Method for Maturing Somatic Embryos for Planting in Natural Environments

Technical Field

The present invention relates generally to the asexual reproduction of plants for agriculture, such as crop, ornamental, and forestry plant production. More specifically, the invention relates to a method for the development of tissue culture-derived propagules that can be planted in a fashion similar to true seed.

Background of the Invention

Vegetative or asexual propagation has been used in agriculture throughout recorded history. The advantage of vegetative propagation over seed propagation is that only mitotic cell division occurs, so that each daughter cell contains the same genetic information as the parent. Hence, the progeny are clones of the parent. In contrast, seed propagation involves eiotic division and genetic recombination, such that the progeny contain genetic information from both parents. Asexual propagation is especially important for heterozygous crops, such as bananas, conifers, figs, oranges, grapes, and many others. See Hartmann, H.T. and D. Kester, Plant Propagation. Prentice-Hall, Englewood Cliffs, New Jersey (1975) . However, vegetative propagation is usually labor-intensive and slow, involving large areas of greenhouse or field space to maintain the mother plants. Although vegetative propagation is used for many crops, it is limited to those that have a high per-unit value. As a result another method, plant tissue culture, has been proposed and used for clonal propagation.

Tissue culture methods were developed because of the faster propagation rate and the requirement for significantly less space for maintaining and producing plants. Tissue culture techniques, including somatic embryogenesis and organogenesis, have been used extensively for clonal propagation of plants. The basic tissue culture process involves the following steps: 1) excise pieces of tissue from intact plants, 2) surface sterilize and place the tissue under a sterile environment with supplemental nutrients, carbohydrates such as sucrose, and other necessary growth components, 3) regenerate plants from the original tissue or from callus derived from the tissue via organogenesis or somatic embryogenesis, 4) grow the plants in vitro until sufficient leaves are developed for subsequent autotrophic growth, 5) harden the plants under controlled laboratory, growth chamber, or greenhouse conditions where the humidity can be regulated, and 6) transplant the resultant, hardened plant to a greenhouse or field environment for crop production. See Handbook of Plant Cell Culture, Evans, D.A. et al. editors, Vol. 1, Mac illan Publishing Co., New York (1983).

Once again, tissue culture technology, as with vegetative propagation, has been useful for many plants. However, the tissue culture process has several limitations; it requires many separate manipulations, the production of plants must be carefully performed in vitro to prevent premature water loss and wilting, and the process, though not as costly as vegetative propagation, is still too expensive for a broad variety of crops, particularly those for which the per-unit value (such as cost of seed) is medium to low. Additionally, it has not been possible to plant tissue culture-derived organogenic propagules or somatic

e bryos naturally and directly in the field, as one would plant true botanic seed.

The first somatic embryos were produced in 1958 from carrot tissue. See Reinert, J., "Mσrphogenese und Ihre Kontrolle an Gewebekulturen aus Carotten,"

Naturwissenschaft 4_5:344-345 (1958); and Steward, F. et al. , "Growth and Organized Development of Cultured Cells II. Organization in Cultures Grown from Freely Suspended Cells," Am. J. Bot. .45:705-708 (1958). Subsequently, somatic embryos have been produced from many plant species, including alfalfa, corn, cotton, conifers, and soybean. See Vasil, I., Cell Culture and Somatic Cell Genetics of Plants, Volumes 1-4, Academic Press, Orlando, Florida (1984, 1985, 1986 & 1987) and Bonga, J. and D. Durzan, Cell and Tissue Culture in Forestry. Volumes 1-3, Martinus Nijhoff Publishers, Dordrecht, Netherlands (1987) . Although somatic embryos have been produced for a large number of species, for only a portion of these have complete plants been produced. See Redenbaugh, K. , D. Slade, and J. Fujii, "Artificial Seeds," In: Int. Conf. on In Vitro Selection and Propagation of Economic Plants (Ilahi, I. and K. Hughes, eds.), University of Peshawar, Pakistan (1988).

Furthermore, success with plant production from tissue culture-derived propagules has generally been achieved only in an .in vitro environment. What has become apparent is that, although somatic embryos can be produced for many species, the developmental maturation of the embryos is incomplete and falls far short of the . maturation of zygotic embryos within true seeds.

Somatic embryos, after formation, undergo a form of precocious germination when placed on the appropriate culture medium. Consequently, somatic embryos have not been handled and planted naturally under greenhouse conditions, with the exception of a few examples in

which a small number of plants were produced. See Redenbaugh, K. , D. Slade, P. Viss, and J. Fujii, "Encapsulation of Somatic Embryos in Synthetic Seed Coats," HortSci. .22:803-809 (1987); Redenbaugh, K. B. Paasch, J. Nichol, M. Kossler, P. Viss, and . Walker, "Somatic Seeds: Encapsulation of Asexual Embryos," Bio/technology 4_:797-801 (1986). No plants are known to have been produced from somatic embryos planted naturally and directly under field conditions for any plant species.

To aid in the production of somatic embryos, researchers have adapted knowledge gained from zygotic embryogeny research. The premise has been that somatic embryogeny is and will be analogous to zygotic embryogeny. One of the important observations was that immature zygotic embryos of many species germinate precociously when removed from the seed and cultured in vitro. Inclusion of abscisic acid (ABA) in the in vitro medium is known to prevent this precocious germination. Since ABA also accumulates naturally within the developing zygotic embryos, it has been hypothesized that ABA functions in vivo to inhibit precocious germination. See Morris, P.C. et al. , "Determination of Endogenous Abscisic Acid Levels in Immature Cereal Embryos During in Vitro Culture," Planta 173:110-116 (1988).

