Patent Application: US-64580696-A

Abstract:
the invention relates to a method for regeneration of plants of the genus pinus by culturing explants of immature zygotic embryos on culture initiation medium containing abscisic acid , nutrients , growth hormones , sugar and a gelling agent to grow embryogenic tissue for cryopreservation . culturing of the embryogenic tissue is continued on culture maintenance medium , embryo development medium , and germination medium . the germinated embryos are further converted to acclimatized plants for field planting . the method is well suited for producing clonal planting stock useful for reforestation .

Description:
the present invention is a method for initiating embryogenic cell cultures of plants selected from the group consisting of pinus taeda , pinus serotina , pinus palustris , pinus elliottii , pinus rigida , and hybrids thereof , said method comprising culturing a suitable explant containing immature zygotic embryos on culture initiation media containing a sufficient amount of nutrients and growth hormones , a level of gelling agent , and abscisic acid . as noted above , the somatic embryogenesis process utilized with conifers ( particularly the pinus species ) can be divided into seven general steps : 1 ) culture initiation , 2 ) culture maintenance , 3 ) embryo development , 4 ) embryo maturation , 5 ) embryo germination , 6 ) conversion , and 7 ) plant growth ( field planting ). the present invention improves upon traditional somatic embryogenesis processes by including aba in the first step ( the culture initiation step ). thus , the methods taught in u . s . pat . nos . 5 , 413 , 930 and 5 , 506 , 136 are improved by replacing the standard culture initiation step with the following improved culture initiation step , which comprises : 1 . placing a suitable explant selected from the group consisting of immature zygotic embryos and megagametophytes containing immature zygotic embryos on culture initiation medium containing a sufficient amount of nutrients , 0 . 1 to 5 . 0 mg / l of auxin , 0 . 1 to 1 . 0 mg / l of cytokinin , 10 . 0 to 100 . 0 g / l of a sugar selected from the group consisting of glucose , maltose , sucrose , melezitose , and combinations thereof , a level of gelling agent selected from the group consisting of 2 . 5 to 9 . 0 g / l of agar , 0 . 5 to 4 . 0 g / l of gellan gum , 3 . 0 to 10 . 0 g / l of agarose , 1 . 5 to 5 . 0 g / l of agargel , and combinations thereof , and 0 . 1 to 100 . 0 mg of abscisic acid per liter of medium ( mg / l ), for 2 to 14 weeks under suitable environmental conditions to grow a culture containing embryogenic tissue . the present method significantly improves this culture initiation step by incorporating phytohormonic abscisic acid into the culture initiation media . this addition significantly increases , across a range of genotypes , the amount and number of viable tissues that are extruded and initiated . this improved step for producing embryogenic tissue masses is employed with the remaining method steps ( culture maintenance , embryo development , embryo maturation , embryo germination , conversion , and plant growth ) taught in u . s . pat . nos . 5 , 413 , 930 and 5 , 506 , 136 to create improved methods for producing coniferous plants via somatic embryogenesis . to practice the improved method one follows these additional steps : 2 . transferring the embryogenic tissue culture to culture maintenance medium containing a sufficient amount of nutrients , 0 . 1 to 5 . 0 mg / l of auxin , 0 . 1 to 1 . 0 mg / l of cytokinin , 10 . 0 to 40 . 0 g / l of a sugar selected from the group consisting of glucose , maltose , sucrose , melezitose and combinations thereof , and a level of gelling agent selected from the group consisting of 6 . 0 to 9 . 0 g / l of agar , 1 . 75 to 3 . 5 g / l of gellan gum , 6 . 0 to 8 . 0 g / l of agarose , 3 . 5 to 5 . 0 g / l of agargel , and combinations thereof , for a sufficient amount of time under suitable environmental conditions to develop a mass of embryogenic tissue having a minimum weight of at least 100 . 0 mg ; 3 . transferring at least about 100 . 0 mg of the mass of embryogenic tissue to embryo development medium containing 5 . 