AU703582B2 - An improved method for regeneration of coniferous plants by somatic embryogenesis employing polyethylene glycol - Google Patents

Description

Ics Case Docket No. FSL 96-2 AN IMPROVED METHOD FOR REGENERATION OF CONIFEROUS

PLANTS

BY SOMATIC EMB OGENESS POLYETHYLENE

GLYCOL

FIELD OF INVENTION This invention relates to a method for regeneration of coniferous plants. In particular, t.is inventionu relates to an improved method for producing and developing somatic embryos for somatic embryogenesis processes for plants of the genus Pinus and Pinus interspecies hybrids by including polyethylene glycol in the development media. This method is well suited for producing clonal planting stock useful for reforestation.

BACKGROUND OF THE INVENTION Propagation by somatic embryogenesis refers to methods whereby embryos are produced in vitro from small pieces of plant tissue or individual cells. The embryos are referred to as somatic because they are derived from the somatic (vegetative) tissue, rather than from the sexual process. Vegetative propagation via somatic embryogenesis has the capability to capture all genetic gain of highly desirable genotypes. Furthermore, these methods are readily amenable to automation and mechanization. These qualities endow somatic embryogenesis processes with the potential to produce large numbers of individual clones for reforestation purposes.

-2 0 It was not until 1985 that somatic embryogenesis was discovered in conifers (Hakman et al. 1985) and the first viable plantlets were regenerated from conifer somatic embryos (Hakman and von Arnold 1985). Since 1985, conifer tissue culture workers throughout the world have pursued the development of somatic embryogenesis systems capable of regenerating plants. The goal of much of this work is to develop conifer somatic embryogenesis as an efficient propagation system for producing clonal planting stock en masse. Additionally, the embryogenic system interfaces very well with genetic engineering i techniques for production of transgenic clonal planting stock of conifers.

Case Docket No. FSL 96-2 The two most economically important conifer genera are Picea (spruce) and Pinus (pine). Those working in conifer somatic embryogenesis have found that there is a striking difference between Picea conifers and Pinus conifers as to the ease with which somatic embryogenesis can be induced and plants regenerated (Tautorus et al. 1991). In fact, when one measures the respective levels of achievement in the art of conifer somatic embryogenesis among species of these two important genera, it is clear that significantly more success has been obtained with Picea than with Pinus. Indeed, the recalcitrance of emhrvyogen.; cultues of Pinus species is well documented. This is especially true for pines commonly found in the southeastern United States (known in the industry as Southern yellow pines).

Nevertheless, researchers working with Pinus species plants have recently achieved some important advances. In U. S. Patents Nos. 5,413,930 and 5,506,136 Becwar et al.

disclose multi-step methods that are able to complete the entire somatic embryogenesis regenerative process, from explant collection to planting, for historically recalcitrant Southern yellow pines Pinus taeda, Pinus serotina, Pinuspalustris, and Pinus elliottii), Pinus rigida, and hybrids thereof.

In U. S. Patent No. 5,491,090 Handley et al. improved upon the above-noted processes by teaching a method which enables its practitioners to replace the semi-solid maintenance culture media taught by Becwar et al. with liquid suspension culture media.

While the methods taught in U. S. Patents Nos. 5,413,930, 5,491,090, and 5,506,136 have achieved considerable success in both establishing embryogenic cultures of Pinus and in producing large numbers of field grown plants, these methods have proven to be somewhat limited by low embryo development frequencies experienced by different genetic families.

Indeed, one of the limiting factors in achieving clonal forestry in these pines has been the inability to produce sufficient numbers of developed embryos from some of the very best 25 genetic material and, subsequently, production of somatic embryo plants for field testing and eventual clonal deployment (Handley et al. 1995). Simply put, a major problem limiting commercial development of the above-noted methods is that they tend to exhibit relatively low embryo development frequencies in certain cell lines due to the genetic specificity of the lines.

2 3 Case Docket No. FSL 96-2 Having a low frequency of embryo development can severely limit the potential applications of somatic embryogenesis in Pinus species for large-scale production of genetically improved conifers for the following reason. Skilled artisans in the conifer tissue culture field recognize that the use of embryogenic cultures derived from juvenile explants zygotic embryos derived from seed) necessitate that the resulting regenerated plants be field tested prior to large scale production. Only selected genotypes which show the potential for producing significant genetic gain in such field tests will subsequently be p-ropagated+I somatic embryogenesis. It will, therefore, be necessary to screen numerous genotypes from desirable parents, initiate embryogenic cultures, cryopreserve each genetically different culture, regenerate and develop embryos and plants from each genetically different culture, field test plants from each genotype, and choose select genotypes for large scale production via somatic embryogenesis.

Low culture development frequencies pose a severe limitation for implementing this strategy in that when few embryos are produced per gram of embryogenic callus or suspension culture cells, it is almost impossible to obtain sufficient embryos for field planting. For example, embryo production at the level of around 10 to 15 embryos per clump of tissue is not commercially acceptable. Indeed, lines having embryo development rates this low are usually eliminated from testing (as it is extremely difficult to get these to produce .i enough embryos for a field test and later production).

0 As noted above, another major problem plaguing current somatic embryogenesis methods is that it has been extremely difficult to establish sufficient numbers of embryos o. from some of the best genetic families of Southern yellow pine. A series of experiments have shown that even when propagated via the above-noted patented methods a large percentage of those embryogenic lines exhibited relatively low production numbers.

5 Somatic embryogenesis processes utilized with conifers (particularly the Pinus "species) have traditionally involved seven general steps: 1) culture initiation, 2) culture maintenance, 3) embryo development, 4) embryo maturation, 5) embryo germination, 6) conversion, and 7) plant growth (field planting). The culture media utilized in the different steps are key components of effective somatic embryogenesis regeneration systems.

3 Case Docket No. FSL 96-2 U. S. Patents Nos. 5,413,930, 5,491,090, and 5,506,136 teach the use of semi-solid culture media during the embryo development step. These culture development media are generally composed of seven groups of ingredients: inorganic nutrients, vitamins, organic supplements, a carbohydrate source, phytohormones abscisic acid), and a gelling agent.

A significant step was made towards solving the above-noted problems when it was determined that the addition of polyethylene glycol (PEG) to development media containing either standard or very high levels of abscisic acid (ABA) resulted in a significant increase in the number of Pinus somatic embryos produced when employing the methods taught in U. S.

Patents Nos. 5,413,930, 5,491,090, and 5,506,136.

However, this improvement unveiled an additional problem. When PEG was included in the development media, the somatic embryos produced on this media failed to germinate even after being subjected to standard germination protocols. While inclusion of PEG in development media at the levels taught in the present method significantly increased embryo production, the PEG also induced a germination block which prevented the resulting somatic embryos from germinating.

This problem was solved via the adoption of novel development protocols. Under these protocols the somatic embryos are transferred from a first development media (which contains PEG) to a second development media (which does not contain PEG). The employment of these steps results in significantly increased numbers of embryos produced oand, very importantly, the produced embryos can germinate into viable plants.

Therefore, an object of the present invention is to provide an improved method for the *.regeneration of coniferous plants by somatic embryogenesis.

Another object of the present invention is to provide an improved method for producing somatic embryos for use in somatic embryogenesis processes for plants of the genus Pinus and Pinus interspecies hybrids.

Yet another object of the present invention is to provide a method for overcoming the o* germination block resulting from the inclusion of polyethylene glycol in embryo development S"media.

4 9-9

E

Case Docket No. FSL 96-2 A further object of the present invention is to provide an improved method for the production and germination of somatic embryos from embryogenic tissue cultures from plants of the genus Pinus and Pinus interspecies hybrids so that these embryos can converted to yield viable plants for field planting.

SUMMARY OF THE INVENTION The above objectives are achieved by the use of an improved method for developing embryos for use in somatic embryogenesis processes employing embryogenic tissues from plants of the genus Pinus and Pinus interspecies hybrids. This method allows the practitioner to develop and germinate viable Pinus embryos from a wide range of genetic backgrounds.

This improved method teaches the addition of polyethylene glycol (PEG) to embryo development media containing either standard or very high levels of abscisic acid (ABA).

While this combination of PEG and ABA significant increases the numbers of somatic embryos produced, these resulting embryos will not germinate. To correct this problem the improved method also teaches novel development protocols which overcome the block and allow the embryos to germinate.

This method results in improved stage 3 embryo development frequencies which allow many more vigorous embryos to be obtained (which can, in turn, be successfully carried through subsequent stages of the somatic embryogenesis process) and germinated.

Furthermore, the method makes it feasible to include more genotypes from families of high 2*0 genetic value. Somatic plants produced from these families can be planted in clonal field tests and thereby increase the likelihood of being able to select highly productive genotypes.

In addition, more genotypes can be quickly proliferated via this method for rapid production of clonal planting stock from many selected parents. The employment of previous methods often resulted in a significant number of embryogenic lines exhibiting very low (or zero) 5 embryo development frequencies. While one may still expect that a few lines will fail to S° produce sufficient numbers of embryos even when using the present improved method, the overall embryo development response across lines is substantially increased.

Case Docket No. FSL 96-2 DESCRIPTION OF THE PREFERRED

EMBODIMENT

As noted above, traditional somatic embryogenesis process utilized with conifers (particularly the Pinus species) can be divided into seven general steps: 1) culture initiation, 2) culture maintenance, 3) embryo development, 4) embryo maturation, 5) embryo germination, 6) conversion, and 7) plant growth (field planting). The present invention improves upon the methods taught in U. S. Patents Nos. 5.411 ,10 5 491,090 C .and -T v V i tl lU 1 DV 0 by replacing the third step (the embryo development step) in those methods with two separate embryo development steps.

