD + 10 mg/L ABA
BMD + 15 mg/L ABA
6 BMD + 10 g/L activated charcoal 7 BMD + 5 mg/L ABA
PROCEDURES ~ RESULTS
25 First Method -- ABA/Activated Chsrcoal Medium 1.0 mL of settled late stage proembryos was pipetted directly onto replicate 5 cm plates of media 1 and 2 above and cultured in the dsrk at about 22 ~C for six weeks.
Embryos Produced Genotype A Genotype B
Medium 1 54.0 26.3 Medium 2 83.0 92.5 These embryos had improved apical domes, hypocotyl region ~nd 35 root primordia when compared with embryos cultured on a development medium using ABA alone. Also a greater number of embryos were produced using the charcoal containing m~dia. Ultimately the germin~tion rate and successful growth into plantlets W85 also incre~sed ov~r a con~rol WO 91/0~;854 2 0~ ~ ~ &~ PCI/US90/06057 group grown without Qctivated charcoal.
Second Method -- Activated Chsrcoal Coated Embryos on ABA Medium Settled late stage proembryos were suspended in \qedium 3 5 above, settled, and then 1.0 mL was pipetted onto replicate 5 cm plates of media 4 and 5 above. Most of the activated charcoal in the rinse medium was retained with the settled proembryos. These were also cul-tured in the dark for six weeks at about 22~C
~ Embryos Produced Genotype A Genotype B
Medium 4 0.0 1.0 Medium 5 11.8 35.8 These embryos atso had the improved morphology noted for those 15 produced by the first method.
Third Method -- Activated Charcoal Medium then ABA Medium 1.0 mL of settled late stage proembryos was pipetted directly onto replicate 5 cm plates of medium 6 (activated charcoal only). These 20 were cultured for one week at 22~C in the dark then were trQnsferred to medium 7 (ABA only) for five weeks. No cotyledonary embryos Nere produced for either genotype.
Fourth Method -- ABA Medium With and Without Transfers 25 = 1.0 mL of settled late stage proembryos was pipetted directly onto replicate 5 cm plates of medium 7 (ABA only). These were cultured in the dark at about 22~C. One subset was maintained on the original medium for the entire six week period. The other subset was Iransferred to fresh medium of the same composition at the end of the second and 30 fourth weeks, then maintained on the last medium until the end of the test.
Embryos Produced Genotype A Genotype B
l~ot transferred 0.0 0.0 35 -. Transferred 2.0 3.3 The few cotyledonllry embryos that developed were of poorer quality than those de~eloped using the first and second methods. They - _~

WO91/OS854 ~ 9~ PCI/US90/06057 did not show the prominent apicQI domes And had a shorter, orten bulg-ing, shape.
It is very evident that the combination of ABA and activated charcoal in the Embryo Development Medium is highly advantageous from 5 the standpoint of both numbers and quality of embryos produced. It appears that the charcoal is most e~fective when it is uniformly dispersed throughout the medium. However, it is also advantageous when it is localized around the growing embryos. It is believed that the charcoal could also be localized on the upper surface of the medium with 10 similar good results. For example the activated charcoal could be on a filter paper or other type porous membrane which might also be used as a support surface ~or the growing embryos. It should be noted here that none of the media compositions used are represented as being optimized for any of the genotypes employed.
To the present time, using the procedures just outlined, about 3800 plantlets of Picea abies have been produced from 20 different genotypes of the spec~es and established in soil.
The use of the combined ABA-activated charcoal systems gave an additionul entirely, ted advantage. Somatic embryos generated on 20 these media could be stored for extended periods of time at 4~-5~C while remaining on the Embryo Development Medium. A number of genotypes of both Pinus taeda and Picea abies have now been stored for_over three _ months without any evident deterioration or loss of vigor. Embryos of PseudotsuFa menziesii have been stored only for shorter periods at the 25 present time but they too seem to have retained full vigor. These stored embryos develop normally into plantlets when moved to a rooting medium~ Storage temperatures from just above the freezing point of the embryos (approximately 0~C) to about 10~C appear to be very satisfac-tory. It would appear that the temperature needs to be lowered only 30 sufficiently to essentially inhibit metabolic action within the embryos.
Storage is preferebly done in the dark or on low light conditions.
Prior to the present time long term storage usually required desiccation (e g., see Buchheim, Colburn and Elanch 1989) or alginate gel encapsula-tion (e.g., Gupta and Durzan 198~).
-WO 91/058S4 2 0 6 ~ ~q~ ,PCI~/US90/060S7 DOUGLAS FIR CULTUFE _ _ - A number of observations in the 12boratory led to the hypo-thesis that considersbly raised osmotic levels would be advantegeous in the embryo development stage of Douglas-fir, and perhaps other difficult S to culture species as well. Very high osmotic levels would tend to gradually remove moisture from the developing embryos. This might pro-vide an analogous situation to the later stages of embryo development experienced in the formation of a natural seed. Prior to this time Douglas-fir cultures used a development medium containing 2-3% sucrose 10 and having an osmotic potential in the 130-160 mM/kg range. The result-ing somatic embryos were generally of poor quality and the conversion rate to rooted plants was low. The experiments outlined in the follow-ing examples were designed to test the above hypothesis and to optimize conditions if results were ~II;OUI~~ g.
lS ~ : ~ As t~oted in the background discussion, the embryogeny of Douglas-fir is quite different from trees such as the spruces or pines.
One of these differences is seen when early stage proembryos are placed in or on a late stage proembryo development medium. Instead of single Iflte stage embryos, Douglas-fir develops tight clumps of these embryos, 20 as is shown in the photomicrograph of Figure 6. Upon further develop-ment into cotyledonary embryos, these clumps remain united snd the resulting product is useless for further conversion (Figure ~). This phenomenon had apparently been recognized earlier by Durzsn and Gupta (1987) who, while they did not discuss it specifically, transferred 25 their embryonal-suspensor masses to a liquid shake culture containing 0.5 ~uM abscisic acid. They note that under the influence of ABA, indi-vidual bipolar embryos were produced which were then tr~nsferred to a development medium without ABA. The present method utilizes a liquid sllake culture with reduced osmotic level and added abscisic acid between 30 late proembryo development and cotyledonary embryo d~velopment to achieve the necessary singulation.
A reformulated basal culture medium hfls been developed by the present inventors specifically to give more successful initiation and multiplication of Douglas-fir. Preferred media compositions are given 3S in the following tables. A number of ingredients, such as those that affect the level and balance betwe~n organic and inorgenic nitrogen, are varied in quantity depending on the respons~ of individual genotypes.
This response cannot b~ reAdil~ predict~d and m~dia optimization must WO 91/05854 2 ~ 6 9 9 ~ ~ P~/US9ff/06057 largely be achieved by a combination of in~uition and ~rial and error.
Tsble 10 Pseudotsu~ Menziesli Basic Culture Media Constituent Concentration, mg/L
WTC(I) BMG(2) BASAL SALTS
NH4NO3 -- 206.3 10 KNO3 v~ries(l) 1170.0 CACl2 6H2O 200.0 220.0 Ca(N03)2 2H20~ varies(l) -- --KH2PO4 340.0 85.0 MgSO4 7H2O 400.0 185.0 15 MnSO4~H2o 20.8 8.45 znSO4 7H2O 8 0 4.30 CuSO4~sH2o 0.024 0.013 FeSO4 7H2O 27.85 13.93 Na2EDTA 37.25 18.63 20 H3BO3 5 3.10 NaMOO4~2H2o 0.20 0.125 CoC12 6H2O 0.025 0.0125 Kl 1 00 0.42 AIC13 0.02 ORGANIC ADDITIVES
myo-lnositol Y~ries(l) 100.0 Thiamine-HCI 1.00 1.00 Nicotinic acid 0-50 0-50 30 PyridoYine- HCI 0.50 0.50 Glycine 2.00 2.00 L-Glutamine Yaries 450.0 Casem~no acids 500.0 Sucrose varies 20,000 35 pH 5.7 5.7 (1) Usage varies according to culturing stage and g~notype.
(2) Modified Gupta and Durzan medium EM3 (1986b)- M~dium B 'SG ~
application Seri~l No. 426,331.

