Patent Application: US-959099-A

Abstract:
a method is disclosed for producing a transgenic cotton plant by agrobacterium - mediated transformation of petiole tissue . the method comprises the steps of obtaining cotton petiole explants , exposing the petiole explants to a culture of agrobacterium tumefaciens that harbors a vector comprising an exogenous gene and a selectable marker , the agrobacterium being capable of effecting the stable transfer of the exogenous gene and selection agent resistance gene to the genome of the cells of the petiole explant , culturing the petiole explants to induce callus formation , selecting transformed callus that expresses the exogenous gene , culturing the selected callus in suspension culture to induce formation of embryoids , regenerating the embryoids into whole transgenic cotton plants .

Description:
an efficient method is disclosed for genetic transformation of cotton plants , including elite lines , using cotton petiole as an explant . by using petiole explants , plus a set of improved media , transformation efficiency is significantly enhanced and the time required from transformation to regeneration is shortened in comparison to previously reported methods . by using the methods of the present invention , the whole process from agrobacterium transformation to the regeneration of transgenic plantlets can take about 6 - 7 months . the reported hypocotyl and cotyledon methods usually required 7 - 9 months or longer to complete the same process ( cousins et al ., 1991 ; chen et al ., unreported observation ). another two months were required for growing the small plantlets to a suitable size for potting in soil . techniques for introducing exogenous genes into agrobacterium such that they will be transferred stably to a plant or plant tissue exposed to the agrobacterium are well - known in the art and do not form part of the present invention . it is advantageous to use a so - called “ disarmed ” strain of agrobacterium or ti plasmid , that is , a strain or plasmid wherein the genes responsible for the formation of the tumor characteristic of the crown gall disease caused by wild - type agrobacterium are removed or deactivated . numerous examples of disarmed agrobacterium strains are found in the literature ( e . g ., pal4404 , peha101 and peh 105 ( walkerpeach & amp ; veltern , 1994 )). it is further advantageous to use a so - called binary vector system , such as that described in schilperoort et al ., 1990 , 1995 . a binary vector system allows for manipulation in e . coli of the plasmid carrying the exogenous gene to be introduced into the plant , making the process of vector construction much easier to carry out . similarly , vector construction , including the construction of chimeric genes comprising the exogenous gene that one desires to introduce into the plant , can be carried out using techniques well - known in the art and does not form part of the present invention . chimeric genes should comprise promoters that have activity in the host in which expression is desired . for example , it is advantageous to have a series of selectable markers for selection of transformed cells at various stages in the transformation process . a selectable marker ( for example a gene conferring resistance to an antibiotic such as kanamycin , cefotaxime or streptomycin ) linked to a promoter active in bacteria would permit selection of bacteria containing the marker ( i . e ., transformants ). another selectable marker linked to a plant - active promoter , such as the camv 35s promoter or a t - dna promoter such as the npt ii nos promoter , would allow selection of transformed plant cells . the exogenous gene that is desired to be introduced into the plant cell should comprise a plant - active promoter in functional relation to the coding sequence , so that the promoter drives expression of the gene in the transformed plant . again , plant - active promoters , such as the camv 35s , the npt ii nos promoter or any of a number of tissue - specific promoters , are well - known in the art and selection of an appropriate promoter is well within the ordinary skill in the art . the present method can be used to produce transgenic plants expressing any number of exogenous genes , and is not limited by the choice of such a gene . the selection of the desired exogenous gene depends on the goal of the researcher , and numerous examples of desirable genes that could be used with the present invention are known in the art ( e . g ., the family of bacillus thuringiensis toxin genes , herbicide resistance genes such as shikimate synthase genes that confer glyphosate resistance , u . s . pat . no . 5 , 188 , 642 , or a 2 , 4 - d monooxygenase gene that confers resistance to 2 , 4 - dichlorophenoxyacetic acid ( 2 , 4 - d ), bayley et al ., theoretical and applied genetics , vol . 82 , pp . 645 - 49 , male sterility genes such as the antisense genes of u . s . pat . no . 5 , 741 , 684 ( fabijanski , et al . ), or even the elaborate crop protection systems described in u . s . pat . no . 