The invention comprises methods for producing mature desiccated and desiccation-tolerant somatic embryos, particularly conifer embryos. The methods include the application of a growth-promoting hormone such as abscisic acid (ABA) to the immature embryos during development. The embryos are also water stressed during development. The concentration of the ABA undergoes a net increase relative to its initial concentration to reach a peak prior to discontinuing ABA treatment when the embryos have attained a moisture content of between 32 and 55%. The water stressing may remain constant or may undergo a net increase in intensity over the period of time during which it is applied to the embryos. The method may optionally include the further water stressing of the mature embryos to further reduce moisture contents of the embryos preferably to the level at which the embryos are tolerant of freezer storage.

This application claims priority from Canadian Patent Application No. 2,240,135 filed Jun. 5, 1998.
This invention relates to a development treatment for somatic embryos, particularly conifer embryos, including water stressing and growth regulator treatment, preferably including the use of a relatively high molecular weight non-permeating osmoticum and abscisic acid or equivalents, characterized by an increase in the concentration of the growth regulator or the intensity of the water stressing during the course of the growth regulator treatment.
Somatic embryogenesis offers the potential to produce clonally large numbers of plants of many species at low cost. Somatic embryos, develop without the surrounding nutritive tissues and protective seed coat found in zygotic embryos, so considerable research has been devoted to causing somatic embryos to functionally mimic seeds with regard to efficient storage and handling qualities. The development of techniques for somatic embryogenesis in conifers has greatly improved the ability to culture conifer tissues in vitro and now offers the means to propagate clonally commercially valuable conifers of a number of species. However, there is still room in the technology for improvement of the quality and vigour of plants resulting from somatic embryos, including those from all species of conifers.
It has been suggested to use abscisic acid (ABA) or osmoticum or both for enhancing storage levels in plant cells. For example, it was shown that somatic embryos of Theobroma cacao could be induced to accumulate fatty acids approaching the composition of commercial cocoa butter by using a high sucrose concentration in the culture medium (Pence et al. 1981; Physiol. Plant. 53:378-384). Modifying the culture conditions by osmoticum concentration and/or ABA content similarly improved lipid accumulation in Brassica napus L. somatic- (Avjioglu and Knox 1989; Ann. Bot. 63:409-420) and microspore-derived (Taylor et al. 1990; Planta 181: 18-26) embryos as well as somatic embryos of carrot (Dutta and Appelqvist 1989; Plant Sci. 64: 167-177) and celery. Also, the level of storage lipids in P. abies somatic embryos was improved by optimizing the ABA level to between 10-20 xcexcM, but the somatic embryos contained about 4% of the lipid level obtained by zygotic embryos (Feirer et al. 1989; Plant Cell Rep. 8:207-209).
Japanese laid-open patent publication No. 1-218520, issued on Aug. 31, 1989, describes a process for producing plant body regenerative tissue. The process includes a step of cultivating a plant body regenerative tissue in a medium containing ABA and having an osmotic pressure of 180 to 2500 mmol/kg. In-order to control the osmotic pressure within the specific range, a non-toxic substance such as sugar, alcohol, an amino acid or glycol is added.
Water stress plays an important role in maintaining embryos in a maturation state (Kermode 1990, Crit. Res. Plant Sci. 9, 155-194). Kermode suggests that low water content rather than ABA prevents precocious germination during later stages of development. This is important because precocious germination of embryos during development in seeds would be lethal during desiccation.
A conventional way to water stress plant cells grown in vitro is to increase the osmotic concentration of the culture medium through the use of plasmolysing osmotica. For example, increased concentrations of plasmolysing osmotica such as sucrose have been used to promote somatic embryo maturation of many plant species. Sucrose at levels of 3 to 6% was found to improve somatic embryo development of many conifers (Attree and Fowke 1993). It seems that high concentrations generally led to repressed embryo development. Mannitol had a similar effect on maturation of conifer somatic embryos (Roberts 1991; Physiol. Plant. 83; 247-254). Low levels of mannitol (2-6%) led to a doubling of the number of mature embryos recovered at the end of the maturation period; however, the treatment could only be applied as a short pulse (one week) as prolonged maturation treatment with mannitol became detrimental to further embryo maturation.
Poor embryo response using sucrose and mannitol or other simple sugars and salts may be due to the absorption of such plasmolysing, osmotica by the symplast of plant cells. Such absorption facilitates adjustment of tissue osmotic potential (osmotic recovery) without lowering the tissue water content. Additionally, direct or indirect metabolic effects on specific plant metabolites can occur, due to utilization of the solute by the embryo or its toxic effects.
Alternatives to plasmolysing osmotica are non-plasmolysing osmotic stresses as well as other forms of non-plasmolysing stresses which have the same effect as drought conditions. Such stresses can be induced using a controlled environmental relative humidity (r.h.) or, for example, by non-permeating high molecular weight compounds such as polyethylene glycol (PEG) or dextran. These compounds are usually available in a wide range of molecular weights. For example, PEG is available in molecular weights ranging from 200 to 35,000. However, only those with a molecular weight above 1000 would normally be considered to be non-permeating. This is because the large molecular size of these solutes excludes their passage through plant cell walls, so preventing entry into cells and consequently preventing plasmolysis, while still removing water (Carpita et al, 1979). Consequently, their non-plasmolysing effect reduces tissue water content in a manner similar to the effects of water stress observed in cells of plants subjected to drought conditions. The effect is constant and cell turgor can only be restored by cells actively increasing their cellular solute concentrations. PEG has been most commonly used to apply water stress to whole plants, to osmotically prime whole seeds to synchronise germination and improve seedling vigour.
Embryo drying occurs naturally in most seeds, and has a role to play in the developmental transition between maturation and germination. Thus, desiccation leads to enhanced germination of both zygotic and somatic embryos. Desiccation of whole somatic embryos is also an alternative method of germplasm storage. Somatic embryos produced continuously year-round could therefore be dried and stored until the appropriate planting season, or shipped to new locations.
A number of prior patents and publications describe methods for the desiccation of somatic embryos. In U.S. Pat. No. 4,615,141 issued on Oct. 7, 1986, Janick and Kitto describe a method for stimulating desiccation. tolerance to asexual plant embryos which are then desiccated. In this method, the embryos are removed from medium containing auxin and cytokinin to a hormone-free development medium. During subsequent development, the somatic embryos are pre-treated by increasing the sucrose concentration of the development medium from normal levels to high levels, or by applying ABA. The hydrated embryos are then encapsulated in a hydrated coating material. The coating material dries to form a thin, non-toxic film enclosing one or more embryos, protecting the embryos during storage but readily redissolving in an aqueous solution. The use of ABA and increased sucrose during embryo development is suggested to improve subsequent survival of the encapsulated embryos during desiccation. Once the embryos have been encapsulated, they are dried at a temperature ranging from 20 to 30xc2x0 C. for a period of at least five hours.
In U.S. Pat. No. 4,777,762 Oct. 18, 1988, Redenbaugh et al. describe a method for producing desiccated analogs of botanic seeds which are created by providing ABA during the development phase then removing a portion of the water by slow or fast drying so that the plant tissue is no longer saturated with water. The method also involves encapsulating meristematic tissue in a hydrated gel or polymer and removing water by slow or fast drying. The formation of somatic embryos is induced and the embryos are then encapsulated in the gel or polymer followed by drying. Alternatively, the somatic embryos are desiccated to less than complete tissue saturation during, or at the end of, embryo development and then encapsulated.
McKersie et al. (U.S. Pat. No. 5,238,835, issued on Aug. 24, 1993), describe a method through which in vitro formed plant embryos are desiccated following the application of ABA or other types of environmental stress inducing desiccation tolerance. The embryos are induced at the torpedo-shaped stage or later with the source of ABA for a sufficient period of time to cause expression of desiccation tolerance. The induced embryos are then dried to provide stable, viable artificial seeds.
McKersie et al. emphasize the importance of stimulating the embryo at the appropriate stage by the use of signals to develop tolerance to desiccation. It is stressed that if the signals are applied at the incorrect stage of development, the tissue will not respond properly. Angiosperm embryos can undergo maturation in the absence of ABA, and it is suggested that ABA be supplied as late as possible during the maturation protocol and applied for a relatively short period of time. Hence, the timing and duration of ABA application seem to be critical.
Japanese laid-open patent publication. No. 2-031624, issued on Feb. 1, 1990, discloses the use of ABA in plant cultures. ABA is used as part of a process for drying embryos prior to storage.
Senaratna et al., in 1989, Plant Science, 65, pp. 253-259, describe the induction of desiccation tolerance in alfalfa somatic embryos by exogenous application of ABA in the form of a short pulse. Embryos are then dried to 10 to 15% of their moisture content and stored for at least three weeks in the dry state. Senaratna et al. also describe a method by which tolerance to desiccation is induced by exposing the somatic embryos to sub-lethal levels of low temperature, water, nutrient or heat stress prior to desiccation. However, it was demonstrated that some of these stress pre-treatments had deleterious effects on embryo maturation and seedling vigour.
Hence, the prior literature on somatic embryos, and artificial seeds shows that desiccation tolerance was achieved in some plant species such as alfalfa, geraniums, celery, brassica, carrots, corn, lettuce, orchard grass and soybeans. Various methods were suggested, which all appear to revolve around promoting desiccation tolerance by applying ABA and other stresses late in maturation and subsequently reducing the water content of the embryos. However, these methods were not applicable to all species including conifers.
