Abstract:
This invention relates to a method for the non-agricultural production of jojoba wax through the culture of asexual embryos of jojoba.

Description:
RELATED INVENTION 
     This application is a continuation of my co-pending United States patent application Ser. No. 06/266,322 filed May 22, 1981 (now abandoned). 
    
    
     FIELD OF THE INVENTION 
     This invention relates to culture of living plant tissue to produce embryos of jojoba. 
     BACKGROUND OF THE INVENTION 
     U.S. Pat. No. 4,204,366, teaches a method whereby asexual embryos of cacao (Theobroma cacao L.) can be proliferated in vitro to produce useful products, i.e. a cocoa-butter like product. This invention covers a non-agricultural method for producing jojoba wax by tissue culture procedures. In this invention we describe a new system to produce jojoba wax, a wax ester of long chain (20-22 carbon) fatty acids and alcohols produced by the jojoba shrub [Simmondsia chinensis (Link)], which has useful properties as an industrial lubricant. 
    
    
     THE DRAWINGS 
     FIG. 1 is a graphic representation of the results of a gas chromatographic separation of commercial jojoba oil. Identification of peaks is based on known jojoba wax ester composition. The parameters used in the graph are 
     
         ______________________________________column             330° C.detector           400° C.injector           300° C.chart speed        0.5 cm/secattenuation        128carrier gas        30 ml/min3% OV-1 2 m × 2 mm              stainless steel column______________________________________ 
    
     FIG. 2 is a graphic representation of the results of a gas chromatographic separation of wax esters of asexual embryos of jojoba. Identification is based on retention times from FIG. 1. The parameters used in the graph are______________________________________column             330° C.detector           400° C.injector           300° C.chart speed        0.5 cm/secattenuation        128carrier gas        30 ml/min3% OV-1 2 m × 2 mm              stainless steel column______________________________________ 
     DETAILED DESCRIPTION OF THE INVENTION 
     In order to obtain zygotic embryos for further growth and proliferation, a group of immature jojoba fruits are selected and individually cut open and the developing immature zygotic embryos are separated out of each fruit. Zygotic embryos of less than about twenty milligrams by weight are separated out and discarded and none in excess of one hundred milligrams by weight are retained. The zygotic embryos selected as aforesaid, namely those ranging anywhere from twenty milligrams to one hundred milligrams in weight, are then placed on the basal medium described below. 
     The zygotic embryos are then placed on said basal medium and grown thereupon, and eventually they will produce asexual embryos. 
     The maturing asexual embryos each form two cotyledons, thereby resembling a normal seed, but without a seed coat. As the asexual embryos continue to grow, some will eventuate in plant production (and these are discarded) and some grow out to what nearly resemble &#34;coatless&#34; seeds, and it is these embryos that are used for wax production. 
     Immature fruits of jojoba undergo a process of asexual embryogenesis when cultured on a basal medium consisting of salts as described in a paper by Toshio Murashige and Folke Skoog (1962) entitled A revised medium for rapid growth and bioassays with tissue cultures. Physiol. Plant. 15:473-497. 
     The pecise formulation is as follows:______________________________________Components      mg/liter______________________________________Salts I NH 4  NO 3            1650KNO 3        1900CaCl 2 .2H 2  O           440MgSO 4 .7H 2  O           370KH 2  PO 4            170Na.EDTA         373FeSO 4 .7H 2  O           27.8KI              0.83H 3  BO 3            6.2MnSO 4 .7H 2  O           22.3ZnSO 4 .7H 2  O           8.6Na 2  MoO 4 .2H 2  O           0.25CuSO 4 .5H 2  O           0.025CaCl 2 .6H 2  O           0.025______________________________________ 
     The formulation described above is commonly referred to by those skilled in the art as &#34;Murashige and Skoog salts&#34;. 
     The asexual embryos continue to proliferate in culture, mature to a seed-like state, and are capable of germination. 
     Analysis of lipids for asexual embryos have shown the presence of wax esters of long chain fatty acids and alcohols that are identical with commercial jojoba wax as measured by gas-liquid chromatography. The chromatograph tracing of the wax esters from commercial jojoba wax and asexual embryos of jojoba are shown in FIGS. 1 and 2. These results are supported by the data shown in Table 1 as follows: 
     
