Patent Abstract:
a process of producing somatic seedlings from a somatic embryo . the process comprising pre - germinating somatic embryos , placing the pre - germinated somatic embryos into a state of physiological dormancy , sowing the pre - germinated physiologically dormant somatic embryos onto or into germination media , and propagating the sown pre - germinated somatic embryos in environmental conditions manipulated to facilitate imbibition , germination , and development into complete seedlings possessing shoots and roots . advantageously , the process may be carried out with “ naked ” embryos using conventional seed handling equipment , growing mixes , and plant propagation environments . the process does not require the use of aseptic techniques or sterilized media or equipment . the invention also relates to pre - germinated embryos and seedlings produced by the process .

Detailed Description:
in a preferred form , the present invention is generally a multi - step process for ex vitro sowing and germination of plant somatic embryos using conventional horticultural equipment and facilities , comprising , but not restricted to , some or all of the following sequential steps : 1 . sowing the plant somatic embryos onto physical supports placed within containers which contain a liquid germination medium , said embryos placed onto the physical supports in a manner such that the embryos are not submerged in the liquid medium , but instead , such that the liquid medium forms a thin capillary layer around the embryo , said capillary layer also referred to as a film . 2 . incubating the somatic embryos surrounded with a film of germination medium for a period of time ranging between 2 - 30 days such that embryo germination commences as evidenced by the emergence of a shoot and a root . 3 . placing the containers with the somatic germinants into cold storage , said cold storage comprised of temperatures in range of 2 - 15 ° c ., preferably in the range of 4 - 10 ° c ., for at least , but not limited to , 1 day . 4 . conditioning the somatic germinants by transferring them from the containers containing germination medium to containers containing an aba solution in the range of 2 - 100 μm , preferably in the range of 5 - 20 μm , for a period of time ranging between 30 - 180 minutes , preferably in the range of 60 - 90 minutes . 5 . drying the conditioned germinants by transferring them from the containers containing the aba solution , to a drying chamber , said drying chamber maintaining a relative atmospheric humidity in the range of 35 - 99 % rh , preferably in the range of 80 - 99 %, and incubating said conditioned germinants in said drying chamber for a period of time ranging between 12 hours to 7 days , preferably in the range of 18 hrs to 48 hrs . 6 . storing said conditioned and dried embryos in a sealed package in a facility wherein the temperature is maintained in the range of − 85 ° to 30 ° c ., preferably in the range of 2 - 20 ° c . 7 . sowing the pre - germinated plant somatic embryos into nursery containers containing a three - phase substrate , said three phases comprising solids , liquids and air . 8 . placing the nursery containers sown with plant somatic embryos , into a conventional plant propagation environment in which light , temperature , atmospheric humidity , and moisture content of the rooting substrate can be controlled and manipulated to enable and facilitate the re - germination of the somatic embryos and their further development into seedlings . 9 . supplying an aerosol to the surface of the nursery containers sown with somatic embryos , said aerosol containing the necessary carbohydrate compounds required to initiate and sustain the re - germination processes of the somatic embryos . 10 . supplying in the forms of an aerosol and / or a liquid suspension and / or a liquid solution , the micro - and macro - mineral elements required to sustain the re - germination of somatic embryos and their subsequent development into seedlings . 11 . adjusting as required during the somatic embryo re - germination period , the ambient light intensity and diurnal photoperiod , temperature and atmospheric humidity to maintain the development of re - germinated somatic embryos into fully functional seedlings . alternatively , at the completion of step 2 , pre - germinated embryos may be sown directly after harvesting , i . e ., per steps 7 - 11 . a particular advantage of the process , at least in its preferred forms , is that special hygienic and / or aseptic and / or sterile handling methods and / or equipment and / or facilities are not required to successfully handle , sow and germinate , dry , and re - germinate plant somatic embryos . accordingly , these steps may be carried out in non - sterile , unhygienic and / or septic conditions , preferably using “ naked ” embryos ( i . e ., non - encapsulated or otherwise coated embryos ). it is preferable that the invention be practiced with plant somatic embryos that have been dried to moisture contents that approximate those of their corresponding zygotic seeds , i . e ., in the range of 5 - 20 % and more specifically , in the range of 10 - 15 %. however , it is possible to practice the present invention with somatic embryos containing higher moisture contents in the range of 20 - 70 % with the only limitation on the upper limit being the highest level of moisture content that the somatic embryos can be singulated with conventional seeding or seed - handling equipment . it is preferable that the pre - germination step is carried out in a container wherein there is a physical support such as , but not restricted to , filter paper or a screen comprised of a nylon or polypropylene material or other such materials , is placed on the liquid germination medium such that plant somatic embryos are held on the surface or above the surface of the liquid medium such that a thin capillary layer or film of the germination medium is formed around the somatic embryos . alternatively , the somatic embryos can be successfully pre - germinated on discontinuous physical substrates comprised of materials such as but not limited to , vermiculite , perlite , peat , coconut husk fibres and the like , said discontinuous supports containing sufficient liquid germination medium to enable the formation of a thin capillary layer or film of germination medium around the somatic embryos . the somatic embryos can be sown onto the surface of the absorbent material by hand or by the means of a mechanical sowing device such as but not restricted to conventional seeding equipment . however , it is also possible to accomplish the pre - germination of somatic embryos by sowing them with conventional seeding equipment into empty multi - chambered nursery containers exemplified by but not restricted to miniplug trays , said containers having their drainage holes covered by a mesh - like material which will support the somatic embryos after sowing . the containers are then placed onto liquid germination media such that the somatic embryos are in contact with but are not submerged in the liquid media , such that a thin layer of film of germination medium is formed around the somatic embryos . although the pre - germinated and subsequently dried somatic germinants can be sown with all conventional seeding equipment used for sowing zygotic seeds , it is preferred to use equipment that dispenses singulated seed into multi - chambered nursery containers , commonly referred to as miniplug trays or cell - packs , said containers commonly used to produce plant plugs which can be mechanically transplanted into larger containers or into field - growing environments . another important advantage of the present invention , at least in its preferred forms , is that the sowing and propagation of pre - germinated somatic embryos can be practiced with a wide variety of non - sterilized growing substrates commonly used in conventional plant propagation . the preferred growing substrate is peat - based and has been formulated specifically for germination of zygotic seed and is exemplified by mixtures such as ( a ) 15 . 2 cu . ft of peat , 8 cu . ft . of vermiculite , 680 grams of dolomite lime , and 300 grams of micromax ® ( a commercial fertilizer composition comprised of microelements such as but not limited to sulfur , boron , manganese , magnesium , cobalt and iron ), and ( b ) 16 . 2 cu . ft . of peat , 6 . 75 cu . ft . perlite , 4 cu . ft . vermiculite , 6 kilograms of dolomite lime , 1 . 5 kilograms of gypsum , 375 grams of potassium phosphate , 250 grams micromax ®, and 35 grams of wetting agent . alternatively , commercially formulated mixes such as pro - mix - g ® or pro - mix - pgx ® ( premier peat moss ltd . montreal , pq , canada — these are commercial soilless plant growing media comprised of mixtures containing but not limited to peat , perlite , vermiculite and pumice ), sunshine mix # 3 ( sun - gro horticulture inc ., hubbard , oreg ., usa ), and redi - earth ® ( the scotts co ., marysville , ohio , usa — this is a commercial soilless plant growing media comprised of mixtures containing but not limited to peat , perlite , vermiculite and pumice ) can also be used with the present invention . it is preferred that the peat - based growing substrate is moistened to a moisture content in the range of 59 - 75 % and then dispensed into multi - chambered trays commonly used for production of plant plugs . although examples of such trays include styrofoam # 252 miniplug trays manufactured by beaver plastics inc ( edmonton , ab , canada ) and hard plastic # 288 or # 512 miniplug trays manufactured by tlc polyform inc ( plymouth minn ., usa , 55441 ), the present invention can be practiced