Patent Abstract:
protocols for organogenic regeneration of cotton are provided , which makes the in vitro regeneration of mature fertile plants in a reduced amount of time possible . seedlings are the basis for monocotyl or hypocotyl explants which are transferred from the germination medium to a shoot initiation medium which comprises agno 3 . these explants , prior to shoot initiation , may be transformed with exogenous dna , either through inoculation with an agorbacterium agent such as a . tumefaciens , or through biolistic bombardment of the explants with microprojectiles having the exogenous dna adsorbed onto their surface .

Detailed Description:
this invention pertains to methods of regeneration of cotton , using organogenesis , coupled with methods of transformation of this important commercial crop , which can be used to introduce exogenous dna to provide more desirable species , which can be regenerated using the regeneration protocols to provide mature , fertile plants which breed true ( pass on the transformed or exogenous dna and the traits encoded thereby ). the detailed discussion below describes cotton regeneration , together with a method for transformation . it is important to note that cotton can be transformed using either agrobacterium , such as a . tumefaciens coupled with wounding , or through biolistic bombardment . in connection with biolistic bombardment , both nuclear and plastid dna can be targeted . where using dna for introduction into plastids , for transformation , smaller microprojectiles ( 0 . 4 - 0 . 7 microns verses 1 . 0 - 1 . 7 microns ) may be advantageously employed , and dna containing chimeric genes that can be expressed in plastids are utilized . one example , not intended to be limiting , of this type of vector is pzs 197 containing a chimeric aada gene which confers resistance to spectinomycin ( svab & amp ; maliga , 1993 ). a similar plastid expression vector supplied by dr . hans - ulrich koop , botanisches institut , germany , was made available . use of vectors containing the aada gene enables the use of non - lethal selection to identify cells in plants containing transformed plastids . after bombardment , tissues would be grown on shoot initiation media containing spectinomycin and / or streptomycin but otherwise conducted as described herein below . ( 2 ) excision of tissues such as hypocotyl explants for use in transformation and regeneration ( 4 ) selection of transformed tissues in the presence of antibiotic or herbicide to allow selective growth of transformed shoots ( antibiotic or herbicide resistance gene is part of introduced dna ) ( 1 ) seeds a are surface disinfected in a bleach solution ( 25 %, v / v ) containing 0 . 5 % sds ( detergent ) for 20 min ., then rinsed 4 times with sterile distilled water . seeds are placed in / on nitsch & amp ; nitsch ( nh - based ) or murashige & amp ; skoog ( ms - based ) media ( cefotaxime may be added ) for 1 - 2 weeks to allow germination . ( 2 ) explants ( hypocotyl ) are excised and placed on shoot initiation media . ( 3 ) dna is introduced with a . tumefaciens via a modified protocol of those already reported . dna was successfully delivered into cotton coker lines ( commercially unimportant ) by umbeck et al . ( 1987 ; hypocotyl sections ), firoozabady et al . ( 1987 ; cotyledon sections ), and rajasekaran et al . ( 1996 ; both explants ) c . introduction of dna via the pds - 1000 / he apparatus utilizes a modified protocol of those developed and currently used in n . reichert &# 39 ; s lab on other crops . rajasekaran et al . ( 1996 ) successfully introduced dna into embryogenic cotton lines ( coker and acala ) via the pds - 1000 / he d . multiple bombardments may increase transformation efficiencies , as has been demonstrated in cotton ( rajasekaran et al ., 1996 ). dna introduction via a combination of biolistics and a . tumefaciens may also enhance the recovery of transformed cotton tissues . in other plant species , this has been demonstrated to increase a . tumefaciens transformation efficiencies due to enhanced wounding ( bidney et al ., 1992 c ). ( 4 ) selection systems currently in use include the use of geneticin ( g 418 ) and kanamycin ( chimeric nptii gene ), phosphinothriein ( chimeric bar gene ) and hygromycin ( chimeric hph gene ) for selection of transformants . ( 5 ) adventitious shoots emerge from tissues containing introduced dna by growth on media containing a selection agent as discussed above . transformed shoots that arise are cut and placed on a rooting medium . a . commercial cotton varieties such as deltapine 50 , stoneville 474 have been used . b . previously , researchers primarily introduced dna into commercially unimportant lines , and regeneration ( coker and non - coker lines ) was through somatic embryogenesis which entailed a protracted culture period [ up to 24 weeks to generate embryogenic callus ( rajasekaran et al ., 1996 )]. this callus was then transferred to a second medium for production of somatic embryos , which were subsequently transferred to a third medium to achieve germination . regeneration via adventive shoot organogenesis as described works on commercially important varieties and shortens the time it takes to produce transgenic cotton plants . in addition , since a prolonged callus phase will be avoided , there should be less chances for production of mutated cotton plants due to somaclonal variation . c . the tissues rajasekaran et al . ( 1996 ) used in bombardments with the pds1000 / he apparatus were embryogenic callus lines initiated from seedling explants . regeneration then entailed development of somatic embryos from this callus ( u . s . pat . no . 