The improved first embryo development step comprises: transferring at least 100.0 mg of the mass of embryogenic tissue (or 30.0 mg of liquid embryogenic cell culture) to a first embryo development medium containing a sufficient amount of nutrients, a gelling agent selected from the group consisting of 6.0 g/L to 12.0 g/L ofagar, 1.75 g/L to 4.0 g/L ofgellan gum, 6.0 g/L to 8.0 g/L of agarose, and combinations thereof, 20.0 g/L to 70.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, and wherein the improvement comprises the addition of up to about 10.0 g/L of activated carbon, about 10.0 g/L to about 100.0 g/L of polyethylene glycol, about 5.0 mg/L to about 300.0 mg/L of abscisic acid, and the continued maintenance of the abscisic acid concentration at said level; for a sufficient time under suitable environmental conditions to develop pre-stage 3 somatic embryos.

The novel second embryo development step comprises: transferring the pre-stage 3 somatic embryos to a second embryo development medium containing a sufficient amount of nutrients, a level of gelling agent selected from the group consisting of 6.0 g/L to 12.0 g/L of agar, 1.75 g/L to 4.0 g/L of gellan gum, 6.0 g/L to 8.0 g/L of agarose, and combinations thereof, 20.0 g/L to 70.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, up to about 10.0 g/L Case Docket No. FSL 96-2 of activated carbon, about 5.0 mg/L to about 300.0 mg/L of abscisic acid, and the continued maintenance of the abscisic acid concentration at said level; for a sufficient time under suitable environmental conditions to develop stage 3 somatic embryos.

An alternative development protocol may also be employed wherein the embryogenic tissue is maintained on the first embryo development media until stage 3 somatic embryos are actually developed. Afterwards, the developed embryos are transferred to a second embryo development media and maintained for a period of time at a reduced temperature.

Under this cold treatment protocol, the improved first embryo development step comprises: transferring at least 100.0 mg of the mass of embryogenic tissue (or 30.0 mg of liquid embryogenic cell culture) to a first embryo development medium containing a sufficient amount of nutrients, a gelling agent selected from the group consisting of 6.0 g/L to 12.0 g/L of agar, 1.75 g/L to 4.0 g/L of gellan gum, 6.0 g/L to 8.0 g/L of agarose, and combinations thereof, 20.0 g/L to 70.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, and wherein the improvement comprises the addition of up to about 10.0 g/L of activated carbon, about 10.0 g/L to about 100.0 g/L of polyethylene glycol, about 5.0 mg/L to about 300.0 .0 mg/L of abscisic acid, and the continued maintenance of the abscisic acid 9 concentration at said level; for a sufficient time under suitable environmental conditions to develop stage 3 somatic embryos.

The novel second embryo development step comprises: transferring the stage 3 somatic embryos to a second embryo development medium containing a sufficient amount of nutrients, a gelling agent selected from the group consisting of 6.0 g/L to 12.0 g/L of agar, 1.75 g/L to 4.0 g/L of gellan gum, 6.0 g/L to 8.0 g/L of agarose, and combinations thereof, 20.0 g/L °to 70.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, up to about 10.0 g/L of Case Docket No. FSL 96-2 activated carbon, up to about 100.0 mg/L of abscisic acid, and the continued maintenance of the abscisic acid concentration at said level; for a period of about 2 to about 12 weeks at a temperature in the range of about 0°C to about C and under suitable environmental conditions to maintain the viability of the stage 3 somatic embryos.

The present method significantly improves embryo development by incorporating polyethylene glycol and standard to high levels of the phvtohormone abscisic acid into the first embryo development step (and by maintaining the ABA concentration in the first development media). A second embryo development step is added to overcome the germination block caused by the utilization of PEG in the first development media. These changes significantly increase, across a range of genotypes, the amount and number of viable embryos developed.

These development steps are employed with the remaining method steps (culture initiation, culture maintenance, embryo maturation, embryo germination, conversion, and plant growth) taught in U. S. Patents Nos. 5,413,930 and 5,506,136 to create improved methods for producing coniferous plants via somatic embryogenesis. To practice these improved methods one follows the following steps: placing a suitable explant selected from the group consisting of immature zygotic embryos and megagametophytes containing immature zygotic embryos 23 on culture initiation medium containing a sufficient amount of nutrients, 0.1 to 5.0 mg/L of auxin, 0.1 to 1.0 mg/L of cytokinin, 10.0 to 40.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, and a gelling agent selected from the group consisting of 2.5 to 4.5 g/L ofagar, 0.5 to 1.5 g/L ofgellan gum, 3.0 to 5.0 g/L of agarose, and combinations thereof, for 2 to 14 weeks under suitable environmental conditions to grow a culture containing embryogenic tissue; transferring the embryogenic tissue culture to culture maintenance medium Scontaining a sufficient amount of nutrients, 0.1 to 5.0 mg/L of auxin, 0.1 to mg/L of cytokinin, 10.0 to 40.0 g/L of a sugar selected from the group Case Docket No. FSL 96-2 consisting of glucose, maltose, sucrose, melezitose and combinations thereof, and a gelling agent selected from the group consisting of 6.0 to 9.0 g/L of agar, 1.75 to 4.0 g/L of gellan gum, 6.0 to 8.0 g/L of agarose, and combinations thereof, for a sufficient amount of time under suitable environmental conditions to develop a mass of embryogenic tissue having a weight of at least 100.0 mg; transferring at least 100.0 mg of the mass of embryogenic tissue to a first embryo development medium containing a sufficient amount of nutrients, a gelling agent selected from the group consisting of 6.0 g/L to 12.0 g/L of agar, 1.75 g/L to 4.0 g/L of gellan gum, 6.0 g/L to 8.0 g/L of agarose, and combinations thereof, 20.0 g/L to 70.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, and wherein the improvement comprises the addition of up to about 10.0 g/L of activated carbon, about 10.0 g/L to about 100.0 g/L of polyethylene glycol, about 5.0 mg/L to about 300.0 mg/L of abscisic acid, and the continued maintenance of the abscisic acid concentration at said level; for a sufficient time under suitable environmental conditions to develop pre-stage 3 somatic embryos; transferring the pre-stage 3 somatic embryos to a second embryo 0 development medium containing a sufficient amount of nutrients, a level of gelling agent selected from the group consisting of 6.0 g/L to 12.0 g/L of agar, 1.75 g/L to 4.0 g/L of gellan gum, 6.0 g/L to 8.0 g/L of agarose, and combinations thereof, 20.0 g/L to 70.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, 5 up to about 10.0 g/L of activated carbon, about 5.0 mg/L to about 300.0 mg/L of abscisic acid, and the continued maintenance of the abscisic acid concentration at said level; for a sufficient time under suitable environmental conditions to develop stage 3 somatic embryos; Case Docket No. FSL 96-2 separating the stage 3 somatic embryos from the development medium and partially drying the embryos by exposing the embryos to an atmosphere having a high relative humidity for a period of about 2 to 5 weeks; transferring the partially dried somatic embryos to germination medium containing a sufficient amount of nutrients, up to 10.0 g/L of activated carbon, a gelling agent selected from the group consisting of 6.0 to 9.0 g/L of agar, 1.75 to 3.50 g/L of gellan gum, 6.0 to 8.0 g/L of agarose, and combinations thereof, and 20.0 to 40.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, and combinations thereof, for a sufficient time 1 0 under suitable environmental conditions to germinate the partially dried embryos; converting the germinated embryos into acclimatized plants; and field planting the acclimatized plants.

Should one choose to develop stage-3 embryos on the PEG-containing first development media, one may overcome the resulting PEG germination block by employing a cold treatment protocol. To employ this cold treatment protocol in the above-noted method one: maintains the mass of embryogenic tissue [produced in step above] on the first embryo development medium for a sufficient time under suitable 0 environmental conditions to develop stage 3 somatic embryos; and transfers the stage 3 somatic embryos to a second embryo development medium containing a sufficient amount of nutrients, a gelling agent selected from the group consisting of 6.0 g/L to 12.0 g/L of agar, 1.75 g/L to 4.0 g/L of gellan gum, 6.0 g/L to 8.0 g/L of agarose, and combinations thereof, 20.0 g/L 2.5 to 70.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, up to about 10.0 g/L of activated carbon, up to about 100.0 mg/L of abscisic acid, and the continued maintenance of the abscisic acid concentration at said level; for a period of about 2 to about 12 weeks at a temperature in the range of about 0°C to about Case Docket No. FSL 96-2 and under suitable environmental conditions to maintain the viability of the stage 3 somatic embryos; prior to the stage 3 somatic embryos being separated from the development medium and being partially dried [step above].