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~r o ~ c --~O91/05854 20~i PCI/US90/06057 It will be seen by reference to the mediu compositions thatthe features of the earlier inventions described in our parent applica-tions have been advantageously retained. A raised osmotic pulse is still advantageous for good quality late proembryo development. This 5 level will differ somewhat between species and even between genotypes within each species. Similarly, the cotyledonary embryo development medium should cont~in the same combination of abscisic acid and acti-vated charcoal found so desirable with Norway spruce and loblolly pine.
The examples that follow represent steps in the evolutionary 10 process of formulating a Douglas-fir development medium that represents the best mode known at present for culturing this species by somatic embryogenesis. These examples are all directed to the cotyledonary development stage. The steps prior to that time are similar to those used for loblolly pine and Norway spruce with the exceptions of the now essential embryo singulation stage and somewhat reformulated media.
These earlier steps will be briefly outlined.
A preferred explant for Douglas-fir is an immature zygotic embryo. Best results have been realized with embryos selected iust prior to the development of an apical dome up to the time just before cotyledon primord~a become Yisible. The cones are split longitudinally and seeds isolated from young ovuliferous scales. Seeds are sterilized by first being agitated in 10 % Liqui-Nox laboratory cleaner (Alconox, Inc, New York, New York) with a small additional amount of liquid sur-factant for about 10 minutes. They are then rinsed in running tap water for 3û minutes. At this time they are transferred to a sterile hood and agitated in 2096 HgO2 for 10 minutes. Following five rinses in sterile deionized water the seed coat is split and the female gametophyte removed. This is split on one side and the embryo teased out while still remaining attsched to the gametophyte by the suspensor. An explant so prepared is placed on the Stage I solid initiation medium in a 50 m m petri dish. The explants are incubated in the dark from 4-8 weeks. Success in forming an embryonal-suspensor mass (ESM) containing proembryos varies from about 1-796 depending on a number of variable factors which presently are not well understood.
All stages of culture are carried out at temperatures which may vary between about 20-25~C. Temperature is not generAlly critical and may, on occasion be varied so as to fall outsid~ this range.
The embryonal-suspensor masses containing early stag~ pro-WO 91/0~854 ~ ~ ~' ~~ PCr/US90/06057 embryos are transferred to a solid Stage 11 maintenance and multiplica-tion medium containing greatly reduced plant growth hormones and prefer-at~ly a somewhat raised osmotic level. Again, culturing is carried out in the dark with subcultures made at no greater than about two week 5 intervals. The clone can be maintained at this stage for long periods of time.
Early stage proembryos from the multiplication step are trans-ferred to a liquid Stage Ill second maintenance medium Having a signifi-cantly raised osmotic level. This corresponds to the raised osmotic 10 pulse found so beneficial for loblolly pine. It is similarly advanta-geous for Douglas-fir and Norway spruce. However, a slightly lower osmotic level of at least about 170 mM/kg will usually suffice for Douglas-fir although some genotypes may require levels as high as 240 mM/kg. Myo-inositol, which will normally be around 5000 mg/L, may need 15 to be adjusted somewhat depending on the needs of the particular geno-type in order to obtain optimum results. Culture is carried out in the dark and is periodically subcultured, usually weekly. nobust late stage proembryos having 100 or more cells will develop during this time.
Following late proembryo development, the cultures are trans-20 ferred to a Stage IV.liquid medium for the singulation step referred toearlier. This has a reduced osmotic level and is free of auxins and cytokinins. Abscisic acid is a newly added hormone in an initial amount in the range of about 5-15 ppm, more usually the lower level. Cultures are again carried out in the dark. From two to four subcultures are 25 made on a weekly basis. The level of exogenous abscisic acid will drop somewhat during each subculture. It is important that the level of abscisic acid at the beginning of a new subculture not be significantly higher than the level at the end of the previous subculture. This will result in an essentially continuous drop in ABA level over the singula-30 tion period. At this time th~ embryos are ready to begin development tocotyledonary embryos. They are transferred to either a solid or liquid medium with an effective abscisic acid level which again is not signifi-cantly higher than that at the end of the final singulation subculture Most typically this will be about 5-~ ppm effective ABA for cultures on 3~ solid medium but it may be lower. In some cases it is not necessary to add exogenous ABA to the development medium since a sufficient amount will be carried over with the residual m~dium accompanying the embryos w~len the transfer is mad~ from ~he ~ast singulation stag~. However, it :