5 , 244 , 802 ). as stated above , maintenance of cotton tissues in the callus phase for prolonged periods of time will increase the prevalence of mutations in cotton regenerants . d . bidney , d ., c . scelonge , j . martich , m . burrus , l . sims , and g . huffman . 1992 . microprojectile bombardment of plant tissues increases transformation frequency by agrobacterium tumefaciens . plant mol . biol . 18 : 301 - 313 . all culture stages were incubated under a 16 h . photoperiod at room temperature . cotton seeds were soaked for 5 minutes in 70 % ethanol , then surface sterilized for 25 minutes using 25 % commercial bleach and 0 . 5 % sodium dodecyl sulfate ( sds ) on a shaker ( 200 rpm ). the seeds were rinsed 3 times with sterile deionized double distilled water and placed on a growth maintenance medium ( gms ) gmsc [ ms basal salts , 1 . 0 mg / l thiamine - hcl , 0 . 5 mg / l pyridoxine - hcl , 0 . 5 mg / l nicotinic acid , 100 mg / l myo - inositol , 30 g / l glucose , 0 . 8 g / l phytagar , ph 5 . 8 ; plus 500 mg / l cefotaxime ( for bacterial contamination )] for one week . germinated seeds ( with at least the radicle emergent ) were then placed in liquid gmsc ( no phytagar ) for an additional week . after 1 week in liquid medium , the cotyledons and shoot tips were clearly visible . hypocotyl sections ( one per seedling ) were then excised with the acropetal cut made just below the cotyledonary nodes ( the cut was made on a line that is clearly visible on the explants ). the basipetal cut was made 1 . 0 cm below the initial cut hypocotyl sections were placed horizontally on shoot initiation medium ( ga ). explants were maintained for 6 weeks on this medium . shoot primordia were visible on the acropetal ends of hypocotyl sections after 2 weeks . after 6 weeks , leaves were clearly visible . shoot initiation has been observed in 7 commercial cultivars ( deltapine 50 , stoneville 474 , deltapine 5111 , tx 121 , suregrow 125 , fibermax 819 , and paymaster 1215 ) and 3 breeding lines , and the response ranged from 20 - 73 % ( percentage explants capable of initiating shoots per cultivar ). at the end of 6 weeks , the upper 1 . 0 cm portion of the acropetal ends were excised and placed on shoot elongation medium ( gb ). cultures were maintained on this medium for an additional 6 weeks . rooting can occur in this medium . up to 5 elongated shoots per explant have been generated . shoots with a defined shoot pole were excised from shoot clumps for rooting on gc . rooting may take up to 6 weeks . plants can be successfully acclimatized in 2 weeks . ga : semi - solid gms plus an auxin like naa ( 0 . 1 - 3 . 0 mg / l ), a cytokinin like tdz , or ba plus kinetin ( 0 . 1 - 2 . 5 mg / l ) and 1 - 50 mg / l silver nitrate gb : semi - solid gms plus an auxin like naa ( 0 . 1 - 1 . 0 mg / l ), a cytokinin like kinetin plus ba ( 0 . 1 - 1 . 0 mg / l each ), gibberellic acid like ga 3 ( 0 . 1 - 1 . 0 mg / l ) and activated charcoal ( 0 . 1 - 2 . 0 g / l ) gc : semi - solid gms plus an auxin like naa or iba ( 0 . 1 - 1 . 0 mg / l ) and activated charcoal ( 0 . 1 - 2 . 0 g / l ) apical portions were excised from newly cut , 2 , and 4 week old explants . observations on explants that have been newly cut indicated that intact cotyledonary nodes were not present . after 2 weeks , the explant had increased in width and numerous shoot meristems ( densely stained ) were clearly visible , particularly in the central portion of the explant . these shoot meristems arose from the parenchyma cells of the central pith . leaf primordia were forming around the young shoot tip . no vascular tissues were connected to these early shoot meristems , although they were visible in the original lower pith region of the explant . after 4 weeks , the explant had increased tremendously in width . well - defined leaf primordia and shoot tips were visible . the internode region of each shoot had started to elongate with its own epidermal and pith layer ( differentiation into plant tissues was occurring ). vascular tissues were beginning to be formed in the internode region . the effect of the aminoglycosides kanamycin and geneticin on cotton shoot initiation were investigated . kanamycin , at concentrations of 0 . 0 , 50 , 100 , 150 and 200 mg / i , and geneticin at 0 . 0 , 5 . 0 , 10 , 15 and 20 mg / l , were incorporated in medium ga . the experiment consisted of 3 replicate plates / treatment and 4 explants / replicate plates using the cultivar deltapine 50 . results after 1 month indicated that a kanamycin concentration of 50 mg / l was sufficient to inhibit shoot initiation . a geneticin concentration up to 20 mg / l was not inhibitory to shoot initiation . factors affecting gus transient expression were optimized using cultivars deltapine 50 and stoneville 474 . the pds - 1000 / he device was utilized using a vacuum pressure of 25 - 26 mm hg . there were 20 explants / plate and each plate was bombarded once or twice with 750 μg - 10 microprojectiles . gus transient expression ( nine randomly selected explants per cultivar ) was assayed histochemically , 3 days post - bombardment . optimized factors were : pressure of 1350 psi , ⅜ in gap distance , 7 . 5 cm target distance , 2 - 20 μg dna ( pbi121 ) and a preculture time of 1 day . 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