Conifer somatic embryos appear to be different from the somatic embryos of monocotyledonous and dicotyledonous species in that ABA should be supplied as early as possible, to conifer embryos in maturation protocols in order to promote embryo maturation. Merely reducing or eliminating auxin and cytokinin levels, as has been successful for maturation of somatic embryos of many angiosperm species (Ammirato 1983, Handbook of Plant Cell Culture, Vol. 1, pp. 82-123) leads to infrequent or poor maturation in conifer embryos and more often results in browning and death of the immature somatic embryos. Furthermore, it appears that ABA should be applied for longer periods and at higher levels than generally applied to angiosperm somatic embryos in the past.
In U.S. Pat. No. 5,183,757, Roberts (issued on Feb. 2, 1993) describes a process for assisting germination of spruce somatic embryos that comprises partially drying the embryo at humidities of less than about 99.9%. Roberts also suggests that a medium having a sucrose concentration of 2 or 3.4%, which is used between the maturation treatments and the germination media, promotes root and shoot elongation. Roberts mentions that the humidity range that can be used for partial drying of somatic embryos without lethal effect is about 85 to 99.9%, which according to the Roberts method results in only a very small (5-10%) moisture loss.
In a study published in Can. J. Bot., Vol. 68, 1990, pp. 1086-1090, Roberts et al. mention that conifer somatic embryos do not survive desiccation at room humidity, but that partial drying at very high humidity promoted germination up to 90% (as opposed to the 95% to 100% germination described in the examples of the present invention detailed below). Roberts et al. also refer to the fact that drying embryos over a range of r.h. indicated that r.h. of 81% or lower was lethal to conifer embryos. This can be further visualized at Table 3 of the report where the effects of partial drying at different r.h. on germination are shown. It can be seen that very small levels of germination are obtained following drying at a r.h. of 90% and that no germination is observed when r.h. of 81% and 75% are used. Based on those results, Roberts et al. conclude that only a mild drying of the somatic embryos was possible to permit normal germination and that the spruce somatic embryos do not tolerate desiccation to zygotic levels. According to Roberts et al., spruce somatic embryos did survive and undergo improved vigour following a partial drying treatment in an environment of very high humidity (over 95% humidity), during which time only 5% of moisture was removed.
Later, Roberts et al. (J. Plant Physiol., 138, pp. 1-6, 1991) emphasize that somatic embryos from a number of species, including spruce, are sensitive to severe water loss and show decreased survival following desiccation. In this paper, Roberts shows that Sitka spruce somatic embryos do not survive desiccation, even though high frequency and synchronised germination could be obtained following partial drying of the embryos.
Hence, despite attempts to desiccate conifer somatic embryos following ABA maturation, survival was not described until Attree et al (U.S. Pat. No. 5,464,769, issued on Nov. 7, 1995). The important aspect of the invention of Attree et al. resided in the combined use of a non-plasmolysing water stress and ABA during the embryo maturation process to stimulate maturation frequencies and promote further maturation of the embryos, and to increase dry weight and lower moisture content, leading to desiccation tolerance to moisture contents below 36%. Constant levels of ABA were maintained during development of the embryos. With regard to the non-plasmolysing water stress, a non-plasmolysing high molecular weight compound such as PEG having a molecular weight range over 1000 (e.g., PEG 4000) or other high molecular weight polymers such as dextran having a minimum molecular weight over 7000 was preferred, although other non-plasmolysing water stresses such as increased gel strength, or environmental stresses were also suggested. Attree et al. in WO 93/11660 suggest that when using bioreactors for development the environment could be controlled and ABA and water stress levels raised or lowered at the start or end of maturation.
PEG has been included in conifer embryo maturation protocols with varying success. For example, Ilic-Grubor et al., in, pending U.S. patent application Ser. No. 09/096,547 (filed on Jun. 12, 1998), disclose that, in some circumstances, using PEG as a water stress in the presence of a restricted carbon source may enhance embryo development. In the specific context of conifers, Norgard 1997 (Plant Science 124, 211-221) and Li et al. 1997 (In Vitro Cell. Dev. Biol.-Plant 33, 184-189) report that PEG has a positive effect on maturation, while others found negative effects on maturation and germination (Cornu and Geoffrion 1990, Euk. Soc. Bot. Fr. 137, 25-34; Gupta et al., U.S. Pat. No. 5, 036,007; Klimaszewska et al. 1997 Can. J. For. Res. 27, 538-550; Find 1977, Plant Science 128, 75-83.
There has been a trend for using increasingly higher concentrations of ABA to promote the maturation of conifer somatic embryos, probably resulting from a need to inhibit precocious germination late in maturation which has become more apparent following the increasingly longer maturation times used. Thus, ABA was first successfully used by Hakman and von Arnold 1988 (Physiol. Plant. 72:579-587) and von Arnold and Hakman 1988 (J. Plant Physiol. 132:164-169), at 7.6 xcexcM. Dunstan et al. 1988 (Plant Sci. 58:77-84) subsequently found 12 xcexcM ABA to be better. Shortly after, Attree et al. 1990 (Can. J. Bot. 68:2583-2589) reported that 16 xcexcM was optimal. Roberts et al. 1990 (Physiologia Plantarum 78; 355-360) have shown that for some species of spruce, ABA at 30-40xcexcM could be used to promote maturation and yield mature embryos with storage protein polypeptides comparable to zygotic embryos. Such high levels were necessary to prevent precocious germination and allow maturation to proceed to later stages. Dunstan et al. 1991 (Plant Sci. 76:219-228) similarly found that high levels could permit embryo maturation. Unfortunately, high ABA levels used throughout the development period also increased the frequency of developmentally abnormal embryos. In the above reports concerning conifers, increased osmoticum or water stress was not included with the ABA. Recently, much higher ABA concentrations have been described. Becwar et al., U.S. Pat. No. 5,506,136, issued on Apr. 9, 1996, describe ABA in development media at levels up to 120 xcexcM. Dunstan et al., 1997 (Journal of Expt. Bot. 48, 277-287) suggest that a remedy to prevent precocious germination of conifer somatic embryos is to transfer cultures to fresh medium with ABA in the maturation culture period, as is commonly done. It is stated that exposure to fresh ABA is unlikely to lead to greatly improved yields of mature somatic embryos, unless the population of immature embryos remains sizable, but is more likely to lead to improvement in the quality of the mature somatic embryos through deposition of storage product and prevention of precocious germination. Dunstan et al.1997 (J. Plant Physiol.) show that the availability of (+)-ABA decreases during culture, which can lead to precocious germination. They suggest that this is generally attributed to a low concentration of ABA, and also that extending the use of ABA during the maturation phase by periodic transfer to fresh nutrient medium would extend ABA availability. Uddin 1993 (U.S. Pat. No. 5,187,092, issued on Feb. 16, 1993) describes using various combinations and proportions of glucose, maltose, abscisic acid and/or indolebutyric acid to promote maturation of conifer somatic embryos. This patent suggests that conifer somatic embryos should be cultured in the presence of maltose and/or glucose in a total concentration of at least 3%, and at least 10 xcexcM ABA. A two-step process is described in which the preferred concentration of maltose is 6% and the ABA is raised preferably after about four weeks culture from 20 xcexcM to 30 xcexcM. Uddin provides no information on why ABA was raised and whether raising ABA was preferable to maintaining constant ABA, or reducing it in the presence of the permeating osmoticum. It is not disclosed in the patent whether the embryos obtained by means of the Uddin method were viable and capable of vigorous germination. Kapik et al. 1995 (Tree Physiology 15, 485-490), and Kong et al., 1997 (Physiologia Plantarum 101, 23-30) show that endogenous ABA rises during seed and zygotic embryo development then falls during late development. Therefore, the current thinking is that ABA should not be increased, or even maintained at a constant level during development, but should be moderately high at the start of development then decreased throughout development to low levels or to zero at the end of the culture period, promoting germination.
Thus, in U.S. Pat. No. 5,034,326, Pullman et al. (Jul. 23, 1991) describe a method for developing tissue culture-induced coniferous somatic embryos into well-developed cotyledonary embryos. The method comprises a multi-stage culturing process in which early stage embryos are cultured on a late stage medium comprising a significantly higher osmotic potential along with moderately high ABA and an absorbent material to gradually, reduce the level of available ABA over time. A critical aspect of this method lies in the inclusion of the absorbent material in the embryo development medium. Absorbent materials suggested include activated charcoal and silicates. The absorbent is used to slowly reduce the ABA and remove metabolic waste products. The purpose of this reduction in ABA is to follow the natural tendency in embryo development. Pullman et al. suggest that as development approaches completion, the presence of lesser amounts of ABA is required.
Similarly, Gupta et al. in U.S. Pat. No. 5,036,007 (Jul. 30, 1991) describe a similar method. In Douglas fir culture, ABA is reduced from about 10-20 xcexcM at the start of development to less than about 3 xcexcM or even to zero. The method also suggests the use of osmotica to control osmotic potential. A preferred osmoticum suggested is sucrose in amounts in the range of 2 to 3%. Another osmoticum that is suggested by Gupta et al. is PEG. Gupta et al. mention that PEG 8000 was evaluated and found to be a superior osmoticum in the presence of decreasing ABA levels, stating that the reasons for its superior performance compared with other materials is not entirely clear. Gupta et al. also suggest that polyethylene or polypropylene glycols of other molecular weights are believed to be equally useful. According to U.S. Pat. No. 5,036,007, the combination of osmotica is to be modified at some point during the development stage. In fact, the patent teaches that the osmotic concentration is increased during development in conjunction with the decrease in ABA. If development is started at levels around 300-350 mmol/kg, the osmotic level may be increased during development to a final level of about 450 mmol/kg.