=
- 206996~ -WO 91/05854 ~ " PCI/US90/060 is always necessary for some activated charcoal to be present in the development medium. It has been found preferable for Douglas-fir to carry out development cultures entirely in the dark.
Example 8 A basal Douglas-fir development medium was modified by addit-ion of 1, 2, or 3% myo-inositol, 2% sucrose, 2% sorbitol, or 2% mannitol to determine the effect of these osmoticants on embryo development. The above noted sucrose and _yo-inositol was in addition to that normally lO present in the development medium. All media contained û.5 % activated charcoal and 5 ppm abscisic acid and were gelled with 5 g/L tissue cul-ture agar. Each culture plate received 1 m L of settled singulated cells. Five genotypes were cultured using triplicate plates for most genotypes. While successful development was not obtained on all geno-15 types, a clearly superior response was achieved on the media containing 2 % sorbitol.
It is known that activated charcoal is an effective adsorbent of plant hormones in culture media. For example, we showed earlier that charcoal can effectively reduce over time the level of exogenous 20 abscisic acid evailable to developing embryos. Until the present, the rate and magnitude of this effect has not been well known.
ABA concentration in liquid media can be studied using known analytical methods that determine ABA concentration by measurement of ultraviolet absorption. Nitrate is un interfering ion so the liquid 25 mediA for the foL'owing tests were made up without any inorganic nitro-gen present. Present studies h~ve shown that when ABA is added to a charcoal-containing liquid medium there is an imm~diate drop in the level of available ABA. As would be expected, with a constant amount of activated charcoal present, the effect is more pronounced when only 30 small quantities of ABA are added. In the following tests, stirred ~orway spruce liquid development media with nitrstes omitted were sAm-pled at various times after addition of ABA. All media contained û.075%
activated charcoal. The ABA added increased in 5 ppm steps from 5 to 25 ppm total addition.

Wo 91/0~,854 ~ ~ j! .' ' ~' -39-T~ble 12 Percentage of Originally Added Abscisic Acid Remaining Available S - ABA Added, Time after ABA Addition, minutes _-ppm 0 1 5 10 39~ 35 16 9 5~ 44 25 20 It is evident from the above data that when ABA levels are 15 discussed in a charcoal-containing system only available or free ABA
levels should be considered.
Not surprisingly, it was also found that activated charcoal of different grades and sources adsorbed ABA at different rates. In a companion experiment to the above, ch~rcoal from four suppliers was 20 tested. An ABA solution of known concentration was added to a liquid medium with stirring and measurements of free ABA were made over a 24 hour period. In all cases 50 ppm ABA was added. Charcoal concentration was 0.12596. About 250 mL of medium was made up in a 400 mL beaker. The medium was stirred for 10 minutes and then the charcoal was al~owed to 25 settle and the supernatant liquid was sampled at the indicated times.
As was also the case with the first example, all suspended charcoal was immediately filtered from the liquid samples before further analysis.
Results were as follows.
Table 13 ~ :
Amount of Originally Added Abscisic Acid RemaininF Available, ppm Charcoal Time after ARA Addition 35Source 0 min 5 min 10 min 30 min 2i hours ~ . . .