In a similar manner, a high osmotic in vitro environment (for example, high levels of mannitol or sucrose in the medium) is known to inhibit precocious germination of zygotic embryos. hen ABA and mannitol were compared side-by-side with in vitro culture of zygotic embryos, identical responses were measured in morphology, fresh weight, protein content, and levels of agglutinin in the embryos. See Morris, P.C. et al. , "Changes in the Levels of Wheat and Barley-germ

Agglutinin During Embryogenesis In Vivo, In Vitro, and During Germination," Planta 166:407-413 (1985) . One major hypothesis about ABA and embryo development is that control of embryo development is not mediated directly by ABA, but through the action of the growth regulator on water uptake. Furthermore, Morris et al. (Ibid) postulated that when immature embryos are cultured upon media of high osmotic potential, they might adjust their endogenous ABA content so that the combined influence of the external osmoticum and endogenous growth substance inhibit precocious germination and allow continued embryo development. Consequently, ABA and osmotic agents would be expected to have a similar effect on embryo development. A further effect of ABA during mid- to late- embryogeny is a selective inhibition of synthesis and translation of specific mRNA's, apparently those involved with embryogenesis. ABA has been reported not to restrict accumulation of proteins, presumably because mRNA's for storage proteins are transcribed during early embryogenesis. Instead, ABA has been implicated in affecting partitioning of photosynthate and in not inhibiting storage protein mRNA's, thereby increasing levels of storage proteins in the zygotic embryos (Ackerson, R.C., "Regulation of Soybean Embryogenesis by Abscisic Acid," J. Exp. Bot. 3_5:403-413 (1984)).

Based upon these observations, researchers have added ABA to the cell culture medium employed during somatic embryo formation. See Bonga, J. and D. Durzan, Cell and Tissue Culture in Forestry, Volumes 1-3, Martinus Nijhoff Publishers Dordrecht, Netherlands (1987) and Vasil, I., Cell Culture and Somatic Cell Genetics of Plants, Volumes 1-4, Academic Press Orlando, Florida (1984, 1985, 1986 & 1987) . The use of ABA has been found to lead to improved morphology of the

somatic embryos, including enhanced synchrony of development.

However, despite the extensive research done with ABA on zygotic embryogeny, no reports or suggestions have appeared that indicate that ABA might actually improve the ability of embryos, either zygotic or somatic, to germinate under greenhouse or field conditions, outside an in vitro environment.

It has been reported that ABA and high sucrose levels in the media were important components for obtaining survival of carrot somatic embryos which were encapsulated and desiccated. (Janick, J. and S. Kitto, "Process for Encapsulating Asexual Plant Embryos," U.S. Patent 4,615,141) . However, these components were described as hardening treatments which were added to the carrot tissue cultures during the embryo induction phase and were not applied to the regenerated embryos at the precocious germination stage.

Furthermore, although this reference reported that the hardening treatments increased embryo survival after desiccation, the examples do not demonstrate actual plant production, but only that the embryos survived the desiccation. Similarly, plant production was not reported in published articles. See Kitto, S. and J. Janick, "Production of Synthetic Seeds by

Encapsulating Asexual Embryos of Carrot," J. Amer. Soc. Hort. Sci. 110:277-282 (1985) ; and Kitto, S. and J. Janick, "Hardening Treatments Increase Survival of Synthetically-Coated Asexual Embryos of Carrot," J. Amer. Soc. Hort. Sci. 110:283-286 (1985). Finally, no indication is given as to whether these hardening treatments might be useful for germination and conversion of somatic embryos under a natural environment such as a greenhouse or field.

Another aspect of zygotic embryogeny that has been used to enhance true seed germination is to treat seed prior to planting with an osmotic agent, such as polyethylene glycol or mannitol, or with gibberellic acid. Also, simple imbibition of the seeds has been shown to be beneficial for germination. See Bradford, K. , "Manipulation of Seed Water Relations Via Osmotic Priming to Improve Germination Under Stress Conditions," HortSci. .21:1105-1112 (1986) and Bewley, J.D. and M. Black, Physiology and Biochemistry of Seeds. Volume 2, Springer-Verlag, New York (1982) .

Such treatments cause priming (seed is imbibed and ready to complete germination, but no visible germination event such as radicle emergence has occurred) or pregermination (radicle has emerged from the seed coat) of the seeds. However, priming and pregermination of somatic embryos have never been reported, apparently because somatic embryos are at a precocious germination stage after their development and are inherently already at a primed/pregerminated stage. Just as priming/pregermination has never been considered applicable to immature zygotic embryos at a precocious germination stage, neither has priming/pregermination been considered for somatic embryos. Thus, it is an object of this invention to provide a technique whereby somatic embryos can be matured and planted directly in the growth chamber, greenhouse, and field, without requiring an in vitro planting environment. Another object of the invention is to provide methods whereby somatic embryos can be planted in a manner similar to true seed.

Yet another object of the invention is to improve somatic embryo maturation.

A further object of the invention is to control the germination of somatic embryos so that their shelf life is significantly increased.

Still another object is to utilize pregermination of somatic embryos for enhancing conversion of somatic embryos planted directly in the growth chamber, greenhouse, and field.

Disclosure of the Invention These and other objects are achieved in accordance with the present invention, which provides a method for producing mature somatic embryos, capable of being planted in the manner of true seeds, in growth chamber, greenhouse, and field conditions, without the constraints of an .in vitro environment.

In the practice of the invention, plant somatic embryos at the precocious germination developmental stage are subjected to a maturation treatment wherein the somatic embryos are placed on a medium which contains abscisic acid at a concentration sufficient to induce embryo maturation for the times and conditions employed.

The maturation treatment of the present invention is surprisingly effective for somatic embryo germination outside an in vitro environment. Matured somatic embryos produced in accordance with the invention are surprisingly unlike non-treated embryos in appearance as well as germination and conversion ability. The maturation treatments were found to be useful for three plant species and are expected to be equally applicable to other species.

Detailed Description of the Invention

In accordance with the invention, a method is provided for maturing somatic embryos such that they can

then be planted under growth chamber, greenhouse, and field conditions, without requiring a sterile, in vitro environment or added carbohydrates to sustain growth and viability. Such mature somatic embryos are capable of forming complete plants under environments in which previously only true seeds could germinate and grow.

