Abstract:
This invention is directed to a method for producing somatic embryos on plant tissue. Furthermore, somatic embryos may be produced using explants obtained from a broad range of plant species, and using either juvenile or mature tissues. The method involves obtaining a stock tissue culture plantlet by exposing the plant tissue to a medium comprising salts, vitamins and an energy source; preparing an explant from the stock tissue culture plantlet; transferring the explant to a proliferation medium comprising salts, vitamins, an energy source and at least one growth regulator for a period of time sufficient to produce a callused explant; and transferring the callused explant to a medium comprising salts, vitamins, an energy source and at least two growth regulators for a period of time sufficient to produce somatic embryos. Following this method somatic embryos are produce in significantly less time that observed using other somatic embryogenesis protocols. Plants produced from the somatic embryos are easily prepared. Since somatic embryos can be obtained from mature tissues, specific plants of agronomic importance comprising desirable traits, may now be clonally propagated. Furthermore, somatic embryos produced following the method of this invention may be used for the preparation of synthetic seed.

Description:
The present invention relates to a method for the production of somatic embryos in plants. 
     BACKGROUND OF THE INVENTION 
     Somatic embryos are embryos formed in tissue cultures of plants, although there are reports of somatic embryos forming in vivo. The structure and morphology of somatic embryos is like that of zygotic embryos from true seed, that is somatic embryos go through the same development stages--proembryo, globular, heat-shaped and torpedo-shaped morphology. However, somatic embryos lack a seed coat, and also sometimes lack a suspensor. Unless they are specially treated, somatic embryos also lack the dormancy that characterizes true seed (Gray et al., 1995a). 
     Somatic embryos are clonal in origin and thus multiplication using somatic embryos can have the potential for exceedingly high rates of vegetative increase and is therefore of considerable commercial interest. Somatic embryos also lend themselves to large scale production in bioreactors when grown in liquid culture (Bajaj, 1995a) and this form of propagation is of commercial interest when somatic embryos are transformed to provide novel plant forms. 
     Regeneration via somatic embryogenesis is an attractive option for plant tissue culture. Somatic embryos reportedly provide more stable regenerants than shoots. Another advantage of regeneration systems using somatic embryos is their apparent single cell origin. This means that it is unlikely that regenerants are of chimerical origin, since, if a regenerant originates from a cluster of cells rather than a single sell, the plant tissues may be chimerical or unstable and produce off-types. The availability of somatic embryogenesis protocols for potato, or other crop species recalcitrant to somatic embryogenesis, will permit these crops to take advantage of any new artificial seed technology advances. 
     Immature somatic embryos are ideal material for transformation protocols (Ellis, 1995). The induction of somatic embryos from cultured tissues of Vitus vinifera (Goussard and Wiid, 1993) and Citrus (D&#39;Onghia et al., 1979) has led to the elimination of several viruses and viroids. Scorza et al. (1996) disclose the transformation of somatic embryos of `Thompson Seedless` grape. The use of somatic embryo technology for potato and other species recalcitrant to somatic embryogenesis, would open new avenues for research and development. As somatic embryos are reported to be more stable regenerants than adventitious shoots, the use of somatic embryos as transformant tissues may overcome the difficulties with off-types which are inherent with the use of stem internode leaf and microtuber tissue. Somatic embryos are suitable for transformation via Agrobacterium tumefaciens (Mathews et al., 1992), microinjection (Neuhaus et al., 1987) and particle bombardment (Wilde et al., 1992). 
     Many plant species regenerate shoots from tissues cultured in vitro, but the low frequency of shoot regeneration and the lack of stability (trueness-to-type) makes this technology less desirable. This is to be contrasted with the proposed somatic embryo technology described herein. This technology is very productive and results in many embryos being regenerated from one explant. Furthermore, this technology can be readily adapted for use with existing or future transformation technologies. Prior to transforming plant tissues with novel genes, a regeneration technology has to be in place or there is no way to recover transformed plants. Plant tissues regenerated as shoots or somatic embryos in vitro can be used to carry valuable genetic material coding for the production of vaccines, disease resistant plants, superior agronomic characteristics, food processing qualities, etc. 
     Somatic embryos can be cryopreserved using liquid nitrogen without loss of viability. Cryopreservation is an efficient means of maintaining germplasm and enables plant material to be transported over large distances. Furthermore, somatic embryos are suitable for the development of artificial seed technology (U.S. Pat. No. 5,572,827; Bajaj, 1995 a). Regeneration technologies in which somatic embryos are the propagule can produce many more regenerants and lead the way for the production of artificial seed (synseed) technology. Synthetic seed, appropriately coated with products providing nutrients and protection against desiccation and microorganisms may be used within fluid drilling technology to &#34;sow&#34; the synseeds under field conditions. 
     Somatic embryos have been produced from juvenile somatic tissues, or from cultured stocks of juvenile tissues, for a few potato cultivars (Bragd.o slashed.-Aas, 1977; Garcia and Martinez, 1995; Pretova and Dedicova, 1990). Pretova and Dedicova (1992) disclose the production of cotyledonary somatic embryos obtained from zygotic embryo tissues (i.e. juvenile tissues) from potato in media using BAP, dicamba or 2,4-D. They note that friable callus is obtained after 2 weeks. However, a granular callus is not obtained for 6-8 weeks on media containing 2,4-D. After subculturing this callus onto hormone free media supplemented with sucrose, somatic embryos appear within 14-18 days. Bragd.o slashed.-Aas (1977) discloses the formation of callus from potato tuber tissue and the regeneration of plants from this callus tissue. It is noted that there is great variety in the production of callus depending upon the genetic background of the material used. Furthermore, the applicability of this method is limited to certain clonal sources and therefore lacks applicability to a range of cultivars . Hutchinson et al. (1996) teach the production of somatic embryos using explants obtained from six day old etiolated hypocotyls of geranium. The media contained either TZD, or IAA+BAP, and embryo formation was observed over a 21 day period. The production of embryos was not investigated past the 21 day period. 
     Garcia and Martinez (1995) disclose the induction of somatic embryos from cultured stem sections of the potato variety `Bintje`. The protocol employed by these workers requires about 20 to 25 weeks to produce somatic embryos, and another 5 to 6 weeks to produce plantlets from these somatic embryos. However, the method of this invention produces somatic embryos after 4 to 6 weeks, with plantlets forming within 2 to 4 weeks from these somatic embryos. Furthermore, within the prior art, somatic embryos from potato have only been obtained from tetraploid genotypes, however, following the protocol of this invention, somatic embryos have been produced using tetraploid, diploid and monoploid potato cultivars. There are also many other advantages associated with the production of somatic embryos following the method disclosed herein (e.g. see Table 1). 