0 to 33 . 0 mg / l of abscisic acid , a level of gelling agent selected from the group consisting of 6 . 0 to 12 . 0 g / l of agar , 1 . 75 to 4 . 00 g / l of gellan gum , 6 . 0 to 8 . 0 g / l of agarose , 3 . 5 to 6 . 0 g / l of agargel , and combinations thereof , and 20 . 0 to 70 . 0 g / l of a sugar selected from the group consisting of glucose , maltose , sucrose , and combinations thereof , for a sufficient time under suitable environmental conditions to develop stage 3 somatic embryos ; 4 . separating the stage 3 somatic embryos from the development medium and partially drying the embryos by exposing the embryos to an atmosphere having a high relative humidity for sufficient time to permit the embryos to lose about 25 % to 75 % of their pre - dried weight ; 5 . transferring the partially dried somatic embryos to germination medium containing a sufficient amount of nutrients , up to 10 . 0 g / l of activated carbon , a level of gelling agent selected from the group consisting of 6 . 0 to 9 . 0 g / l of agar , 1 . 75 to 3 . 50 g / l of gellan gum , 6 . 0 to 8 . 0 g / l of agarose , 3 . 5 to 5 . 0 g / l of agargel , and combinations thereof , and 20 . 0 to 40 . 0 g / l of a sugar selected from the group consisting of glucose , maltose , sucrose , and combinations thereof , for a sufficient time under suitable environmental conditions to germinate the partially dried embryos ; any somatic tissue explant capable of being employed for somatic embryogenesis is suitable for use in the present method . however , it is preferred that the explant be either an immature whole megagametophyte containing zygotic embryos or an isolated immature dominant zygotic embryo . this method is generally applicable to somatic tissue obtained from the pinus species including , but not limited to , the following : pinus taeda ( loblolly pine ), p . elliottii ( slash pine ), p . palustris ( longleaf pine ), p . serotina ( pond pine ), p . echinata ( shortleaf pine ), p . clausa ( sand pine ), p . glabra ( spruce pine ), p . rigida ( pitch pine ), p . echinata ( shortleaf pine ), p . nigra ( austrian pine ), p . resinosa ( red pine ), p . sylvestris ( scotch pine ), p . banksiana ( jack pine ), p . virginiana ( virginia pine ), p . radiata ( monterey pine ), p . contorta ( shore pine ), p . contorta latifolia ( lodgepole pine ), p . ponderosa ( ponderosa pine ), p . leiophylla ( chihuahua pine ), p . jeffireyi ( jeffrey pine ), and p . engelmannii ( apache pine ), p . strobus ( eastern white pine ), p . monticola ( western white pine ), and p . larnbertiana ( sugar pine ), p . albicaulis ( whitebark pine ), p . flexills ( limber pine ), p . strobiformis ( southwestern white pine ), p . caribaea ( caribbean pine ), p . patula ( mexican weeping pine ), p . tecurnumanii ( tecun uman pine ), p . maximinoi , p . oocarpa ( ocote pine ) and p . chiapensis ( mexican white pine ). in addition , the current invention is specifically applicable to interspecies hybrids of the above mentioned pines including pinus rigida x p . taeda , p . serotina x p . taeda , and reciprocal crosses . it is preferred to utilize the present method with southern yellow pines , pinus rigida , and hybrids thereof . those skilled in the art recognize that several species of pine indigenous to the southeastern united states are closely related and hybridize naturally . taxonomically this group of pines is referred to as &# 34 ; southern yellow pines &# 34 ; and includes pinus taeda , p . serotina , p . palustris , and p . elliottii ( preston 1989 ). in addition to the taxonomically similarity of the above southern yellow pine species , these species have also responded similarly in studies on somatic embryogenesis attempts . for example , all previous reports of somatic embryogenesis with the above species have found the same stage , very early precotyledonary zygotic embryos , to be optimum for embryogenic culture initiation ( see , e . g ., becwar et al ., 1990 , and jain et al . 1989 ). initiation frequencies were similarly low , about 1 . 0 to 5 . 