5 , 723 , 765 ( oliver et al .)). cotton regeneration is considered in the art to be heavily variety - dependant . the coker series of cotton varieties have been shown to be relatively easy to transform . however , dp 5412 , zhongmain 12 and many other varieties still have difficulties associated with regeneration . the situation is the same for g . barbadense and other diploid species . while somatic embryogenesis and regeneration of whole plants is a highly genotype - dependent process in cotton , successful transformation and regeneration of two distinct cotton varieties , i . e . coker 312 from u . s . a . and si - mian 3 from china , has been demonstrated using the methods of the present invention . it this therefor believed that the present invention has wide applicability to transformation of a variety of cotton lines . transgene integration in the genome of cotton produced by the methods of the present invention was confirmed using standard southern hybridization techniques , as can identification of the copy number of the inserted transgene in each transgenic line ( see example 6 , below ). the f1 generation of transgenic cotton can be tested for the presence of the transgene , and inheritance pattern of the transgene in the f1 generation can be analyzed to confirm stability and inheritability . as compared with other reported protocols , the cotton transformation system of the present invention has higher transformation efficiency and survival rate . this is attributable to several factors . in the present invention , petiole was used as an explant for transformation . different types of cotton explants can have significant effects on the efficiencies of plant transformation and regeneration ( firoozabady et al ., 1987 ). induction of somatic embryogenesis from petiole was reported previously . but regeneration was either unsuccessful or very poor ( finer and smith , 1984 ; gawel et al ., 1986 ). with the present invention , the efficiency of regeneration was significantly improved by using the improved media discussed below . in a preferred embodiment , calli of high quality were obtained when tender petioles rich in parenchyma cell in primary vascular bundle tissue were cultured in the mmsi medium ( described below ) with low concentrations of 2 , 4 - d and kinetin . with the present invention , the time for embryo induction in suspension culture can be shortened to 10 - 14 days , from a previously reported 3 weeks ( cousins et al ., 1991 ). it was found that a shortened period of suspension culture treatment is important for high frequency induction of embryogenesis . it is also important for reducing production of abnormal embryos , since a high percentage of vitreous embryos that are poor in regeneration are produced when cotton calli are maintained in suspension culture for too long ( chen et al , unpublished observation ). for maximum cell growth at different stages except at the young plant growing stage , glucose was used as the sole carbon source . the amount of glucose in the media can be from about 10 to about 50 g / l , preferably about 30 g / l . at the young plant growing stage , glucose and sucrose at about 10 g / l respectively as carbon sources are preferable for promotion of healthy plantlets growth . for growth of callus , embryogenesis and callus proliferation , ph range can be from 5 . 8 to 7 . 5 , preferably ph 6 . 2 - 7 . 0 , most preferably at ph 6 . 5 . a medium of ph 7 . 0 is preferable for healthy root growth of plantlets . for effective callus initiation and induction of the potency of embryogenesis , low concentrations of 2 , 4 - d and kinetin in the callus induction and selection medium is important . the amount of 2 , 4 - d can be from 0 to about 0 . 5 mg / l , preferably about 0 . 05 mg / l . the amount of kinetin can be from 0 . 0 mg / l to about 1 . 0 mg / l , preferably about 0 . 1 mg / l . in the callus differentiation stage and embryoid germination stage , best result were obtained when no plant hormone was added to the media . the amino acids asparagine and glutamine are better nitrogen sources than inorganic ammonia nitrogen for specifically supporting embryoids germination and root development . in the embryoid germination medium , the amount of asparagine can be about 200 to about 1000 mg / l , preferably about 500 mg / l . the amount of glutamine can be about 500 to about 2000 mg / l , preferably about 1000 mg / l . with these optimized nitrogen sources , the growth of non - embryogenic calli was inhibited while the germination , growth and root development of embryoids were preferentially promoted . at different stages of cotton transformation except co - culture with agrobacterium , plant tissue and callus are preferably maintained at 28 ° c . but can be varied from 25 - 35 ° c . for effective transformation , temperature in co - culture stage should not be higher than 28 ° c . a light condition of 16 hrs . light ( 60 - 90 , μem − 2 s − 1 ) and 8 hrs . dark per day is preferable for all stages of cotton transformation and regeneration . unlike previously reported transformation and regeneration protocols ( umbeck et al ., 1987 ; firoozabady et al ., 1987 , cousins et al . ), the media used in the present invention are optimized in several respects : ( a ) glucose is used as a sole carbon source in all culture media except in the medium used to culture young plants previous to planting out in the greenhouse ; ( b ) the media is adjusted to higher ph value ( 6 . 5 - 7 . 0 ); ( c ) lower concentration of 2 , 4 - d ( 0 . 05 mg / l ) and kinetin ( 0 . 