A similar method was described in U.S. Pat. No. 5,236,841, issued on Aug. 17, 1993, by Gupta et al.; however, the described technique relates to the use of gradually decreasing amounts of the abscisic acid during the time when the embryos are further developed into cotyledonary embryos by stepwise subcultures. It was suggested that when transfers to fresh medium are made, the initial ABA level of the fresh medium should not be higher than the final level of the medium at the end of the preceding culture period. In examples in which activated charcoal was used, exogenous ABA levels were required to be an order of magnitude higher due to the ability of charcoal to rapidly absorb ABA.
More recently, however, Gupta et al. in U.S. Pat. No. 5,482,857 (Jan. 9, 1996) have found that, when using activated charcoal, ABA was not necessary for cotyledonary embryo development of Douglas fir. Similar methods to those above are also described in the more recent patents by Pullman and Gupta (U.S. Pat. No. 5,294,549, Mar. 15, 1994) and Gupta (U.S. Pat. No. 5,563,061, Oct. 8, 1996). The aforementioned U.S. Pat. Nos. 5,294,549, 5,563,061, and 5,236,841 all suggest that it is advantageous to use one combination of osmotica at the beginning of development and transfer embryos to a medium having a different combination during development. In U.S. Pat. No. 5,036,007, Gupta et al. also suggest the replacement of PEG with an alternative osmoticum such as lactose or sorbitol mid-way through development as embryos tended to deteriorate over time in the presence of PEG. Similarly, U.S. Pat. Nos. 5,731,191 and 5,731,204 (both issued on Mar. 24, 1998) report that the use of PEG throughout development was found to cause a xe2x80x9cgermination blockxe2x80x9d. To overcome this xe2x80x9cgermination blockxe2x80x9d, these patents teach the use of PEG for only-the first part of development and the use of a cold treatment during development, respectively. U.S. Pat. No. 5,731,203, also issued on Mar. 24, 1998, teaches the avoidance of the use of PEG altogether, and instead teaches the use of high levels of ABA throughout development.
Desiccation of conifer somatic embryos is desirable to enable somatic embryos to be stored for very long periods. Storage times of desiccated embryos may be further extended by storing frozen embryos. The ability to survive prolonged storage is important given the long life cycles of conifers and the length of time required to evaluate in vitro produced trees. Tissues able to survive freezing in liquid nitrogen are considered to be capable of survival following storage for indefinite periods. Dronne et al. 1997 (Physiologia Plantarum, 99:433-438) recently showed that desiccation decreases abscisic acid content in hybrid larch somatic embryos, which is consistent with the earlier understanding of abscisic acid as an inhibitor of precocious germination.
In conclusion, most available techniques within the prior state of coniferous somatic embryo development technology have failed to provide optimally vigorous and viable conifer somatic embryos and especially, viable desiccated conifer somatic embryos, although the previous Attree et al. work mentioned above has carried the technology forward considerably. Conifer somatic embryos require particular combinations of hormonal and water stressing conditions in order to develop. Current methods call for the application of moderate to high levels of ABA to be applied at the start of development, then for the application of fresh medium in which exogenous ABA is maintained at a constant level in order to control precocious germination at the end of development, or in which ABA is decreasing from the point at which it is first applied, or is even absent throughout development in order to attempt to match the zygotic pattern of ABA levels. In the latter method, precocious germination is controlled by increasing the osmotic concentration of the medium. High levels of ABA have been reported to lead to developmental abnormalities, while high levels of osmotica have been shown in some instances to be detrimental. Decreasing ABA and sub-optimal water stress teaches away from the conditions required for successful desiccation tolerance. Therefore, applying high levels of ABA throughout development, or decreasing ABA, in association with permeating osmotica is not desirable for conifers. More suitable methods for culturing plant embryos are therefore required.
Moreover, some prior reports suggest that water stressing using an osmoticum, or particular osmotica, is unreliable, at least over certain moisture content reduction ranges. The poor response with osmoticum, and PEG in particular, reported by some possibly results from a number of factors, such as unduly prolonged exposure to PEG/ABA, inadequate desiccation or inadequate removal of endogenous ABA prior to germination, sub-optimal PEG/ABA levels during culture, incorrect preparation of PEG/ABA media, incompatible gelling agents or incompatible. molecular weight of PEG, and incorrect combinations of osmotica. As shown in the Examples detailed below, when media are prepared correctly, ABA and water stressing may be increased to very high levels towards the end of development, which can lead to superior embryo quality showing that non-plasmolysing water stressing at correct levels for at least a substantial part of development in combination with optimal levels of ABA is most suitable for conifer embryos.
The invention comprises methods of culturing immature somatic embryos in the presence of a growth regulator and water stressing to produce mature desiccation tolerant somatic embryos. The somatic embryo culturing processes of the present invention are similar in their objectives and steps to the various processes described in prior allowed U.S. patent application Ser. No. 08/244,725 (Attree et al.) filed on Aug. 18, 1994 and allowed on Jun. 20, 1997, counterpart to pending Canadian patent application Serial No. 2,125,410 filed on Dec. 18, 1992, but differ in that the present invention is characterized by increasing concentrations of growth regulator or intensities of water stressing during the course of development and produces higher yields of higher quality desiccation-tolerant embryos, which convert (that is, exhibit, both needle development and a viable root) into somatic seedlings with improved vigour compared to those resulting from production methods known previously.
The invention comprises three principal variants, the common characteristic of which is that the level of stress hormone growth regulator (such as abscisic acid or its precursors, derivatives, or analogs) or the intensity of the water stressing applied to the embryos over a selected period of time is raised during the development (maturation) of the immature embryos to the late cotyledonary stage. As timing is relevant to both the growth regulator and water stressing treatments, it is to be understood that, unless otherwise stated, the early part of embryo development refers to the period from the immature/suspensor stage to the globular stage, the middle refers to the period from the club-shaped stage to the early cotyledonary stage, and the late part of development is the remaining period in which the cotyledons become fully developed. Of course, these are not exact definitions, and it is to be understood as well that there is often significant overlap between these periods.
In this description, such terms as xe2x80x9cABAxe2x80x9d and xe2x80x9cABA treatmentxe2x80x9d encompass any of the family of stress hormone growth regulators such as those mentioned in the preceding paragraph, or treatment by such growth regulators, as the case may be. The mature somatic desiccation-tolerant embryos produced in accordance with the methods of this invention may be desiccated to low moisture contents (which are indicated herein as a percentage of total embryo weight), preferably to moisture contents low enough to permit the embryos to survive freezing, and/or stored for extended periods of time, and/or germinated to produce somatic seedlings of high quality and good vigour. A further aspect of the invention is that the application of exogenous abscisic acid (ABA) to the embryos may be commenced at a suitable selected time during the development/maturation of the immature embryos, the selection of the commencement time depending upon such variables as the species and genotype being cultured, the length of the maturation culture period, the initial concentration of ABA, and other applicable factors. Additionally, the water stressing may also commence at a suitable time, selected with regard to the aforementioned factors, during the development/maturation of the immature embryos, although the commencement of the ABA application and the commencement of the water stressing do not have to coincide.
In accordance with the first two variants, the ABA treatment preferably commences at some point from the immature suspensor stage to the club-shaped stage, and the concentration of the exogenous ABA is progressively increased as the development of the immature embryos progresses, preferably to a cotyledonary stage although the application of ABA may be discontinued prior to the attainment of that stage. Ideally, the increase in ABA concentration would be continuous, however, it is expected that in practice the increase will be effected in a stepwise or incremental fashion. In the context of the present invention, the preferred progressive increase in exogenous ABA concentration refers to the general upward trend of ABA concentration levels over time, which encompasses the possibility of interim declines or plateaus in the ABA concentration curve relative to time, as well as ever-increasing concentration of exogenous ABA.
In accordance with a first variant of the invention, the water stressing does not increase in intensity during development. Alternatively, in accordance with a second variant, the immature somatic embryos are cultured in the presence of a suitable water stress and ABA, the intensity of the water stressing as well as the concentration of the ABA increasing during the development period. However, it is not necessary to correlate (i) the duration of the increasing water stressing, (ii) the timing of the increase of water stressing, or (iii) the magnitude of the increase of water stressing with the counterpart parameters applicable to the increase of the ABA concentration. As the embryos are susceptible to developmental abnormalities if excessively high water stress is exerted upon the embryos too early in development, the second variant of the invention provides the advantage of increasing yields of mature desiccation-tolerant embryos associated with high rates of embryo water loss without severe risk of causing developmental abnormalities including precocious germination. Finally, in accordance with the third variant, the level of ABA remains substantially constant while the intensity of the water stressing increases over the course of development. As in the context of the increasing ABA, the increase in water stressing contemplated in the second and third variants is ideally continuous, but is expected to be effected in a stepwise fashion, and encompasses the possibility of plateaus and declines punctuating the general upward trend. In any of these variants of the invention, it is advantageous to discontinue the application of exogenous ABA or remove the exogenous ABA prior to the embryos"" attainment of moisture contents of less than about 30%, and preferably when the embryos reach between about 55% and about 30% moisture content, although the discontinuance or removal may be effected prior to the attainment of about 55% moisture content.