0 %, among these species . the present invention lies in the incorporation of abscisic acid ( aba ) into media formulations used to initiate conifer embryogenic cell cultures . a suitable level of aba for use in improving the initiation media for the methods taught in u . s . pat . nos . 5 , 413 , 930 and 5 , 506 , 136 is from about 0 . 1 to about 100 . 0 milligrams per liter of medium ( mg / l ) of medium . the preferred aba level is about 1 . 0 to about 60 . 0 mg / l ; with the most preferred level of aba being about 5 . 0 to about 30 . 0 mg / l . in u . s . pat . nos . 5 , 413 , 930 and 5 , 506 , 136 , abscisic acid is utilized in combination with an auxin and a cytokinin . in the improved methods for these patents the culture initiation media must contain from about 0 . 1 to 5 . 0 mg / l of auxin and from about 0 . 1 to 1 . 0 mg / l of cytokinin . auxins suitable for use in these improved methods are 2 , 4 - d ( 2 , 4 - dichlorophenoxy acetic acid ), naa ( α - naphthaleneacetic acid ), and the like . cytokinins suitable for use in the present method are bap ( n 6 - benzylamino - purine ), kinetin ( 6 - furfurylaminopurine ), zeatin ( 6 - 4 - hydroxy - 3 - methylbut - 2 - enylamino ! purine ), and the like . in addition to these growth regulators , the medium also requires sufficient amounts of nutrients to allow the culture to remain viable . however , the present method is not limited to any single culture nutrient medium formulation . for example , four common basal culture media formulations which were used in examples 1 - 4 ( designated dcr , sh , wv5 , and msg ) are listed in table i below . although the listed basal media gave excellent results when employed in the present method , it should be understood that any nutrient media commonly used in pinus somatic embryogenesis will be suitable for use with this invention . table i______________________________________formulations of basal culture media dcr . sup . a sh . sup . b wv5 . sup . c msg . sup . dcomponent concentration , mg / l______________________________________inorganic saltsnh . sub . 4 no . sub . 3 400 . 00 -- 700 . 00 -- kno . sub . 3 340 . 00 2500 . 00 259 . 00 100 . 00ca ( no . sub . 3 ). sub . 2 . 4h . sub . 2 o 556 . 00 -- 963 . 00 -- mgso . sub . 4 . 7h . sub . 2 o 370 . 00 400 . 00 1850 . 00 370 . 00kh . sub . 2 po . sub . 4 170 . 00 -- 270 . 00 170 . 00nh . sub . 4 h . sub . 2 po . sub . 4 -- 300 . 00 -- -- cacl . sub . 2 . 2h . sub . 2 o 85 . 00 200 . 00 -- 440 . 00kcl -- -- 1327 . 00 745 . 00ki 0 . 83 1 . 00 0 . 83 0 . 83h . sub . 3 bo . sub . 3 6 . 20 5 . 00 31 . 00 6 . 20mnso . sub . 4 . h . sub . 2 o 22 . 30 10 . 00 15 . 16 16 . 90znso . sub . 4 . 7h . sub . 2 o 8 . 60 1 . 00 8 . 60 8 . 60na . sub . 2 moo . sub . 4 . 2h . sub . 2 o 0 . 25 0 . 10 0 . 25 0 . 25cuso . sub . 4 . 5h . sub . 2 o 0 . 25 0 . 20 0 . 25 0 . 03cocl . sub . 2 . 6h . sub . 2 o 0 . 03 0 . 10 0 . 03 0 . 03nicl . sub . 2 . 6h . sub . 2 o 0 . 03 -- -- -- feso . sub . 4 . 7h . sub . 2 o 27 . 80 15 . 00 27 . 80 27 . 80na . sub . 2 edta 37 . 30 20 . 00 37 . 30 37 . 30vitamins , aminoacidsnicotinic acid 0 . 50 0 . 5 0 . 50 0 . 50pyridoxine hcl 0 . 50 0 . 5 0 . 50 0 . 10thiamine hcl 1 . 00 1 . 00 1 . 00 0 . 10glycine 2 . 00 2 . 00 2 . 00 -- ______________________________________ . sup . a according to gupta and durzan ( 1985 ). . sup . b according to schenk and hildebrandt ( 1972 ). . sup . c according to coke ( 1996 ). . sup . d according to becwar et al . ( 1990 ). suitable media for use in improving the culture initiation step for the methods taught in u . s . pat . nos . 5 , 413 , 930 and 5 , 506 , 136 contain from about 10 . 0 to about 100 . 0 grams per liter ( g / l ) of a sugar selected from the group consisting of glucose , maltose , sucrose , melezitose , and combinations thereof . the preferred sugar content for the media is from about 15 . 0 to about 40 . 0 g / l ; with the most preferred content being from about 20 . 0 to about 30 . 0 g / l . suitable media for use in improving the culture initiation step for the methods taught in u . s . pat . nos . 5 , 413 , 930 and 5 , 506 , 136 contain a level of gelling agent selected from the group consisting of 2 . 5 to 9 . 0 g / l of agar , 0 . 5 to 4 . 0 g / l of gellan gum , 3 . 0 to 10 . 0 g / l of agarose , 1 . 5 to 5 . 