One may also utilize liquid suspension cultures to produce somatic embryos by modifying the above-noted development steps and combining them with the remaining method steps taught in U. S. Patent No. 5,491,090. To employ liquird susen.son cultus i.

the above-noted method one: transfers the embryogenic tissue culture [produced in step above] to 1 0 liquid suspension culture maintenance medium containing a sufficient amount of nutrients, 0.1 to 100.0 mg/L of auxin, 0.05 to 10.0 mg/L of cytokinin, 5.0 to 100.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose and combinations thereof, and about 0.1 to 10.0 g/L of activated carbon, for a sufficient amount of time under suitable environmental conditions to develop a liquid embryogenic cell culture; transfers at least 30.0 mg of the liquid embryogenic cell culture to a first embryo development medium containing a sufficient amount of nutrients, a gelling agent selected from the group consisting of 6.0 g/L to 12.0 g/L of agar, 1.75 g/L to 4.0 g/L of gellan gum, 6.0 g/L to 8.0 g/L ofagarose, and combinations thereof, 20.0 g/L to 70.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, the addition of up to about 10.0 g/L of activated carbon, about 10.0 g/L to :.about 100.0 g/L of polyethylene glycol, about 5.0 mg/L to about 300.0 mg/L of abscisic acid, and the continued maintenance of the abscisic acid concentration 2.5 at said level; for a sufficient time under suitable environmental conditions to develop the pre-stage 3 somatic embryos; and transfers the pre-stage 3 somatic embryos to the second embryo development medium [as taught in step above].

12 Case Docket No. FSL 96-2 This method is generally applicable to somatic tissue obtained from the Pinus species including, but not limited to, the following: Pinus taeda (loblolly pine), P. elliottii (slash pine), P. palustris (longleafpine), P. serotina (pond pine), P. echinata (shortleaf pine), P.

clausa (sand pine), P. glabra (spruce pine), P. rigida (pitch pine), P. echinata (shortleaf pine), P. nigra (Austrian pine), P. resinosa (red pine), P. sylvestris (Scotch pine), P.

banksiana (jack pine), P. virginiana (Virginia pine), P. radiata (Monterey pine), P. contorta (shore pine), P. contorta latifolia (lodgepole nine), P. ponderosa (ponderosa pine), P. eiophylla (Chihuahua pine), P. jeffreyi (Jeffrey pine), and P. engelmannii (Apache pine), P.

strobus (eastern white pine), P. monticola (western white pine), and P. lambertiana (sugar pine), P. albicaulis (whitebark pine), P. flexilis (limber pine), P. strobiformis (southwestern white pine), P. caribaea (Caribbean pine), P. patula (Mexican weeping pine), P. tecumumanii (Tecun Uman pine), P. maximinoi, P. oocarpa (Ocote Pine) and P. chiapensis (Mexican White pine). In addition, the current invention is specifically applicable to interspecies hybrids of the above mentioned pines including Pinus rigida x P. taeda, P. serotina x P.

taeda, and reciprocal crosses.

It is preferred to utilize the present method with Southern yellow pines, Pinus rigida, and hybrids thereof. Those skilled in the art recognize that several species of pine indigenous to the Southeastern United States are closely related and hybridize naturally. Taxonomically :i this group of pines is referred to as "Southern yellow pines" and includes Pinus taeda, P.

2 0 serotina, P. palustris, and P. elliottii (Preston 1989).

An essential element of the present invention is the incorporation of abscisic acid (ABA) into media formulations used to develop stage 3 somatic embryos from conifer embryogenic cell cultures. A suitable level of ABA for use in the present methods is from about 5.0 to about 300.0 milligrams per liter (mg/L) of medium. The preferred ABA level is 5 in the range of about 125 mg/L to about 250 mg/L.

The use of ABA in the second development media of the cold treatment protocol is optional. If employed, the suitable level for ABA is in the range of up to about 100 mg/L.

Yet another essential element of the present invention is the maintenance of the ABA levels in the embryo development media. The preferred method of maintaining the ABA Case Docket No. FSL 96-2 concentration is to transfer the tissues containing the developing embryos onto fresh development medium containing an amount of abscisic acid equal to or greater than the amount present in the initial development medium. However, it is also possible to add additional ABA directly to the initial embryo development medium as needed in order to maintain the desired concentrations.

Another essential element of the present invention is the use of polyethylene glycol (PEG) in the first of the two development steps. That is, PEG is incorporated into the first media development formulations used to develop the somatic embryos from conifer embryogenic cell cultures, but not in the second development media formulations. A suitable level of PEG for use in the first embryo development media is from about 10 to about 100 grams per liter of medium. The preferred PEG level is in the range of about 50 g/L to about 80 g/L.

This combination of PEG and ABA proved effective both with and without the presence of activated carbon in the development media. When employed, the suitable levels of activated carbon for use in the embryo development media range up to about 10.0 g/L. The preferred activated carbon level is in the range of about 0.5 g/L to about 5.0 g/L.

In addition to PEG and ABA, the embryo development media also require sufficient amounts of nutrients to allow the culture to remain viable. However, the present method is not limited to any single culture nutrient medium formulation. For example, four common 0 basal culture media formulations which are suitable for use in the present method are listed in Table I below. However, it should be understood that any nutrient media commonly used in Pinus somatic embryogenesis will be suitable for use with this invention.

Oq a.

o« *oo 1'i- Case Docket No. FSL 96-.2 TABLE I COMPONENT DCR W5 M S G INORGAN IC SALTS CONCENTRATION, mg/L

NH

4

NO

3 40.0-- 700.00

KNO

3 34.0 2500.00 259.00 100.00 \Ca(NOX4 2 1 556.00 A 963.00 MgSO 4 -71- 2 0 370.0 400.00-' 1850.00 300

KH-

2 P0 4 170.00 270.00 170.00

NJ-

4

H

2 P0 4 300.00 CaCI 2 2H 2 0 85.00 200.00 440.00 KCI 1327.0 7-45.00 KI 0.31.00 0.83- 0.83

H

3 B0 3 6.05.00 31.00 6.20 MnSO 4 2 0 22.3 10.00 15.16 16.90 ZnS0 4 -7H,0 8.6 1.00 8.60 8.60 Na 2 Mo0 4 2H 2 O 0.25 0.10 0.25 0.25 CUS0 4 -5H 2 O 0.25 0.20 0.25 0.03 CoCI 2 -6H 2 O 0.0 0.10 0.0 0.03 QNiCl 2 -6H 2 0 0.03 FeSO 4 -7H 2 0 27.80 15.00 27.80 27.80 N2DA37.30 20.00 37.30 37.30 .Nicotinic acid 0.5 0.5 0.50 0.50 Pyridoxine-HCI 0.50 0.5 0.50 0.10 1.00 1.00 1.00 0.10 Glycine 2.00 2.00 2.00 a) According -to Gupta and Durzan (1985).

'3 Jb) According to Schenk and Hildebrandt (1972).

c) According to Coke (1996).

d) According to Becwar et al. (1990).

Suitable media for use in the embryo development steps in the present methods contain from about 20.0 to about 70.0 grams per liter of a sugar selected from the group 14 Case Docket No. FSL 96-2 consisting of glucose, maltose, sucrose, melezitose, and combinations thereof. The preferred sugar content for the media is from about 15.0 to about 40.0 g/L.

Suitable media for use in the embryo development steps in the present methods contain a level of gelling agent selected from the group consisting of 6.0 to 12.0 g/L of agar, 1.75 to 4.0 g/L ofgellan gum, 6.0 to 8.0 g/L of agarose, 3.5 to 6.0 g/L of AGARGEL® (an agar/gellan gum mixture commercially available from Sigma Chemical Company), and combinations thereof.

A suitable period of time for developing pre-stage 3 somatic embryos on the first IrNj u e v e l o p m ent media is in the range of about 2 to about 12 weeks. A suitable period of time for developing the stage 3 somatic embryos on the second embryo development media is in the range of about 1 to about 10 weeks.

A suitable period of time for developing stage 3 somatic embryos on the first embryo development media of the cold treatment protocol is in the range of about 3 to about 18 weeks. A suitable period for cold-treating the stage 3 somatic embryos on the second embryo development media is in the range of about 2 to about 12 weeks, with the preferred period being about 3 to 6 weeks..

The present methods allow embryos to be developed from embryogenic tissue or suspension cultures which has been cryopreserved.

A number of terms are known to have differing meanings when used in the literature.

The following 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 specification.

S"A "cell line" is a culture that arises from an individual explant.

S"Clone" when used in the context of plant propagation refers to a collection of individuals having the same genetic makeup.

5 -"Corrosion cavity" is the cavity within the megagametophyte tissue of conifers formed by the growth and enlargement of the zygotic embryos.

"Conversion" refers to the acclimatization process that in vitro derived germinating somatic embryos undergo in order to survive under ex vitro (nonaxenic) conditions, and subsequent continued growth under ex vitro conditions.

"Cryopreservation" is storing cultures or tissues at ultra-low temperatures (below Sabout -120 0 C) in a liquid nitrogen chamber for indefinite maintenance to prevent aging and loss of viability.

An "embryogenic culture" is a plant cell or tissue culture capable of forming somatic embryos and regenerating plants via somatic embryogenesis.

"Embryogenic tissue", in conifers, is a mass of tissue and cells comprised of very early stage somatic embryos and suspensor-like cells embedded in a mucilaginous matrix. The Case Docket No. FSL 96-2 level of differentiation may vary significantly among embryogenic conifer cultures. In some cases, rather than containing well-formed somatic embryos, the embryogenic tissue may contain small, dense clusters of cells capable of forming somatic embryos. This has also been referred to as "embryogenic suspensor masses" by some researchers and is also called "embryogenic callus" in some of the conifer somatic embryogenesis literature; but most researchers believe it is not a true callus.

An "established" embryogenic liquid suspension culture is considered to be any culture that grows and can be maintained in a viable embryogenic state.