     Similarly, somatic embryos from seedling tissues, such as cotyledons, which are juvenile in a physiological sense, of tomato (Chen and Adachi, 1994; Gill et al., 1995) and lettuce (Zhou et al., 1992) have been prepared. Furthermore, somatic embryos have been prepared using hypocotyl sections of juvenile tomato seedlings. However, the ability to produce somatic embryos from mature tissues is in general lacking. This is also true with conifer somatic embryo technology as well, which is directed to juvenile (zygotic embryo) tissues. The preparation of somatic embryos using zygotic (seed) embryos is a common way of achieving embryogenesis. However, this system has the disadvantage that the variability of a heterozygous species produces unreliable propagules for industrial applications. 
     There is a real need within the art to be able to propagate a plant that has already been selected for a set of desirable traits. Current breeding practices typically require sexual crossing, grafting or other procedures that results in the offspring never being identical to the parent. Since the ability to propagate the exact same individual does not exist with these techniques, a breeder must then rescreen the offspring in order to reselect and validate the stock material for the desired set of characteristics. The ability to propagate a plant comprising the same set of characteristics as the parent, is available through clonal propagation. However, the use of juvenile tissues for the production of somatic embryos, does not improve this situation as the material used for somatic embryogenesis has itself not been subject to the screening process. The ability to produce somatic embryos from mature tissues, however, provides the ability to generate clonal propagules from an individual plant that has been subject to a screening process, so that the propagule comprised the same set of characteristics as the pre-selected parent. 
     The regeneration of tissues from mature plants of potato, tomato and lettuce via somatic embryogenesis would enable industry to rapidly propagate elite plants of horticultural value. By contrast, propagating tissues from zygotic embryos is not feasible in heterozygous plants such as potato which are vegetatively propagated because of the variability of seedling material. Furthermore, somatic embryos lend themselves to transformation of valuable genotypes. 
     By using the method of the present invention: 
     somatic embryos have been obtained on a plurality of potato (Solanum tuberosum L.) cultivars, of a variety of ploidy levels in vitro. This protocol can be used for clonal multiplication of selected potato genotypes, regeneration after gene transformation or bombardment, large-scale production of somatic embryos for artificial seed production, etc.; 
     somatic embryos are obtained within significantly shorter time frames than prior art methods; 
     somatic embryos can be induced to form on various somatic tissues of the potato, including stem internodes, roots, leaves, microtuber slices; 
     somatic embryos can be produced on stem internode tissue from plantlets in vitro of tomato; 
     Somatic embryos have been produced on seedling leaf and stem tissue of lettuce in vitro. Somatic Embryos have also been produced on mature tissues of lettuce in vitro. This is of commercial interest because superior genotypes can be multiplied clonally. 
     SUMMARY OF THE INVENTION 
     The present invention relates to a method for the production of somatic embryos in plants. 
     The method of the present invention involves the use of several different culture media and the brief exposure of a tissue explant to these media. By so doing, the overall time required to obtain somatic embryos is dramatically reduced, and somatic embryos can be produced from typically recalcitrant species. Furthermore, somatic embryos can be prepared from mature tissues in a wide variety of species. 
     According to the present invention there is provided a method (A) for producing somatic embryos on plant tissue comprising: obtaining a stock tissue culture plantlet by exposing the plant tissue to a medium (medium #1) comprising salts, vitamins and an energy source; preparing an explant from the stock tissue culture plantlet; transferring the explant to a proliferation medium (medium #2) comprising salts, vitamins, an energy source and at least one growth regulator for a period of time sufficient to produce a callused explant; and transferring the callused explant to a medium (medium #3) comprising salts, vitamins, an energy source and at least two growth regulators for a period of time sufficient to produce somatic embryos. 
     This invention is also directed at a method as described above, wherein the stock tissue culture plantlet is maintained from about 4 to about 5 weeks prior to transfer to the proliferation medium. This invention is encompasses the above method, wherein the period of time the explant is maintained in the proliferation medium is from about 1 to about 4 weeks, and wherein the period of time within the medium comprising at least two growth regulators is from about 2 to about 6 weeks. 
     This invention also provides a method as described above, wherein the growth regulator of the proliferation medium is selected from the group consisting of Thidiazuron, BAP, or Indole acetic acid, or a combination thereof Furthermore, the growth regulators within the medium comprising at least two growth regulators are selected from the group consisting of Zeatin, Indole acetic acid, or Gibberellic acid, or a combination thereof 
     This invention also embraces the above method wherein the plant tissue is obtained from potato, tomato or lettuce, and wherein the plant tissue is obtained from mature tissue including stem internode, root, leaf, microtuber tissue, leaf, or axilary bud tissue. 
     This invention is also directed to the above method wherein the somatic embryo is grown into a plant, cryopreserved, or treated for use as a synthetic seed. 
     This invention also provides for a somatic embryo produced using the method as described above, as well as a plant obtained using a somatic embryo obtained using the method described above. 
     Furthermore, this invention describes a method (B) of maintaining a plant comprising a desired set of characteristics comprising: screening a library of plants for a desired set of characteristics; selecting a plant having the desired set of characteristics; preparing a somatic embryo, from tissue obtained from the selected plant, using the method (A); and either storing the somatic embryo, or growing the somatic embryo into a plant. 
     This invention also encompasses a method of obtaining a transgenic plant with a desired set of characteristics comprising: transforming a plant with a gene of interest; screening transformed plants to obtain a plant with the desired set of characteristics; obtaining an explant from the transformed plant; and preparing somatic embryos using the method (A) as defined above. Also considered within the scope of the present invention is a method of preparing a transgenic plant comprising: preparing somatic embryos according to the method (A) defined above; introducing a gene of interest into the somatic embryo; screening the transformed somatic embryos for the presence of the transgene; growing the selected somatic embryo into a plant; and optionally, propagating the selected somatic embryo or plant. 
     This invention is directed at a method (A) for producing somatic embryos as defined above wherein: 
     the stock tissue culture plantlets are maintained on a medium (Medium #1) comprising: 
     