B 31 20 17.5 17.5 12.5 C 41 30.5 30 29.5 = 25 It is evident that different sources and/or grades of charcoal behave in very different manners in regard to adsorption rates of abscisic acid. Thus the type or brand of activ~ted charcoal should be 45 p--cisely specifi~d if consistcnt resuI~s ar~ to b~ expected. Charcoal WO91/05854 20699~-'t:.~a~ PCI/IJS90/060~i7 A was used in the preceeding example and throughout all of the work described herein. It is availAble from Sigma Chemical Co., St~ Louis, Missouri as their CAtAlog No. C-4381i And is described as HCI washed.
This is not intended to be sn endorsement of this product over others that would undoubtedly be equally suitable but merely sets the speclfic identify of the product used in the examples.
Measurement of A8A in solid media presented a much more diffi-cult problem thAn meAsurement in e liquid system. Normally it would involve a tedlous And complex extr~ction process. However, the extrac-tion method would not be suitAble in an environment where the A8A con-centration was chAnging rapidly. The following method has been devel-oped and hes proved very suitable. A smull amount of tritium labeled abscisic acid is added to the normal abscisic acid used for making up the media. 3H labeled A8A is available from AmershAm Corp., Arlington ileights, Illinois. After pouring the plates And Allowing the media to solidify and cool, a quAdrAnt (1/4 circle) of A 42.4 mm diameter filter pAper is plAced on the surface of the gelled medium for Approximately 10 - seconds. In this period of time the filter pAper will imbibe about 0.43-0.4~ g of liquid from the medium. The moist paper is removed with tweezers And plAced in A viAI suitable for counting in a scintillation counter. All sAmples Are normalized to A pickup of 0.45 g of liquid medium. The Amount of radioactivity on the filter pAper cAn be can be relAted to the totAI Amount of avAilable or free Abscisic acid in the medium.
It was Assumed thAt rAtes of A8A Adsorption by activated char-coAI would be different in solid And liquid media. The following exper-iment WAS designed to show free ABA in a development medium solidified by 3% Gelrite Gellan Gum. Gelrite gum is A microbially produced hetero-polysAcchAride and is AvAilable form Chemical Dynamics Corp., South PlAinfield, New Jersey. One medium was made using 0.125% activated chArcoal. An equivAlent medium was made without chArcoAI for compar-ison. The media were formulated with the gellant and sterilized by AUtoCIAving. After cooling to e bout 55-60~C, a filter sterili~ed solu-tion equivalent to 40 ppm of A8A in the medium was added. The A8A
35 solution included An Appropriate Aliquot of the tritiated A8A. The mixture wAS then stirred for either I minute or 10 minutes berore being pipetted into 50 m m petri dishes and sllowed to cool. Abscisic acid content of the gelled medium w~s measared 2 hours and 24 hours after 8~4 2 0 6 9 9 6 4: Pcr/us90/o6057 pouring and agAin after 5 days. ~esults were as follows.
Table 1 4 Abscisic Acid Availability Over Time in Solid Culture Media ~ _ Time of Stirring Effective ABA Concentration, ppm ~ Measurement Time, min With Charcoal Without Charcoai -10 2 hrs 10 9.0 -40 2 hrs 1 14.5 -40 24 hrs 10 5.~ -40 24 hrs 1 6.6 -40 days 10 4.2 -40 The short term drop in abscisic acid in the charcoal contain-ing samples is again quite dramatic. i~lo loss of ABA was seen in the medium without activated charcoal. It is clearly evident that in any charcoal containing medium it is the effective or free amount of 20 abscisic acid, and undoubtedly other hormones as well, that must be considered. The amount of added hormone is meaningful only when all other parameters are defined.
Osmotic potential of the various media is measured using a Wescor 5500 Vapor Pressure Osmometer. This is available from Wescor, Z5 Inc., Logan, Utah. Osmotic potential of liquid media is measured by placing a 6.5 mm circle of filter paper on the sample tray of the instrument and adding a measured 10 ~uL of medium. For solid media, the fll~er paper circles are placed on the surface of the gelled media where they imbibe a sufficient amount of liquid for measurement.
Exa m ple 9 ~ ~ -Further experiments carried out on the basis of the results described in the previous example confirmed the beneficial effects of 2% =~
sorbitol used in the development media. However, sorbitol concentra-35 tions in the 3-4% range, while giving lower embryo yields in terms of numbers, did appear to improve quality. The embryos were more similar in appearance to zygotic embryos.
It had been observed elsewhere that the use of a gelling material other than agar in the development cultures improved embryo 40 yield and quallty. In the following tests tissue culture agar had been replaced with Gelrite 5ellan Gurr,. Its use as s m~dium g~llant in tissuc culture is not new, aithouFh it is not belleved to have been used before .