Formation of somatic embryos and other meristematic tissue is now a widely occurring phenomenon among many plant genera. Numerous important crop and horticultural species, including alfalfa, celery, carrot and lettuce, have been shown to be capable of propagation through tissue culture and somatic embryogenesis. For recent lists of such species, see Evans, D.A. et aJL. , "Growth and Behavior of Cell Cultures: Embryogenesis and Organogenesis," in Plant Tissue Culture: Methods and Applications in Agriculture, Thorpe, ed. , Academic Press, pg. 45 et seq. (1981); Ammirato, P., "Embryogenesis," In: Handbook of Plant Cell Culture (Evans, D. et aj _^ . , eds.), Macmillan Publishing Co, New York, pp. 82-123 (1983); and Redenbaugh, K. , "Analogs of Botanic Seeds," U.S. Patent 4,562,663. These lists, however, are by no means exhaustive, and the practice of the present invention will be found to be beneficial in the propagation of species which are subsequently shown to be capable of undergoing somatic embryogenesis. Somatic embryos have the general morphology, appearance, and biochemistry of zygotic embryos. See D. Stuart, J. Nelsen, C. McCall, S. Strickland, and K. Walker, "Physiology of the Development of Somatic Embryos in Cell Cultures of Alfalfa and Celery," In: Biotechnology in Plant Science (Zaitlin, M. et al . , eds.), Academic Press, Inc., New York, pp. 35-47 (1985); and D. Stuart, J. Nelsen, S. Strickland, and J. Nichol, "Factors Affecting Developmental Processes in Alfalfa Cell Cultures," In: Tissue Culture in Forestry and

Agriculture (Henke, R. et al. , eds.), Plenum Publ. Corp., New York, pp. 59-73 (1985). Germination and plant production from somatic embryos has not been studied extensively, and for most species the frequency of plant production (conversion) from somatic embryos is very low. Furthermore, despite considerable efforts on somatic embryogeny throughout the world, no success has been reported with planting somatic embryos directly in the field. The handling of somatic embryos in the manner of true seed has not heretofore been possible. It has been recognized that the quality and appearance of somatic embryos can be controlled by external factors, such as environmental conditions and media components. However, the focus and objectives of previous research has been on the synchronous production of uniform somatic embryos that have an appearance similar to zygotic embryos. Whether the somatic embryos could germinate at high frequencies in a non-in vitro environment, such as the field, has not been an area of investigation. The principle limitation to the commercial success of somatic embryos has been that, despite the production of somatic embryos that appeared like zygotic embryos, the performance of such somatic embryos was limited to in vitro germination and conversion, hardly a natural environment.

As used herein, an in vitro environment is taken to mean one in which the sterility is maintained and a metabolizable carbon source is added for cell nutrition. In addition, generally the temperature, humidity, and/or photoperiod are capable of being regulated in an in vitro environment to prevent their fluctuation with varying ambient conditions.

In distinction, a natural planting environment is taken to mean one in which efforts are not taken to maintain the sterility and a carbon source is not added.

Abscisic acid (ABA) has been used to improve the quality and appearance of somatic embryos via its addition to the plant cell culture medium during embryo formation. ABA has been reported to permit embryo maturation, inhibit abnormal secondary embryo production, and repress precocious germination. See Ammirato, P., "Embryogenesis," In: Handbook of Plant Cell Culture (Evans, D. et al., eds.), Macmillan Publishing Co, New York, pp. 82-123 (1983) . Surprisingly, what was not examined or realized was the effect of ABA on embryo performance. In fact, when ABA was tested according to the protocols of Ammirato (Ibid) , it did not have a beneficial effect on in vitro conversion. The use of ABA during the production of alfalfa somatic embryos at concentrations of 0.01 to lOμM gave in. vitro conversion frequencies of 7-24%, not significantly different than the control frequency of 17%, with no ABA. See Redenbaugh, K. , P. Viss, D. Slade, and J. Fujii, "Scale-up: Artificial Seeds," In: Plant Tissue and Cell Culture (Green, C. et al. , eds.), Alan Liss, Inc., New York, pp. 473-493 (1987). Although ABA has been known to be important for somatic embryogeny and researchers have speculated that ABA is involved in inhibiting precocious germination and allowing the embryos to continue the maturation process, it has not been known that, surprisingly, ABA treatment in accordance with the present invention provides improved embryo maturation and allows somatic embryos which do not possess leaves, secondary roots or extended shoot apex to be planted naturally, as are true seeds.

In accordance with another aspect of the invention, the somatic embryos prepared in accordance with the present method can be simultaneously or sequentially subjected to a pregermination treatment. As used

herein, pregermination is taken in a generic sense to mean any method to begin the biochemical or physiological processes of germination before planting of the embryos. Other terms which are also used for this process include priming, osmoconditioning, vigorizing, chitting, etc.

Just as pregermination has never been considered applicable to immature zygotic embryos at a precocious germination stage, neither has pregermination been considered for somatic embryos. It has unexpectedly been discovered that the ABA maturation treatment would shift somatic embryogeny into maturation and quiescent stages and that removal of the ABA by pregermination, with or without the addition of gibberellic acid, would stimulate somatic embryo germination and increase the frequency of plant production.

Modes of Practicing the Invention

In accordance with the present invention, callus is initiated from surface sterilized explant tissue from a selected plant species capable of undergoing somatic embryogenesis. Typically, leaf petioles will be employed to provide the explant tissue. Other explants which may be used for callus initiation are immature zygotic embryos, unfertilized ovules, anthers, young inflorescence, leaf sheaths, and somatic embryos.

The selected explant tissue is plated on a culture medium containing mineral salts, a carbon source, a plant growth regulator and at least one auxin. The culture media which are utilized in various stages of the present method include any nutrient media known and developed for regeneration of whole plants from callus tissue. Culture media useful in the present invention are generally composed of mineral salts,

vitamins, carbohydrate, and, in certain stages of somatic embryogenesis, one or more hormones.