         ______________________________________Murashige and Skoog salts, full strengthsucrose              30     g/lmyo-inositol         100    g/lvitaminsNicotinic acid       0.5    mg/lPantothenic acid     2.5    mg/lPyridoxine.HCl       1.0    mg/lThiamine.HCl         0.5    mg/lpH5.8agar                 8      g/l;______________________________________ 
    
     the proliferation medium, medium #2, comprises: 
     
         ______________________________________Murashige and Skoog salts, full strengthsucrose              30     g/lmyo-inositol         100    mg/lvitaminsNicotinic acid       0.5    mg/lPantothenic acid     2.5    mg/lPyridoxine           1.0    mg/lThiamine.HCl         0.5    mg/lpH 5.8Agar                 6.5    g/l;______________________________________ 
    
     and further comprises the constituents selected from the group consisting of; 
     
         ______________________________________Medium 2a:Thidiazuron          0.15   μMIndole acetic acid   19     μM;Medium 2bThidiazuron          0.15   μM;Medium 2cBAP                  0.15   μMIAA                  19     μM; orMedium 2dBAP                  0.15   μM;______________________________________ 
    
     and the medium comprising at least two growth regulators, medium #3, comprises: 
     
         ______________________________________Murashige and Skoog salts, full strengthsucrose              30     g/lmyo-inositol         100    mg/lvitaminsnicotinic acid       1.0    mg/lthiamine.HCl         0.5    mg/lpantothenic acid     0.5    mg/lpyridoxine           1.0    mg/lZeatin               12.0   μMIndole acetic acid   0.05   μMGibberellic acid     0.55   μMpH 5.8agar                 8      g/l______________________________________ 
    
     Many advantages are noted between the above described method and variations thereof, and prior art methods for obtaining somatic embryos. These advantages include: 
     obtaining somatic embryos within shorter time frames than prior art methods (weeks to months shorter than previously published methods); 
     adaptability to a range a wide variety of plant species, and cultivars that typically have not been amenable to the production of somatic embryos; 
     effectiveness in obtaining somatic embryos from both mature or juvenile tissues. Prior art methods are typically limited to juvenile tissues; 
     with regards to potato cultivars, somatic embryos have been produced using tetraploid, diploid and monoploid tissues. All prior art methods have only been successful using tetraploid tissues, and, to the best of our knowledge, no somatic embryos have previously been reported using diploid or monoploid potato tissues. As a results of these advantages, mature plants may be selected for desirable properties and clonally propagated to ensure maintenance of the genotype and phenotype of the stock tissues. This last advantage has not been previously available within this field since prior art methods for obtaining somatic embryos require juvenile tissues. Any selection of plants with desirable traits, therefore had to place using plants obtained from somatic embryos. This requires producing large amounts of somatic embryos from which plants are regenerated, and the screening of these libraries of regenerated plant stocks. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein: 
     FIGS. 1A-1H are a composite print of somatic embryos forming on potato (Solanum tuberosum L.) cultivar `Russet Burbank`. 
     FIG. 1A--non-embryogenic callus cells; 
     FIG. 1B--overview of non-embryogenic callus; 
     FIG. 1C--germinating somatic embryo with two additional somatic embryos forming at basal area; 
     FIG. 1D--torpedo stage somatic embryo with hear-shaped somatic embryo forming nearby; 
     FIG. 1E--slightly different view of embryos in plate FIG. D; 
     FIG. 1F--detail of heart-shaped somatic embryo shown in FIG. 1D and FIG. 1E, note large callus cells nearby; 
     FIG. 1G and FIG. 1H--late heart-shaped somatic embryo showing unequal sized cotyledons and external arrangement of epidermal cells. 
    
    
     DESCRIPTION OF PREFERRED EMBODIMENT 
     The present invention relates to a method for the production of somatic embryos in plants. 
     Following the method of this invention, somatic embryos are observed using mature tissues as the material from which somatic embryos are produced. Somatic embryos have been obtained using a large range of potato germplasm as well as several other species of plants, for example but not limited to, tomato (all genotypes tested) and lettuce (all genotypes tested). Furthermore, preliminary tests indicate that other species are amenable to the production of somatic embryos following the method of this invention. Globular, heart-shaped and torpedo somatic embryos are observed in all cultures, and embryos excised from these embryos have been grown as plants. Emblings and fully established plants are easily grown from somatic embryos produced using the method of this invention. An analysis of plants regenerated from emblings (that is the small plantlet grown from a somatic embryo) indicates that off-types are not always observed. However, the frequency of observed off-types appears to be influenced by the genotype from which the somatic embryo was obtained, since several genotypes exhibited higher rates of off-types than others. 
     The method of this invention involves the exposure of explant tissues to short-period treatments of at least two media formulations comprising different compositions of growth regulators. As a result of the use of the method described herein, somatic embryos are obtained within significantly shorter time-frame than prior art methods. Furthermore, the present method is readily adaptable to a range of plant species and cultivars that other wise have not been amenable to the production of somatic embryos, and has proven effective using both mature or juvenile tissues in order to produce somatic embryos. Furthermore, reculture of somatic embryos is possible using the method of this invention. 
     The basic method is outlined below, however, it is to be understood that variations within this method are readily within the skill of one of skill within the art, and alterations and variations to the over all protocol of this invention are to be considered within the scope of the method disclosed below. In order to exemplify the method and possible medium compositions used, the production of somatic embryos from potato is first outlined, however, variations in this protocol have also been successfully used to obtain somatic embryos from a range of potato cultivars including tetraploid, diploid and monoploid mature tissue, as well as several cultivars of tomato and lettuce. 
     Induction of Somatic Embryos of Potato 
     Somatic embryos have been obtained from juvenile potato tissue within the prior art (Petrova and Dedicova, 1992) as well as cultured plant material (Garcia and Martinez, 1995), however, the protocol used is time consuming. For example Garcia and Martinez report that their method requires about 20 to 25 weeks, that is, 5 weeks for callus formation, 12 weeks for embryogenic callus formation and a further 8 weeks for high frequency somatic embryo production in potato (see page 529 of Garcia and Martinez, 1995). Furthermore, their method and has several drawbacks including abnormal embryo development. The method of this invention produces high frequency embryos in potato within 4 to 6 weeks, with no abnormal embryo development. Other comparisons are provided in Table 1, below. 
     Stock Material for Excision of Explants 
     Stock tissue culture plantlets of potato cultivars may be propagated using any suitable means. For example, cultivars may be propagated by single-node cuttings established from shoot tips excised from potato sprouts, or root, leaf or microtuber tissues. Cultures are grown in vitro under any suitable day/night regime, and the growth temperature should be maintained from about 12° C. to about 23° C. Without wishing to limit the method of this invention in any manner, the following conditions have proved to be suitable for the preparation of stock material: 
     standard cool white fluorescent lamps supplying a suitable light intensity, for example of 25 w/m 2   
     a day/night regime comprising 16 hour photoperiod, 8 hour dark period 
     a growth temperature of 19° C.±1. The basal culture medium can be selected from several available within the art, for example Murashige and Skoog (1962), however, other media as known to those of skill in the art may also be used. For example suitable media may include White&#39;s Medium (White 1963), or Linsmayer and Skoog&#39;s medium (Linsmayer and Skoog 1965) or the like may also be used. This medium should comprise a salt solution, vitamins, and an energy source, for example sucrose, with no added growth regulators. The following medium (Medium #1), which is not to be considered limiting in any manner, may be used to culture the stock plants from which the subsequent explant material such as a stem intenode explants (or root, leaf, or microtuber derived explants), are obtained. 
     