with other such multi - chambered trays , or alternatively , with individual pots . it should be noted that the practice of the present invention is not restricted to peat - based mixtures , but also includes other substrate such as jiffy - 7 peat plugs , composted or shredded coconut husk fibres commonly referred to as “ cor ” or “ coir ” ( 1993 crystal co ., st . louis , mo ., usa ), polymerized substrates ( grow tech inc ., san juan bautista , calif . usa ; preforma inc ., oberlin , ohio usa ), extruded foams such as oasis ® ( smithers - oasis ltd ., kent , ohio , usa — this is a commercial expanded foam product comprised of urea formaldehyde ), rock wool ( rockwool international a / s , hovedgaden 584 , dk - 2640 , denmark ) and the like . regardless of the rooting substrate chosen , it &# 39 ; s physical characteristics should enable development and maintenance of a high relative humidity in the gaseous phase , i . e ., in excess of 75 % rh , within the substrate while minimizing saturation of the substrate with the liquid phase . after the pre - germinated somatic embryos are sown onto the surfaces of the rooting substrates , if desired , the embryos may be covered with a thin layer of additional rooting substrate that may be comprised of the same material underneath the embryos or alternatively , with a different type of material . one non - limiting example is sowing the pre - germinated embryos onto pro - mix - pgx medium , then overlaying the embryos with a thin layer of coconut husk fibres . nursery containers sown with pre - germinated somatic embryos are preferentially placed into a conventional plant propagation environment wherein the conditions are within but not limited to the ranges of temperatures of 15 - 35 ° c ., relative humidities of 75 - 100 %, light intensities of 10 - 500 foot candles , and diurnal cycles of 6 h day / 18 h night — 22 h day / 2 h night . it is preferable to maintain a very high level of atmospheric humidity around the nursery containers sown with pre - germinated somatic embryos , i . e ., greater than 90 % rh , for the first 3 - 7days after sowing to facilitate somatic embryo imbibition and germination . a number of methods can be used to maintain the atmospheric humidity at these levels including but not restricted to placing the containers in a greenhouse environment with misting or fogging equipment which is deployed at controlled intervals , placing the containers in a fogging or misting tent or chamber , placing clear plastic domes over the nursery containers and then removing domes periodically to mist or fog the sown embryos and replacing the domes immediately thereafter . another non - limiting method is to provide a space ranging between 2 mm and 10 mm above the surface of the rooting substrate onto which the embryos are sown and the top of the container , and then covering the top of the nursery container with a plastic film which is removed to enable misting or fogging of the sown embryos and then immediately replaced . after somatic embryo germination is established as evidenced by development of epicotyl and root structures , the germinants can be weaned from the high relative humidity environments and integrated into conventional nursery cultural practices by gradually reducing the amount of misting / fogging applied and / or by extending the periods of time between the misting or fogging steps . it is preferable to maintain the sown pre - germinated embryos in a high relative humidity environment , i . e ., greater than 90 % rh , for a period of , but not restricted to , 3 - 7 days after sowing to facilitate embryo imbibition , prior to supplying exogenous nutrients required for embryo germination . another important feature of the invention , at least in preferred forms , is that the exogenous nutrients , including but not restricted to carbohydrates and minerals , required for successful somatic embryo re - germination and subsequent growth and development may be applied as aerosols . the nutrient solutions may be applied with , but not restricted to , conventional misting and / or fogging equipment . although , the nutrients can be applied individually or combined into one solution , it is preferred to supply the carbohydrates as one solution and the remaining nutrients as a separate solution . a non - limiting example of how this can be practiced is by applying a 3 % sucrose solution as a mist to the surface of the growing substrate containing a sown pre - germinated embryo , and then applying at a later time , a solution containing a mixture of mineral nutrients formulated to deliver 454 mg / l nitrogen , 81 mg / l phosphorus , 704 mg / l potassium , 50 mg / l calcium , 39 mg / l magnesium , 193 mg / l sulfur , 3 mg / l manganese , 0 . 5 mg / l zinc , 89 mg / l chlorine , 3 mg / l iron , 0 . 7 mg / l iodine , 0 . 6 mg / l boron , 0 . 