An "eplant" is the organ, tissue, or cells derived from a plant and cultured in vitro for the purpose of starting a plant cell or tissue culture.

"Extrusion" is the process by which zygotic embryos and/or embryogenic tissue derived from zygotic embryos emerges or extrudes from the corrosion cavity of the megagametophyte of conifer seeds via the opening in the micropylar end, when placed in culture.

"Field planting" is the establishment of laboratory, greenhouse, nursery, or similarly grown planting stock under field conditions.

"Genotype" is the genetic constitution of an organism; the sum total of the genetic information contained in the chromosomes of an organism.

"Germination" is the emergence of the radicle or root from the embryo.

"Initiation" is the initial cellular proliferation or morphogenic development that eventually results in the establishment of a culture from an explant.

S "Megagametophyte" is haploid nutritive tissue of the conifer seed, of maternal origin, within which the conifer zygotic embryos develop.

"Micropyle" is the small opening in the end of the conifer seed where the pollen tube 5 enters the ovule during fertilization, and where embryogenic tissue extrudes from the megagametophyte during culture initiation.

"Nutrients" are the inorganics nitrogen), vitamins, organic supplements, and carbon sources necessary for the nourishment of the culture.

A "plantlet" is a small germinating plant derived from a somatic embryo.

"Regeneration", in plant tissue culture, is a morphogenic response to a stimuli that S: results in the production of organs, embryos, or whole plants.

"Stage 1 embryos" are small embryos consisting of an embryonic region of small, densely cytoplasmic cells subtended by a suspensor comprised of long and highly vacuolated cells.

"Stage 2 embryos" are embryos with a prominent (bullet shaped) embryonic region that is more opaque and with a more smooth and glossy surface than stage 1 embryos.

1"- Case Docket No. FSL 96-2 "Stage 3 embryos" are embryos with an elongated embryonic region with sfall cotyledons visible.

"Somatic embryogenesis" is the process of initiation and development of embryos in vitro from somatic cells and tissues.

A "somatic embryo" is an embryo formed in vitro from vegetative (somatic) cells by mitotic division of cells. Early stage somatic embryos are morphologically similar to immature zygotic embryos; a region of small embryonal cells subtended by elongated suspensor cells. The embryonal cells develop into the mature somatic embryo.

0A "suspension culture" is a culture composed of cells suspended in a liquid medium, usually agitated on a gyrotory shaker. An embryogenic suspension culture in conifers is usually composed of early stage somatic embryos with well formed suspensor cells and dense cytoplasmic head cells that float freely in the liquid medium.

A "suspensor cell" is an extension of the base of the embryo that physically pushes the embryo into the megagametophyte in conifer seeds and is comprised of elongated and highly vacuolated cells. In a somatic embryo these elongated cells are clustered in rows and extend from the base of the dense cytoplasmic cells at the head or apex.

A "zygotic embryo" is an embryo derived from the sexual fusion of gametic cells.

The following examples are provided to further illustrate the present invention and are not to be construed as limiting the invention in any manner.

EXAMPLE 1 The methods claimed in U. S. Patents Nos. 5,413,930 and 5,506,136 were followed in this example. However, evaluations were conducted to determine what effect the addition of polyethylene glycol to the embryo development media might have on the production of stage 3 somatic embryos.

Immature seed cones were collected from several different loblolly pine (Pinus taeda sources located in Westvaco's South Carolina coastal breeding orchards near Charleston, South Carolina. The seed cones were collected when the dominant zygotic embryo was at the precotyledonary stage of development. Using the classification system of von Arnold and S-Hakman (1988), the dominant zygotic embryo at this stage is referred to as being at stage 2; .0 that is, an embryo with a prominent embryonic region with a smooth and glossy surface, subtended by elongated suspensor cells which are highly vacuolated. However, zygotic embryos at an earlier stage of development (stage 1) may also be used effectively to initiate embryogenic cultures.

17 -)9i) 1% Case Docket No. FSL 96-2 Seed cones were harvested from selected trees, placed in plastic bags and stored at 4 0 C until used for culture initiation. If the cones were stored for more than two weeks at 4 C, they were aired and dried out weekly (placed at 23 ambient laboratory conditions for 2-3 hours) to prevent growth of fungi on the surface of the cones and concomitant deterioration of seed quality.

For culture initiation, intact seeds removed from seed cones were surface sterilized by treatment in a 10 to 20% commercial bleach solution (equivalent of a 0.525% to 1.050% sodium hypochlorite solution) for 15 minutes followed by three sterile water rinses (each of 10 T,111ULm uuation). seeds were continuously stirred during the sterilization and rinsing process.

Megagametophytes containing developing zygotic embryos were used as the explant for culture initiation. The seed coats of individual seeds were cracked open under a laminarflow hood with the use of a sterile hemostat. The intact megagametophyte (which contains the developing zygotic embryos) was removed from the opened seed coat with forceps.

Tissues attached to the megagametophyte, such as the megagametophyte membrane and the nucellus, were removed from the megagametophyte and discarded. The megagametophyte was placed on culture medium (longitudinal axis of megagametophyte parallel to the surface of culture medium) with forceps. The micropyle end of the megagametophyte was placed in contact with (but not submerged in) the culture medium.

Basal salt mixtures which have proven effective for culture initiation include the basal salts formulations listed in Table I. (The complete formulations of the media used in the Examples are listed in Table The pH of the medium was adjusted to 5.8 with KOH and HCl prior to autoclaving at 110 kPa (16 psi) and 121 C for 20 minutes. Aqueous stock solutions ofL-glutamine were filter sterilized and added to warm (about 60°C) medium prior to pouring the medium into culture dishes. Approximately 20 ml of medium was poured into 100 x 15 mm sterile plastic petri dishes.

C.

18

-N)

I

19 Case Docket No. FSL 96-2 TABLE II Comosition Of IMainte ce MediComonl Used In The ample Initiation Semi-Sotid Liquid Medium Maintenance Maintenance COMPONENT DCRI Medium Medium

DCR

1 DCR, Basal medium a DCR CR r DCR

DCR

CONCENTRATION (g/L) Inositol 0.50 0.50 0.50 50 ___0.50 Casein 0.50 0 hydroysate 0.50 0.50 Sucrose 30.00 30.00 30.00 o 30.00 10 GELRITEb 1.25 2.00 o Activated Carbon 0 0 CONCENTRATION (mg/L) Auxinc 3.00 3.00 3.00 3 -3 0 3.00 Cytokinind 0.50 O50.5 0.50 a) Refer to Table I for composition of basal medium.

b) GELRITE (gellan gum manufactured by Merck, Inc.) c) 2 4 -dichlorophenoxyacetic acid d) N 6 -benzylaminopurine [or N 6 -benzyladenine

(BAP)].

After megagametophyte explants were placed in culture, the perimeter of the dishes were sealed with two wraps of PARAFILM® (manufactured by American Can The dishes were incubated in the dark at a constant temperature of 23C. After about 7 to 21 days, embryogenic tissue extruded from the micropyle of the megagametophyte explants.

After 28 days in culture embryogenic tissue was removed from responsive megagametophyte explants and moved to a new position on the same culture dish, or the embryogenic tissue was transferred to a new culture dish containing the same culture medium as used for initiation.

Each individual culture derived from an individual megagametophyte explant was kept separate and assigned a cell line identification code.

Cultures were maintained on semi-solid medium DCR, (Table II) by subculturing masses ofembryogenic tissue every 14 to 21 days to fresh medium. Culture maintenance conditions were the same as for culture initiation, except that the gelling agent levels contained in the culture maintenance media were increased.

Case Docket No. FSL 96-2 At the end of a two to three week period of subculturing on DCR maintenafice medium, masses of embryogenic tissue (about 200 mg each) were transferred to a MSG 1 development medium (see Table III below) containing 11 mg/L of ABA and no activated carbon. All cultures were incubated at 23 'C in the dark. It is preferred that the cultures be incubated in the dark rather than light condition. The embryogenic tissue was transferred to fresh embryo development medium as often as needed in order to keep the concentration of ABA in the media from being reduced. After two passages on the MSG, medium, cotyledonary somatic embryos (stage 3) were visible on the surface of the embryogenic tissue.

Typically, multiple harvests of cotyledonary somatic embros were made at the end of the vy au 1 d t me end oI the second and third transfers, and sometimes after the fourth transfer onto MSG, medium.

Subsequently the embryogenic tissue became necrotic and produced very few, if any, cotyledonary somatic embryos on MSG, medium and the embryogenic tissue was discarded.

a a *a* Case Docket No. FSL 96-2 TABLE III Composition of Development and Germination MediasedIn The Examp Development Development Development Germination Medium 1 Medium 2 Medium 3 Medium MSG, MSG, MSG3 MSG IVlVJ 4

COMPONENT

Basal medium MSG MSG MSG

MSG

CONCENTRATION (g/L) Ammonium 0 nitrate 0 Inositol 0.10 0.10 0.10 0.10 L-glutamine 1.45 1.45 1.45 Sucrose 3 30.00 Maltose 60.00 60.00 60.00 GELRITE b 2.00 2.00 2.00 2.00 Activated 0-1.25 0-1.25 0-1.25 5.00 carbon PEG 0-100.00 CONCENTRATION (mg/L)

ABA

d 11-150 125 21a) Refer to Table I for composition of basal medium.

b) GELRITE® (gellan gum manufactured by Merck, Inc.).

20 c) Polyethylene glycol (molecular weight of 4000).

d) Abscisic acid.