0 g / l of agargel ® ( an agar / gellan gum mixture commercially available from sigma chemical company ), and combinations thereof . preferred media would contain a level of gelling agent selected from the group consisting of 2 . 5 to 4 . 5 g / l of agar , 0 . 5 to 1 . 5 g / l of gellan gum , 3 . 0 to 5 . 0 g / l of agarose , 1 . 5 to 3 . 0 g / l of agargel , and combinations thereof . suitable culture initiation periods for use in the improved culture initiation step for the methods taught in u . s . pat . nos . 5 , 413 , 930 and 5 , 506 , 136 lasts for about 2 to 14 weeks , with the preferred period being 3 to 10 weeks . after initiation the embryogenic tissue is transferred for maintenance to culture maintenance media . the embryogenic tissue can be maintained by sub - culturing at regular intervals ( usually every 2 to 3 weeks ) to new maintenance medium . alternatively , the present method also allows embryogenic cultures initiated on aba - containing media to be cryopreserved for future use via standard methods . a number of terms are known to have differing meanings when used in the literature . the following definitions are believed to be the ones most generally used in the field of botany and are consistent with the usage of the terms in the present specification . a &# 34 ; cell line &# 34 ; is a culture that arises from an individual explant . &# 34 ; clone &# 34 ; when used in the context of plant propagation refers to a collection of individuals having the same genetic makeup . &# 34 ; corrosion cavity &# 34 ; is the cavity within the megagametophyte tissue of conifers formed by the growth and enlargement of the zygotic embryos . &# 34 ; conversion &# 34 ; refers to the acclimatization process that in vitro derived germinating somatic embryos undergo in order to survive under ex vitro ( nonaxenic ) conditions , and subsequent continued growth under ex vitro conditions . an &# 34 ; embryogenic culture &# 34 ; is a plant cell or tissue culture capable of forming somatic embryos and regenerating plants via somatic embryogenesis . &# 34 ; embryogenic tissue &# 34 ;, in conifers , is a mass of tissue and cells comprised of very early stage somatic embryos and suspensor - like cells embedded in a mucilaginous matrix . the level of differentiation may vary significantly among embryogenic conifer cultures . in some cases , rather than containing well - formed somatic embryos , the embryogenic tissue may contain small , dense clusters of cells capable of forming somatic embryos . this has also been referred to as &# 34 ; embryogenic suspensor masses &# 34 ; by some researchers and is also called &# 34 ; embryogenic callus &# 34 ; in some of the conifer somatic embryogenesis literature ; but most researchers believe it is not a true callus . an &# 34 ; explant &# 34 ; is the organ , tissue , or cells derived from a plant and cultured in vitro for the purpose of starting a plant cell or tissue culture . &# 34 ; extrusion &# 34 ; is the process by which zygotic embryos and / or embryogenic tissue derived from zygotic embryos emerges or extrudes from the corrosion cavity of the megagametophyte of conifer seeds via the opening in the micropylar end , when placed in culture . &# 34 ; field planting &# 34 ; is the establishment of laboratory , greenhouse , nursery , or similarly grown planting stock under field conditions . &# 34 ; genotype &# 34 ; is the genetic constitution of an organism ; the sum total of the genetic information contained in the dna of an organism . &# 34 ; initiation &# 34 ; is the initial cellular proliferation or morphogenic development that eventually results in the establishment of a culture from an explant . &# 34 ; megagametophyte &# 34 ; is haploid nutritive tissue of the conifer seed , of maternal origin , within which the conifer zygotic embryos develop . &# 34 ; micropyle &# 34 ; is the small opening in the end of the conifer seed where the pollen tube enters the ovule during fertilization , and where embryogenic tissue extrudes from the megagametophyte during culture initiation . &# 34 ; nutrients &# 34 ; are the inorganics ( e . g ., nitrogen ), vitamins , organic supplements , and carbon sources necessary for the nourishment of the culture . a &# 34 ; plantlet &# 