01 mg / l molybdenum , 0 . 01 mg / l cobalt , and 0 . 01 mg / i copper . alternatively , the macronutrients can be supplied as a commercial formulation such as but not restricted to plantprod ® plant starter fertilizer 10 - 52 - 10 ( nitrogen - phosphate - potassium ) or plantprod ® forestry seedling starter 11 - 41 - 8 ( nitrogen - phosphate - potassium ) ( plant products ltd ., brampton , on , canada — these are commercial water - soluble fertilizers containing mineral nutrients such as nitrogen , phosphorus and potassium , and a dye ). an alternative non - limited means of supplying exogenous nutrients to pre - germinated somatic embryos sown onto three - phase growing media within nursery containers is to irrigate or “ drench ” the media with nutrient solutions formulated as previously described . this is preferably done just before the embryos are sown in the three - phase growing media . since microorganisms such as fungi , bacteria , yeast , and algae , are ubiquitous in conventional plant propagation substrates , equipment , containers and growing environments , a wide variety of chemical and biological pesticide products are available to control and eradicate plant pathogens . it has surprisingly been found that aseptic handling procedures and sterilized growing substrates , nursery containers and environments are not required to successfully germinate and grow plant somatic embryos . indeed , the invention can be practiced in conventional plant propagation environments using only the standard commercial methods of hygiene . furthermore , we have surprisingly found that pesticides such as benlate ® ( a commercial fungicide composition containing a chemical active ingredient ), rovril ® ( a commercial fungicide composition containing a chemical active ingredient ), trumpet ® ( a commercial insecticide composition containing a chemical active ingredient ) and the like , which are registered for pest control in plant crops , can be used in conjunction with somatic embryos pre - germinated and subsequently sown with the present novel multi - step procedure . the following examples are provided to further illustrate the present invention and are not to be construed as limiting the invention in any manner . somatic embryos of interior spruce ( picea glauca ( moench ) voss x p . engelmannii parry ex engelm .) lines i - 1026 , and 10 - 1418 were produced and dried with a high - relative - humidity treatment ( hrht ) according to the methods of roberts et al . ( 1990a ; 1990b ) and webster et al . ( 1990 ). two groups of dried embryos for each of the two genotypes , were germinated on whatman # 1 filter paper on a thin layer of cotton in polycarbonate boxes . the liquid germination medium was modified gmd medium ( nh 4 + — n / no 3 − — n ratio was 1 . 4 ; phosphate concentration was 6 mm ; 3 % sucrose ). after the embryos were sown onto the filter paper , the liquid level in the germination boxes was adjusted to provide a liquid film surrounding the surface of embryos situated at the interface with the solid support . the boxes were sealed with parafilm and placed in a controlled environment room with the temperature maintained at a constant 23 ° c . the first group of somatic embryos was germinated for 7 days after which , the polycarbonate boxes containing the germinants were transferred into refrigerated storage ( i . e ., temperatures were within a range of 8 - 12 ° c .) for 72 hours . the germinants were then removed from the cold treatment and subsequently received the various aba treatments described below . the germinants which received the cold treatment are hereafter referred to as “ stored germinants ”. the second group of somatic embryos did not receive a cold treatment but instead , received a 10 - day germination period . this group of germinants is referred to hereafter as “ fresh germinants ”. the stored and fresh germinants of each genotype were carefully paired into four groups , on wetted paper towel disks in separate 10 cm petri dishes and received four aba treatments , said aba treatments being concentrations of 0 , 5 , 10 , 20 μm , respectively . the aba treatments were applied in darkness for 90 min . after the completion of the aba treatments , the somatic germinants were blot - dried on paper towels . the germinants were then transferred to fresh 5 cm petri dish . the dishes , two per drying chamber , were placed in drying chambers containing a saturated kcl solution , which provided an atmospheric humidity of 85 % rh over the liquid surface in a sealed chamber at 20 ° c . the somatic germinants were dried for 24 hr in a refrigerated room maintained between 5 - 8 ° c . after which , the drying chambers were transferred into a controlled environment room maintained at 20 ° c . for another 15 h . after the hrht treatment was completed , 3 samples of 3 germinants were taken from each