All of these variants tend to produce substantially increased yields of mature, fully developed somatic embryos (as defined below), with high desiccation tolerance and improved vigour relative to embryos produced in accordance with previously known methods, while late, unwanted embryogenic tissue proliferation (that can occur in the absence of somatic embryo maturation) and precocious germination are prevented or inhibited.
Immature somatic embryos that have been cultured in the presence of a suitable water stress and ABA, in accordance with either of the first two variants discussed above, may then be subjected to severe water stressing (desiccation) to low moisture content, which severe water stressing may occur either in the presence or absence of culture medium, depending on how the embryos are desiccated. The desiccated mature embryos produced by this modification may have moisture contents as low as about 5-10%, and preferably at least low enough that the embryos are sufficiently devoid of unbound water to permit them to tolerate being frozen and stored. Moisture content levels of such embryos are usually less than about 36% at the. upper limit of the range. Such desiccated embryos may be stored indefinitely at a range of storage temperatures, from about room temperature to very low temperatures approaching about xe2x88x92200xc2x0 C., and a relatively high percentage of such embryos are subsequently typically able to germinate to produce vigorous plantlets. As a very high water stress too early in development is detrimental and causes embryo abnormalities, it is preferable that the intensity of the water stressing during the development time period contemplated in the first two variants of the invention be lower than that of the water stressing exerted upon the embryos to severely desiccate them once they have reached the desiccation-tolerant stage.
Although low-intensity water stressing of at least about xe2x88x920.1 MPa may be employed in preculturing the embryos (along with hormone levels that are, reduced from those initially used to induce somatic embryogenesis, such as about a tenth or less of the concentrations of auxin and cytokinin), water potentials for the development treatment per se can range from xe2x88x920.3 MPa at the start of the water stressing treatment to preferably between about xe2x88x92100 to xe2x88x92500 MPa at the end of desiccation. The term xe2x80x9cwater stressingxe2x80x9d includes stressing the embryos by subjecting them to low water potential drought conditions (such as a relative humidity environment, higher relative osmotic pressure (such as by the inclusion of osmotica in the substrate upon which they are grown), and any other forms of water stress (moisture stress) that tend to lower the moisture content of the embryos. Non-plasmolysing water stressing is preferred, although under some circumstances (notably as the embryos become increasingly mature), the embryos are to a greater extent resistant to potentially plasmolysing water stress that at an earlier stage of development might lead to sufficiently severe plasmolysis to kill the embryo. It is to be understood that, in addition to non-permeating osmotica and environmental stress, non-plasmolysing water stressing may be effected by, inter alia, permeating osmotica at sufficiently low concentrations to avoid plasmolysis of the embryonic cells. That is, any concentration of a metabolizable carbon source that exceeds the amount of nutrient which is utilized by the embryos for nutrition will have an osmotic effect which exerts water stress upon the embryos. For example, even a 3% concentration of sucrose may be sufficient to exceed embryos"" nutritional needs and exert a water stress, while being sufficiently low (see, for example, Ilic-Grubor et. al., supra) to avoid causing permanent developmental damage by plasmolysis. Moreover, it is to be understood that non-plasmolysing water stressing encompasses water stressing that may cause some relatively minor reversible plasmolysis from which the embryos are able to recover while suffering little reduction of their overall viability and vigour. Although the lower the moisture content of the embryos, the higher the concentration of permeating osmotica necessary to cause plasmolysis, non-permeating osmotica are still preferred as the primary means of water stressing the embryos during at least the early part of development, as such osmotica are prevented or impeded from entering the cells, thereby reducing toxic effects. Permeating osmotica can be applied later in development, when the embryonic cells have already lost some of their moisture and are less likely to be susceptible to irreversible plasmolysis. As osmotica having sizes of about 30 Angstrom units (xc3x85) or more cannot pass through the cell wall, this is the minimum size of the preferred non-permeating osmotica. In this respect, polyethylene glycol (PEG) is suggested as a suitable water stressing agent, which can be substituted by any other suitable polyalkylene glycol, or alternatively by any other suitable high-molecular-weight water stressing agent. The preferred minimum molecular weight of PEG to be used in accordance with this invention is about 1000. Gels may also provide a non-permeating water stress; for example, 1% or higher w/v PHYTAGEL(trademark) is suitable for water stressing. Moreover, it is not necessary to use only one means of water stressing throughout the development period and/or further water stressing period; rather, the means used may be varied. As relative water stress levels or intensities may not admit of easy quantitative determination, an empirical results-oriented approach may be taken; if following a particular water-stressing treatment it appears that the embryos were not optimally water-stressed to reach a particular target moisture content and target viability percentage, the water-stressing environment may be suitably modified, as by varying the amount of osmoticum, the composition or relative drying effect of gel in the medium, the relative humidity in the vicinity of the embryos, any other water stressing applied to the embryos, or the time during which any particular water-stressing effect is applied.
For embryo nourishment during development, a suitable metabolizable carbon source is preferably restricted to less than about 90 mM (or about 3%) for sucrose and equivalent well-metabolised carbon sources, but this is not essential. Moreover, it is noted that both the metabolizable carbon source(s) and other components of the medium may increase the relative osmotic pressure of the medium.
In any of the foregoing alternative processes according to the invention, the exposure of the embryos to one or both of ABA and water stress should preferably begin by the time the embryos reach the globular stage, although it may begin as early as the immature (i e., suspensor) stage, and continue through the club-shaped stage to the early and late cotyledonary stages. It is to be understood that the water stressing of and/or application of ABA to the embryos may be interrupted or decreased at any time, as long as there is a net increase in the level of exogenous ABA at any point prior to the final discontinuation of the ABA application (in the case of the first two variants described above) or, in the third variant, in the intensity of water stressing prior to the end of the water stressing treatment. After the ABA treatment is discontinued, it is preferable to continue to water stress the embryos to a severely desiccated state (that is, to moisture contents less than about 30% to 36%), during which the exogenous ABA drops to low levels or to zero. Such severe desiccation promotes survivability following long-term embryo storage and tends to render the embryos freezer storage-tolerant.
It is not necessary to apply the ABA and water stress to the embryos concurrently, nor is it necessary to raise the levels of one or both of these factors consistently throughout the process. However, both should preferably be at relatively higher levels later in culture, such as at or near the end of culture, prior to the point of the removal or discontinuance of exogenous ABA and/or water stress. Preferably both ABA and water stressing should be increased throughout the early cotyledonary stages of development. The magnitude of the increase of either the ABA concentration or of the intensity of water stressing may be as little as about 5% above the initial level, but may also be many times higher than the initial level, depending on the species chosen and the initial level of the ABA or water stressing that is applied. In the discussion of increases in intensity of water stressing, the incremental increase relates to the increase in the magnitude of the water potential, which in turn relates to the rate of embryo water loss. Thus, if the initial water potential is, say, xe2x88x92100 MPa, a 5% increase in the intensity of water stressing would result in the water potential decreasing to xe2x88x92105 MPa. In this specification, xe2x80x9cincreasingxe2x80x9d or xe2x80x9craisingxe2x80x9d water potential means increasing the absolute value of the numerical value expressed, regardless of whether the value is expressed in positive or negative terms. For the purposes of this discussion, a mature embryo (that is, one having successfully reached the end of development) may be defined as having full desiccation tolerance, having a moisture content of less than 55% (preferably between about 30% and 55%), and having achieved the late cotyledonary stage and being capable of developing into a plant. However, it is to be noted that further changes occur during desiccation that enhance the vigour of the desiccated embryos, relative to those mature embryos that are not water stressed to low moisture contents.
It can be understood from, the foregoing discussion that two variants of this invention comprise a method for producing viable mature conifer somatic embryos comprising water stressing the somatic embryos, preferably beginning earlier than the globular stage, in medium containing ABA whose concentration in medium increases during development, including such increase towards the mid-point of cotyledonary development of the embryos when the tendency for precocious germination is the highest, prior to the water content of the embryos becoming sufficiently low to inhibit precocious germination. (At low moisture content of the embryos below about 40%, the embryos naturally resist precocious germination, so any further increase in ABA levels beyond such stage of desiccation of the embryos is unhelpful; ABA treatment may normally cease at such stage.) Water stressing also begins during development, although it does not have to coincide with the timing of the initial application of exogenous ABA. In one variant of the method of the invention, the water stressing is maintained at a substantially constant level during its application, in order to maintain a substantially constant water potential which, in turn, causes the reduction of the embryos"" water content at a substantially constant rate. Preferably, water stressing continues at least to the end of the development period, although it may be discontinued prior to that point. Desiccation of the embryos may then be carried out.
As mentioned, also contemplated within the scope of the invention is an alternative method in which the intensity of water stressing (and therefore the magnitude of the water potential, which is discussed further-below) rises throughout development and in which ABA concentrations also rise during the period of ABA application. Finally, the third alternative method is that in which the intensity of the water stressing rises throughout development while the ABA concentration remains constant throughout the ABA treatment period. Preferred ABA concentrations for all of the alternatives discussed thus far may be in the range of 0.1 xcexcM to 200 xcexcM.