                                           TABLE 1__________________________________________________________________________Gas Chromatographic Analysis of AsexualEmbryo Lipids as Compared to Jojoba OilSource of   Ester Carbon Lengthwax         36 38 40 42  44 Total                           % wax__________________________________________________________________________ Jojoba Oil  Area.sup.1       376          2059             9927                15025                    280                       27668                           100%  Area %        1.4           7.4             35.9                54.3                     1.0Asexual  Area tr tr  975                 2766                    tr  3742                           13.5Embryo.sup.2  Area %       &lt;0.1          &lt;0.1             26.1                73.9                    &lt;0.1__________________________________________________________________________ .sup.1 Determined by Hewlett Packard 3880 S Integrator/1000 .sup.2 2.46 g fresh weight, 0.33 g dry weight, 32.4 mg lipid, 9.8% lipid 
    
     In a subsequent study initialed in May 1982, zygotic asexual embryos of jojoba, each between the size of 20 and 100 mg, were cultured on the identical basal medium previously described with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The results indicate that asexual embryogenesis was stimulated by the addition of 1 mg/liter 2,4-D as shown in Table 2 below: 
     
                       TABLE 2______________________________________The effects of 2,4-dichlorophenoxyacetic acid on theinduction of asexual embryogensis in zygotic embryosof jojoba.                     Cultures with2,4-D      No.            asexual embryos(mg/liter) cultures       No.    %______________________________________0          60             0      00.1        60             0      01          60             9      1510         60             0      0______________________________________ 
    
     CONCLUSION 
     These results demonstrate that immature asexual embryos of Simmondsia chinesis can be induced to proliferate asexual embryos and that these asexual embryos will produce wax esters in tissue culture similar to the wax esters produced by jojoba under normal field culture. 
    
    
     Physiol. Plant. 15:473-497. 
     The pecise formulation is as follows: 
     
         ______________________________________Components      mg/liter______________________________________Salts.sup.INH.sub.4 NO.sub.3           1650KNO.sub.3       1900CaCl.sub.2.2H.sub.2 O           440MgSO.sub.4.7H.sub.2 O           370KH.sub.2 PO.sub.4           170Na.EDTA         373FeSO.sub.4.7H.sub.2 O           27.8KI              0.83H.sub.3 BO.sub.3           6.2MnSO.sub.4.7H.sub.2 O           22.3ZnSO.sub.4.7H.sub.2 O           8.6Na.sub.2 MoO.sub.4.2H.sub.2 O           0.25CuSO.sub.4.5H.sub.2 O           0.025CaCl.sub.2.6H.sub.2 O           0.025______________________________________ 
    
     The formulation described above is commonly referred to by those skilled in the art as &#34;Murashige and Skoog salts&#34;. 
     The asexual embryos continue to proliferate in culture, mature to a seed-like state, and are capable of germination. 
     Analysis of lipids for asexual embryos have shown the presence of wax esters of long chain fatty acids and alcohols that are identical with commercial jojoba wax as measured by gas-liquid chromatography. The chromatograph tracing of the wax esters from commercial jojoba wax and asexual embryos of jojoba are shown in FIGS. 1 and 2. These results are supported by the data shown in Table 1 as follows: 
     
                                           TABLE 1__________________________________________________________________________Gas Chromatographic Analysis of AsexualEmbryo Lipids as Compared to Jojoba OilSource of   Ester Carbon Lengthwax         36 38 40 42  44 Total                           % wax__________________________________________________________________________ Jojoba Oil  Area.sup.1       376          2059             9927                15025                    280                       27668                           100%  Area %        1.4           7.4             35.9                54.3                     1.0Asexual  Area tr tr  975                 2766                    tr  3742                           13.5Embryo.sup.2  Area %       &lt;0.1          &lt;0.1             26.1                73.9                    &lt;0.1__________________________________________________________________________ .sup.1 Determined by Hewlett Packard 3880 S Integrator/1000 .sup.2 2.46 g fresh weight, 0.33 g dry weight, 32.4 mg lipid, 9.8% lipid 
    
     In a subsequent study initialed in May 1982, zygotic asexual embryos of jojoba, each between the size of 20 and 100 mg, were cultured on the identical basal medium previously described with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The results indicate that asexual embryogenesis was stimulated by the addition of 1 mg/liter 2,4-D as shown in Table 2 below: 
     
                       TABLE 2______________________________________The effects of 2,4-dichlorophenoxyacetic acid on theinduction of asexual embryogensis in zygotic embryosof jojoba.                     Cultures with2,4-D      No.            asexual embryos(mg/liter) cultures       No.    %______________________________________0          60             0      00.1        60             0      01          60             9      1510         60             0      0______________________________________ 
    
     CONCLUSION 
     These results demonstrate that immature asexual embryos of Simmondsia chinesis can be induced to proliferate asexual embryos and that these asexual embryos will produce wax esters in tissue culture similar to the wax esters produced by jojoba under normal field culture.