The mineral salts used in the media of the invention are well-known materials in the art, and are comprised of macroelements and microelements. The mineral salt macroelements and microelements used in the induction medium are well known materials generally selected from the following compounds: ammonium sulfate, potassium nitrate, monopotassium phosphate, magnesium sulfate heptahydrate, manganese sulfate dihydrate, zinc sulfate heptahydrate, boric acid, potassium iodine, calcium chloride dihydrate, ferrous sulfate heptahydrate, ethylenediamine tetraacetic acid (diεodium salt) . Other combinations of mineral salts may also be used. Representative culture media mineral salts include, but are not limited to: Schenk- Hildebrandt salts (SH) , Schenk, R.U. and A.C. Hildebrandt, Can. J. Bot. .50:199-204 (1972) , or Murashige-Skoog salts (MS) , Murashige, T. and F.K. Skoog, Physiol. Plant. 15:473-497 (1962) . For example, SH mineral salts comprise (in milligrams per liter) : potassium nitrate (2500) , calcium chloride dihydrate (200) , magnesium sulfate heptahydrate (400) , ammonium dihydrogen phosphate (300) , potassium iodide (1.0) , boric acid (5.0) , manganese sulfate monohydrate (10) , zinc sulfate heptahydrate (1.0), sodium molybdate dihydrate (0.1) , cupric sulfate pentahydrate (0.2) , cobalt chloride hexahydrate (0.1), ferrous sulfate heptahydrate (15) , disodium ethylenediamine tetraacetic acid (20) . As another example, MS mineral salts comprise (in milligrams per liter) :

ammonium nitrate (1650) , potassium nitrate (1900) , calcium chloride dihydrate (440) , magnesium sulfate heptahydrate (370) , cupric sulfate pentahydrate (0.025), manganese sulfate monohydrate (16.9), zinc sulfate heptahydrate (8.6), potassium phosphate (170) , boric acid (6.2) , potassium iodine (0.83), sodium molybdate dihydrate (0.25), cobalt chloride hexahydrate (0.025), disodium ethylenediamine tetraacetic acid (37.3), ferrous sulfate heptahydrate (27.8). It will be readily understood that other combinations of mineral salts comprised of macroelements and microelements may be known or hereafter developed and incorporated into the practice of the invention.

Vitamins are also generally included in the medium. Typical vitamins, such as inositol, nicotinic acid, pyridoxine hydrochloride, and thiamine hydrochloride, among others, are included in plant tissue culture medium, in accordance with known techniques.

A carbon source, generally consisting of readily metabolizable carbohydrate, is also included in the medium. The most commonly used carbon source is the disaccharide sucrose. Other saccharides, including fructose or maltose, can be employed in the medium at, e.g., approximately 5 to lOOg per liter to provide acceptable cell culture production. The carbon source most often employed in the media of the invention is usually sucrose, in a concentration of approximately 30g per liter of medium.

An optional plant growth regulator such as a cytokinin is often included in plant growth media at a concentration sufficient to stimulate the growth of the plant tissue. Typically, kinetin (Sigma Chemical Co. ; Hoechst) can be employed as a plant growth regulator and will generally be used at a concentration of

approximately 0.1 to 20μM, more usually at an optimum range of from approximately 1 to 15μM.

Hormones, such as "auxins," are known to be useful during somatic embryogenesis and are employed in the media of the present invention including, e.g., 2,4-D (2,4-dichlorophenoxyacetic acid), picloram (4-amino-3,5,6-trichloro picolinic acid), DICAMBA (2,6- dichloro-o-anisic acid) , IAA (indole-3-acetic acid) and NAA (naphthaleneacetic acid) may also be used, either alone or in combination, in the practice of the invention.

The auxin employed in the media used in the practice of the invention to produce and grow callus tissue is generally a known auxin such as a- naphthaleneacetic acid, at a concentration of approximately 0.1 to 500μM, with an optimum range of from approximately 1 to 50μM, either alone or in combination with other auxins.

Cells are induced to form embryos by incubation in an induction medium, such as SH medium + 50μM 2,4-D, for approximately four days, after which time the cells are transferred to a regeneration medium.

The embryo regeneration medium employed in the invention is also generally a medium well-known in the art, consisting of mineral salts and other components, as described above, but generally without auxins. Alternatively, auxins may be present at concentrations much lower than for the induction medium. See Stuart, D. and S. Strickland, "Somatic Embryogenesis From Cell Cultures of Medicago sativa L. I. The Role of Amino

Acid Additions to the Regeneration Medium," Plant Sci. Lett. 3_4:165-174 (1984); and Stuart, D. and S. Strickland, "Somatic Embryogenesis From Cell Cultures of Medicago sativa L. II. The Interaction of Amino Acids with Ammonium," Plant Sci. Lett. 3.4:175-181 (1984).

Further, the regeneration medium may contain other beneficial substances desirable for plant cell growth and development, such as one or more amino acids.

It is known that amino acids are grouped generally in accordance with certain characteristics of particular subclasses. Amino acid residues can be generally classified into four major subclasses as follows:

Acidic - i.e., the residue has a negative charge due to loss of H ion at physiological pH; Basic - i.e., the residue has a positive charge due to association with H ion at physiological pH;

Neutral/non-polar, i.e., the residues are not charged at physiological pH and the residue is repelled by aqueous solution; and Neutral/polar, i.e., the residues are not charged at physiological pH and the residue is attracted by aqueous solution .

It is understood, of course, that in a statistical collection of individual residue molecules some molecules will be charged, and some not. To fit the definition of charged, a significant percentage (at least approximately 25%) of the individual molecules are charged at physiological pH.

For the naturally occurring protein amino acids, subclassification according to the foregoing scheme is as follows:

Acidic: Aspartic acid and Glutamic acid;

Basic: Arginine, Histidine and Lysine;

Neutral/polar: Glycine, Serine, Cysteine, Threonine, Asparagine, Glutamine, Tyrosine;

Neutral/non-polar: Alanine, Valine, Isoleucine,

Leucine, Methionine, Phenylal nine, Proline and

Tryptophan. In the present application, the L-form of any amino acid

residue having an optical isomer is intended unless otherwise expressly indicated.

In certain presently preferred embodiments of the present invention, the regeneration medium can contain an addition of one or more neutral/non-polar amino acids. Of such amino acids, particularly preferred are L-proline and L-alanine.