In accordance with the first two (rising-ABA) variants, the preferred initial concentrations are in the range 1-40 xcexcM (although higher concentrations may be used in some instances), and more preferably 5-30 xcexcM, which then increase during development to a peak of preferably about 30-60 xcexcM. Concentrations of ABA greater than 100 xcexcM later in development may be preferable in some instances, as will be described below. The increase can be at any developmental point or throughout development. At the uppermost level, ABA should be present at a concentration of 30-300 xcexcM or possibly even higher, but most preferably 30-100 xcexcM. Moreover, as variables such as the quality, purity, and source of ABA as well as the presence of an adsorbent also bear upon the effectiveness of the exogenous ABA on bringing about the desired embryonic development activity, an empirical approach may require that concentrations even higher than about 300 xcexcM be used in accordance with this invention. Activated charcoal or some other adsorbent may be used to remove toxic compounds from the medium; however, as an adsorbent for toxins would also tend to absorb exogenous ABA, the ABA levels must be increased sufficiently to maintain a net increase of exogenous ABA during the increasing-ABA phase of the invention. (As will be discussed in more detail below, ABA may be maintained at constant levels at the beginning or the end of development, and may also be reduced somewhat at the end.)
In any variant, to circumvent unwanted adsorption of ABA during treatment of the embryos, the selected adsorbent may be first saturated with ABA prior to addition to the culture, to inhibit ABA absorption during embryo development while still permitting removal of toxins. Alternatively, filtration systems such as, dialysis or molecular sieves may be used instead of adsorbents to remove toxins, in such a manner as to maintain the net increase of ABA during the increasing-ABA phase of the invention. (In addition, dialysis membranes and molecular sieves may fulfill other functions, such as to provide physical support to the embryos while allowing the embryos access to nutrients in media and to lessen or prevent the contact of the embryos with toxins that may otherwise play a useful role in the culture, such as, for instance, high molecular weight osmotica that may have a toxic effect if in contact with the embryos.) Frequent replacement of the medium when increasing ABA can also serve tolerance toxins. Of course, it is to be understood that more than one toxin removal means may be used in combination or in series.
Also within the scope of the invention are methods of the foregoing character involving increasing-ABA (which, as already mentioned, includes increasing equivalent growth regulator) treatment or increasing-water stressing of the somatic embryos in combination with subsequent desiccation of the somatic embryos to a moisture content of less than about 40%, and preferably less than about 30%. Embryos having moisture contents of less than about 30% may be considered fully desiccated, although embryos having moisture contents of as low as about 5-10% may survive storage and germinate successfully. In this last modification of the methods in accordance with the invention, moderately low ABA and water stress (e g., less than about 30 xcexcM ABA, with the concentration of non-permeating PEG (polyethylene glycol) adjusted to provide a water potential of the medium of less than about 350 mmol/kg, and preferably in the range of 250-350 mmol/kg) are preferably applied within the first 1-4 weeks of the maturation culture. In variants in which the ABA is to be increased, it is preferably increased up until about the point of the development period when the embryos are. entering the early to mid-cotyledonary stages.
The increase of ABA and/or water stress from the initial (non-zero) levels to the final desired levels can be accomplished in multiple steps of whatever increment the user prefers, or in one transfer. The increase in ABA, may be effected, for example, by medium replacement or by simply adding concentrated ABA to the medium to effect the. final desired rise in ABA. To be most effective, exogenous ABA should preferably rise throughout the majority of the early- to mid-culture period so that ABA levels are high close to the end of development, particularly at the mid to late cotyledonary stage. Subsequent additions of ABA provide the conditions required at the middle stages of culture suitable for suppressing precocious germination, promoting development and providing optimal desiccation tolerance late in development. It is not necessary to apply ABA in one step to high levels at the start of development and then continuously decrease the levels throughout development, as has been suggested in the prior art. As discussed, the magnitude of the incremental increase may be as little as 5% of the initial level to as much as many times the initial level, depending on the species to which the embryo belongs and the initial level of ABA or water stress that is chosen. Moreover, the incremental increases in ABA concentration do not have to remain the same, rather the increments may be varied. By way of example, the first incremental increase may be 5% of the initial ABA concentration, while the second increase may be 7% or 10% of the initial ABA concentration. Thus, a graphic representation of the period during which ABA concentration is increased does not necessarily have to be a straight line nor an approximation of same. In addition, the concentration of ABA may be maintained at the initial level over some duration of the development period, prior to being raised, and the uppermost concentration of ABA may similarly be maintained at that level over time. There may even be some drop from the uppermost concentration to the final concentration of ABA in the development period (effected in one or more incremental reductions) as long as there is a net increase in exogenous ABA concentration from the commencement to the discontinuation of ABA application.
Although somatic embryos may be cultured on gelled medium, bioreactors, which are highly suitable for use with liquid medium and so me types of which allow the relative humidity to be controlled, may be used, as the one practising the method will presumably apply understanding already well known in the technology, including the modification of the medium during development, e g., in the manner for medium replacement effected in a bioreactor as described by Attree et al. (1994, Plant Cell Rep, 13:601-606). The change in levels of exogenous ABA causes changes in the embryos"" endogenous ABA levels, thus effecting the desired developmental changes that are analogous to those occurring naturally in the development of zygotic embryos. Prior to the application of the exogenous ABA, endogenous ABA levels may be as low as zero, although there is usually some naturally-occurring endogenous ABA. In the method of this invention, as exogenous ABA is applied to the embryos, endogenous levels rise. It is not usually necessary to begin increasing the exogenous concentration of ABA until the increase in levels of endogenous ABA is desired. The concentration of endogenous ABA peaks at around the mid-cotyledonary stage, after which it begins to fall again as the embryos"" moisture contents decrease. Without limiting the generality of the foregoing, it is preferred that, over a given time period in the growth regulator treatment, smaller and more frequent increases of ABA concentration be applied to the embryos rather than larger and less frequent increases. For example, three applications of 10 xcexcM increases is preferable to one application of a 30 xcexcM increase over the same time period.
It appears that it is necessary to reduce or eliminate ABA only near the end of development or after development, and preferably when moisture contents are in the range of 30-55%, particularly when further desiccating to less than 30% moisture content. It is desirable that the application of exogenous ABA be restricted to the initial levels or discontinued altogether before the embryonic moisture contents approach about 30% to 36%, as the continued application of a high concentration of exogenous ABA may in some instances raise endogenous ABA thereby inhibiting the proper germination of the desiccated embryos. However, the application of exogenous ABA may be restricted or discontinued prior to this point, and should preferably be restricted or discontinued during the late cotyledonary stages (although it may be done even earlier). In terms of moisture contents, the restriction or discontinuation of exogenous ABA may preferably occur when the embryos have attained a 40% moisture content, or a 55% moisture content or higher. As mentioned earlier, the tendency for precocious germination decreases as the embryo moisture content decreases, such that the germination-inhibiting effect of ABA becomes unnecessary at low enough moisture contents. As the desirable influence of ABA is therefore ineffective when the embryos no longer tend to germinate precociously, the completion of the ABA treatment may coincide with the natural inhibition of precocious germination. Likewise, the completion of the ABA treatment may be brought about by the removal of the embryos from the germination-inhibiting influence of ABA. It is therefore to be understood that some ABA may even remain in contact with embryos at the completion of the ABA treatment, as long as the remaining ABA is insufficient or unnecessary to adversely inhibit germination.
The discontinuance of ABA application may be effected in one step by the complete removal of the maturation medium from the embryos, or in several steps, for example as the ABA in the medium is successively diluted to zero, as the embryos are transferred to fresh medium with progressively lower. ABA concentrations, or with the addition of an adsorbent such as activated charcoal to the medium. As contemplated in the preferred modification of the three variants, water stressing continues after the restriction/discontinuance of exogenous ABA application, in order to desiccate the embryos further. It is to be understood that water stressing for further desiccation may. also be interrupted or otherwise modified prior to completion. Embryos may undergo the growth regulator treatment on supports in medium, at the end of which the embryos may be removed with their supports from the medium and then desiccated on the supports, or removed from supports and placed on fresh supports wetted with a solution of ABA at the final concentration. During desiccation, the embryos and their supports dry together, thereby restricting the amount of available exogenous ABA. Alternatively, the medium may be completely removed from the embryos, which may then be dried.
During development, it is preferable to maintain a substantially non-plasmolysing water stress until a fully desiccated embryo is obtained. Furthermore, providing a rapid rise to water potentials of high magnitude together with a rapid rise in ABA very early in maturation prior to meristem development and early cotyledon development may be the cause of developmental abnormalities during late stage development, and consequently may result in embryos of poorer quality and fewer mature embryos overall. Equally, one should try to avoid overstressing the embryos; better results are obtained if the embryos are given adequate time to respond to the changes in their environment. Initially, at the beginning of development, the application of moderate to low ABA (e.g., less than about 40 xcexcM ABA, and preferably about 5-30 xcexcM), and moderate water stress, preferably comprising a non-permeating component of water potential of less than about 350 mmol/kg, and preferably in the range of 250-350 mmol/kg or less, is most preferred. Non-permeating PEG present in the medium is suitable to apply the water stress. Alternatively, the water stressing may be applied to the embryos in the form of environmental stressing (by, for example, controlling the relative humidity of the culture vessel to provide the requisite level of water stress), or in the form of physical or chemical stress (by, for example, the application of relatively firm gels), or a combination of any of the foregoing.