34 ; is a small germinating plant derived from a somatic embryo . &# 34 ; regeneration &# 34 ;, in plant tissue culture , is a morphogenic response to a stimuli that results in the production of organs , embryos , or whole plants . &# 34 ; stage 1 embryos &# 34 ; are small embryos consisting of an embryonic region of small , densely cytoplasmic cells subtended by a suspensor comprised of long and highly vacuolated cells . &# 34 ; stage 2 embryos &# 34 ; are embryos with a prominent ( bullet shaped ) embryonic region that is more opaque and with a more smooth and glossy surface than stage 1 embryos . &# 34 ; stage 3 embryos &# 34 ; are embryos with an elongated embryonic region with small cotyledons visible . &# 34 ; somatic embryogenesis &# 34 ; is the process of initiation and development of embryos in vitro from somatic cells and tissues . a &# 34 ; somatic embryo &# 34 ; is an embryo formed in vitro from vegetative ( somatic ) cells by mitotic division of cells . early stage somatic embryos are morphologically similar to immature zygotic embryos ; a region of small embryonal cells subtended by elongated suspensor cells . the embryonal cells develop into the mature somatic embryo . a &# 34 ; suspensor cell &# 34 ; is an extension of the base of the embryo that physically pushes the embryo into the megagametophyte in conifer seeds and is comprised of elongated and highly vacuolated cells . in a somatic embryo these elongated cells are clustered in rows and extend from the base of the dense cytoplasmic cells at the head or apex . a &# 34 ; zygotic embryo &# 34 ; is an embryo derived from the sexual fusion of gametic cells . the following examples are provided to further illustrate the present invention and are not to be construed as limiting the invention in any manner . immature seed cones were collected from several different loblolly pine ( pinus taeda l .) sources located in westvaco &# 39 ; s south carolina coastal breeding orchards near charleston , s . c . the seed cones were collected when the dominant zygotic embryo was at the precotyledonary stage of development . using the classification system of von arnold and hakman ( 1988 ), the dominant zygotic embryo at this stage is referred to as being at stage 2 ; that is , an embryo with a prominent embryonic region with a smooth and glossy surface , subtended by elongated suspensor cells which are highly vacuolated . however , zygotic embryos at an earlier stage of development ( stage 1 ) may also be used effectively to initiate embryogenic cultures . seed cones were harvested from selected trees , placed in plastic bags and stored at 4 ° c . until used for culture initiation . if the cones were stored for more than two weeks at 4 ° c ., they were aired and dried out weekly ( placed at 23 ° c ., ambient laboratory conditions for 2 - 3 hours ) to prevent growth of fungi on the surface of the cones and concomitant deterioration of seed quality . for culture initiation , intact seeds removed from seed cones were surface sterilized by treatment in a 10 to 20 % commercial bleach solution ( equivalent of a 0 . 525 % to 1 . 050 % sodium hypochlorite solution ) for 15 minutes followed by three sterile water rinses ( each of five minutes duration ). seeds were continuously stirred during the sterilization and rinsing process . megagametophytes containing developing zygotic embryos were used as the explant for culture initiation . the seed coats of individual seeds were cracked open under a laminar - flow hood with the use of a sterile hemostat . the intact megagametophyte ( which contains the developing zygotic embryos ) was removed from the opened seed coat with forceps . tissues attached to the megagametophyte , such as the megagametophyte membrane and the nucellus , were removed from the megagametophyte and discarded . the megagametophyte was placed on culture medium ( longitudinal axis of megagametophyte parallel to the surface of culture medium ) with forceps . the micropyle end of the megagametophyte was placed in contact with ( but not submerged in ) the culture medium ( see table i , dcr ). basal salt mixtures which have proven effective for culture initiation include dcr , sh and wv5 basal salts formulation listed in table i . ( the complete formulations of the media used in the examples are listed in table ii .). the ph of the medium was adjusted to 5 . 