Plant cells are incubated for approximately three weeks in the regeneration medium, after which the formed embryos are usually aseptically transferred to a solidified conversion medium consisting of half-strength mineral salts, a carbon source such as carbohydrate, preferably maltose at approximately 1.5% (w/v), and with reduced concentration or no auxins. Somatic embryo quality is observed after approximately 30 days, by counting the percent of embryos which ultimately form plants with roots and shoots.

In one embodiment of the present invention, a plant species can be selected and somatic embryos formed having a bipolar structure consisting of a root

(radicle) end and a shoot apex end with a vascular connection between the ends. Such embryos are at or near the developmental stage where precocious germination can occur. At this stage, the somatic embryos may be able to germinate .in vitro and produce whole plants, but they are unable to produce plants at any consequential frequency in an uncontrolled environment, such as that found in the field.

At the precocious germination developmental stage, the somatic embryos are then placed on a medium, such as an agar medium, a liquid suspension, or a humid environment, which contains abscisic acid (ABA) at a concentration ranging from 0.001 to lOOOμM, more usually 0.01 to 100μM, and preferably 0.1 to lOμM. The medium may also contain nutrients and carbohydrates as desired

to promote the growth and viability of a particular species. Generally, the medium will consist of those components necessary for basic growth of tissue cultures, such as Murashige and Skoog medium (MS salts plus the components recited above) or Schenk and

Hildebrandt (SH salts plus the components recited above) .

Furthermore, while ABA is presently considered to be the preferred embodiment for the maturation treatment of the present invention, it will be readily appreciated that other compounds with ABA-like activity may be employed as either alternatives or supplements to ABA.

For example, numerous ABA-analog compounds, including:

Phaseic acid (PA) Dihydrophaseic acid (DPA)

6*-hydroxymethyl abscisic acid (HM-ABA) beta-hydroxy abscisic acid beta-methylglutaryl abscisic acid beta-hydroxy-beta-methylglutarylhydroxy abscisic acid 4'-desoxy abscisic acid abscisic acid beta-D-glucose ester

2-2(2-p-chlorophenvl-trans-ethyl)cyclopropane carboxylic acid are known to possess some ABA-like activity, and may therefore be employed in the practice of the invention with varying degrees of success. See Ladyman, J. et al. , "The biological activity of a novel analog of abscisic acid," Plant Physiol 8j$.:68 (1988) and Walton, D. , "Biochemistry and physiology of abscisic acid," Ann. Rev. Plant Physiol. 3.1:453-489 (1980). Therefore, while reference is made to ABA in describing the maturation treatment, such ABA-analogs which display ABA-like activity will be recognized as equivalent to ABA. The somatic embryos are matured on ABA for 1 day to

1 year, more usually 5 days to 3 months, and preferably

7 days to 2 months. The temperature will be 0-40°C, more usually 10-30°C, and preferably 15-25°C. The

embryos will be matured in light or dark conditions, more preferably dark.

After ABA maturation, the embryos will have a hardier, more robust appearance. The embryos can be stored on the ABA medium further, if greenhouse and field conditions are not suitable for planting.

Prior to planting, the embryos are removed from the ABA medium and placed on a medium, such as an agar medium, a liquid suspension, or a humid environment, to promote priming and pregermination of the somatic embryos. This treatment also allows for ABA to leach out of the embryos and for the embryos to begin the process of germination away from the inhibitory effects of ABA. The medium will be a basic medium such as SH or MS. Alternatively, the medium may consist solely of water to promote embryo germination.

Certain embodiments of the present invention include a pregermination treatment, administered either simultaneously or sequentially with the maturation treatment of the invention. In one form of pregermination treatment, an osmotic agent in an aqueous solution of sufficient concentration to inhibit root and shoot growth is added to the medium at the appropriate time. A typically useful osmotic agent such as mannitol, generally administered at 1 to 15%, will serve to control root emergence. Other known pregermination agents include a monovalent salt. Many monovalent salts are useful, such as potassium nitrate (KNO ₃ ) . Potassium nitrate inhibits germination at concentrations between 0.3 and 1.0M, preferably 0.4 to 0.6M. Small molecular weight organic molecules can also serve as an osmoticum.

An additional, optional pregermination agent, which can be added as a component of the maturation medium of the invention, is one or more gibberellins, preferably

GA ₃ , GA ₄ , or GA ₇ , depending on the plant species employed to generate somatic embryos. The gibberellin component is usually added at concentrations of 0.001 to lOOOμM, more usually 0.01 to 500μM, and preferably 0.1 to 300μM.

The somatic embryos are primed/pregerminated for 1 hour to 1 month, more usually 12 hours to 1 week, and preferably 1 to 5 days. The temperature will be 0-40°C, more usually 10-30°C, and preferably 15-25 ^β C. The embryos will be in light or dark, more preferably light. After preparing the somatic embryos in accordance with the invention, they will ordinarily be planted in a growth chamber, greenhouse or directly in the field. In one presently preferred embodiment, the present somatic embryos are planted in the greenhouse or field and covered with a translucent moisture barrier, generally in accordance with the teachings of U.S. Patent No. 4,612,725, the relevant portions of which are incorporated herein by this reference. The somatic embryo-derived plantlet will be maintained under the moisture barrier until the plantlet is capable of withstanding the less controlled planting environment. In order to illustrate various embodiments of the invention, the following demonstrations were carried out with a variety of species. Although it was expected that the effect of ABA and of pregermination might be confined to only one species, alfalfa, it was subsequently found to be a universal effect, useful for all plant species tested. Consequently, the ABA and pregermination effects are not to be construed to be limited only to the examples presented, but to be widely applicable in plant somatic embryogenesis.

Experimental

The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, all weights are given in grams (g) or milligrams (mg) , all concentrations are given as illimolar (mM) or micromolar (μM) and all volumes are given in liters (L) or milliliters (mL) unless otherwise indicated.

EXAMPLE A (Alfalfa, Medicago sativa)

A.l. Production of Alfalfa Somatic Embryos. Callus was produced from alfalfa leaf petioles placed on Schenk and Hildebrandt medium (SH) , supplemented with 25μM α-naphthaleneacetic acid (NAA) and lOμM kinetin. The callus was then incubated for 3 weeks on SH medium with potassium nitrate replaced by 20mM potassium citrate and 25mM glutamine.