If the concentration of ABA is to be raised in accordance with the invention then, during development, the ABA concentration should be increased over at least a portion of the development period. In the case of conifer somatic embryos, the ABA should preferably be increased prior to the last few weeks of development, that is, when the embryos have reached the cotyledonary stages. In such instances, the ABA should be increased to about 30 to 200 xcexcM ABA or even higher, most preferably to 30 to 100 xcexcM ABA.
In accordance with either the second or third preferred variant of the invention involving increasing water stress along with either increasing or constant growth regulator, respectively, the water stress preferably should be increased prior to the last few weeks of development, that is, prior to the time when the embryos have reached the cotyledonary stages, and most preferably throughout development from the immature suspensor stage, through the globular and, in gymnosperm embryos, club stages, to the cotyledonary stage. The water stressing during these stages should preferably be non-plasmolysing (which, of course, may include permeating osmotica at non-plasmolysing concentrations). For example, the absolute value of the magnitude of the water potential should rise to about 800 mmol/kg or greater, preferably to about 400-700 mmol/kg, and most preferably to about 500-600 mmol/kg.
The novel combinations of increasing ABA with a constant level of water stressing, increasing the intensity of water stressing with a constant concentration of ABA, and increasing both the intensity of water stressing and the concentration of ABA during development produces high frequencies of mature desiccation-tolerant embryos from initially immature embryos, promotes the development of normal looking somatic embryos (including those of conifer species), and inhibits precocious germination of these embryos.
Mature embryos obtained from the processes leading to desiccation tolerance according to the invention may be germinated directly, or because of desiccation tolerance, may be further desiccated in accordance with the further desiccation option of the invention, which may lead to further improvement in plant vigour. The further desiccation preferably occurs in the absence of a replenishing source of ABA. Water potentials during desiccation may typically reach xe2x88x92100 to xe2x88x92500 MPa to desiccate the embryos to moisture contents below about 30%, although embryo moisture contents may fall to as low as 5% or even less. Preferably the somatic embryos are desiccated after maturation to a moisture content at which there is no unbound water (so that the somatic embryos may be frozen and stored), which is usually below about 30-35%, at which moisture content water potentials preferably are less than about xe2x88x922 to xe2x88x922.4 MPa. Fully desiccated somatic embryos may then be germinated or stored indefinitely and then germinated.
Desiccation to low moisture contents may not always be necessary or desired. Desiccation in the absence of exogenous ABA naturally reduces endogenous ABA levels, thereby promoting germination vigour, however, alternative methods to reduce high endogenous ABA may be employed or combined with desiccation, such as stratification of embryos at low temperature, osmotic priming treatments, or ABA inhibitors. Alternatively, such methods may not be required if, for example, the endogenous ABA levels fall with moisture content levels regardless of whether exogenous ABA is being applied. The development times may also be varied to enhance desiccation tolerance, or to compensate for development temperature variations, development time requirements for different species, and so forth. Varying the development temperatures, by, for example, culturing at lower temperature or using a high/low temperature fluctuation, would lead to longer development times; the principles of the present invention would continue to apply, but one would have to take into account the slower expected reaction time of the embryos at lower temperatures to changes in ambient conditions and other effects that low temperatures might incur. For example, the preferred temperature range in which to develop the embryos is: from about 0xc2x0 C. to about 35xc2x0 C., although the range of about 0xc2x0 C. to about 12xc2x0 C. may be suitable for at least part of development. Similarly, some species such as pines develop slower than other species such as spruces, so that, for example, instead of employing a six- to seven-week development period that may be used with spruces, a nine- to fifteen-week period may be necessary or desirable for pines. The temperature for desiccation may also be varied preferably between the ranges of 0-35xc2x0 C. (and most preferably between about 0-12xc2x0 C.), as might the relative humidity at which desiccation occurs. Environmental or physical methods for desiccation may be employed, and the rate of desiccation may be varied. Mature somatic embryos may be desiccated and/or converted into artificial seeds. All these modifications are considered to be within the scope of the present invention. These methods have been found to be advantageous for a range of conifers, such as spruces (white, black, Norway), Douglas fir, lodgepole pine, and western larch, so are considered advantageous for all conifers, including loblolly pine. Mature somatic embryos obtained by means of this invention which are then germinated show increased vigour over those obtained through conventional treatments that omit the preferred procedures of the present invention. Embryos prepared according to the invention undergo rapid shoot development and growth in soil.
The Examples to be detailed below are limited to application of variants of the methods according to the invention to embryos of coniferous species. However, the expected and predicted reactions to the inventive methods of other embryos indicates the broad utility of the inventive methods for somatic embryo development, without a necessary restriction to conifers. In particular, the response of a given embryo to increases in exogenous ABA concentration levels is, based on known response characteristics of both gymnosperm and angiosperm embryos, expected to be parallel for angiosperm and other gymnosperm species to the responses of specific coniferous embryos to ABA concentration increases detailed below in the Examples. Of course, preferred parameters to be selected for any given embryo development project will vary considerably from case to case, depending not only on the embryos selected for development but also on other factors, such as ambient temperature and humidity, choice of growth medium, timing of commencement of development relative to pre-development proliferation of embryos, the target for terminal moisture content, and other aspects of the development conditions, including the type of bioreactor or containment vessel used and the physical characteristics of the support for the embryos within the vessel, and the quantity of embryos being simultaneously developed. As always, an empirical approach will be necessary to optimize the selection of variable parameters.

Increasing levels of growth regulator and/or water stress during embryo development


Calcined ammonium salts of phosphotungstic acid salts are used to polymerize C2-C30 xcex1-olefins and derivatives thereof to form highly reactive products having a high content of terminal vinylidene groups and xcex2-isomers in resonance therewith.

The present invention provides a method for preparing high vinylidene polyisobutylene by polymerizing isobutylene with a solid catalyst of a calcined ammonium salt of a heteropolyacid.
It is well known to polymerize olefins using boron trifluoride (BF3). The polymers so produced are highly reactive due to a large percentage of their terminal groups having vinylidene structure. The reactivity of polyolefins has been related to double bond content and the location thereof in the polymer.
U.S. Pat. No. 4,152,499, Boerzel et al., May 1, 1979, discloses the synthesis of polyisobutylene polymers having a degree of polymerization of 10 to 100 units and a high proportion of reactive double bonds. The isobutene is polymerized with boron trifluoride as the initiator at xe2x88x9250xc2x0 C. to +30xc2x0 C. The polymer contains a proportion of double bonds capable of reacting with maleic anhydride of 60 to 90% of theory. The adducts from the polyisobutylene/maleic anhydride are reacted with polyamines to form products useful as lubricating oil additives.
Polyolefins have also been prepared by polymerization catalyzed with heteropolyacids. U.S. Pat. No. 5,710,225, Johnson et al., Jan. 20, 1998, discloses a method for producing polymers by polymerization of olefins, by contacting a C2-C30 olefin or derivative thereof with a heteropolyacid. The heteropolyacid catalyst can be a partially or fully exchanged with cations from the elements in groups IA, IIA and IIIA of the periodic chart, Group IB-VIIB elements and Group VIII metals, including manganese, iron, cobalt, nickel, copper, silver, zinc, boron, aluminum, bismuth, or ammonium or hydrocarbyl-substituted ammonium salt. The heteropolyacids can be used in their initial hydrated form or they can be treated (calcined) to remove some or all of the water of hydration. The calcining is preferably conducted in air at a temperature of, for instance, up to 375xc2x0 C.; temperatures much over 350xc2x0 C. do not generally provide much advantage. In the resulting polymers, the combined terminal vinylidene and xcex2-isomer content is preferably at least 30%.
U.S. Pat. No. 2,982,799 (Klinkenberg, May 2, 1961) reports that at about 20-200xc2x0 C. isobutylene can be polymerized by use of a specially prepared heteropolyacid catalyst. The catalyst consisted of a heteropolyacid deposited on a solid carrier. The solid carrier had an alkali (including ammonium) content of less than one milliequivalent per 100 grams of carrier and a silico-tungstic acid concentration of 0.5-8% by weight of the total catalyst. The system resulted in oligomers up to C16 from isobutylene, or a degree of polymerization of four. The temperature used was above 20xc2x0 C.
It is believed to be desirable to use highly reactive polyolefins to prepare hydrocarbyl-substituted acylating agents (e.g., anhydrides) by way of a thermal route rather than a chlorine catalyzed route. The thermal route avoids products containing chlorine. The reactivity of the polyolefin is believed to be related to the end group in the polymer with terminal olefins (terminal vinylidene) and terminal groups capable of being isomerized thereto being identified as the reactive species. The groups capable of being isomerized to the terminal vinylidene (I) group are the xcex2-isomers (II) of Table 1.
As used herein, the term xe2x80x9chydrocarbyl substituentxe2x80x9d or xe2x80x9chydrocarbyl groupxe2x80x9d is used in its ordinary sense, which is well-known to those skilled in the art. Specifically, it refers to a group having a carbon atom directly attached to the remainder of the molecule and having predominantly hydrocarbon character. Examples of hydrocarbyl groups include hydrocarbon substituents, substituted hydrocarbon substituents, and hetero substituents, that is, substituents which, while having a predominantly hydrocarbon character, contain other than carbon in a ring or chain otherwise composed of carbon atoms. In general, no more than two, preferably no more than one, non-hydrocarbon substituents will be present for every ten carbon atoms in the hydrocarbyl group; typically, there will be no non-hydrocarbon substituents in the hydrocarbyl group.