8 with koh and hcl prior to autoclaving at 110 kpa ( 16 psi ) and 121 ° c . for 20 minutes . aqueous stock solutions of l - glutamine were filter sterilized and added to warm ( about 60 ° c .) medium prior to pouring the medium into culture dishes . approximately 20 ml of medium was poured into 100 × 15 mm sterile plastic petri dishes . the basal media modified for each of the culture stages are listed in tables ii , iii , and iv below . table ii______________________________________composition of initiation media commonly used in the examples initiation initiation initiation medium medium mediumcomponent dcr . sub . 1 sh . sub . 1 wv5 . sub . 1______________________________________basal medium . sup . a dcr sh wv5concentration ( g / l ) inositol 0 . 50 0 . 50 0 . 50casein 0 . 50 0 . 50 0 . 50hydrolysatesucrose or maltose 30 . 00 . sup . b 30 . 00 . sup . b 30 . 00 . sup . bgelrite 1 . 00 - 2 . 00 1 . 00 - 2 . 00 1 . 00 - 2 . 00concentration ( mg / l ) auxin . sup . c 3 . 00 3 . 00 3 . 00cytokinin . sup . d 0 . 50 0 . 50 0 . 50aba . sup . e 0 . 1 - 100 . 0 0 . 1 - 100 . 0 0 . 1 - 100 . 0______________________________________ . sup . a refer to table i for composition of basal medium . . sup . b see example 3 below , where either sucrose or maltose was added to the basal media . . sup . c 2 , 4dichlorophenoxyacetic acid ( 2 , 4d ). . sup . d n . sup . 6benzylaminopurine or n . sup . 6benzyladenine ( bap )!. . sup . e abscisic acid . table iii______________________________________composition of maintenance media used in the examples maintenance maintenance maintenance medium medium mediumcomponent dcr . sub . 1 sh . sub . 1 wv5 . sub . 1______________________________________basal medium . sup . a dcr sh wv5concentration ( g / l ) inositol 0 . 50 0 . 50 0 . 50casein 0 . 50 0 . 50 0 . 50hydrolysatel - glutamine 0 . 25 0 . 25 0 . 25sucrose or maltose 30 . 00 . sup . b 30 . 00 . sup . b 30 . 00 . sup . bgelrite 2 . 00 2 . 00 2 . 00concentration ( mg / l ) auxin . sup . c 3 . 00 3 . 00 3 . 00cytokinin . sup . d 0 . 50 0 . 50 0 . 50aba . sup . e 30 . 0 - 90 . 0 30 . 0 - 90 . 0 30 . 0 - 90 . 0______________________________________ . sup . a refer to table i for composition of basal medium . . sup . b see example 3 below , where either sucrose or maltose was added to the basal media . . sup . c 2 , 4dichlorophenoxyacetic acid ( 2 , 4d ). . sup . d n . sup . 6benzylaminopurine or n . sup . 6benzyladenine ( bap )!. . sup . e abscisic acid ( example 5 only ). table iv______________________________________compositon of development andgermination media used in the examples development germination medium mediumcomponent msg . sub . 1 msg . sub . g______________________________________basal medium . sup . a msg msgconcentration ( g / l ) ammonium -- 0 . 80nitrateinositol 0 . 10 0 . 10l - glutamine 1 . 45 -- sucrose -- 30 . 00maltose 60 . 00 -- gelrite 2 . 00 2 . 00activated -- 5 . 00carbonpolyethylene 70 . 00 -- glycol 4000concentration ( mg / l ) aba . sup . b 125 . 00 -- ______________________________________ . sup . a refer to table i for composition of basal medium . . sup . b abscisic acid after megagametophyte explants were placed in culture , the perimeter of the dish was sealed with two wraps of parafilm ® ( manufactured by american can co .). the dishes were incubated in the dark at a constant temperature of 23 ° c . after about 7 to 21 days , embryogenic tissue extruded from the micropyle of the megagametophyte explants . after 28 days in culture , embryogenic tissue was removed from responsive megagametophyte explants and moved to a new position on the same culture dish , or the embryogenic tissue was transferred to a new culture dish containing the same culture medium as used for initiation . each individual culture derived from an individual megagametophyte explant was kept separate and assigned a cell line identification code . subcultures were made to fresh medium at 3 week intervals after that . explants were initially placed on initiation medium containing 0 . 