Subsequently, the callus was induced to alter its developmental progression by being cultured on SH medium with 50μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5μM kinetin for 3 days. The induced callus was placed on SH medium with lOmM ammonium nitrate and 30mM proline, causing numerous somatic embryos to form after three weeks at 24°C. See Stuart, D. and S. Strickland, "Somatic Embryogenesis From Cell Cultures of Medicago sativa L. I. The Role of Amino Acid Additions to the Regeneration Medium," Plant Sci. Lett. 34:165-174 (1984) ; and Stuart, D. and S. Strickland, "Somatic Embryogenesis From Cell Cultures of Medicago sativa L. II. The Interaction of Amino Acids with Ammonium," Plant Sci. Lett. 3_4:175-181 (1984).

A.2. Conversion of Alfalfa Somatic Embryos to Plants. Somatic embryos produced as described in Example A.1 were planted in a potting mix (10:9:1 ratio of peat:vermiculite:perlite, obtained from McCalif Growers Supplies, Inc., San Jose, CA) , without sucrose and without efforts to maintain a sterile environment, using a process similar to that used for true seed.

The embryos were placed in shallow furrows, with cotyledons facing upright, and without any potting mix covering. The pan of potting mix was covered by a clear plastic cover, slightly ajar, and placed into a growth chamber at 25°C under fluorescent lights (12-hour days) . The number of plantlets formed from embryos was counted six weeks after planting.

A.3. ABA Effect on Embryo Conversion in Growth Chamber.

A.3.a. Somatic embryos produced as described in

Example A.l were stored at 4°C in the dark for one day.

Selected embryos were placed on SH medium containing 0, 1, 2.5, 5, 7.5 or lOμM ABA (maturation medium) and cultured for 14 days at 24°C in the dark.

After 14 days, the somatic embryos were removed from the media and placed on conversion medium as described in Example A.2. At this time, the ABA matured somatic embryos had significantly changed in their color and appearance. The embryos had gone from a green to white color and had become bulkier, heavier and more robust. All treatments that received an ABA treatment formed plantlets in potting mix with plantlet formation ranging from 36-54% (of total embryos planted) . Optimum ABA concentrations under these conditions were between 1 and 7.5μM. The embryos that received no ABA exposure did not form any plants.

A.3.b. Somatic embryos produced as described in Example A.l were placed onto SH + 2.5μM ABA medium for

various lengths of time at 24°C in the dark to determine the optimum maturation period. The embryos were removed from the medium and placed on conversion medium as described in Example A.2. Maximum conversion of 40-44% was obtained when the ABA exposure length was 20 to 31 days. The lowest conversion (8-14%) was obtained when the ABA exposure was less than 14 days. For maturation periods greater than 31 days, conversion decreased slightly (30-37%) , indicating that a 20-31 day period was optimum. Without ABA treatment, no plants formed. A.3.σ. Somatic embryos were produced as described in Example A.l. The embryos were placed into liquid SH + ABA (5, 10, or 20μM) in a flask and shaken on a rotary shaker at 24°C in the dark for 20 days. Other embryos were placed onto semi-solid medium SH + ABA (5, 10, or 20μM) at 24 ° C in the dark for 20 days. The embryos which were matured in the liquid culture medium (at all 3 ABA concentrations) had the same high quality appearance as embryos matured on the semi-solid medium, indicating that the ABA exposure is equally effective when done in liquid as in semi-solid medium.

A.3.d. Somatic embryos produced as described in Example A.l were matured under one of two in vitro environments: SH + 5μM ABA at room temperature or on the original regeneration medium at 4°C for 30 days. Embryos were subsequently removed from the culture treatment and some of the embryos were examined for changes in fresh and dry weights, while other embryos were converted as described in Example A.2. ABA-treated embryos displayed an increase of fresh weight from 2mg/embryo to 6mg/embryo and a dry weight increase of 0.3mg/embryo to 2mg/embryo, while cold-treated embryos showed no increase in fresh or dry weights. ABA-treated embryos increased in average percent dry matter from 14% to 31%, while cold-treated

embryos had no increase. Finally, ABA-treated embryos had 47% conversion while cold-treated embryos had 0% conversion.

A.3.e. Somatic embryos produced as described in Example A.l were matured as described in Example A.3.d, with the exception that the maturation period was 25 days. The embryos were analyzed for starch content (See Huber, S.C. et al.. , "Effect of Photoperiod on Photosynthate Partitioning and Diurnal Rhythms in Sucrose Phosphate Synthase Activity in Leaves of Soybean (Glycine max L. [Merr.]) and Tobacco (Nicotiana tabacum L.), ⁿ Plant Physiol. 7_5:1080-1084 (1984) and Boehringer Mannheim GMBH Starch Biochemical Analysis, Catalog No. 207748 (1986)). ABA-matured embryos displayed an increase in starch content from 10μg starch/embryo (or 4μg starch/g fresh weight) to 164μg starch/embryo (or 22μg starch/g fresh weight) , whereas cold-matured embryos increased starch content from lOμg starch/embryo (or 4μg starch/g fresh weight) to 16μg starch/embryo (or 6μg starch/g fresh weight) . The somatic embryos were converted as per Example A.2. ABA-matured embryos had 83% conversion, whereas cold-matured embryos had 5% conversion.

A.3.f. Somatic embryos produced as described in Example A.l were matured on SH + 5μM ABA for 3-12 weeks. The embryos were removed from the maturation medium and converted to plants as described in Example A.2. The conversion frequency remained unchanged over the maturation period at 56-66%.