Conventional polyolefin synthesis involves Friedel-Crafts type catalysts reacting with terminal olefins such as isobutene or mixtures of compounds such as a C4 raffinate from a cat cracker or an ethylene plant butane/butene stream. The polyolefins so synthesized are not noted for having high terminal vinylidene contents and are thus not the reagents of choice to use the thermal route to forming polyolefin substituted succinic anhydrides. Conventional polyisobutylene (xe2x80x9cPIBxe2x80x9d) when used in thermal condensation procedures with maleic anhydride give low yields and high tar contents and low succination ratios. The thermal route to substituted succinic anhydrides using highly reactive PIB""s has been discussed in detail in U.S. Pat. Nos. 5,071,919, 5,137,978, 5,137,980 and 5,241,003, all issued to Ethyl Petroleum Additives, Inc.
The isomer content of a conventional (AlCl3) and high terminal vinylidene PIB""s are shown in Table 1. Conventional PIB has terminal vinylidene content of roughly 5%. The terminal isomer groups of conventional PIB and high vinylidene PIB are given below in Table 1 and those published in EPO 0355 895. However, in this invention polyisobutylene containing relatively high content of vinylidene and xcex2-isomers can be formed. Such materials can contain at least 30 percent terminal vinylidene (I) and xcex2-isomer (II) groups, as shown below. In preferred cases the polyisobutylene can contain at least 30 percent terminal vinylidene (I) groups, and more preferably at least 60 percent terminal vinylidene groups.
As can be seen from the structures in Table 1, conventional PIB is characterized by very low terminal vinylidene groups (I) and species capable of isomerization therewith (II). Conventional PIB further comprises a distinct tri-substituted terminal olefin group (III) which is nearly absent or present in only a low level in high vinylidene PIB. The distinct terminal group III is a 2-butene in which the 2-carbon is tri-substituted.
Structure IVA of Table 1 is an acid-catalyzed rearrangement product of IV while V is an internal vinylidene group. The terminal group content of conventional and high vinylidene PIBs have been determined by NMR analysis. Conventional PIBs are commercially available under various tradenames including Parapol(copyright) from Exxon, Lubrizol(copyright) 3104, 3108 from Lubrizol and Indopol(copyright) from Amoco and Hyvis(copyright) from BP. Conventional PIBs have number average molecular weight in the range of 300-5000, but the preferred number average molecular weight is in the range of 500-2000.
The present invention provides a method for producing polymers by polymerization of at least one olefin, the method comprising:
contacting (a) at least one C2-C30 olefin or polymerizable derivatives thereof with (b) a catalyst comprising a partially or fully neutralized ammonium salt of a heteropolyacid,
wherein said catalyst has been calcined at above 350xc2x0 C. to 500xc2x0 C.;
whereby the efficiency of polymerization is increased compared to the efficiency in the absence of such calcining.
The present invention further provides polymers of isobutylene having a {overscore (M)}n of at least 1500, {overscore (M)}w/{overscore (M)}n of greater than 4, preferably 7.5 to 20, and preferably at least 30% terminal vinylidene (I) groups.
Heteropolyacid catalysts can exist as the free acid or as a salt of a heteropolyanion. Heteropolyanions are polymeric oxoanions formed by a condensation reaction of two or more different oxoanions, e.g.,
12WO42xe2x88x92+HPO42xe2x88x92+23H+xe2x86x92(PW12O40)3xe2x88x92+12H2O
A variety of structures are known for these materials; they can have, for instance, the so-called Keggin structure, wherein twelve WO6 octahedra surround a central PO4 tetrahedron (in the case where phosphorus is employed). Other structures and related formulas are also known, including PW12O42, PW18O62, P2W5O23, PW9O32, PW6O24, P2W18O62, PW11O39, and P2W17O61, where P and W are taken as representative elements and the indicated structure is an ion with the appropriate charge. The central atom of the Keggin structure, which is typically phosphorus, as shown, can also be any of the Group IIIA to Group VIIA (ACS numbering) metalloids or non-transition metals, including P, As, Si, Ge, B, Al, Sb, and Te. The tungsten (W) in the above formula fills the role known as the xe2x80x9cpoly atom,xe2x80x9d which can be any of the Group VB or VIB transition metals, including W, V, Cr, Nb, Mo, or Ta. Thus suitable materials include preferably phosphomolybdates, phosphotungstates, silicomolybdates, and silicotungstates. Other combinations selected from among the above elements are also possible, including borotungstates, titanotungstates, stannotungstates, arsenomolybdates, teluromolbydates, aluminomolybdates, and phosphovanadyltungstates, the latter representing a mixed material having a formula (for the anion portion) of PW11VO40. The preferred material is a phosphotungstate, which term generally encompasses both the acid and the various salts, described below.
The heteropoly catalysts are active both as their acid form, in which the anion is associated with the corresponding number of hydrogen ions, in the fully salt form, in which the hydrogen ions have been replaced by other cations such as metal ions, or in the partially exchanged salt form, in which a portion of the hydrogen ions have been thus replaced. For more detailed information on the structures of heteropoly catalysts, attention is directed to Misono, xe2x80x9cHeterogeneous Catalysis by Heteropoly Compounds of Molybdenum and Tungsten,xe2x80x9d Catal. Rev.xe2x80x94Sci. Eng., 29(2and3), 269-321 (1987), in particular, pages 270-27 and 278-280. In the present invention, the hydrogen ions have been partially or fully replaced by ammonium, that is the catalyst is a partially or fully neutralized ammonium salt of a heteropolyacid. Moreover, the catalyst has been calcined at above 350xc2x0 C. to 500xc2x0 C.
Heteropoly acids are commercially available materials, (e.g., Aldrich Chemical Company, #22,420-0). The salts are similarly commercially available. Alternatively, they can be prepared from the acid materials by neutralization with an appropriate amount of base. Heteropoly acids are generally received in a hydrated form. They can be successfully employed in this form (uncalcined) or as in the present invention, they can be treated (calcined) to remove some or all of the water of hydration, that is, to provide a dehydrated or otherwise modified species, which in the context of the present invention exhibits improved reactivity. Calcining can be conducted by simply heating the hydrated material to a suitable temperature to drive off the desired amount of water. The heating can be under ambient pressure or reduced pressure, or it can be under a flow of air or an inert gas such as nitrogen. The use of air ensures that the acid is in a high oxidation state. The flow of air can be across the surface of the catalyst, or for greater efficiency, it can be through the bulk of the catalyst. The length of time required for calcining is related to the equipment and scale, but in one broad embodiment the calcining can be conducted over the course of 5 minutes to 16 hours, more typically 30 minutes to 8 hours, and preferably 1 hour, 2 hours or even 3 hours, up to 4 hours. The upper limits of time are defined largely by the economics of the process; times in excess of about 5 hours do not generally provide much advantage.
The material which is calcined to prepare the catalysts for use in the present invention is preferably an ammonium salt of H3PW12O40. Typical ammonium salts include (NH4)3PW12O40 and (NH4)2.5H0.5PW12O40. While generally the temperature of calcining will be in the range of above 350xc2x0 C. to 500xc2x0 C. and preferably 375 to 475xc2x0 C., the optimum conditions will depend to some extent on the particular ammonium salt which is selected. When the starting salt is (NH4)3PW12O40, it has been found that relatively higher temperatures are desirable for obtaining the most active catalyst. Therefore, such material is preferably calcined at 450 to 475xc2x0 C. When the starting salt is (NH4)2.5H0.5PW12O40, desirable calcining temperatures can be somewhat lower, namely, above 350 to 475xc2x0 C. and preferably above 375 to 475xc2x0 C. When the calcining temperature is too low, the catalysts may be largely or entirely inactive. For instance, when (NH4)3PW12O40 is treated at below 350xc2x0 C., it is generally found to be substantially inactive to provide the polymers of the present invention. This phenomenon is not fully understood; but, without intending to limit the generality or scope of the invention, it is believed that the high temperature calcining serves to remove a portion of the ammonia from the catalyst, thereby leading to a more active species. The time and temperature of the calcining are believed to be interrelated to some extent, so that use of temperatures in the lower ranges can be more effective when the calcining is conducted for a longer period of time, and vice versa, as will be apparent to the person skilled in the art.
The catalyst can be employed as particles of the pure salt, or it can be provided on a solid support of an inert material such as alumina, silica/alumina, an aluminophosphate, a zeolite, carbon, clay, or, preferably, silica. The source of the solid silica support can be a colloidal silica, which is subsequently precipitated during the catalyst preparation, or a silica which has already been preformed into a solid material. The catalyst can be coated onto the support by well-known catalyst impregnation techniques, e.g., by applying the catalysts as a solution, followed by drying, such as by spray drying or evaporation. If a support such as silica is employed, the ratio of the active catalyst component to the silica support will preferably be in the range of 0.5:99.5 to 50:50 by weight, preferably 3:97 to 40:60 by weight, and more preferably 10:90 to 30:70 by weight.