         ______________________________________Medium #1______________________________________Murashige and Skoog (1962) salts, full strengthenergy source, sucrose 30     g/lmyo-inositol           100    g/lvitaminsNicotinic acid         0.5    mg/lPantothenic acid       2.5    mg/lPyridoxine.HCl         1.0    mg/lThiamine.HCl           0.5    mg/lpH 5.8TC agar (JRH Biosciences Ltd.)                  8      g/l______________________________________ 
    
     Induction of Nodular Callus 
     When stock plantlets have been incubated under the above conditions from about 2 to about 20 weeks, or preferably from about 3 to about 10 weeks, or more preferably, from about four to about five weeks, sections of stem internode are excised aseptically and placed onto Proliferation medium (medium #2). The length of stem internode may vary per cultivar used, a suitable length, for example, may be from about 0.5 to about 1.5 cm. Best results in producing the maximum number of somatic embryos per explant are observed if healthy, vigorous stock tissue culture plantlets material is used. 
     The proliferation medium, medium #2, is typically a semi-solid medium, however, other media exhibiting a range of consistencies may also be found to be effective. Suitable proliferation media, without intending to limit the use of other media for this purpose, may comprise Thidiazuron or BAP, and optionally Indole acetic acid. Several compositions of Medium 2 are listed below as examples of Proliferation media, however, variations of the composition of medium #2 have been found to be effective in producing somatic embryos with different cultivars (see Medium 2 a to 2 d--below), therefore, other variations of the composition of medium #2 may also be effective as a proliferation medium: 
     
         ______________________________________Medium #2, Proliferation Medium______________________________________Medium 2aMurashige and Skoog (1962) salts, full strengthenergy source, sucrose    30     g/lmyo-inositol              100    mg/lvitaminsNicotinic acid            0.5    mg/lPantothenic acid          2.5    mg/lPyridoxine                1.0    mg/lThiamine.HCl              0.5    mg/l*Thidiazuron              0.15   μM*Indole acetic acid       19     μMpH 5.8TC Agar                   6.5    g/lMedium #2bMurashige and Skoog (1962) salts, full strengthenergy source, sucrose    30     g/lmyo-inositol              100    mg/lvitaminsnicotinic acid            0.5    mg/lpantothenic acid          2.5    mg/lpyridoxine                1.0    mg/lthiamine                  0.5    mg/l*Thidiazuron              0.15   μMpH 5.8TC agar                   65.    g/lMedium #2cMurashige and Skoog (1965) salts, full strengthenergy source, sucrose    30     glmyo-inositol              100    mg/lvitaminsnicotinic acid            0.5    mg/lpantothenic acid          2.5    mg/lpyridoxine                1.0    mg/lthiamine                  0.5    mg/l*BAP                      0.15   μM*IAA                      19     μMpH 5.8TC agar                   6.5    g/lMedium #2dMurashige and Skoog (1965) salts, full strengthenergy source, sucrose    30     g/lmyo-inositol              100    mg/lvitaminsnicotinic acid            0.5    mg/lpantothenic acid          2.5    mg/lpyridoxine                1.0    mg/lthiamine                  0.5    mg/l*BAP                      0.15   μMpH 5.8TC agar                   6.5    g/l______________________________________ *Filter sterilized 
    
     Explants are maintained on medium #2 from about 3 days to about 4 weeks, or more preferably from about 6 to 15 days. A suitable time for maintaining explants on this medium, which should not be considered limiting in any manner, is of about 7 days. Tissues maintained on medium #2 increase in size, and the cut surfaces of the tissues has been found to suberize. Routinely, an increase in size of about five-fold is observed. A nodular, callus forms on the cut surface of the explants, typically in 1 to 2 weeks after incubation on medium #2. 
     Following this step, the explants are transferred to medium #3. An example of Medium # is described below, however, variations in the composition of this medium may be used and this composition is not to be considered limiting in any manner: 
     
         ______________________________________Medium #3______________________________________Murashige and Skoog (1962) salts, full strengthenergy source, sucrose    30     g/lmyo-inositol              100    mg/lvitaminsnicotinic acid            1.0    mg/lthiamine.HCl              0.5    mg/lpantothenic acid          0.5    mg/lpyridoxine                1.0    mg/l*Zeatin                   12.0   μM*Indole acetic acid       0.05   μM*Gibberellic acid         0.55   μMpH 5.8TC agar                   8      g/l______________________________________ *Filter sterilized 
    
     Somatic embryos typically form from about two to about six weeks after explants are transferred to medium #3, and have been observed first on the edge of cut surfaces and then to form over the callused cut surface. Following the above protocol, over 100 somatic embryos per 1 cm of stem internode tissue have been observed. 
     It is preferred that fresh explant material be cultured to produce more embryos, however, embryogenic callus may be subcultured to produce somatic embryos if required. 
     A comparison of the method of this invention with that of the prior art is provided in Table 1. 
     