A.4. GA ₃ Effect on Embryo Conversion in Growth Chamber. A.4.a. Somatic embryos produced as described in Example A.l, except that 50μM 2,4-D was replaced with lOOμM 2-(2,4-dichlorophenoxy) propanoic acid. The embryos were placed onto SH + 5μM ABA for 21 days at 24°C. The embryos were then transferred to SH, containing various levels of GA ₃ (0, 25, 50, 100, 150μM) for 2 days at 24°C (pregermination treatment). The embryos were planted in potting mix as described in Example A.2, except the embryos were covered by a thin layer of potting mix. This planting method was harsher than in previous examples, as the embryos were required to germinate and grow through a covering of potting mix. The highest conversion (19%) was obtained when the pregermination treatment consisted of SH + lOOμM GA3. However, it was found that somatic embryos that had received higher or lower levels of GA ₃ also converted to plants, but at a lower frequency (5-10%) than embryos that received the 100μM GA ₃ treatment. Embryos that did not receive a pregermination treatment did not convert at all, even those that had an ABA maturation treatment. A.4.b. Somatic embryos produced as described in Example A.4.a and treated with SH medium + 5μM ABA for 21 days were placed into liquid SH medium + lOOμM GA3, either shaking on a rotary shaker or without shaking, or on semi-solid medium containing agar. Conversion of all the treatments was done as described in Example A.2 and the frequencies were similar (64-78%) , indicating that the pregermination treatment can be used equally well with semi-solid medium as well as liquid medium.

A.5. Effect of Sequence of Exposure to ABA and GA ₃ on Conversion in the Growth Chamber.

Somatic embryos were produced as described in

Example A.l and the sequence of embryo exposure to SH + 5μM ABA (24 days at 24°C, dark) and SH + lOOμM GA ₃ (2 days at 24°C, dark) was investigated. Embryos were planted in potting mix as described in Example A.2 or in vitro on 0.5X SH medium, with no sucrose plus 1.5% maltose and 25μM GA ₃ . The n vitro conversion frequency was the same for all embryos regardless of whether they had received only the ABA treatment, only the GA ₃ treatment, or the ABA followed by the GA ₃ treatment. In contrast, when the embryos were converted on potting mix in a growth chamber, embryos that received only the ABA treatment converted at a frequency of 10%, embryos that received only the GA ₃ treatment converted at 0%, and the embryos that received the ABA followed by the GA ₃ treatment converted at 32%. Thus, while the ABA and GA ₃ treatments, individually or when used together, gave equal conversion in vitro, the highest conversion in potting mix was obtained when the GA ₃ treatment followed the ABA treatment.

A.6. Effect of Osmotica on Conversion in Growth Chamber. A.6.a. Somatic embryos produced as described in Example A.l were placed onto SH media containing various levels of ABA and 8% mannitol for 16 days at 24°C in the dark. Subsequently, the somatic embryos were converted as described in Example A.2.

The use of mannitol alone (without ^" ABA) promoted conversion to 16%. However, the highest potting mix conversion obtained was 32% when using 10μM ABA without mannitol. Whenever mannitol was present, the conversion was lower than without mannitol, indicating that the ABA

effect was not osmotic control induced by ABA, but due to the growth regulator properties of ABA itself.

A.6.b. Somatic embryos produced as described in Example A.l were placed onto SH media containing lμM ABA with 11% sucrose or 5μM ABA without sucrose for 21 days at 24°C in the dark. Subsequently, the somatic embryos were converted as described in Example A.2.

The use of sucrose with ABA gave a low conversion frequency of 4%, while with ABA alone, the conversion was 48%. Sucrose was not sufficient as a substitute for ABA during embryo maturation.

A.7. ABA Effect on Embryo Conversion in the Greenhouse. Somatic embryos produced as described in Example A.l were placed onto SH medium + 5μM ABA for 21 days at 24°C in the dark. The embryos were removed from the medium and placed on SH medium + lOOμM GA ₃ for 2-4 days at 24°C in the light. The embryos were then planted directly in the greenhouse using a natural process common to planting true seed. The embryos were sown in McCalif mix inside a humidity tent (containing a humidifier to maintain high humidity) . After 6 weeks, the conversion frequency was 64%. Embryos that were not treated with ABA had a conversion between 0 and 18%.

A.8. ABA Effect on Embryo Conversion in the Field.

Somatic embryos produced as described in Example A.l were placed onto SH medium + 5μM ABA for 21 days at 24°C in the dark. The embryos were removed from the medium and placed on SH medium + 100μM GA ₃ for 2-4 days at 24°C in the light. The embryos were then planted directly in the field using a natural process common to planting true seed. The embryos were sown and then left uncovered, covered by a styrofoam cup, covered with a white plastic sheet, or covered with a cloth sheet

(white polyester from McCalif Growers Supplies, Inc., San Jose, CA) . After 6 weeks, conversion was measured.

Conversion was 3% for direct planting, 29% under the cups, 12% under plastic, and 13% under the cloth. Embryos that were not treated with ABA would not survive in the field under any of these conditions.

EXAMPLE B (Celery, Apium graveolens

B.l. Production of Celery Somatic Embryos. Callus was produced from celery leaf petioles placed on SH medium supplemented with 7.5μM 2,4-D and 0.5μM kinetin for 3 weeks at 25°C in the dark. The explants and callus were then transferred to SH medium containing 2.25μM 2,4-D and 0.5μM kinetin and incubated for another 3 weeks at 25°C in the dark.

The callus was further maintained on SH medium containing l.OμM 2,4-D and 0.5μM kinetin at 25°C in the dark. Somatic embryos were formed after 3 weeks incubation at 25°C under fluorescent lights by placing the callus onto SH medium with the ammonium level reduced to 5mM, the sucrose reduced to 1%, and with the addition of 8% mannitol.

B.2. Acclimation of Celery Somatic Embryos. Somatic embryos produced as described in Example

B.l were placed in flasks of SH medium containing 1% sucrose, 3% mannitol, and 5mM ammonium (liquid acclimation medium) on a rotary shaker for 4 days at 24"C. With the acclimation treatment, in vitro conversion of celery was significantly increased over that for embryos that did not have this treatment.

B.3. Conversion of Celery Somatic Embryos.

Somatic embryos produced as described in Example B.l were planted in a potting mix (10:9:1 ratio of peat:vermiculite:perlite; obtained from McCalif Growers Supplies, Inc., San Jose, CA) , without sucrose and without efforts to maintain a sterile environment, using a process similar to that used for true seed.