The effect of the level of polyethylene glycol (PEG) contained in the development medium on the production of harvestable stage 3 somatic embryos (SEs) of Pinus taeda were evaluated using four different embryogenic culture genotypes (listed as A, B, C, and and the results recorded in Table IV below. The mean values listed in Table IV are based on the results obtained using three culture plates for each different PEG level and culture line.

21 22- Case Docket No. FSL 96-2 TABLE IV Effect Of Polyethylene Gvlcol Levels On Somatic Embro PEG Levels Mean Number of Stage 3 SEs Harvested Per Culture Plate A B C D 0 0 4 4 3 2 14 25 6 3 32 20 66 9 2 35 11 39 The results listed in Table IV clearly show that the inclusion of PEG in the embryo development media resulted in the production of significantly higher numbers of stage 3 somatic embryos. For example, embryo production was increased on these culture lines by an average of 225% when a 6% level of PEG was included in the development media.

EXAMPLE 2 The methods claimed in U. S. Patents No. 5,491,090 were followed in this example.

However, evaluations were conducted to determine what effect the addition of polyethylene glycol to the embryo development media might have on the production of stage 3 somatic embryos.

Immature seed cones were collected from several different loblolly pine (Pinus taeda .20 sources located in Westvaco's South Carolina coastal breeding orchards near Charleston, South Carolina. The seed cones were collected when the dominant zygotic embryo was at the precotyledonary stage of development. Using the classification system of von Arnold and Hakman (1988), the dominant zygotic embryo at this stage is referred to as being at stage 2; *:that is, an embryo with a prominent embryonic region with a smooth and glossy surface, subtended by elongated suspensor cells which are highly vacuolated. However, zygotic embryos at an earlier stage of development (stage 1) may also be used effectively to initiate embryogenic cultures.

Seed cones were harvested from selected trees, placed in plastic bags and stored at 4C until used for culture initiation. If the cones were stored for more than two weeks at 30 4 0 C, they were aired and dried out weekly (placed at 23 C, ambient laboratory conditions for 23 Case Docket No. FSL 96-2 2-3 hours) to prevent growth of fungi on the surface of the cones and concomitant deterioration of seed quality.

For culture initiation, intact seeds removed from seed cones were surface sterilized by treatment in a 10 to 20% commercial bleach solution (equivalent of a 0.525% to 1.050% sodium hypochlorite solution) for 15 minutes followed by three sterile water rinses (each of five minutes duration). Seeds were continuously stirred during the sterilization and rinsing process.

Megagametophytes containing developing zygotic embryos were used as the explant for culture initiation. The seed cot. of individual were cracked open under a lamarcracKeu open under a lam inarflow hood with the use of a sterile hemostat. The intact megagametophyte (which contains the developing zygotic embryos) was removed from the opened seed coat with forceps.

Tissues attached to the megagametophyte, such as the megagametophyte membrane and the nucellus, were removed from the megagametophyte and discarded. The megagametophyte was placed on culture medium (longitudinal axis of megagametophyte parallel to the surface of culture medium) with forceps. The micropyle end of the megagametophyte was placed in contact with (but not submerged in) the culture medium.

Basal salt mixtures which have proven effective for culture initiation include the basal salts formulations listed in Table I. (The complete formulations of the media used in the Examples are listed in Table The pH of the medium was adjusted to 5.8 with KOH and HCI prior to autoclaving at 110 kPa (16 psi) and 121 C for 20 minutes. Aqueous stock solutions of L-glutamine were filter sterilized and added to warm (about 60°C) medium prior to pouring the medium into culture dishes. Approximately 20 ml of medium was poured into 100 x 15 mm sterile plastic petri dishes. The basal media modified for each of the culture i stages are listed in Tables II and III above.

Embryogenic tissue cultures from two loblolly pine sources were initiated on semisolid DCRi medium containing 3.0 mg/L 2,4-D, 0.5 mg/L BAP, and 0.125% GELRITE.

Once cultures were extruded and subcultured, they were kept on the above medium but with the GELRITE concentration increased to After 10-22 months on this semi-solid maintenance medium, the callus clumps were placed in DCR 2 liquid maintenance medium 0 containing 3 mg/L 2,4-D, 0.5 mg/L BAP, and 0.5 g/L activated carbon (as taught in U. S.

Patent No. 5,491,090). These were maintained by subculturing to fresh DCR 2 liquid medium every 1 to 2 weeks.

After 16 weeks in liquid culture, 5 lines from these two sources were plated on GELRITE-solidified MSG, development media in which the levels of PEG, ABA, and 3 5 activated carbon were varied in order to assess the ability of the respective cultures to develop high quality harvestable stage 3 embryos. A sterile 90 mm sterile NITEX nylon membrane Case Docket No. FSL 96-2 disk (#3-35/16XX, commercially available from Tetko, Inc.) was placed in a sterile Buchner funnel. Three 40 mm nylon disks were placed on top of this larger nylon disk in the funnel equidistant from one another but not touching. One ml of suspension culture cells and medium were pipetted onto each of the 40 mm disks. The liquid medium was suctioned from the cells using a mild vacuum. Each 40 mm nylon disk with cells was removed from the Buchner funnel and placed on GELRITE solidified MSG, development medium (see Table III) in 100 X 25 mm plastic petri dishes. Dishes were incubated in a dark growth chamber at 23 C. The nylon disks were then transferred to new petri dishes containing fresh medium every 3 weekq Th11) 1- 1 every 3e There re 12 disks u f uspension culture cells from each line placed on each treatment medium.

Between weeks 6 and 12, stage 3 embryos were counted and those deemed suitable for germination were harvested. The results are listed in Table V below.

TABLE V Effect Of Polyethylene Glycol Levels On Somatic Embr Development Media' Harvest 1 2 3 4 5 6 Total Tota Harvest 1 95 647 136 426 780 1291 3375 Harvest 2 731 944 313 1054 1135 2008 6185 Harvest 3 186 314 162 499 74 697 1882 Sum 1012 1905 611 1929 1989 3996 11442 *Media Formulations: 1 MSG basal, no PEG, 21 mg/L ofABA, no activated carbon.

2 MSG basal, 7% PEG, 21 mg/L of ABA, no activated carbon.

3 MSG basal, no PEG, 125 mg/L of ABA, no activated carbon.

4 MSG basal, 7% PEG, 125 mg/L ofABA, no activated carbon.

5 MSG basal, no PEG, 125 mg/L of ABA, 1.25 g/L of activated carbon.

6 MSG basal, 7% PEG, 125 mg/L of ABA, 1.25 g/L of activated carbon.

The results listed in Table V show that the inclusion of PEG in the embryo development media resulted in the production of significantly higher numbers of stage 3 somatic embryos when compared to the number of embryos produced on media not S'3 0 containing

PEG.

24

J

Case Docket No. FSL 96-2 EXAMPLE 3 Germination of the stage 3 somatic embryos produced in Examples 1 and 2 from both the PEG-containing embryo development media and the control media (which did not contain PEG) was attempted utilizing the methods claimed in U. S. Patents Nos.5,413,930, 5,491,090, and 5,506,136. First, the harvested stage 3 somatic embryos were transferred with forceps to sterile NITEX nylon membranes and were then partially dried by exposing the embryos to an atmosphere having a high relative humidity for a period of about 2 to 5 weeks.

The partially dried somatic embryos were then placed horizontally on the surface of

MSG

4 medium. The medium was in 100 x 15 mm sterile plastic petri plates. Typically, about 16 to 25 somatic embryos were placed in each plate. The perimeter of plates were wrapped twice with PARAFILM. Plates with embryos were incubated in the dark at 23 0 C in an attempt to initiate germination. After about 10 to 14 days a number of the embryos produced using the control (no PEG) development media proceeded to elongate to approximately 1 to 2 cm. At this time the germination process had begun, with the emergence of the radicle (root) on some somatic embryos. Plates with the germinating somatic embryos were then transferred to a 16-hour fluorescent light and 8-hour dark photoperiod at However, no germination occurred in those somatic embryos which had been produced on PEG-containing development media. These unexpected results showed that the use of PEG at the noted levels in culture media throughout the development step somehow caused the resulting stage 3 somatic embryos to exhibit a germination block. To overcome oothis germination block, a novel method which utilizes two separate embryo development steps was developed (see Examples 4-10 below).

EXAMPLE 4 o.*,25 Immature seed cones were collected from a hybrid pine (Pinus rigida x Pinus taeda) seed source located in Westvaco's South Carolina coastal breading orchard near Charleston, 0 South Carolina. These seed cones were utilized to produce embryogenic tissue in liquid so suspension via the method taught in Example 2 above.

Suspension culture cells were established to nylon disks as taught in Example 2 above and transferred to different embryo development media for testing. All cultures were ~incubated at 23 C in the dark.

One culture group (a control designated Group 1) was developed on a MSG development media (MSG, Table III above) containing 125 mg/L ABA and no PEG for a period of 9 Case Docket No. FSL 96-2 weeks. A second culture group (a control designated Group 4) was developed on the same MSG, development media, but containing 7% PEG, for the same amount of time.

Another group (Group 2) was cultured on a first embryo development medium (MSG,) which contained 7% PEG for a 3 week period. The resulting pre-stage 3 somatic embryos were then transferred to a second embryo development medium

(MSG

2 containing no PEG for a period of 6 weeks.