20~96~
WO91/05854 t ~ ' ' '' - PCI/US9~/tK057 in a medium similar to the present embryo development medium. As one hypothesis for its superior performance, Its faster gelling rate, com-p~red w~th ~gar, is believed to reduce the initial adsorption r~te of J~bscisic acid by the activated charcoal present in the medium.
Experiments were then carried out in which from 2-6% sorbitol was used in a basal Douglas-fir development medium in combination with 0.3% Gelrite gum. Other variable components were 2500 ppm KNO3, 750 ppm I~glutamine, S ppm abscisic acid, and 0.05% activated charcoal. The media were mixed one minute after the addition of abscisic acid and poured into petri dishes. Embryos were plated approximately 24 hours later. Pive genotypes were used with four replicates per genotype per treatment. Summary results are given in the following table.
Table 15 Osmotic Average Number of Somatic Embryos Formed Sorbttol, Potential, Genotypes %mM/kF 738 742 735 676 733 ~ 2277 0~8 6.0 14.5 0.0 0.3 20 3 341 12.0 4.0 18.3 2.5 0.0 4402 30.3 19.5 19.5 4.0 0.0 4.5 426 26.8 9.0 9.0 2.0 0.3 5471 37.7 26.3 22.0 2.8 0.0 6523 7.8 15.5 22.5 0.0 0.0 Depending on genotype, it appears that in combination with Gelrite gum, sorbitol is beneficial as an osmoticant at concentrations ~t least as high as 6%. Both yield and quality were Improved. Embryos were compact and yellow and more similar to zygotic embryos than those 30 developed previously. Osmotic levels above 400 mM/kg were the highest investigated to that time. The performance in this higher range tends to support the earlier stated hypothesis.
Exa m ple 10 Sorbitol is known to be poorly metabolized by embryos in cul-ture. Its effect noted above is believed to be primarily osmotic.
However, it also apparently presents a favorable chemical environment as well since the other osmoticant materials screened earlier (Example 8) showed definitely inferior performance. Subsequent to the work just reported, polyethylene glycol (PEG), with an average molecular weight of ~bout 8000, was evaluated snd found to h~ a superior osmotic~nt. It WO91/05854 2 ~ 699 6 4; ,1 ;~ P,~/US90,06057 is assumed tha~ there is no metabolism of the PEG by the developing plant and the reasons for its superior performance, compared with other materials, is not entirely clear. Very high levels of PEG, up to about 30%, have been found useful in liquid media. When more than about 12%
5 PEG is used in a solid medium there is a tendency to inhibit gelation.
Polyethylene or polypropylene glycols of other molecular weights are believed to be equally useful. Upper and lower limits of molecular weight which are useful have not yet been determined.
In order to determine the optimum level of PEG a Stage V liq-lO uid development medium having 2500 ppm KNO3, 1000 ppm L-glutamine, and 0.1% Activated charcoal but no abscisic acid was made up with polyethyl-ene glycol 8000 percentages varying between 15 and 3û% (15û,û00-300,000 mg/L?. A 40 X 4û mm polyester pad was dipped into each medium so as to pick up about 5-5.5 mL of the medium which was kept continuously mixed 15 to keep the charcoal in uniform suspension. The pads were cut from polyester batting having a thickness of about 4 mm and a basis weight of about 150 g/m2. Each saturated pad was placed in a 120 mm petri dish.
Singulated l)ouglas-fir late stage proembryos were settled and excess medium removed. The cells were rinsed with an equal volume of 20 the development medium of similar composition to that on which they were to be placed. The rinse liquid did not contain any charcoal or PEG but did contain 2.5 ppm ABA.. The cells were again settled and half of the supernatant liquid removed (l/4 of the total volume). The cells were again resuspended in the remaining liquid and 1.5 mL was placed on the 25 medium saturated polyester pad. The effect of the contained liquid transferred with the cells was to dilute the osmoticant contained within the pad. This dilution is taken into account in determining the effec-ti~e osmotic level after dilution. The only exogenous ABA supplied was that which was transferred with the cells from the rinse liquid. Table 16 whlch ~llow~ ~ experimen~al ~on~itions an~ resulls.

WO91/0~8~4 2069~e(~ PCI/US90/06057 T~ble 16 Initial Osmolality Finul Osmo-PEG in Osmol~lity after Dil- Embryo Embryo lality S Medium, % m ~ J ution, mM/kg Yield Quality mM/kg 15.0 315 -- ~ 50+ Note 1 17.5 372 -- 50+ Note 1 ---20.0 465 -- 100+ Note 1 10 22.5 543 -- 100+ Note 1 25.0 657 450 100+ Note 2 415 26.0 731 490 100+ Note 2 505 27.0 798 621 100+ Note 2 578 28.0 861 -- 100+ Note 3 ---15 29.0 93I -- 100+ Note 3 ---30.0 101I -- 200+ Note 3 ---~l) Measured before transfer of cells.
Note 1. Embryos were slightly green and small with swollen hypocotyls.
Note 2. Best quality embryos, yellow in color and with well developed cotyledons. An elongated hypocotyl region similar to zygotic embryos.
Note 3. Good quality embryos but about half the size of embryos produced in 25-27% PEG
Embryos produced using 25-27% polyethylene glycol are shown in the photomicrograph of Figure 9~ These have a close resemblance to 30 zygotic embryos and have a high rate of germination and conversion to plants.
In some instances there has been evidence of nutrient exhuus-tion when the pad system is used with liquid medium. This hus been overcome by using a pad-on-pad culture which effectively doubles the 35 amount of medium present.
Exa m ple l l A serles of experiments was made to compare the effectiveness of solid development media containing vurious ternary combin~tions of 40 polyethylene glycol 8000, sorbitol, snd lactose all having a similar osmolality of about 450 mM/kg. These were compared with a control med-ium having only sorbitol and with snother having sorbitol and polyethyl-ene glycol 8000. Stage 5 medium lsee Tables 10 and 11~ was used with 2500mgtL KNO3, 75~0 mg/L L-glutamine, 30 mg/L initial abscisic ucid, and 45 0.125% Sigma activated churcoal. The osmoticunts, in addition to the 2 sucrose in the basal medium, wer~ as follows.

WO91/05854 ~~ '9 9c6~; PCI/US90/06057 Table 17 Medium Sorbitol, PEG, Lactose, % Osmolality, Osmolar Ratio No.' % % % mM/kg S:P:L
4656 3.0 0.0 0.0 386 1:0:0 4982 3.5 6.0 0.0 439 7:1:0 4984 2.0 11.0 0.0 422 1:1:0 4983 3.0 6.0 1.0 438 6:1:1 10 4985 2.0 8.0 2.0 445 2:1:1 ~4986 1.25 10.0 2.5 458 3:3:3 4987 0.0 12.0 4.0 447 0:1:1 The media were made by combining all ingredients except abscisic 15 acid and heat sterilizing. The temperature was reduced to about 60-65~C
and filter sterilized A8A was added with stirring for 10 minutes before plates were poured and cooled. Medium 4984 gelled somewhat prematurely but produced useable plates. About 24 hours later 1 mL of settled cells (from Stage 4) of five genotypes was plated onto the test media. After 20 four weeks the cultures were examined and cotyledonary embryos counted alld graded. The following average numbers of well deYeloped embryos per plate were obt~ined. One genotype (703) did not produce embryos in uny of th~ media tested here.
~5 ~ Table 18 Medium Genotype Genotype Genotyp~ Genotype ~lo. 735 711 742 _ 676 30 4656 9.8 0.8 5.5 4.3 4982 47.0 31.5 0.3 8.0 4984 71.3 61.3 5.7 17.7 4983 36.0 74.3 4.0 17.3 4985 100.0 143.3 ~ 27.3 9.0 35 4986 136.0 162.8 1.3 10.3 4987 125.0 91.3 21.0 18.3 It is evident that the best results were obtain~d from media 4985, 4986 and 4987 which were either tern~ry combinations or binary combinat-40 ic~ns of polyethylene glycol 8000 and lactose without any sorbitol. Theeffect of medium composition on performance of individual genotypes appears most markedly with Genotype 742. However, the trial on Medium I'~o. 4986 should be replicated with this genotype before firm ~ ' as to its perform~nce are drawn.