The embryos were placed in shallow furrows, with cotyledons facing upright, and without any potting mix covering. The pan of potting mix was covered by a clear plastic cover, slightly ajar, and placed into a growth chamber at 25°C under fluorescent lights (12-hour days) . The number of plantlets formed from embryos was counted six weeks after planting.

B.4. ABA Effect on Embryo Conversion to Plants.

B.4.a. Somatic embryos produced as described in Example B.l were exposed to liquid acclimation medium as described in Example B.2. and planted in potting mix as described in Example B.3. No conversion to plants was obtained.

B.4.b. Somatic embryos produced as described in Example B.l were matured at 4°C in the dark for three days on the original regeneration petri dishes. Selected embryos were placed on SH media containing 1, 10 or lOOμM ABA for 5 days at 24"C in the dark. The somatic embryos were then converted as described in Example B.3. All ABA-treated embryos formed plants in potting mix (at frequencies of from 7-40%) with the greatest frequency of plant formation when the embryos were matured on SH + lμM ABA (40%) . Somatic embryos that had not been treated with ABA had no conversion in the potting mix.

B.4.c. Somatic embryos produced as described in Example B.l were matured at 4°C in the dark for three

days on the original regeneration petri dishes. Selected embryos were placed on SH medium containing 10, 20, 30, 40 or 50μM ABA for 5 days at 24°C in the dark.

The somatic embryos were then converted as per Example B.3. All ABA treatments formed plants in potting mix (15-23%) with the greatest plant formation when celery somatic embryos were matured on SH + 30μM ABA (23%) . Somatic embryos that had not been treated with ABA had no conversion in the potting mix.

B.5. GA ₃ Effect on Embryo Conversion to Plants.

B.5.a. Somatic embryos produced as described in

Example B.l were placed onto SH medium + 40μM ABA for 21 days at 24°C. The embryos were then transferred to SH medium containing various levels of GA ₃ (0, 50, 100,

150, or 250μM) for 2 days at 24°C (pregermination treatment) .

After the ABA and GA ₃ treatments the somatic embryos were converted as described in Example B.3. Embryos that did not receive the pregermination treatment of GA ₃ converted at a frequency of 8%. The highest conversion (54%) was achieved using the SH medium + 250μM GA ₃ . All of the embryos that had been treated with pregermination medium had significantly higher conversion frequencies than embryos that did not receive any pregermination treatment.

B.5.b. Somatic embryos produced as described in

Example B.l were placed onto SH medium + 40μM ABA for 21 days at 24°C. The embryos were then transferred to SH medium containing various levels of GA ₃ (0, 25, 50, 100,

150, or 200μM) for 2 days at 24°C.

After the ABA and GA3 treatments the somatic embryos were converted as described in Example B.3.

Embryos that did not receive the pregermination treatment of GA ₃ converted at a frequency of 5%. The

highest conversion frequency (56%) was achieved using SH medium with no GA ₃ . All of the other embryos that had been pregerminated had significantly higher conversion frequencies than embryos that did not receive any pregermination treatment.

B.6. Effect of Mannitol on Conversion.

Somatic embryos produced as described in Example B.l were placed onto SH medium + 30μM ABA, containing various levels of mannitol (0, 2, 4, or 6%), for 10 days at 24°C in the dark. The embryos were then pregerminated on SH for 2 days at 24°C in the dark. Subsequently, the somatic embryos were converted as described in Example B.3. The highest conversion frequency of 52% was obtained in the treatment using the ABA medium containing 4% mannitol. Embryos matured on the ABA medium without the addition of mannitol converted at a frequency of 32%.

EXAMPLE C (Carrot, Daucus carota)

C.l. Production of Carrot Somatic Embryos.

Carrot seeds were surface sterilized and plated onto MS medium for ten days at 25°C in the dark. Petioles were excised and placed into static liquid culture in Lin & Staba medium (LS) (Fuji ura, T. and A. Komamine, "Effects of Various Growth Regulators on the Embryogenesis in Carrot Cell Suspension Culture," Plant Science Lett. .5:359-354 (1975)), containing lpp 2,4-D, for one month at 25°C in the dark. Cell clusters were sieved and fractions under 300μm in size were cultured in liquid LS medium containing O.lppm 2,4-D for one month at 25° C, in the dark, on a rotary shaker at 80 rpm. Cells under 300μm were again collected and suspended in liquid LS medium containing O.lppm 2,4-D for two weeks.

Cells from 73μm to 37μm were collected and washed five times with LS medium. The cells were centrifuged at 600rpm for 10 minutes then placed onto LS medium containing 0.2ppm zeatin and 0.2ppm ABA and shaken at 80rpm for one month at 25°C. Embryos between 3 and 4mm in length were collected.

C.2. Conversion of Carrot Somatic Embryos.

Somatic embryos produced as described in Example C.l were planted in a potting mix (5:2:1 ratio of peat:vermiculite:perlite; obtained from McCalif Growers Supplies, Inc., San Jose, CA) , without sucrose and without efforts to maintain a sterile environment, using a process similar to that used for true seed. The embryos were placed in shallow furrows, with cotyledons facing upright, and without any potting mix covering. The pan of potting mix was covered by a clear plastic cover, slightly ajar, and placed into an growth chamber at 25°C under fluorescent lights (12-hour days) . The number of plantlets formed from the embryos was counted six weeks after planting.

C.3. ABA Effect on Embryo Conversion in Growth Chamber. C.3.a. Somatic embryos produced as described in Example C.l were matured at 4°C in the dark for eight days. Selected embryos were placed on SH medium containing 1 or 10μM ABA for 7 days at 24°C in the dark. These and other embryos that had not been matured with ABA were planted in potting mix as described in Example C.2.

The highest conversion to plantlets in potting mix was 26.7% from the ABA treatment of SH medium + lμM ABA. Embryos that did not receive an ABA treatment converted at 3.3%, which was significantly lower. ABA was

therefore crucial in increasing the conversion of somatic embryos in potting mix.

It should be understood that various alternatives to the methods and materials herein disclosed may be employed in practicing the present invention. It is intended that the following claims define the invention, and that the materials and methods within the scope of these claims and their equivalents be covered thereby.