The final group (Group 3) was cultured on a first embryo development medium (MSG,) which contained 7% PEG for a 6 week period. The resulting pre-stage 3 somatic em.b.. were ten ultnferred to a second embryo development medium

(MSG

2 containing no PEG for a 3 week period.

After the end of the 9 weeks, the number of stage 3 somatic embryos produced by each group were tallied. Germination of these stage 3 somatic embryos was then attempted utilizing the methods outlined in Example 3 above. The results are listed in Table VI below.

TABLE

VI

Effect Of Embryo Development Steps On Somatic Embryo Production and Germinatin PEG Treatment of Embryos Germination (weeks) Developed Group 1 12 55.7 20 Group 2 27 60.9 Group 3 68 33.0 Group 4 67 The results listed in Table VI show that cultures grown on development media containing PEG (Groups 2, 3, and 4) produced significantly higher numbers of stage 3 somatic embryos than did the cultures grown on media which did not contain PEG (Group 1).

Also, the embryos developed for the full 9 weeks on PEG containing media (Group 4) exhibited the germination block. However, the embryos produced utilizing the novel two step development method (Groups 2 and 3) overcame the block and exhibited improved germination characteristics.

26 Case Docket No. FSL 96-2 EXAMPLE Immature seed cones were collected from one loblolly pine (Pinus taeda seed source located in Westvaco's South Carolina coastal breading orchard near Charleston, South Carolina. These seed cones were utilized to produce embryogenic tissue masses via the method taught in Example 2 above.

Suspension culture cells were established to nylon disks as taught in Example 2 above and transferred to different embryo development media for testing. All cultures were incubated at 23 °C in the dark.

One culture group (a control designated Group 1) was developed on MSG development media (MSG 2 Table III above) containing 125 mg/L ABA and no PEG for a period of 9 weeks. A second culture group (a control designated Group 4) was developed on the same MSG, development media, but containing 7% PEG, for the same amount of time.

Another group (Group 2) was cultured on a first embryo development medium (MSG,) which contained 7% PEG for a 3 week period. The resulting pre-stage 3 somatic embryos were then transferred to a second embryo development medium (MSG) containing no PEG for a period of 6 weeks.

.:The final group (Group 3) was cultured on a first embryo development medium (MSG,) which contained 7% PEG for a 6 week period. The resulting pre-stage 3 somatic embryos were then transferred to a second embryo development medium

(MSG

2 containing no PEG for a 3 week period.

After the end of the 9 weeks, the number of stage 3 somatic embryos produced by each group were tallied. Germination of these stage 3 somatic embryos was then attempted 0utilizing the methods outlined in Example 3 above. The results are listed in Table VII below.

TABLE VII S. febDevelo ent Stes n SomaticEmb Prductio PEG Treatment of Embryos Germination 0.0o4 "(weeks) Developed Group 1(0) 31 28.5 Group 2 29 36.1 Group 3 25 28.8 Group 4 44 0.0 Case Docket No. FSL 96-2 The results listed in Table VII again show that the embryos developed for the full 9 weeks on PEG containing media (Group 4) exhibited the germination block. However, the embryos produced utilizing the novel two step development method (Groups 2 and 3) overcame the block and exhibited improved germination characteristics.

EXAMPLE 6 Immature seed cones were collected from one loblolly pine (Pinus taeda seed source located in Westvaco s South Carolina coastal breading orchard near Charleston, South Carolina. These seed cones were utilized to produce embryogenic tissue masses via the method taught in Example 2 above.

1 0 o Suspension culture cells were established to nylon disks as taught in Example 2 above and transferred to different embryo development media for testing. All cultures were incubated at 23 °C in the dark.

One culture group (a control designated Group 1) was developed on MSG development media (MSG 2 Table III above) containing 125 mg/L ABA and no PEG for a period of 9 weeks. A second culture group (a control designated Group 4) was developed on the same MSG, development media, but containing 7% PEG, for the same amount of time.

Another group (Group 2) was cultured on a first embryo development medium (MSG,) which contained 7% PEG for a 3 week period. The resulting pre-stage 3 somatic embryos were then transferred to a second embryo development medium (MSG 2 containing no PEG for a period of 6 weeks.

The final group (Group 3) was cultured on a first embryo development medium (MSG,) which contained 7% PEG for a 6 week period. The resulting pre-stage 3 somatic embryos were then transferred to a second embryo development medium (MSG 2 containing no PEG for a 3 week period.

After the end of the 9 weeks, the number of stage 3 somatic embryos produced by each group were tallied. Germination of these stage 3 somatic embryos was then attempted utilizing the methods outlined in Example 3 above. The results are listed in Table VIII below.

28 Case Docket No. FSL 96-2 TABLE VIII Effect Of Emry Develoment Steps On Somatic Embryo Production PEG Treatment of Embryos Germination (weeks) Developed Group 1(0) 23 Group 2 48 12.0 Group 3 64 18.4 Group 4 84 5.3 The results listed in Table VIII show that cultures grown on development media containing PEG (Groups 2, 3, and 4) produced significantly higher numbers of stage 3 somatic embryos than did cultures grown on development media which did not contain PEG (Group Also, the embryos developed for the full 9 weeks on PEG containing media (Group 4) again exhibited a germination block. However, the embryos produced utilizing the novel two step development method (Groups 2 and 3) overcame the block and exhibited improved germination characteristics.

EXAMPLE 7 A number of the stage 3 somatic embryos from Example 2 which had been cultured on development media containing PEG Groups 2, 4, and 6) were divided into two s samples. The first sample was immediately subjected to the partial drying protocol and the germination protocol taught in Example 3 above.

The second sample, however, was subjected instead to a cold treatment protocol prior to undergoing the partial drying and germination steps. This cold treatment step consisted of culturing the somatic embryos in the dark on a second development medium (MSG 3 Table SIII) which contained 21 mg/1 of ABA, no PEG, and no activated carbon, for four weeks at a temperature of 4°C.

After the respective samples had undergone the germination protocol for about 8 weeks, the somatic embryos were examined for germination. The results were tallied and are listed in Table IX below.

Case Docket No. FSL 96-2 TABLE IX Efet eatme on the Germination o

GERMINATION

Development Media No Cold Treatment Cold Treatment 2 0 46 4 0 42 6 0 34 Average 0 41 *Media Formulations: 2 MSG basal, 7% PEG, 21 mg/1 of ABA, no activated carbon.

4 MSG basal, 7% PEG, 125 mg/l of ABA, no activated carbon.

6 MSG basal, 7% PEG, 125 mg/1 of ABA, 1.25 g/1 of activated carbon.

The PEG block is clearly illustrated by the fact that no germination occurred in those somatic embryos which had not undergone the cold treatment. However, germination was achieved in a significant number of those somatic embryos produced on PEG-containing development media when these embryos were subjected to the additional cold treatment step.

EXAMPLE 8 Immature seed cones were collected from a hybrid pine (Pinus rigida x Pinus taeda) seed source located in Westvaco's South Carolina coastal breading orchard near Charleston, South Carolina. The seed cones were collected when the dominant zygotic embryo was at the precotyledonary stage of development. Using the classification system of von Arnold and Hakman (1988), the dominant zygotic embryo at this stage is referred to as being at stage 2; that is, an embryo with a prominent embryonic region with a smooth and glossy surface, subtended by elongated suspensor cells which are highly vacuolated. However, zygotic embryos at an earlier stage of development (stage 1) may also be used effectively to initiate embryogenic cultures.

Seed cones were harvested from selected trees, placed in plastic bags and stored at 4'C until used for culture initiation. If the cones were stored for more than two weeks at 4 C, they were aired and dried out weekly (placed at 23 ambient laboratory conditions for 2-3 hours) to prevent growth of fungi on the surface of the cones and concomitant deterioration of seed quality.

Case Docket No. FSL 96-2 For culture initiation, intact seeds removed from seed cones were surface sterilized by treatment in a 10 to 20% commercial bleach solution (equivalent of a 0.525% to 1.050% sodium hypochlorite solution) for 15 minutes followed by three sterile water rinses (each of five minutes duration). Seeds were continuously stirred during the sterilization and rinsing process.

Megagametophytes containing developing zygotic embryos were used as the explant for culture initiation. The seed coats of individual seeds were cracked open under a laminarflow hood with the use of a sterile hemostat. The intact megagametophyte (which contains the developing ygotic embryos) was removed from the opened seed coat with forceps.

Tissues attached to the megagametophyte, such as the megagametophyte membrane and the nucellus, were removed from the megagametophyte and discarded. The megagametophyte was placed on culture medium (longitudinal axis of megagametophyte parallel to the surface of culture medium) with forceps. The micropyle end of the megagametophyte was placed in contact with (but not submerged in) the culture medium.

Basal salt mixtures which have proven effective for culture initiation include the basal salts formulations listed in Table I. (The complete formulations of the media used in the Examples are listed in Table The pH of the medium was adjusted to 5.8 with KOH and HCI prior to autoclaving at 110 kPa (16 psi) and 121 C for 20 minutes. Aqueous stock solutions of L-glutamine were filter sterilized and added to warm (about 60 C) medium prior to pouring the medium into culture dishes. Approximately 20 ml of medium was poured into S100 x 15 mm sterile plastic petri dishes.

After megagametophyte explants were placed in culture, the perimeter of the dishes were sealed with two wraps of PARAFILM® (manufactured by American Can The dishes were incubated in the dark at a constant temperature of 23 C. After about 7 to 21 5 days, embryogenic tissue extruded from the micropyle of the megagametophyte explants.