125 % gelrite . at the first subculture the gelrite concentration was increased to 0 . 2 %. this example examined the incorporation of aba at five different levels in standard culture initiation media containing dcr salts and vitamins , along with 3 mg / l 2 , 4 - d and 0 . 5 mg / l bap . the control in this study was 0 mg / l aba . a total of 49 explants were cultured per family per aba level . the extrusion of embryogenic tissues and the number of embryogenic lines produced after 9 weeks were recorded , and the results are shown in table v below . table v______________________________________the effect of abscisic acid onembryogenic culture initiation aba contained in initiation media in mg / lfamily 0 15 30 45 60______________________________________a 2 7 4 8 9b 4 11 14 15 10c 16 27 23 34 30total 23 45 41 57 49percent (%) 15 . 0 30 . 6 27 . 9 38 . 8 33 . 3extrusion______________________________________ as shown above , in the majority of cases the embryo extrusion and proliferation on culture initiation media containing aba more than doubled the results achieved from the control initiation media which contained no aba . following the procedures taught in example 1 above , embryogenic tissue cultures from three loblolly pine sources were initiated on semi - solid dcr 1 medium with 3 . 0 mg / l 2 , 4 - d , 0 . 5 mg / l bap , 0 . 125 % gelrite with four different levels of aba . once cultures were extruded and subcultured they were kept on the above four media but with the gelrite concentration increased to 0 . 2 %. a total of 96 explants were cultured per family per aba level . the cultures were initiated for four weeks and then subsequently subcultured at three - week intervals by plating the newly formed , mucilaginous tissue on to fresh medium . the extrusion and proliferation of embryogenic tissues and the number of embryogenic lines produced after 10 weeks were recorded , and the results are shown in table vi below . table vi______________________________________the effect of abscisic acid onembryogenic culture initiation aba mg / lfamily 0 30 60 90______________________________________d 16 . 8 31 . 2 31 . 2 23 . 9e 19 . 8 33 . 3 34 . 4 32 . 3f 21 . 9 37 . 5 28 . 1 36 . 4average percent (%) 19 . 4 34 . 0 31 . 2 30 . 9proliferation______________________________________ the results clearly show that the percent extrusion and proliferation at 10 weeks on media containing aba again increased significantly from the control containing no aba . it should be noted that family d is a superior genetic family containing excellent genetic potential . following the procedures taught in example 1 above , embryogenic tissue cultures from six loblolly pine sources were initiated on three basal salt media formulations ( semi - solid dcr 1 , wv5 and sh media ) with 3 . 0 mg / l 2 , 4 - d , 0 . 5 mg / l bap , 0 . 125 % gelrite with either sucrose or maltose as the carbohydrate and either 0 or 30 mg / l aba . there were 96 megagametophytes cultured per family per treatment combination . once cultures were extruded and subcultured they were kept on the above media but with the gelrite concentration increased to 0 . 2 %. the cultures were initiated for four weeks . the cultures were then subsequently subcultured at three - week intervals by plating the newly formed , mucilaginous tissue on to culture maintenance media . the extrusion and proliferation of embryogenic tissues and the number of embryogenic lines produced after 13 weeks were recorded , and the results are shown in table vii below . table vii______________________________________the effect of abscisic acid onembryogenic culture initiation carbohydrate sucrose maltosebasal salts aba mg / l ( 30 g / l ) ( 30 g / l ) ______________________________________dcr 0 2 . 1 1 . 6 30 6 . 1 9 . 3wv5 0 4 . 8 9 . 8 30 10 . 0 13 . 6sh 0 5 . 6 6 . 3 30 9 . 6 12 . 