The temperatures used in this invention for the polymerization of olefins is preferably below 20xc2x0 C. and more preferably below 10xc2x0 C. Preferred temperature ranges are xe2x88x9230 to 20xc2x0 C., more preferably xe2x88x9220 to 10xc2x0 C. and most preferably about xe2x88x925xc2x0 C., which is the approximate reflux temperature of isobutylene. The polymerization can be conducted in a batch apparatus or using continuous apparatus, such as a continuous stirred tank reactor or a tubular reactor, as will be apparent to those skilled in the art. The residence time of the polymerization reaction will vary with conditions including the type of reactor. Generally suitable residence times of 5 or 10 to 60 minutes, preferably 20 to 40 minutes. The polymerization can be conducted neat but is preferably conducted in the presence of a substantially inert hydrocarbon solvent or diluent, such as isobutane, pentane, hexane, octane, decane, kerosene, or Stoddard Solvent, which will normally be removed by conventional means at the conclusion of the reaction. The reaction using the catalysts of the present invention will generally provide at least a 10% conversion under these conditions, and preferably at least 20 or 25% conversion to polymer.
The preferred products are polyisobutylenes having {overscore (M)}n greater than 300. For the C4 isobutylene, this would correspond to an average degree of polymerization (dp) of about 5.3. The preferred {overscore (M)}n of polyisobutylene is at least 500 and more preferably at least 1000 or 1500, and up to 5,000, preferably in the range of 2000 to 5000. Such materials are particularly useful when used in reactions to alkylate maleic anhydride and for subsequent derivatization to form, e.g., imides, for use as additives for lubricants, as is well known to those skilled in the art. As well as isobutylenes, other C2-C30 olefins and derivatives thereof may be used in this invention as well as styrene and derivatives thereof, conjugated dienes such as butadiene and isoprene and non-conjugated polyenes. The reaction to produce polymers may be run with mixtures of starting olefins to form copolymers. The mole ratio of olefin substrate to catalyst in this invention ranges from 1,000:1 to 100,000 to 1.
The polymers produced by the process of this invention are derived from C2-C30 olefin monomers and mixtures thereof and derivatives thereof. Under this terminology, styrene and derivatives would be a C2-olefin substituted by a phenyl group.
Useful olefin monomers from which the polyolefins of this invention can be derived are polymerizable olefin monomers characterized by the presence of one or more unsaturated double bonds (i.e.,  greater than Cxe2x95x90C less than ); that is, they are monoolefinic monomers such as ethylene, propylene, butene-1, isobutylene, and octene-1 or polyolefinic monomers (usually diolefinic monomers) such as butadiene-1,3 and isoprene.
These olefin monomers are preferably polymerizable terminal olefins; that is, olefins characterized by the presence in their structure of the group xe2x80x94Rxe2x80x2xe2x80x94CHxe2x95x90CH2, where Rxe2x80x2 is H or a hydrocarbyl group. However, polymerizable internal olefin monomers (sometimes referred to in the patent literature as medial olefins) characterized by the presence within their structure of the group:
can also be used to form the polyalkenes. When internal olefin monomers are employed, they normally will be employed with terminal olefins to produce polyalkenes which are interpolymers. For purposes of this invention, when a particular polymerized olefin monomer can be classified as both a terminal olefin and an internal olefin, it will be deemed to be a terminal olefin. Thus, for example, pentadiene-1,3 (i.e., piperylene) is deemed to be a terminal olefin for purposes of this invention.
While the polyalkenes of this invention generally are hydrocarbon polyalkenes, they can contain substituted hydrocarbon groups such as lower alkoxy, and carbonyl, provided the non-hydrocarbon moieties do not substantially interfere with the functionalization reactions of this invention. Preferably, such substituted hydrocarbon groups normally will not contribute more than 10% by weight of the total weight of the polyalkenes. Since the polyalkene can contain such non-hydrocarbon substituents, it is apparent that the olefin monomers from which the polyalkenes are made can also contain such substituents. Normally, however, as a matter of practicality and expense, the olefin monomers and the polyalkenes will be free from non-hydrocarbon groupsxe2x80x94(as used herein, the term xe2x80x9clowerxe2x80x9d when used with a chemical group such as in xe2x80x9clower alkylxe2x80x9d or xe2x80x9clower alkoxyxe2x80x9d is intended to describe groups having up to seven carbon atoms.)
Although the polyalkenes of this invention may include aromatic groups (especially phenyl groups and lower alkyl- and/or lower alkoxy-substituted phenyl groups such as para-(tert-butyl)phenyl) and cycloaliphatic groups such as would be obtained from polymerizable cyclic olefins or cycloaliphatic substituted-polymerizable acrylic olefins, the polyalkenes usually will be free from such groups. Again, because aromatic and cycloaliphatic groups can be present, the olefin monomers from which the polyalkenes are prepared can contain aromatic and cycloaliphatic groups.
There is a general preference for polyalkenes which are derived from the group consisting of homopolymers and interpolymers of terminal hydrogen olefins of 2 to 16 carbon atoms. A more preferred class of polyalkenes are those selected from the group consisting of homopolymers and interpolymers of terminal olefins of 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms.
Specific examples of terminal and internal olefin monomers which can be used to prepare the polyalkenes of this invention include propylene; butene-1; butene-2; isobutylene; pentene-1; hexene-1; heptene-1; octene-1; nonene-1; decene-1; pentene-2; propylene-tetramer; diisobutylene; isobutylene trimer; butadiene-1,2; butadiene-1,3; pentadiene-1,2; pentadiene-1,3; isoprene; hexadiene-1,5; 2-chloro-butadiene-1,2; 2-methyl-heptene-1; 3-cyclohexylbutene-1; 2-methyl-5-propyl-hexene-1; pentene-3; octene-4; 3,3-dimethyl-pentene-1; styrene; 2,4-dichlorostyrene; divinylbenzene; vinyl acetate; allyl alcohol; 1-methyl-vinyl acetate; ethyl vinyl ether; and methyl vinyl ketone. Of these, the hydrocarbon polymerizable monomers are preferred and of these hydrocarbon monomers, the terminal olefin monomers are particularly preferred.
Useful polymers formed in this invention include alpha-olefin homopolymers and interpolymers, and ethylene/alpha-olefin copolymers and terpolymers. Specific examples of polyalkenes include polypropylene, polybutene, ethylene-propylene copolymer, ethylene-butene copolymer, propylene-butene copolymer, styrene-isobutylene copolymer, isobutylene-butadiene-1,3 copolymer, propene-isoprene copolymer, isobutylenechloroprene copolymer, isobutylene-(para-methyl)styrene copolymer, copolymer of hexene-1 with hexadiene-1,3, copolymer of octene-1, copolymer of 3,3-dimethyl-pentene-1 with hexene-1, and terpolymer of isobutylene, styrene and piperylene. More specific examples of such interpolymers include copolymer of 95% (by weight) of isobutylene with 5% (by weight) of styrene; terpolymer of 98% of isobutylene with 1% of piperylene and 1% of chloroprene; terpolymer of 95% of isobutylene with 2% of butene-1 and 3% of hexene-1; terpolymer of 60% of isobutylene with 20% of pentene-1; and 20% of octene-1; terpolymer of 90% of isobutylene with 2% of cyclohexene and 8% of propylene; and copolymer of 80% of ethylene and 20% of propylene. U.S. Pat. No. 5,334,775 describes polyolefin based polymers of many types and their monomer precursors and is herein incorporated by reference for such disclosure.
Relative amounts of end units in conventional and high vinylidene polyisobutylenes are determined from NMR spectra made using a Burker AMX 300 or 500 instrument and UXNMRP software to work up the spectra. The spectra are determined at 300 or 500 MHz in CDCl3. Band assignments in the NMR for the various isomers as parts per million (ppm) down field shift from tetramethyl silane are: terminal vinylidene 4.68 and 4.89, xcex2-isomer 5.18, tri-substituted 5.17 and 5.35, tetra 2.88.
The molecular weight of the isomers are typically determined by GPC on a Waters 150 instrument run with tetrahydrofuran solvent. The columns are Waters ultra-styrogel of pore size 104 xc3x85, 103 xc3x85, 500 xc3x85, and 300 xc3x85 which have been calibrated with PIB standards. Styrene molecular weight standards are also useful. {overscore (M)}n and {overscore (M)}w are determined from comparative elution volume data. Molecular weight values of the polymers produced by the method of this invention will vary according to their degree of polymerization. The dp range for products of this invention typically range from 6 to 350 or even higher.
The polydispersity of the products of this invention as determined by the ratio of {overscore (M)}w/{overscore (M)}n may have a value of up to 20 depending upon reaction conditions. At any given reaction temperature, the {overscore (M)}w/{overscore (M)}n is controlled by the chemical nature of the catalyst as well as the contact time of the olefin with the catalyst and the concentration of the olefin during the reaction. Use of the calcined ammonium catalysts of the present invention in the polymerization of isobutylene leads to polyisobutylene having a polydispersity typically greater than 4, or 5, often 7.5 to 20, more commonly 8 to 19 or 18. The present invention provides a way of preparing such materials directly, from a single polymerization reaction, as opposed to by blending of different batches prepared from separate polymerization reactions. It is, of course, well known to prepare polymeric mixtures of high polydispersity by physical admixture of samples of polymers of significantly different molecular weights. Such blending, however tends to produce polymeric mixtures which are polymodal (including bimodal) or otherwise non-uniform in their molecular weight distribution. The process of the present invention, on the other hand, can lead to polymers having a relatively uniform or monomodal molecular weight distribution, yet having the present high degree of polydispersity.

Ammonium heteropolyacid catalized polymerization of olefins