germinant type of each genotype in each aba treatment , for determination of their relative water contents ( rwc ). the dried germinants were sown into 288 - cavity miniplug trays ( tlc polyform inc ,. plymouth minn ., usa , 55441 ) filled with a peat - based soilless mix comprised of 60 % peat and 40 % coarse horticultural vermiculite . each germinant type of each genotype in each aba treatment was randomly sown into 5 minitrays in blocks of 20 germinants . after sowing , the germinants were covered with a thin layer of coir ( 1993 crystal co ., st . louis , mo ., usa ). the trays were wrapped in black plastic bags for 18 hrs to allow slow rehydration of germinants . then , the trays were misted with a modified gmd nutrient solution ( webster et al ., 1990 ) containing 3 % sucrose until the soilless mix and coir became well wetted . the trays were placed in two misting chambers in a controlled environment germination room under light reduced by a 50 % shade cloth for the first week . the shade cloth was removed at the end of the first week and the germinants were exposed to a light intensity of 180 μmol m − 2 s − 1 photosynthetic photon flux . the chambers were frequently misted to keep the soilless mix moistened . modified gmd solution with 3 % sucrose was sprayed onto the germinants daily . after 7 days , germinants were fertilized weekly with 20 - 20 - 20 fertilizer ( plant products ltd ., brampton , on , canada ) to deliver the equivalent of 100 ppm n . the effects of the cold storage and aba treatments on re - germination of desiccated , pre - germinated spruce somatic embryos are summarized in table 1 . somatic embryos of interior spruce ( picea glauca ( moench ) voss x p . engelmannii parry ex engelm .) lines 1 - 1278 , 4 - 2809 , 107 - 1917 , 143 - 2695 , and i - 1026 were produced and dried with a high - relative - humidity treatment ( hrht ) according to the methods of roberts et al . ( 1990a ; 1990b ) and webster et al . ( 1990 ). the embryos were then incubated in a liquid nutrient solution containing 3 % sucrose for 3 days , then germinated for 4 days in polycarbonate boxes ( sigma - aldrich canada ltd ., 2149 winston park dr ., oakville , ont ., canada ; catalog # c8062 ). the sterile pre - germination substrate in each box consisted of 150 ml of coarse vermiculite plus 150 ml of modified gmd nutrient solution amended with 3 % sucrose ( roberts et al ., 1990b ; webster , 1990 ). the 4 - day - old pregerminated somatic embryos were harvested from the vermiculite , then hand - sown into soilless mixes composing of 50 % screened peat and 50 % fine perlite in tlc polyform 288 - cell miniplug trays ( 10 ml / cavity ). after sowings were completed , the trays were placed in a humidified growth chamber for 1 week under the following environmental conditions : 95 - 98 % rh , day / night air temperatures of 25 / 20 ° c ., an 18 h light / 6 h dark diurnal period , and light intensity of 30 μmol m − 2 s − 1 photosynthetic photon flux . a modified nutrient germination liquid medium containing 3 % sucrose was sprayed onto the trays once every weekday . the trays were then moved to misting chambers with similar environmental conditions except light intensities were increased to 120 - 150 μmol m − 2 s − 1 photosynthetic photon flux . the misting frequencies were 4 seconds at 2 . 5 h intervals during the day and 2 seconds at 3 h intervals at night . the trays were sprayed with a modified nutrient germination liquid medium containing 3 % sucrose once every weekday for one additional week . thereafter , a 0 . 5 g / 1 plantprod ® forestry starter fertilizer solution ( n - p - k ; 11 - 41 - 8 ) was applied twice weekly . the re - germination successes of the different lines of pre - germinated interior spruce se were assessed at different time periods ranging between 2 - 11 weeks after sowing . the results are summarized in table 2 . somatic embryos ( se ) of interior spruce line 1 - 1281 were produced according to the methods of roberts et al . ( 1990a ; 1990b ) and webster et al . ( 1990 ). after harvesting , the se were dried using the hrht method . post - hrht embryos were germinated on whatman # 1 filter paper placed on a thin layer of cotton in polycarbonate boxes . the liquid germination medium was modified gmd medium ( nh 4 + — n / no 3 − — n ratio was 1 . 4 ; phosphate concentration was 6 mm ; 3 % sucrose ). after the embryos were sown onto the filter paper , the liquid level in the germination boxes was adjusted to provide a liquid film surrounding the surface of embryos situated at the interface with the solid support . the boxes were scaled with parafilm and placed in a controlled environment room with the temperature maintained at a constant 23 ° c . after a 7 - day in vitro germination period , the polycarbonate boxes containing the germinants were transferred to cold storage ( 8 - 10 ° c .) for 56 hours . the germinants were then transferred to clean petri dishes and were incubated in a 10 μmol aba solution for 1 . 