                       TABLE 1______________________________________Comparison between the method of this invention and a prior artmethod on somatic embryogenesis of potato (Solanum tuberosum L.).CHARACTERISTIC       Garcia AND Martinez                     Method of this invention______________________________________Stock material       Tissue culture                     Tissue culture plantlets       plantletsNutrient media for       MS salts, vitamins,                     MS salts, vitamins, nostock plantlets       GA.sub.3, 0.1 mg/l.,                     growth regulators, 30 g/l       2.5 g/l sucrose                     sucrose (medium #1)Explant tissue       Nodal sections (i.e.                     Internodal sections of       sections of tissue                     mature in vitro plantlets       including the axillary                     established in vitro for       bud subtended by leaf                     2 years, responsive       petiole) of mature                     explants are excised from       in vitro plantlets                     anywhere along the       established in vitro,                     plantlet. Formation of       close to shoot apex                     somatic embryos mostly                     on lower portion (distal)                     of internode stem                     section, although this                     appears to be under                     genotypic control.                     Somatic embryos also                     from on leaf, root and                     microtuber (in vitro                     formed tubers) - although                     somewhat less                     frequently.Callus phase       5 weeks for callus                     callus formation in 1-2       formation     weeks on medium #2.Callus morphology       Callus was friable &amp;                     Callus is nodular, light       green for first month,                     green to yellow,       then became   compact, and forms       &#34;compact, lobate                     directly on cut end of       &amp; brownish&#34;   stem internodes.Culture media       Solidified with gelrite                     Solidified with agar       (a resin gum) (natural product)Media components       Induction medium:                     Medium #2: Thidiazuron       MS, 2,4-D2.0 mg/l                     or BAP (0.15 μM); with       (0.15 μM) and yeast                     or without Indole acetic       extract. Multiplication                     acid (19 μM).       medium: MS 2 mg/l                     Medium #3 Zeatin       2,4D, yeast extract                     (12 μM); IAA (50 nM);       Differentiation                     GA, (550 nM)       medium: MS GA, (0.1       mg/l) or BA (1 mg/l) -       no 2,1D nor yeast       extract.Somatic embryo       Report that three                     Somatic embryoformation   months is required                     observed after 2-4 weeks       to produce embryo-                     on medium #3. High       genic callus. Two                     frequency somatic       more months for                     embryo formation       high frequency                     immediately for most       somatic embryo                     cultivars tested.       production.   Genotypic variability for                     time to embryo formation                     and numbers of embryos.Cultivars reported to       Desiree,      AC Novachip, Atlanticform somatic embryos      Brador, Caribe, Desiree,,in culture                Katahdin, Kennebec,                     Royal Gold, Ruby Gold,                     Russet Burbank, Russet                     Norkotah, Saginaw Gold,                     Shepody, Superior,                     Temagami Yukon Gold,                     F80054, F83065.                     Somatic embryos have                     been observed on every                     cultivar tested.Synchrony of       Non-synchronous                     Some synchronydevelopment development reported.                     observed. But all                     three development                     stages (globular, heart-                     shaped and torpedo) can                     be found at one timeSecondary somatic       Secondary somatic                     No secondary embryosembryo formation       embryos observed on                     observed.       hypocotyl of torpedo-       shaped somatic       embryos.Developmental stages       All developmental                     All developmental stages       stages reported.                     observed. Somatic       Somatic embryos                     embryos appear to be       appear to be  embedded in surrounding       embedded in   tissues - no suspensor-       surrounding   like structures observed.       tissues - no       suspensor-like       structure observed.Abnormalities       Some abnormal No abnormal somatic       somatic embryos                     embryos observed.       reported in cultures.                     Mature somatic embryos       Necessary to transfer                     (late torpedo state,       all three stages                     sometimes curved like       to MS medium with                     zygotic embryos) when       no growth regulators                     transferred onto MS       for normal develop-                     growth regulator-       ment of `emblings`                     free medium develop       in plantlets. into emblings which                     resemble morphology                     of potato seedlings.Embling development       Emblings develop into                     Emblings develop intofrom somatic embryos       plantlets (presumably                     plants in vitro rapidly.       in vitro) after                     From globular embryo       45 days.      to 1 cm high embling takes                     2-4 weeks.Environmental       Temperature:  Temperature: 19 C. ± 1conditions for       23 C. ± 1  Light: 16 h photoperiodtissue culture       Light: continuous light                     Light intensity: 120 μmol       Light intensity: 50                     .sup.- 2 s.sup.- 1 provided by Cool-       μmol m.sup.- 2 s.sup.- 1                     white and Agrolite                     fluorescent lamps (1:1)Root development       No reported   Explants will produceon explants               many roots if left on                     medium #1 more than                     5-10 days. This is                     cultivar dependent.                     Presence of root                     initials appears to be                     no problem on explants                     producing somatic                     embryos because roots                     do not develop on                     medium #2.______________________________________ 
    