~ s ~: ~
WO91/05854 ~ ~jt~ r~ PCI/US90/06057 Example 12 After long periods o~ time on a development medium containing polyethylene glycol Douglas-fir embryos often show signs of deteriora-tion over time. It has not been clear whether this deterioration was 5 due to nutrient depletion, a decrease in osmotic levels, or a negative response to PEG. The following study was made in order to determine the effect on this deterioration by making a medium and osmoticant change during development. Cultures were made using the liquid medium-polyester pad system described in Example 10.
A development medium was made us~ng 25D0 mg/L KNO3, 750 mg/L
L~lutamine, 260 g/L polyethylene glycol 8000, and 0.1% activated char-coal. ~o abscisic acid was included in the medium. The polyester pads were dipped into this medium, as described in Example 10, and picked up about 5.5 g of the medium.
A rinse medium was made in similar fashion to the above with the exception that the PEG and charcoal were omitted and 2.5 ppm of ABA
was included. Embryos and associated cells from the final singulation medium were settled and the supernatant liquid removed. One volume of the rinse medium was added and mixed well. The cells and embryos were 20 again settled and half of the supernatant liquid was removed. The embryos and remaining liquid were again mixed and 1.5 mL was pipetted onto the pads. Five genotypes were used with four replicates per treat men t.
Osmotic level of the medium over the cells after the rinse was 25 about 200 mM/kg whereas the development medium before plating the cells was about 490 mM/kg. Duplicate osmolality readings were taken from the media on the pads at 3 and 4 weeks after th-e initial plating.
To attempt to find an answer to the questions posed above, after three weeks 2.0 mL of the original development medium, now diluted 30 with the transferred rinse medium, was pipetted from three groups of plates (5 genotypes X 4 replicates X 3 sets) and replaced with an equiv-alent amount of fresh medium made up as follows. The original medium was kept on one set (Set 1) (5 genotypes X 4 replicates) as a control.
~one of the replacement media contained activated charcoal.
35 Sufficient charcoal remained with the original meli~ still on the pads.

206g~
WO 91/05854 ~ t ~, PCI/US90/06057 __ - Set 2 lleplacement Medium -- Original 260 g/L PEG replaced with 221 g/L PEG~ Osmolality 512 mM/kg.
Set 3 Replacement Medium -- Original 260 g/L PEG replaced with 61 g/L sorbitol. Osmolality 502 m M/kg.
Set 4 Replacement Medium -- Original 260 g/l PEG replaced with - 120 g/L lactose. Osmolality 516 mM/kg.
Table 19 Osmolality, 3 Weeks After Genotype Initial plating, mM/kg 15 ~ 742 632 Table 20 ~ --~
Somatic Embryos Developed for Different Genotypes(1) Set ~o. 735 711 742 703 676 (1) Average of 4 plates counted 12 days after medium replacement.
Quality was evaluated as follows:
Set 1 -- Small und declining embryos.
Set 2 -- Somewhat larger and better appearing embryos.
Set 3 -- Large, smoother embryos.
Set 4 -- Largest, smoothest, and best sppearing embryos.
While this test is still in progress, it appears that replace-ment of PEG with lactose or sorbitol midw~y during development will be advantageous. Osmolality has not yet been measured after the replace-40 ment medium was added but is expected to be in the range of 450 mM/kg or ab3ve at the end of the development period.
Example 13 It appears possible to develop Dougl~s-fir embryos without any 45 exogenous abscisic acld in the development medium environment. Good 2p~
WO 91/05854 . . ~ - : PCI/US90/06057 quality embryos have been formed even when no ABA w~s added to the medium during makeup and little or none csrried along with the embryos and assochted ceLs from the singulation step. HoweYer, u small amount of actiYated charcoal; e.g., 0.02~, appears to be essential. This may be due to a high level of endogenous ABA within the embryos at the end 5 of singulation, although analytical work has not yet been done to con-firm this. In the following experiment three solid media were made up.
Each had 1000 mg/L of L-glutamine, 2500 mg/L of KNO3, and 150 g/L of polyethylene glycol 8000.
Table 21 Medium l~lo.