5 h after which , they were blot - dried on paper towels . the germinants were separated into 7 groups and then randomly assigned to 1 of 7 desiccation treatments ( the rh values were : 75 . 8 %; 81 . 9 %; 84 . 6 %; 86 . 3 %; 88 . 7 %; 92 . 6 %; 96 . 7 %). the germinants were desiccated for 48 h at 5 ° c . after the desiccation treatments were completed , subsamples of the germinated , then desiccated spruce somatic embryos ( i . e ., pre - germinated embryos ), were taken for determination of their water contents . the dried germinants were sown into 252 - cavity miniplug trays ( beaver plastics ltd ., edmonton , ab , canada ) filled with a peat - based soilless mix comprised of 60 % peat and 40 % coarse horticultural vermiculite ( lightly pre - moistened with a dilute fertilizer solution , 20 - 20 - 20 , 100 ppm n equivalent ). four replicates of 20 germinants per treatment were randomly assigned to four miniplug trays . after sowing , the germinants were covered with a thin layer of coir ( 1993 crystal co ., st . louis , mo ., usa ). the trays were wrapped in black plastic bags overnight to allow slow rehydration of the desiccated germinants . the next day , the trays were sprayed with a ½ strength gmd solution containing 3 % sucrose ( roberts et al ., 1990b ), until the soilless mix and the coir became well moistened . the trays were then placed into a misting chamber under a light intensity of 30 μmol m − 2 s − 1 for the first week , 55 μmol m − 2 s − 1 for the second week and 165 μmol m − 2 s − 1 for the remainder of the experiment . the light levels were adjusted by placing shading cloth underneath the lighting bulb . the chamber was misted 3 sec per hour for the day and 2 second per 2 hours for the night . a modified gmd solution containing 3 % sucrose was sprayed onto the germinants once every day on weekdays for the first three weeks . the effects of the different desiccation treatments on re - germination of pre - germinated , then desiccated somatic embryos from spruce line 1 - 1281 are summarized in table 3 . somatic embryos ( se ) of interior spruce ( picea glauca engelmannii complex ) line 1 - 1281 were produced according to the methods of roberts et al . ( 1990a ; 1990b ) and webster et al . ( 1990 ). after harvesting , the se were dried using the hrht method . post - hrht embryos were germinated in sterile conditions on whatman # 1 filter paper placed on a thin layer of cotton in polycarbonate boxes . the liquid germination medium was modified gmd medium ( nh 4 + — n / no 3 − — n ratio was 1 . 4 ; phosphate concentration was 6 mm ; 3 % sucrose ). after the embryos were sown onto the filter paper , the liquid level in the germination boxes was adjusted to provide a liquid film surrounding the surface of embryos situated at the interface with the solid support . the boxes were sealed with parafilm and placed in a controlled environment room with the temperature maintained at a constant 23 ° c . after a 10 - day in vitro germination period , the polycarbonate boxes containing the germinants were transferred to cold storage ( 8 - 10 ° c .) for 30 hours . the germinants were then transferred to clean petri dishes and were incubated in a 10 μmol aba solution for 75 min after which , they were blot - dried on paper towels . the germinants were separated into 7 groups and then randomly assigned to 1 of 9 desiccation treatments ( group a : 75 . 7 %; 81 . 5 %; 84 . 2 % rh at 5 ° c . ; group b : 84 . 2 %, 86 . 0 %; 88 . 5 % rh at 12 ° c . ; group c : 88 . 5 %, 92 . 4 %; 96 . 7 % rh at 20 ° c .). the germinants were desiccated for 48 h . after the desiccation treatments were completed , subsamples of the germinated , then desiccated spruce somatic embryos ( i . e ., pre - germinated embryos ), were taken for determination of their water contents . the dried germinants were sown into 252 - cavity miniplug trays ( beaver plastics ltd ., edmonton , ab , canada ) filled with a peat - based soilless mix comprised of 60 % peat and 40 % coarse vermiculite ( lightly pre - moistened with a dilute fertilizer solution , 20 - 20 - 20 , 100 ppm n equivalent ). four replicates of 20 germinants per treatment were randomly assigned to four miniplug trays . after sowing , the germinants were covered with a thin layer of coir ( 1993 crystal co ., st . louis , mo ., usa ). the trays were wrapped in black plastic bags overnight to allow slow rehydration of the desiccated germinants . the next day , the trays were sprayed with a modified gmd solution containing 3 % sucrose ( roberts et al ., 1990b ), until the soilless mix and the coir became well moistened . the trays were then placed into a misting chamber under a light intensity of 30 μmol m − 2 s − 1 for the first week , 55 μmol m − 2 s − 1 for