5______________________________________ the shown above , the percent extrusion and proliferation at 13 weeks on media containing aba again increased significantly from the control containing no aba ( dcr 30 g / l sucrose , 0 mg / l aba ). proliferation increased from 2 percent for the control to as much as 13 percent with 30 mg / l aba . proliferation using aba was higher with either sucrose or maltose as the carbon source . following the procedures taught in example 1 above , embryogenic tissue cultures were derived from two loblolly pine sources using cones collected from tres barras brazil and shipped to the united states . explants were placed on 10 different initiation media . all media contained wv5 salts and vitamins , 30 g / l maltose , 0 . 125 % gelrite along with 3 mg / l 2 , 4 - d and 0 . 5 mg / l bap and 10 different levels of aba . the control in this experiment was the 0 mg / l level of aba . once tissues were extruded they were maintained on the above media but with gelrite level increased to 0 . 2 %. the cultures were initiated for four weeks and then subsequently subcultured at three - week intervals by plating the newly formed , mucilaginous tissue on to fresh medium . the extrusion and proliferation of embryogenic tissues and the number of embryogenic lines produced after 10 weeks were recorded , and the results are shown in table viii below . table viii__________________________________________________________________________the effect of abscisic acid onembryogenic culture initiationaba mg / lfamily 0 . 00 5 10 30 60 90 120 180 240 300__________________________________________________________________________d 2 . 1 11 . 5 18 . 8 5 . 7 6 . 2 9 . 3 3 . 7 3 . 4 0 0e 6 . 8 8 . 3 18 . 8 8 . 3 5 . 2 4 . 1 4 . 1 1 . 0 0 0ave . percent 5 . 4 15 . 6 28 . 1 9 . 8 8 . 8 11 . 5 5 . 8 3 . 9 0 0proliferation__________________________________________________________________________ the results in table viii above clearly show that the percent extrusion and proliferation at 10 weeks on media containing aba again increased significantly from the control containing no aba . proliferation increased from around 5 percent for the 0 aba control to up to 28 percent with 10 mg / l aba in the medium . in order to ascertain the effect on embryo development of subsequent maintenance on aba - containing media , a sample of the lines produced in example 1 were taken through the development step . at week 12 following the initial plating of explants the tissues were transferred and divided between culture maintenance media which contained high levels of aba and standard culture maintenance medium not containing aba . three clumps of embryogenic callus per line were plated on the respective media . the lines were cultured for 13 more weeks on the two different media and were then placed on development medium . the results are shown in table ix below . table ix______________________________________the effect of abscisic acid onembryogenic culture maintenance total number embryos average numberlines maintenance medium produced embryos / clump______________________________________b1 , 2 dcr 3d / 0 . 5b / 30a 170 28 dcr 3d / 0 . 5b 67 11a1 dcr 3d / 0 . 5b / 60a 152 8b3 , 4 , 5 , 6 dcr 3d / 0 . 5b 184 10c1b7 dcr 3d / 0 . 5b / 90a 264 44c2 dcr 3d / 0 . 5b 262 44______________________________________ average embryo production / clump without aba = 21 . 6 average embryo production / clump with aba = 26 . 6 the results contained in table ix above indicate that the inclusion of aba in the culture initiation media was not detrimental to later embryo development for these cultures . many modifications and variations of the present invention will be apparent to one of ordinary skill in the art in light of the above teachings . it is therefore understood that the scope of the invention is not to be limited by the foregoing description , but rather is to be defined by the claims appended hereto . becwar , m . r ., e . e . chesick , l . w . handley , m . r . rutter . method for regeneration of coniferous plants by somatic embryogenesis . u . s . pat . no . 5 , 413 , 930 -- issued may 9 , 1995 . becwar , m . r ., e . e . chesick , l . w . handley , m . r . rutter . method for regeneration of coniferous plants by somatic embryogenesis . u . s . pat . no . 5 , 506 , 136 -- issued apr . 9 , 1996 . becwar , m . r ., r . nagmani , and s . r . wann . initiation of embryogenic cultures and somatic embryo development in loblolly pine ( pinus taeda ). canadian journal of forest research 20 : 810 - 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