After 28 days in culture embryogenic tissue was removed from responsive megagametophyte Sexplants and moved to a new position on the same culture dish, or the embryogenic tissue was transferred to a new culture dish containing the same culture medium as used for initiation.

Each individual culture derived from an individual megagametophyte explant was kept "30 separate and assigned a cell line identification code.

Cultures were maintained on semi-solid medium, DCR, (Table II) by subculturing masses of embryogenic tissue every 14 to 21 days to fresh medium. Culture maintenance conditions were the same as for culture initiation, except that the gelling agent levels contained in the culture maintenance media were increased.

After 10-22 months on this semi-solid maintenance medium, the callus clumps were placed in DCR 2 liquid maintenance medium containing 3 mg/1 2,4-D, 0.5 mg/l BAP, and 31 z^u 32- Case Docket No. FSL 96-2 g/l activated carbon (as taught in U. S. Patent No. 5,491,090). These were maintained by subculturing to fresh DCR, liquid medium every 1 to 2 weeks. After 16 weeks in liquid culture, suspension culture cells were harvested. A sterile 90 mm sterile NITEX nylon membrane disk (#3-35/16XX, commercially available from Tetko, Inc.) was placed in a sterile Buchner funnel. Three 40 mm nylon disks were placed on top of this larger nylon disk in the funnel equidistant from one another but not touching. One ml of suspension culture cells and medium were pipetted onto each of the 40 mm disks. The liquid medium was suctioned from the cells using a mild vacuum.

Three 40 mm disks were transferred to ptri lates containing MSG medium (Table III above) containing 7% PEG, 125 mg/I ABA and 1.25 g/l activated carbon. All cultures were incubated at 23 °C in the dark. The embryogenic tissue was transferred to fresh embryo development medium as often as needed in order to keep the concentration of ABA in the media from being reduced. After two passages on the MSG, medium, cotyledonary somatic embryos (stage 3) were visible on the surface of the embryogenic tissue. Typically, multiple harvests of cotyledonary somatic embryos were made at the end of the second and third passage, and sometimes after the fourth passage on MSG, medium.

The stage 3 somatic embryos were divided into two groups for testing. The first group of stage 3 somatic embryos were first partially dried in a high relative humidity environment for three weeks, then germinated directly.

The partially dried somatic embryos were placed horizontally on the surface of MSG 4 medium (Table III). The medium was in 100 x 15 mm sterile plastic petri plates. Typically, about 25 somatic embryos were placed in each plate. The perimeter of plates were wrapped twice with PARAFILM. Plates with embryos were incubated in the dark at 23°C in an attempt to initiate germination.

The second group was subjected to a cold treatment consisting of culturing the somatic embryos in the dark on a second development medium (MSG 3 Table III) containing .i 21 mg/l ABA, no PEG and no activated carbon for four weeks at a temperature of 4°C.

Afterwards, these somatic embryos were subjected to the same partial drying and germination treatments as had the first group. The results were tallied and are listed in Table X below.

a.3 32 jj Case Docket No. FSL 96-2 TABLE X Effect of Cold Treatmentn the Germinatin of Somatic Embryos

GERMINATION

Development Media No Cold Treatment Cold Treatment MSG (basal) 7% PEG 0.5 49.0 _49.0 The PEG block allowed almost no germination to occur in those somatic embryos not subjected to the cold treatment step. However, germination was achieved in a high frequency of the somatic embryos which underwent the additional cold treatment step.

EXAMPLE 9 Immature seed cones were collected from loblolly pines located in Westvaco's South SCarolina coastal breeding orchard near Charleston, South Carolina. Following the procedures Staught in Example 2 above, these seed sources were used to produce stage 3 somatic embryos from five different cell lines. These embryos were developed on development media (MSG,) which contained 7% PEG, 125 mg/l ABA and 1.25 g/1 activated carbon. The embryos were then split into five different groups and subsequently subjected to differing periods of cold treatment on MSG 3 medium with 21 mg/1 ABA, but without PEG or activated carbon.

Harvested embryos were partially dried, and germinated. The results were tallied and are listed in Table XI below.

3 *3 33 9-) Case Docket No. FSL 96-2 TABLE XI Effect of Cold Treatment on the Germination of Somatic Embryo Treatment Number of weeks Germination Malformed Groups in Cold Treatment 1 0 0 2 1 14 18 3 2 31 4 4 38 0 6 36 0 The results in Table XI clearly show the effects of the germination block caused by PEG as well as the successful surmounting of that block via the employment of the cold treatment step. Malformed germinants were twisted and of a red color, typical of embryos developed on PEG medium that do not germinate.

Plants were raised from all treatments using methods described in Example 10 below and planted in the field.

EXAMPLE Immature seed cones were collected from six different loblolly pine and six different hybrid pine (Pinus rigida x Pinus taeda) seed sources located in Westvaco's South Carolina coastal breading orchard near Charleston, South Carolina. These seed sources were used to produce stage 3 somatic embryos by following the procedures taught in Examples 2, 3, and 4 above. These embryos were developed on three different development media, all of which contained 7% PEG. The stage 3 embryos were subsequently cultured in the dark on a second development medium (MSG 3 containing 21 mg/l of ABA (but with no PEG or activated carbon) for four weeks at a temperature of 4°C. The embryos were partially dried, germinated, and tallied (with the results listed in Table XII).

a Case Docket No. FSL 96-2 TABLE XII Embryo Production pr 0.3 ml of EmbrYvo.geni Tissue

LINES

1 EMBRYO DEVELOPMENT MEDIA 2 A B C Lob 1 36.6 96.7 149.2 Lob 2 3.9 1.3 9.7 LobD 39.2 48.3 99.9 Lob 4 8.0 7.0 17.4 Lob 5 20.5 17.3 34.8 Lob 6 1.7 0.7 14.2 PxL 1 61.3 150.3 121.9 PxL 2 44.4 105.4 111.8 PxL 3 8.8 17.7 11.4 PxL 4 68.0 77.0 116.2 PxL 5 23.4 15.6 25.3 PxL6 10.6 5.3 46.9 Total 367.4 667.6 883.7 1. Lob Loblolly pine.

PxL Pitch pine x loblolly pine hybrid.

2. A MSG basal, 7% PEG, 21 mg/1 of ABA, no activated carbon.

0 B MSG basal, 7% PEG, 125 mg/l of ABA, no activated carbon.

C MSG basal, 7% PEG, 125 mg/1 of ABA, 1.25 g/l of activated carbon.

S.i: Following the tally, several of the germinated embryos were converted into acclimatized plants and field planted in the following manner. When the length of the roots reached about 2 to 3 cm, the germinating plantlets from four of the loblolly lines and six of the pitch x loblolly lines were aseptically removed from the plates and planted into sterilized potting mix in MAGENTA BOXES (containers manufactured by Magenta Corp.). The boxes containing plantlets were sealed with PARAFILM and placed in a growth chamber with a 16hour fluorescent and incandescent light and an 8-hour dark photoperiod at 23 C.

When the plantlets formed epicotyls (newly formed shoots approximately 2 to 4 cm), they were transferred to leach tubes (RAY LEACH "CONE-TAINERS"®

#SSCUV

manufactured by Stuewe Sons, Inc.). Plantlets in boxes were transplanted into leach tubes 39) Case Docket No. FSL 96-2 containing a potting mix (2:1:2 peat:perlite:vermiculite, containing 602 g/m' OSMOCOTE® fertilizer (18-6-12), 340 g/m 3 dolomitic lime and 78 g/m 3 MICRO-MAX® micronutrient mixture (manufactured by Sierra Chem. The leach tubes were placed in a greenhouse mist chamber. The environmental conditions in the mist chamber are as follows: Mist Swas applied for 30 seconds every 30 minutes from 6:00 a.m. to 6:30 and for 30 seconds every 60 minutes from 6:30 p.m. to 6:00 Temperature was maintained at 26 to 31 C during the day and at 18 to 20°C at night; and Ambient light was admitted through black polypropylene shade cloth (51% shade) covering the greenhouse. Supplemental light from high pressure sodium bulbs was nrovided to nroduce a total photoperiod of about 16 hours.

When the plantlets had grown to approximately 8 to 16 cm in height, trays containing the resulting plants in leach tubes were removed from the mist chamber and placed on an open bench in the greenhouse for at least two weeks for acclimatization. Subsequently, the plants in leach tube trays were moved to a shadehouse (framed structure covered with black polypropylene shade cloth) for approximately two weeks, and then to ambient outdoor conditions for an additional two weeks. Acclimatized plants were planted to a prepared field site.

Many modifications and variations of the present invention will be apparent to one of ordinary skill in the art in light of the above teachings. It is therefore understood that the scope of the invention is not to be limited by the foregoing description, but rather is to be 2 0 defined by the claims appended hereto.

S

*e~A, Case Docket No. FSL 96-2

BIBLIOGRAPHY

Becwar, E. E. Chesick, L. W. Handley, M. R. Rutter. Method for regeneration of coniferous plants by somatic embryogenesis U. S. Patent No. 5,413,930 issued May 9, 1995.

Becwar, E. E. Chesick, L. W. Handley, M. R. Rutter. Method for regeneration of coniferous plants by somatic embryogenesis. U. S. Patent No. 5,506,136 issued April 9, 1996.

Becwar, M. R. Nagmani, and S. R. Wann. Initiation ofembryogenic cultures and somatic embryo development in loblolly pine (Pinus taeda). Canadian Journal ofForest Research 20:810-817, 1990.