the second week and 165 μmol m − 2 s − 1 for the remainder of the experiment . the light levels were adjusted by placing shading cloth underneath the lighting bulb . the chamber was misted 3 sec per hour for the day and 2 second per 2 hours for the night . a modified gmd solution containing 3 % sucrose was sprayed onto the germinants once every day on weekdays for the first three weeks . the effects of the different desiccation treatments on the survival and re - germination of pre - germinated somatic embryos from spruce line 1 - 1281 were determined after 4 weeks , and are summarized in table 4 . post - hrht treatment somatic embryos of pinus taeda line c - 3419 were germinated for 7 days in sterile conditions on whatman # 1 filter paper placed onto a thin layer of cotton in polycarbonate boxes . the liquid germination medium was modified gmd medium ( nh 4 + — n / no 3 − — n ratio was 1 . 4 ; phosphate concentration was 6 mm ; 3 % sucrose ). after the embryos were sown onto the filter paper , the liquid level in the germination boxes was adjusted to provide a liquid film surrounding the surface of embryos situated at the interface with the solid support . the boxes were sealed with parafilm and placed in a controlled environment room with the temperature maintained at a constant 23 ° c . after a 7 - day in vitro germination period , the polycarbonate boxes containing the germinants were transferred to cold storage ( 8 - 10c ) for 48 hours . the germinants were then transferred to clean petri dishes and were incubated in a 10 μmol aba solution for 1 . 5 h after which , they were blot - dried on paper towels . the germinants were separated into 7 groups and then randomly assigned to 1 of 7 desiccation treatments ( the rh values were : 75 . 8 %; 81 . 9 %; 84 . 6 %; 86 . 3 %; 88 . 7 %; 92 . 6 %; 96 . 7 %). the germinants were desiccated for 48 h at 5 ° c . after the desiccation treatments were completed , subsamples of the germinated , then desiccated pinus taeda somatic embryos ( i . e ., pre - germinated embryos ), were taken for determination of their water contents . the dried germinants were sown into 252 - cavity miniplug trays ( beaver plastics ltd ., edmonton , ab , canada ) filled with a peat - based soilless mix comprised of 60 % peat and 40 % coarse horticultural vermiculite ( lightly pre - moistened with a dilute fertilizer solution , 20 - 20 - 20 , 100 ppm n equivalent ). four replicates of 20 germinants per treatment were randomly assigned to four miniplug trays . after sowing , the germinants were covered with a thin layer of coir ( 1993 crystal co ., st . louis , mo ., usa ). the trays were wrapped in black plastic bags overnight to allow slow rehydration of the desiccated germinants . the next day , the trays were sprayed with a modified gmd solution containing 3 % sucrose ( roberts et al ., 1990b ), until the soilless mix and the coir became well moistened . the trays were then placed into a misting chamber under a light intensity of 30 μmol m − 2 s − 1 for the first week , 55 μmol m − 2 s − 1 for the second week and 165 μmol m − 2 s − 1 for the remainder of the experiment . the light levels were adjusted by placing shading cloth underneath the lighting bulb . the chamber was misted 3 sec per hour for the day and 2 second per 2 hours for the night . a ½ strength gmd solution containing 3 % sucrose was sprayed onto the germinants once every day on weekdays for the first three weeks . effects of the different desiccation treatments on survival and re - germination of pre - germinated somatic embryos from pinus taeda line c - 3419 are summarized in table 5 . these data demonstrate that somatic embryos of pinus taeda can be germinated in vitro , desiccated and then successfully re - germinated ex vitro in non - sterile peat - based growing substrate if the pre - germinated se are not overly desiccated . somatic embryos of pinus radiata lines pr - 6552 and pr - 6804 were pre - germinated in vitro for 23 days in polycarbonate boxes ( sigma - aldrich canada ltd ., 2149 winston park dr ., oakville , ont ., canada ; catalog # c8062 ). the sterile pre - germination substrate in each box consisted of 150 ml of coarse vermiculite plus 150 ml of modified gmd nutrient solution amended with 3 % sucrose ( roberts et al ., 1990b ; webster , 1990 ). after the pre - germination period was completed , the germinants were sorted into 7 groups with each group placed into a separate petri dish . the 7 groups of germinants were then simultaneously incubated for 1 h in 10 μm aba solutions . after the aba treatment was completed , each group of germinants was surface - dried by blotting with paper towels . then , each group of germinants was randomly assigned to 1 of 7 desiccation treatments ( the rh values were : 75 . 8 %; 81 . 9 %; 84 . 6 %; 86 . 3 %; 88 . 7 %; 92 . 6 %; 96 . 