15 Activated charcoal, % 0.02 0.04 0.04 Abscisic acid, mg/L -- ~ 8 8 Sorbitol, % 2 2 Three genotypes (7L1, 735, 771) of Douglas-for singulated 20 embryos were used in the trials. These were not washed but 1.5 mL was transferred in the singulation medium to the culture plates. A low level of ABA would have been present at the end of singulation. ~ive replicates were made at each test condition. After four weeks over lO0 embryos had formed on each medium for each genotype. After five weeks 25 the embryos formed on the medium without ABA showed some elongation indicative of premature germination. This was not observed for the embryos formed on the medis containing ABA.
It would appear that an osmotic level above about 450 mM/kg may be a threshold value for Douglas-fir embryo development media ir the 30 best yield and quality are to be obtained. This leYel is one measured at the end of the development period rather thsn the beginning.
Ideally, it is believed advantageous for the osmotic level to rise some-what over the period of development. It is possible to control osmotic level during development by periodic medium changes. To facilitate this 35 without ~ b.~hCC to the developing embryos they may be supported on a material such as filter paper which is placed directly on either 8 solid culture medium or on the saturated pad of a liquid medium.
Similarly, it appears inportant to hsve a level of available exogenous abscisic acid that drops essentially continuously rrom the 40 initial usage at the beginning Or the singulation stage to the end of WO 91/0!;8~4 2 0 6 9 9 ~ ~ t- Pcr/usgo/o6o57 ~, -49-the development period. An initial level At the beginning of singulA-tion of About 5-15 ppm Appears suitabie. This will decrease to Q low level at the end of the development stage. Exact measurements have not been possible at the end of development due to the limitations of avail-S able Analytical techniques.
Following embryo development the somatic embryos may be retained for some period of time in cold storage. They may be converted into artificial seeds for field or nursery plsntlng. Alternatively, they may be placed immediately on a germination medium such as Medium 10 BMG (TAble 10) for converslon into plantlets prlor to planting in soil.
Figure 10 contalns somewhat idealized curves for Douglas-fir showing the osmotic levels and abscisic acid level at each of the var-ious stages from initiation through germination. It hQs been observed that the osmotic ~evel will increase somewhat during each llquld shake 15 stage of late proembryo development and singulation. The opposite phen-omenon seems to occur during development, probably due to utillzatlon of sucrose and other nutrients. Taking this drop into account is necessary in adjusting the initial osmotic level of the development media. Here the solid portion of the curve represents the normal course of osmotic 20 level if no media changes are made. The dotted portion shows how osmotic level can be increased if one or more transfers to fresh media are made.
To date 446 converted seedlings from 13 genotypes of Douglas-fir are growing in soil.
25 =~ ~-lt should be recognized that there is not one single set of culturing conditions that will be suitable for achieving somatic embryo-genesis of all species or for all genotypes within a species. Tissue culture as a whole is a highly unpredictable science. This statement has even greater applicability to somatic em~ . Adjustments in 30 the mineral and plant hormone constituents or the culture media must frequently be made depending on the particular species and genotype being cultured. This applies to each of the various stuges of culturing from explants to plantlets. These adjustments are considered to be within the routine experimental capability or those skilled in the art 35 of tissue culture. The important discovery of the present inventlon is the use of A combination of abscisic Acid and an Adsorbent such as activated charcoal durinF the growth of late stage proembryos to cotyle-donary embryos. This has given results thAt are far superior in terms of WO 91/05854 2 ~ i . PCrtVS90/06057 _5~_ success and consistency thQn any process reported heretofore. The pro-cess has been successfully applied to all of the several species and many genotypes of coniferous plants studied to date and appears to be of general use for all coniferous species.
It will be understood that many Yariations can be made in the procedures described for the various culturing stages while still remaining within the spirit of the present inYentiOn. It is the inten-tion of the inventors that such variations should be included within the scope of their invention if found defined within the following claims.

BIBLIOGRAPH Y
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20 Becwar, M.R., S.R. Wann, and R. Nagmani 1988 A survey of initiation frequency of embryogenic callus among ten families of P~nus taeda ~loblolly pine). Abstracts, 4th International Conifer Tissue Culture Work Group, August 8-12, 1988, Saskatoon, Saskatchewan, Canada.
25 Boulay, M. P., P. K. Gupta, P. Krogstrup, and D. J. Durzan 1988 Developtnent of somatic embryos from cell suspension cultures of Norway spruce (Picea abies Karst.). Plant Cell Reports 7: 134-137.
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35 Durzan, D.J. and P.K. Gupta 1987 Somatic embryogenesis and polyembryogenesis in Douglas-fir cell suspenslon cultures. Plant Science 52: 229-235 .. . ... . .

WO 91/05854 2 0 6~ Pcr/USgo/06057 51 - ~
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.. . .. ... _ . .. .. . _ . . .. .. . ..... .... . . .. .. . . .

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Claims (35)

CLAIMS:

1. A method of reproducing coniferous plants by somatic embryogenesis which comprises:
placing a suitable explant on an induction culture medium containing sufficient amounts of nutrients and plant growth hormones to induce and grow a culture containing proembryos; and transferring the proembryos to a cotyledonary embryo development medium having a sufficient initial amount of exogenous abscisic acid and a sufficient amount of an adsorbent material for a sufficient time and under suitable environmental conditions to enable development of cotyledonary embryos, said adsorbent gradually reducing the level of available abscisic acid over time, said combination of abscisic acid and adsorbent in the development medium yielding greater quantities of cotyledonary embryos of improved quality than media lacking the adsorbent material.

2. The method of claim 1 which further includes transferring the proembryos from the induction culture to an intermediate maintenance and multiplication culture medium having a significantly reduced level of plant growth hormones prior to transferring the proembroyos to the cotyledonary embryo development medium.

3. The method of claim 2 which further includes transferring the proembryos from the maintenance and multiplication medium to a culture medium having an osmotic potential of at least about 170 mM/kg, in order to induce late stage proembryo development prior to transferring the proembryos to the cotyledonary embryo development medium.