     Somatic embryos produced using the method of this invention may be stored or manipulated for the generation of emblings and fully established plants (see below). If desired, somatic embryos may be cryopreserved using established techniques (e.g. Bajaj et al., 1995b), or dried and encapsulated with a synthetic endosperm and used as synthetic seed for mechanical handling and planting (e.g. Bajaj 1995 a; Litz and Gray, 1995; Senaratna, 1992; Redenbaugh et al., 1991 and 1992). The ability to maintain somatic embryos for long periods of time helps in ensuring a constant supply of germplasm of agronomically important species. 
     Furthermore, the maintenance, and propagation of transgenic plants, comprising a gene of interest is feasible using the method described herein. Following the transformation of a plant using standard transformation protocols (e.g. Agrobacterium mediated transfer, particle bombardment etc.), desired plants may be propagated via somatic embryogenesis using the method described herein. This method would involve: 
     i) transforming a plant with a gene of interest; 
     ii) screening transformed plants to obtain a plant with the desired set of characteristics; 
     iii) obtaining an explant from the transformed plant; and 
     iv) preparing somatic embryos using the method of this invention as described above. 
     Somatic embryos may themselves be used for the preparation of a transgenic plant using established protocols including Agrobacterium (Mathews et al., 1992), microinjection (Neuhaus et al., 1987), particle bombardment (Wilde et al., 1992) or other protocols as would be evident to one of skill in the art. This method would involve: 
     i) preparing somatic embryos according to the method of this invention; 
     ii) introducing a gene of interest into the somatic embryo; 
     iii) screening the transformed somatic embryos for the presence of the gene of interest; 
     iv) growing the selected somatic embryo into a plant; and optionally 
     v) propagating the selected somatic embryo or plant. 
     To aid in identification of transformed plant cells, constructs comprising the gene of interest may include plant selectable markers. Useful selectable markers include enzymes which provide for resistance to an antibiotic such as gentamycin, hygromycin, kanamycin, and the like. Similarly, enzymes providing for production of a compound identifiable by colour change such as GUS (β-glucuronidase), or luminescence, such as luciferase are useful. 
     Regeneration of Somatic Embryos 
     After a short incubation period of one to two weeks on medium #2 (described above), tissues are transferred to medium #3 (which is also termed Regeneration medium) comprising Zeatin, indole acetic acid, Gibberellic acid, solidified with agar (see above). Note that auxin can be omitted from medium #2. Somatic embryos generally form in 2 to 4 weeks of incubation of medium #3. We have observed that some cultivars of potato are slower to respond than others (e.g. Shepody&#39;). Both distal and proximal cut surfaces of the stem internode explants may produce somatic embryos, but they are more prevalent on the distal (lower) surface, although this may be cultivar dependent. Occasionally, somatic embryos will form on the lateral surface of the stem explants. 
     Regeneration of Somatic Embryos--Genotypic Differences 
     All potato cultivars, including tetraploid (48 chromosomes), diploid (24 chromosomes) and monoploid (12 chromosomes) of selected Solanum genotypes, obtained and publicly available from the Fredericton Research Centre, tested to date have regenerated somatic embryos in vitro. To the best of our knowledge, no previous report has been found in the literature reporting the induction of somatic embryos on potato germplasm with less than the tetraploid complement of 48 chromosomes. 
     Without wishing to be bound by theory, it appears that the productivity of somatic embryos is under genetic control. The time to form somatic embryos, the number of explants responding to induction techniques, and the number of somatic embryos per explant all vary with the cultivar tested. 
     Clonal differences in the number of stem internodes which produced somatic embryos are also evident (Tables 2 and 3). Most cultivars produce somatic embryos on all explants, but potato cultivars Caribe (22%), Ruby Gold (9%), Shepody (9%), Temagami (17%), and Yukon Gold (51%) have been observed to only produce somatic embryos on a portion of the cultured explants (Table 2 and 3). Changes in media formulation may be required to enhance or produce somatic embryos on some potato cultivars, such minor modifications would be evident to one of skill in the art. For instance, Ruby Gold and Temagami form more somatic embryos when placed on medium 2b (described above). 
     Previous workers have reported that auxin is mandatory for the induction of somatic embryos in most plant systems (e.g. Raghavan 1986). However, the results presented herein demonstrate that auxin is not required in all stages of this process. For example, medium #2, lacking auxin is effective in producing somatic embryos. Furthermore, several cultivars produce more somatic embryos if medium #2 lacks auxin (e.g. Royal Gold, Ruby Gold and Temagami). 
     
                       TABLE 2______________________________________Number of embryos produced per stem internode explant of potato(Solanum tuberosum L) in vitro on medium #2 (one week)and 3 (9 weeks).     Number of      Mean Number of EmbryosCultivar  Explants with Embryos                    per Explant______________________________________AC Novachip     139/140        13.6 ± 8.6Atlantic  119/119        15.9 ± 8.3Brador    120/120        23.1 ± 11.2Caribe     38/109        6.5 ± 7.8Desiree,  40/40          36.1 ± 20.7F80054    110/110        25.1 ± 18.7F83065    123/123        44.5 ± 23.5Katahdin  134/147        14.3 ± 9.8Kennebec  156/160        10.2 ± 6.7Royal Gold     24/30          17.4 ± 12.2Ruby Gold  3/32          3.0 ± 2.2Russet Burbank     72/72          28.4 ± 17Russet Norkotah     111/127        10.5 ± 8.8Saginaw Gold     102/113        8.5 ± 6.2Shepody    17/120        3.9 ± 4.0Superior  121/121        10.7 ± 5.9Temagami  35579          4.1 ± 3.2Yukon Gold      58/114        2.2 ± 1.8______________________________________ 
    