7 %). the germinants were desiccated for 48 hours in a refrigerated environment ( 5 °- 8 ° c .). after the desiccation treatments were completed , subsamples of the germinated , then desiccated pinus radiata somatic embryos ( i . e ., pre - germinated embryos ), were taken for determination of their water contents . their “ relative water contents ” were calculated using the mean water content of 5 blotted samples after aba treatment as the saturated base line . the desiccated pre - germinated pinus radiata somatic embryos were sown into 288 - cell miniplug flats manufactured by tlc polyform inc ( plymouth minn ., usa , 55441 ). the soilless growing media used was comprised of 1 : 1 mixture of screened peat and fine perlite . after sowing was completed , the flats were placed into a high - humidity growth chamber (+ 85 % rh ) for 4 weeks and then moved into a misting chamber for 8 additional weeks . the germinants were fertilized weekly with an 11 - 41 - 8 ( n - p - k ) forestry starter fertilizer solution ( plant products ltd ., brampton , on , canada ) over the 12 - week re - germination period . the effects of the different desiccation treatments on the survival and re - germination of pre - germinated pinus radiata somatic embryos are summarized in table 6 . these data demonstrate that pinus radiata somatic embryos can be germinated for a period of time , then desiccated and subsequently re - germinated . furthermore , these data indicate that the re - germination of desiccated pre - germinated pinus radiata somatic embryos can be accomplished ex vitro in non - sterile soilless mixes commonly used in conventional horticulture practice . matured canola ( brassica napus l .) somatic embryos were harvested and conditioned in nln - 13 liquid medium ( lichter , 1982 ) for one week as follows . embryos were placed in 250 - ml baffled erlenmeyer flasks containing 100 ml of medium . the flasks were then placed onto a shaker ( 60 rpm ) under constant ( 24 h / day ) illumination at 20 - 30 μmol m − 2 s − 1 of photosynthetic photon flux . the conditioned embryos were then germinated in vitro for a period of 7 days at 23 ° c ., in polycarbonate boxes ( sigma - aldrich canada ltd ., 2149 winston park dr ., oakville , ont ., canada ; catalog # c8062 ) containing 150 ml of coarse vermiculite and 150 of a liquid germination medium amended with 3 % sucrose . after the 7 - day pre - germination period , the boxes containing the germinated canola somatic embryos were given a 3 - day “ cold treatment ” at 5 ° c . after the pre - germination period was completed , the germinants were sorted into 7 groups with each group placed into a separate petri dish . the 7 groups of germinants were then simultaneously incubated for 1 h in 10 μm aba solutions . after the aba treatment was completed , each group of germinants was surface - dried by blotting with paper towels . then , each group of germinants was randomly assigned to 1 of 7 desiccation treatments ( the rh values were : 75 . 8 %; 81 . 9 %; 84 . 6 %; 86 . 3 %; 88 . 7 %; 92 . 6 %; 96 . 7 %). the germinants were desiccated for 4 days at 23 ° c . after the desiccation treatments were completed , subsamples of the germinated , then desiccated canola somatic embryos ( i . e ., pre - germinated embryos ), were taken for determination of their water contents . the desiccated pre - germinated canola somatic embryos were sown into 288 - cell miniplug flats . the soilless growing media used was comprised of 3 : 2 mixture of screened peat and fine perlite . after sowing was completed , the flats were placed into a misting chamber for 3 weeks . the germinants were fertilized weekly with an 11 - 41 - 8 ( n - p - k ) forestry starter fertilizer solution ( plant products ltd ., brampton , on , canada ). the effects of the different desiccation treatments on the survival and re - germination of pre - germinated canola somatic embryos are summarized in table 7 . these data demonstrate that that somatic embryos from an angiosperm species , brassica napus , can be germinated for a period of time , then desiccated and subsequently re - germinated . furthermore , these data indicate that the re - germination of desiccated pre - germinated brassica napus somatic embryos can be accomplished ex vitro in non - sterile soilless mixes commonly used in conventional horticulture practice . 1 . carlson , w . c . and j . e . hartle . 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( 1995 ) delivery systems for tissue culture by encapsulation . in automation and environmental control in plant tissue culture . j . aitken - christie , t . kozai , and m . l . a . smith , kluwer academic publishers , dordrecht , the netherlands . pp . 215 - 243 . 7 . webster , f . b ., d . r . roberts , s . m . mcinnis , and b . c . s . sutton ( 1990 ) propagation of interior spruce by somatic embryogenesis . can . j . for . res . 20 : 1759 - 1765 .