4. The method of claim 1 in which the adsorbent material in the development medium is selected from the group consisting of activated charcoal, silica gel, activated alumina, poly(vinylpyrrolidone), molecular sieves, and mixtures thereof.

5. The method of claim 4 in which the adsorbent material is activated charcoal present in a range of about 0.1-5.0 g/L of culture medium.

6. The method of claims 1 or 5 in which abscisic acid is present in a range of about 5-100 mg/L of culture medium.

7. The method of claim 1 in which the coniferous plant is Picea abies.

8. The method of claim 1 in which the coniferous plant is Pinus taeda.

9. The method of claim 1 in which the coniferous plant is Pseudotsuga menziessi.

10. The method of claims 1, 2, or 3 in which the development medium includes sufficient osmoticants to provide an osmotic potential of at least about 350 mM/kg.

11. The method of claim 10 in which the osmotic potential is controlled by a mixture of materials which combine at least one osmoticant providing a readily metabolized carbohydrate energy source and at least one additional osmoticant poorly metabolized by the developing embryos.

12. The method of claim 11 in which the readily metabolized carbohydrates are selected from the group consisting of sucrose, glucose, fructose, maltose, galactose, and mixtures thereof.

13. The method of claim 11 in which the poorly metabolized osmoticants are selected from the group consisting of sorbitol, lactose, a polyalkylene glycol, and mixtures thereof.

14. A method suitable for further developing tissue culture induced coniferous plant species somatic proembryos into well developed cotyledonary embryos which comprises:
placing a suitable explant on an induction culture medium containing sufficient amounts of nutrients and plant growth hormones to induce and grow a culture containing proembryos; and transferring and further culturing the proembryos using a cotyledonary embryo development medium having sufficient amounts of mineral and organic nutrient materials, said medium having sufficient osmoticants to provide an osmotic potential of at least about 350 mM/kg, so as to enable and promote the development and growth of robust cotyledonary embryos having a high potential for normal germination and plant development.

15. The method of claim 14 which further includes transferring the proembryos from an induction culture medium to an intermediate maintenance and multiplication culture medium having a significantly reduced level of plant growth hormones prior to transferring the proembryos to the cotyledonary embryo development medium.

16. The method of claim 15 which further includes transferring the proembryos from the maintenance and multiplication medium to a culture medium having an osmotic potential of at least about 170 mM/kg, in order to induce late stage proembryo development prior to transferring the proembryos to the cotyledonary embryo development medium.

17. The method of claims 14, 15, or 16 in which the development medium further contains a sufficient initial amount of abscisic acid along with a sufficient amount of an adsorbent material to gradually reduce the level of available abscisic acid over time.

18. The method of claim 17 in which the adsorbent material is activated charcoal in a range of about 0.1-5.0 g/L of culture medium.

19. The method of claim 17 in which abscisic acid is present in the range of about 5-100 mg/L of culture medium.

20. The method of claim 17 in which the adsorbent material in the development medium is selected from the group consisting of activated charcoal, silica gel, activated alumina, poly(vinylpyrrolidone), molecular sieves, and mixtures thereof.

21. The method of claim 14 in which the osmotic potential of the development medium is controlled by a mixture of materials which combine at least one osmoticant providing a readily metabolized carbohydrate energy source and at least one additional osmoticant poorly metabolized by the developing embryos.

22. The method of claim 21 in which the readily metabolized osmoticant is selected from the group consisting of sucrose, glucose, fructose, maltose, galactose, and mixtures thereof.

23. The method of claim 21 in which the poorly metabolized osmoticant is selected from the group consisting of sorbitol, lactose, a polyalkylene glycol, and mixtures thereof.

24. The method of claim 14 in which the embryo culture growing in the development medium is transferred at least once to a fresh development medium containing osmoticants which may be different from those in the preceding medium, said fresh development medium also having an osmotic potential which may differ from that of the preceding medium.

25. The method of claim 14 in which the osmotic potential of the development medium is above a level of about 400 mM/kg.

26. The method of claim 24 in which the osmotic potential of the development media are always above a level of about 400 mM/kg.

27. The method of claim 24 in which the osmotic potential of each successive fresh development medium is raised over that of the preceding development medium.

28. The method of claim 14 in which the plant is Douglas-fir (Pseudot-suga menzieii), in which the proembryos are initiated from a zygotic embryo extracted from an immature seed.

29. The method of claim 28 which includes, prior to transfer of proembryos to the development medium, the further step of transferring the proembryos to a liquid culture medium having a reduced osmotic potential and containing a sufficient amount of exogenous abscisic acid in order to cause singulation of said proembryos.

30. The method of claim 29 in which the osmotic potential of the singulation medium is below about 150 mM/kg.

31. The method of claim 29 in which the abscisic acid level in the singulation medium is initially in the range of about 5-15 mM/kg

32. The method of claim 29 in which at least one transfer to fresh medium is made during the singulation step, said fresh medium having a lower concentration of abscisic acid than that initially present in the previous medium, said lower concentration being no greater than about that present in the previous medium immediately prior to the transfer.

33. The method of claims 29, 30, 31, or 32 in which the initial available exogenous abscisic acid in the development medium is no greater than that present in the singulation medium immediately prior to transfer into the development medium.

34. The method of claim 33 in which the development medium lacks exogenous abscisic acid entirely and a sufficient amount of abscisic acid is transferred with the embryos as entrained or endogenous abscisic acid from the singulation step.

35. The method of claim 29 in which the concentration of abscisic acid to which the embryos are exposed is reduced continuously from the beginning of the singulation period until the end of the development period.