     
                                           TABLE 3__________________________________________________________________________Somatic embryo production on stem internode explants of various cultivarsof potato(Solanum tuberosum L.) in vitro over 10 weeks. Explants were placed onMedium 1 for1 week and then transferred to Medium 2 for the remaining 9 weeks.Weeks on Media       1 2 3   4   5   6   7   8   9   10__________________________________________________________________________AC Novachip# Explants with Embryos       0 0 35/140               106/140                   125/140                       139/140                           139/140                               139/140                                   139/140                                       139/140% Explants with Embryos       0 0   25%               75.7%                   89.3%                       99.3%                           99.3%                               99.3%                                   99.3%                                       99.3%Atlantic# Explants with Embryos       0 0  2/119                87/119                   119/119                       119/119                           119/119                               119/119                                   119/119                                       119/119% Explants with Embryos       0 0  1.7%               73.1%                    100%                        100%                            100%                               100  100%                                        100%Brador# Explants with Embryos       0 0 0    83/150                   114/120                       120/120                           120/120                               120/120                                   120/120                                       120/120% Explants with Embryos       0 0 0     55%                   83.3%                        100%                            100%                                100%                                    100%                                        100%Caribe# Explants with Embryos       0 0 0    1/111                    1/111                        3/111                            4/109                                11/109                                    21/109                                        25/109% Explants with Embryos                .9%                    .9%                        2.7%                            3.7%                               10.1%                                   19.3%                                       22.9%Desiree,# Explants with Embryos       0 0 0    5/40                    40/40                        40/40                            40/40                                40/40                                    40/40                                        40/40% Explants with Embryos               12.5%                    100%                        100%                            100%                                100%                                    100%                                        100%F80054# Explants with Embryos       0 0 0    14/110                    92/110                       109/110                           110/110                               110/110                                   110/110                                       110/110% Explants with Embryos       0 0 0   12.7%                   83.6%                       99.1%                            100%                                100%                                    100%                                        100%F83065# Explants with Embryos       0 0 0    8/125                    16/125                        53/125                            81/125                               122/123                                   123/123                                       123/123% Explants with Embryos       0 0 0    6.5%                   12.8%                       42.4%                           64.8%                               99.2%                                    100%                                        100%Katahdin# Explants with Embryos       0 0  1/147               116/147                   125/147                       128/147                           134/147                               134/147                                   134/147                                       134/147% Explants with Embryos       0 0  .7%               78.9%                     85%                         87%                           91.2%                               91.2%                                   91.2%                                       91.2%Kennebec# Explants with Embryos       0 0 29/160               117/160                   147/160                       151/160                           156/160                               156/160                                   156/&#39;60                                       156/160% Explants with Embryos       0 0 18.1%               73.1%                   91.8%                       94.3%                           97.5%                               97.5%                                   97.5%                                       97.5%Royal Gold# Explants with Embryos       0 0 0   35763                   17/30                       18/30                           24/30                               24/30                                   24/30                                       24/30% Explants with Embryos       0 0 0   36.7                   56.7                         60%                             80%                                 80%                                     80%                                         80%Ruby Gold# Explants with Embryos       0 0 0   0    1/32                        3/32                            3/32                                3/32                                    3/32                                        3/32% Explants with Embryos       0 0 0   0    3.1%                        9.4%                            9.4%                                9.4%                                    9.4%                                        9.4%Russet Burbank# Explants with Embryos       0 0 0   4/72                   36/72                       61/72                           69/72                               72/72                                   72/72                                       72/72% Explants with Embryos       0 0 0    5.4%                     50%                       84.7%                           95.8%                                100%                                    100%                                        100%Russet Norkotah# Explants with Embryos       0 0 0    36/129                    82/129                        96/129                           102/129                               106/127                                   111/127                                       111/127% Explants with Embryos       0 0 0   27.9%                   63.6%                       77.5%                           79.1%                               83.4%                                   87.4%                                       87.4%Saginaw Gold# Explants with Embryos       0 0 0    8/115                    56/115                        98/115                            98/113                               100/113                                   102/113                                       102/113% Explants with Embryos       0 0 0    6.9%                   48.7%                       85.2%                           86.7%                               88.5%                                   90.2%                                       90.2%Shepody# Explants with Embryos       0 0 0    2/120                    2/120                        6/120                            6/120                                8/120                                    8/120                                        11/120% Explants with Embryos       0 0 0    1.7%                    1.7%                         5%                             5%                                6.7%                                    6.7%                                        9.2%Superior# Explants with Embryos       0 0 18/121               102/121                   121/121                       121/121                           121/121                               121/121                                   121/121                                       121/121% Explants with Embryos       0 0 14.9%               84.3%                    100%                        100%                            100%                                100%                                    100%                                        100%Temagami# Explants with Embryos       0 0 0   0   0    2/30                            2/30                                2/30                                   35518                                       35579% Explants with Embryos       0 0 0   0   0    6.7%                            6.7%                                6.7%                                     10%                                       16.7%Yukon Gold# Explants with Embryos       0 0  18/114                34/114                    44/114                        57/114                            58/114                                58/114                                    58/114                                        58/114% Explants with Embryos       0 0 15.8%               29.8%                   38.6%                         50%                           50.9%                               50.9%                                   50.9%                                       50.9%__________________________________________________________________________ 
    
     Potato clones vary in the time to produce somatic embryos (Table 3). The cultivars which readily form somatic embryos in culture; AC Novachip, Atlantic, Brador, Desiree, F80054, Kennebec, Russet Norkotah, Russet Burbank, Saginaw Gold and Superior (Table 3), generally produce somatic embryos on all of the explants in 3-4 weeks on medium #3. Notable exceptions were Ruby Gold and Temagami. Ruby Gold may require about 5 weeks to produce one or more explants out of forming somatic embryos. Temagami may require about six weeks to produce one or explants out of forming somatic embryos. 
     The mean number of somatic embryos produced per explant also may be under genotypic control. The rankings for the cultivars tested are expressed as mean number of somatic embryos per explant: F83065 (45), Desiree (36), Russet Burbank (28), F80054 (25), Brador (23), Royal Gold (17), Atlantic (16), Katahdin (14), AC Novachip (14), Superior (11), Russet Norkotah (11), Kennebec (10), Saginaw Gold (9), Caribe (7), Temagami (4), Ruby Gold (3), and Yukon Gold (2) (Table 2). 
     Chronological and Physiological Age of Stock Plantlets 
     The length of time to form somatic embryos in vitro and the number of embryos produced per explant appears to be partly determined by the vigour of the stock plantlets from which the explant tissues are excised. Without wishing to be bound by theory, a component of plantlet vigour may be the length of time since the last transfer to fresh medium and possibly the environmental regime in the tissue culture growth facility. Changes in the photoperiod, light quality, light intensity, or temperature regime may have effects on plantlet vigour. 
     Formation of Somatic Embryos on a Various Tissues 
     Somatic embryos have been formed on the following tissues cultured in vitro, stem internode explants, leaf blade tissue, roots and microtuber slices. 
     Secondary Embryos 
     Secondary embryos do not readily form in cultures of potato. Somatic embryos have been observed forming at the base of germinating embryos, at various stages of development. A scanning electron microscope study of the embryo forming cultures revealed that this was fairly uncommon (FIG. 1A to FIG. 1F). 
     Re-culture of Somatic Embryos 
     Potato somatic embryos at the torpedo, late torpedo and germination stages of development were re-cultured using the standard somatic embryogenesis protocol above. Some regeneration of new somatic embryos occurred at the basipetal end (root) of the re-cultured somatic embryos after nodular callus formation. Stem internode sections from germinating somatic embryos also have regenerated new somatic embryos. However, this process requires longer periods of time, for example, new somatic embryos have been observed after seven weeks of culture, compared with three to five weeks typically noted for somatic embryos production formed from cultured potato plantlet stem internode explants. 
     Size of Explant and Orientation of Explant in Medium 
     Upon transfer to medium #3, the stem internode explants are generally placed in the nutrient medium in a horizontal position, however, other positions may also be feasible. In this orientation the distal (the stem end away from the shot apex) end has been found to form more nodular callus than the proximal the stem surface closest to the shoot apex end of the stem explant. Somatic embryos have also been found to form on very thin slices of stem internode tissues, for example from about 0.05 mm to about 10 mm. The orientation of the explant, that is whether the distal or proximal end of stem piece is in the medium, affects productivity of somatic embryos from stem pieces. 
     Germination of Somatic Embryos to form Emblings&#39; 
     An embling, is defined herein as a small plantlet grown from a somatic embryo. Frequently the early growth habit of an embling resembles that of a seedling. 
     Somatic embryos produced using the method of this invention are easily dissected from the explant tissue using any suitable means as would be evident to one of skill in the art. They are placed on medium #1 (described above) and germinated. The resulting embling resembles seedlings obtained from true seeds. For potato, emblings have a slightly viney habit, with thin stems and small leaves. Embryos at the early torpedo stage take about 2 to 4 weeks to become fully established emblings. The conversion of torpedo-shaped somatic embryos to emblings is easily accomplished, and no discernible alteration in growth of the plantlets is observed. 
     Apical dominance is apparent in many of the cultures. Frequently, clusters of proembryos, globular and heart-shaped embryos form at the base of a germinating somatic embryo. 
     Phenotypic Stability of Potato Regenerants 
     Germinating somatic embryos from potato cultivars Atlantic, Kennebec, Russet Burbank, Russet Norkotah, and Superior were excised and transferred to medium #1, containing no growth regulators. After 2-3 weeks, the emblings were acclimatized to greenhouse conditions and grown as greenhouse plants. These greenhouse plants were observed for differences in plant habit and morphology compared to sewed-grown plants. Minitubers (greenhouse tubers) were harvested and assessed for trueness-to-type. 
     Off-types have been reported from tissue culture regimes of potato (Harding, 1994, 1995). Similarly, some potato cultivars exhibited off-types as well. Furthermore, some cultivars displayed more off-types than others. Of the cultivars that displayed off-types, approximately 10 to 20 percent of the greenhouse plants exhibited this morphology. Generally, if the greenhouse plant showed aberrations, the minitubers harvested from that plant were also off-type. The presence of off-types appears to be influenced by genotype. 
     Somatic Embryogenesis of Tomato (Lycopersicon esculentum L) in vitro 
     Somatic embryos have been produced using juvenile tissue such as seedling hypocotyl or basal area of tomato requiring a 6 week protocol (Gill et al., 1995). However, somatic embryos have not been produced using other tissues. Using the method of this invention, somatic embryos have been produced on tomato using mature stem internode tissue. The method used involved Medium #2 with thidiazuron only for 1 week, followed by Medium #3 with zeatin, indole acetic acid and gibberellic acid for 4 weeks. Somatic embryos are observed within a 4 week period. 
     All three genotypes tested (Big Beef, Cheyenne, and Viva Italia) produce somatic embryos on stem internode tissues. Some genotypic differences in reaction to the tissue culture protocol are observed. Generally more callus formation is observed on tomato tissues compared with potato. The following table (Table 4) outlines the differences in method described herein with the prior art method of Gill et al. (1995). 
     