Coke, J. E. Basal Nutrient Medium for In Vitro Cultures of Loblolly Pines. U. S. Patent No.

5,534,433 issued July 9, 1996.

Gupta, P. K. and D. J. Durzan. Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Reports 4:177-179, 1985.

Hakman, I. and S. von Arnold. Plantlet regeneration through somatic embryogenesis in Picea abies (Norway spruce). Journal ofPlant Physiology 121:149-158, 1985.

o* Hakman, L. C. Fowke, S. von Arnold, and T. Eriksson. The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies (Norway spruce).

20 Plant Science 38:53-59, 1985.

o Handley, M. R. Becwar, E. E. Chesick, J. E. Coke, A.P Godbey and M. R. Rutter.

Research and Development of Commercial Tissue Culture Systems in Loblolly Pine. TAPPI Journal 78.5 (1995): 169-75.

Handley, L.W. and A.P Godbey. Embryogenic Coniferous Liquid Suspension Cultures. U.

S. Patent No. 5,491,090 issued February 13, 1996.

Case Docket No. FSL 96-2 Preston, R. J. North American Trees, 4th edition. Iowa State Univ. Press, Ames. pp. 4-7, 1989.

Schenk, R. U. and A. C. Hildebrandt. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Canadian Journal of Botany 50:199-204, 1972.

Tautorus, T. L. C. Fowke, and D. I. Dunstan. Somatic embryogenesis in conifers.

Canadian Journal of Botany 69:1873-1899, 1991.

von Arnold, S. and I. Hakman. Regulation of somatic embryo development in Picea abies by abscisic acid (ABA). Journal ofPlant Physiology 132:164-169, 1988.

0 Case Docket No. FSL 96-2 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. An improved method for reproducing plants selected from the group consisting of Pinus taeda, Pinus serotina, Pinus palustris, Pinus elliottii, Pinus rigida, and hybrids thereof, by somatic embryogenesis which comprises: placing a suitable explant selected from the group consisting of immature zygotic embryos and megagametophytes containing immature zygotic embryos on culture initiation medium containing a sufficient amount of nutrients, 0.1 to 5.0 mg/L of auxin, 0.1 to 1.0 mg/L of cytokinin, 10. to 40.0 of a sugar selected from the V 6/ V1. d 6ugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, and a gelling agent selected from the group consisting of 2.5 to 4.5 g/L of agar, 0.5 to g/L of gellan gum, 3.0 to 5.0 g/L of agarose, and combinations thereof, for 2 to 14 weeks under suitable environmental conditions to grow a culture containing embryogenic tissue; transferring the embryogenic tissue culture to culture maintenance medium containing a sufficient amount of nutrients, 0.1 to 5.0 mg/L of auxin, 0.1 to 1.0 mg/L of cytokinin, 10.0 to 40.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose and combinations thereof, and a gelling agent selected from the group consisting of 6.0 to 9.0 g/L of agar, 1.75 to 4.0 g/L ofgellan gum, to 8.0 g/L ofagarose, and combinations thereof, for a sufficient amount of time under suitable environmental conditions to develop a mass of embryogenic tissue having a weight of at least 100.0 mg; transferring at least 100.0 mg of the mass of embryogenic tissue to a first embryo development medium containing a sufficient amount of nutrients, a gelling agent selected from the group consisting of 6.0 g/L to 12.0 g/L ofagar, 1.75 g/L to 4.0 g/L of gellan gum, 6.0 g/L to 8.0 g/L of agarose, and combinations thereof, 20.0 g/L to 70.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, and wherein the improvement comprises the addition of up to about 10.0 g/L of activated carbon, about 10.0 g/L to about 100.0 g/L of polyethylene glycol, about 5.0 mg/L to about 300.0 mg/L of abscisic acid, and the 0 continued maintenance of the abscisic acid concentration at said level; for a sufficient time under suitable environmental conditions to develop pre-stage 3 somatic embryos; transferring the pre-stage 3 somatic embryos to a second embryo development medium containing a sufficient amount of nutrients, a gelling agent selected from the group consisting of 6.0 g/L to 12.0 g/L of agar, 1.75 g/L to 4.0 g/L of gellan gum, g/L to 8.0 g/L of agarose, and combinations thereof, 20.0 g/L to 70.0 g/L of a sugar

Claims (1)

1. 96-2 selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, up to about 10.0 g/L of activated carbon, about 5.0 mg/L to about 300.0 mg/L ofabscisic acid, and the continued maintenance of the abscisic acid concentration at said level; for a sufficient time under suitable environmental conditions to develop stage 3 somatic embryos; separating the stage 3 somatic embryos from the development medium and partially drying the embryos by exposing the embryos to an atmosphere having a high relative humidity for a period of about 2 to 5 weeks; transferring the partially dried somatic embryos to germination medium containing a sufficient amount of nutrients, up to 10.0 g/L of activated carbon, a gelling agent selected from the group consisting of 6.0 to 9.0 g/L of agar, 1.75 to 3.50 g/L of gellan gum, 6.0 to 8.0 g/L of agarose, and combinations thereof, and 20.0 to 40.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, and combinations thereof, for a sufficient time under suitable environmental conditions to germinate the partially dried embryos; converting the germinated embryos into acclimatized plants; and field planting the acclimatized plants. 2. The method of claim 1 wherein: said mass ofembryogenic tissue is maintained on the first embryo development 20 medium for a sufficient time under suitable environmental conditions to develop stage 3 somatic embryos; and the stage 3 somatic embryos are transferred to a second embryo development medium containing a sufficient amount of nutrients, a gelling agent selected from the group consisting of 6.0 g/L to 12.0 g/L of agar, 1.75 g/L to 4.0 g/L of gellan gum, g L to 8.0 g/L of agarose, and combinations thereof, 20.0 g/L to 70.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, up to about 10.0 g/L of activated carbon, up to about 100.0 mg/L of abscisic acid, and the continued maintenance of the abscisic acid concentration at said level; for a period of about 2 to about 12 weeks at a temperature S0 in the range of about 0 0 C to about 10 C and under suitable environmental conditions to maintain the viability of the stage 3 somatic embryos; prior to the stage 3 somatic embryos being separated from the development medium and being partially dried. 41 Case Docket No. FSL 96-2 3. The method of claim 1 wherein said embryogenic tissue culture is transferred to liquid suspension culture maintenance medium containing a sufficient amount of nutrients, 0.1 to 100.0 mg/L of auxin, 0.05 to 10.0 mg/L ofcytokinin, 5.0 to 100.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose and combinations thereof, and about 0.1 to 10.0 g/L of activated carbon, for a sufficient amount of time under suitable environmental conditions to develop a liquid embryogenic cell culture; and at least 30.0 mg of the liquid embryogenic cell culture is transferred to said first embryo development medium. V A AI I L I I I U I Lu l l 4. The method of claim 2 wherein said embryogenic tissue culture is transferred to liquid suspension culture maintenance medium containing a sufficient amount of nutrients, 0.1 to 100.0 mg/L of auxin, 0.05 to 10.0 mg/L ofcytokinin, 5.0 to 100.0 g/L of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose and combinations thereof, and about 0.1 to 10.0 g/L of activated carbon, for a sufficient amount of time under suitable environmental conditions to develop a liquid embryogenic cell culture; and at least 30.0 mg of the liquid embryogenic cell culture is transferred to said first embryo development medium. 5. The method of claim 1 wherein the first embryo development medium contains from about 02 0 50 g/L to about 80 g/L of polyethylene glycol. pp. The method of claim 1 wherein the first embryo development medium contains from about S125 mg/L to about 250 mg/L of abscisic acid. 7. The method of claim 1 wherein the abscisic acid concentrations in the embryo development media are continually maintained over time by again transferring the 25 embryogenic tissue at least once to fresh embryo development media containing an amount of abscisic acid which is at least equal to the amount of abscisic acid present in the initial embryo development medium. 8. The method of claim 1 wherein the abscisic acid concentrations in the embryo development media are continually maintained over time by adding additional abscisic acid to the embryo development media. 42 Case Docket No. FSL 96-2 9. The method of claim 1 wherein the embryo development media contain from about 0.5 g/L to about 5.0 g/L of activated carbon. The method of claim 1 wherein the embryogenic tissue has been cryopreserved. 11. The method of claim 3 wherein the stage 3 somatic embryos are maintained on the second embryo development medium for a period of about 3 to about 6 weeks at a temperature in the range of about OC to about 10 0 C. 12. The method of claim 4 wherein the stage 3 somatic embryos are maintained on the second embryo development medium for a period of about 3 to about 6 weeks at a temperature in the range of about OC to about 10 0 C. Dated this 17th day of December 1997 WESTVACO CORPORATION By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent Attorneys of Australia 00.. 0 S SOS* S r ABSTRACT Case Docket No. FSL 96-2 AN IMPROVED MEHOD FORREGENE N'OFCONIFEROS PANTS BY SOMATIC EMBRYOGENESS EMPLOYIG POLYETHYLENE GLYCOL This invention relates to a method for regeneration of coniferous plants. In particular, this invention relates to an improved method for producing and developing somatic embryos for somatic embryogenesis processes for nlants of the genus s a..nd n.s es s o tmus an rmPinus interspecies hybrids by including polyethylene glycol in the development media. This method is well suited for producing clonal planting stock useful for reforestation. **e9 *9