                       TABLE 4______________________________________Comparison of the method of this invention with the prior artfor the production of somatic embryos ofTomato Lycopersicon esculentum L. in vitro       Method of       this invention                    Gill et al. (1995)______________________________________Cultivars regenerated       Big Beef, Cheyene,                    Campbell 1327VF,       Viva Italia  Crimsonvee VF, Glamour,                    MH 6208, Red Robbin,                    Scocet Million, Sweet                    MillionExplant cultured       Tissue culture                    Intact seeding, hypocotyl       plantlet, stem                    or basal area       internodeGenotypic difference       Yes          Yesin response toculture conditionsTissue culture protocol       Two step somatic                    One step protocol with MS       embryogenesis                    medium + BAP       protocol*Time to produce       from 4 to 6 weeks                    6 weeksEnvironmental       16 hour photoperiodprotocol______________________________________ *Medium # 2 with thidiazuron only  1 week Medium # 3 with zeatin, indole acetic acid and gibberellic acid  4 weeks 
    
     Somatic Embryogenesis of Lettuce (Lactuca sativa L.) in vitro 
     Somatic embryogenesis of lettuce has been reported by Zhou et al. (1992) using seedling cotyledons in vitro. The disadvantage of this technique is that there is considerable variability (heterosis) in the morphology of the propagated material, and such variability is not commercially acceptable in lettuce. Therefore, material propagated from seedling tissues is of uncertain advantage commercially. A clonal propagation technology for lettuce is of great interest to the lettuce industry because currently growers have to rogue out off-types which are not commercially acceptable, and this can be a considerable portion of the crop. Furthermore, lettuce somatic embryos lend themselves to synthetic seed technology and fluid drilling of the synseeds for a clonally propagated crop which reduced the need for field roguing. 
     The method of this invention produced somatic embryos using mature lettuce tissues obtained from stem internode, mature leaves, or axllary buds. The method employed using maintaining the tissue of medium #1 for 1 week, followed by medium #2c (i-e. containing BAP and IAA) for two weeks. Following this protocol, somatic embryos are observed withiin 3 weeks, compared to 7 weeks with the prior art protocol. A comparison of the method of this invention with that of Zhou et al. (1992) is outlined in Table 5. 
     
                       TABLE 5______________________________________Comparison of the method of this invention with that ofthe prior art, for the production of somatic embryos of Lettuce(Lactuca sativa L.) in vitro     Method of this invention                     Zhou et al. (1992)______________________________________Cultivars Butter Crunch, Grand                     Lettuce - no cultivarRegenerated     Rapids, Red sails                     name providedExplantSeedling (10 day old)     Seedling cotyledonsCultured  stem internode and leaves                     in vitro     in vitroExcised axillary buds     from mature, greenhouse-     grown `Red Sails plant.     Bud established in vitro;     leaves (occasionally stem     internode) of cultured     plantlet used as explants.Genotypic Some genotypic differences                     Not stated.Differences     in reaction to media     observed, although all cvs.     Tested produced somatic     embryos.Tissue Culture     Two step somatic                     Callus induction inProtocol  embryogenesis protocol*.                     dark (4 weeks), transfer     16 hour photoperiod                     to light on same medium                     for 3 weeks for somatic                     embryos to form.                     MS salts, BAP &amp; NAATime to Produce     Three Weeks     Seven WeeksSomatic Embryos______________________________________ *Medium # 1 for 1 week, followed by 2 weeks on medium #2c. 
    
     The present invention has been described with regard to preferred embodiments. However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as described herein. 
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