1 mg / l ) is used only at callus initiation stage , no hormone is used at other stages ; ( d ) asparagine and glutamine are used to replace inorganic ammoniac nitrogen in the medium used for embryoid germination . these modifications are adapted for the physiological requirement of cotton embryoid development and plantlet growth . it has been found that healthy embryoid development and plantlet growth , especially root system development , are largely attributable to these optimized media . for example , it has been found that asparagine and glutamine were better nitrogen source than inorganic ammonia nitrogen for supporting embryoid germination and root development . in the preferred mms3 medium ( described below ), which contains asparagine and glutamine as the nitrogen source , the growth of non - embryogenic calli was inhibited while the germination , growth and root development of embryoids were preferentially promoted . because of the healthy root development , the survival rate of potted transgenic cotton plants obtained by the methods of the present invention is almost 100 %. with the reported hypocotyl and cotyledon protocols ( umbeck et al ., 1987 ; firoozabady et al ., 1987 ), poor root development has been regarded as the main reason accounting for poor survival rate of potted transgenic cotton plants . the following are preferred plant tissue culture media used in the examples : the following examples are intended to illustrate the present invention , and not in any way to limit its scope , which is solely defined by the claims . a . tumefaciens strain lba 4404 ( pbi121gfp ) was used for transformation of cotton petiole and young stem . the physical map of pbi121gfp is shown in fig1 , which contains gfp as a reporter gene and nptii gene ( encoding neomycin phosphotransferase ) as a selectable marker . the gfp and nptii genes are under the control of camv 35 s promoter and nos promoter respectively . for construction of pbi121gfp , a 720 bp xbai - ssti fragment of gfp gene from the pgfp2 plasmid ( from dr . n . h . chua , rockefeller university , new york ) was cloned into the same sites in plasmid vector pbi121 ( clontech ) to replace the gus gene . the pbi121gfp plasmid was introduced into a . tumefaciens lba 4404 by electroporation . upland cotton varieties coker 312 from the u . s . a . and si - mian 3 from shanxi cotton research institute in china were used in the experiments . tender petioles were collected from plants 8 - 12 weeks old grown in a greenhouse with low light conditions . the petioles were surface - sterilized with 70 % ethanol for a few seconds , followed by 20 % bleach solution ( clorox co . usa , 1 % available chlorine ) for 20 min . after rinsing five times in sterilized water , the petioles were pre - cultured in ms medium for 3 days . a single colony of a . tumefaciens strain lba 4404 ( pbi121gfp ) was inoculated in liquid lb medium with 50 mg / l rifampicin , 50 mg / l kanamycin and 100 mg / l streptomycin . the bacteria was grown overnight at 28 ° c . in a shaker of 200 rpm . the bacterium cultures were diluted using liquid ms medium to od600 = 0 . 3 . the petiole and young stem were cut into about 2 cm long segments . the segments were soaked in the diluted bacterium suspension for 5 min , then transferred onto plastic plates ( 100 × 25 mm ) containing a filter paper soaked in 50 ml of co - culture medium . the plates were kept in an incubator of 24 ° c . under continuous light for 48 hrs . the co - cultured explants were transferred onto mms1 medium and incubated at 28 ° c . with 16 hrs light ( 60 - 90 , μem − 2 s − 1 ) and 8 hrs dark per day . after 2 - 4 weeks calli were initiated at the cut ends of petiole segments . after 4 - 6 weeks kanamycin resistant calli had appeared , and the number of calli were counted and the expression of gfp gene was examined . under the fluorescence microscope , the untransformed control callus appeared red in colour , while the transformed callus expressing gfp gene displayed distinct green fluorescence . a total of 113 putative transformed calli were examined for gfp activity , the transformation frequency of gfp gene was 39 . 8 % ( table 1 ). when petioles from cotton variety si - mian 3 were used for transformation , 11 calli were found gfp positive from 26 calli tested , transformation efficiency was 42 . 3 %. the calli with vigorous growth and strong expression of gfp were selected and transferred into liquid mms2 medium for suspension culture for 2 weeks . friable cream - colored granular calli were selected and transferred to semi - solid differential medium , mms2 . after about 2 months a large number of embryoids were produced . cytoplasmic dense embryogenic structures were gradually developed and large embryos were produced on the medium within 1 - 2 month . a short time of suspension culture treatment was very important , not only for high frequencies of embryogenesis induction , but also for production of embryoids of good quality . expression of gfp gene was checked again and all were gfp positive . the embryoids and embryogenic calli with strong gfp activity were transferred onto the mms3 medium . after 1 - 2 months the plantlets that were about 1 - 2 cm in height with 1 - 2 true leaves and good root development were transferred to the young plant growing medium for about one month . about one month later , young plants with 6 - 8 leaves and about 10 - 15 cm in height were potted in soil and move to the glasshouse . all 30 potted transgenic plants survived and were found expressing gfp protein . the total time required to obtain transgenic plantlets using was under 7 months , and plantlets were reading for potting out in the greenhouse in about 2 additional months ( see table 2 ). green fluorescence of gfp gene can be easily distinguished in the transformed callus , embryoids , and young plantlets , with the untransformed control appeared red in colour under the fluorescence stereo microscope . the exceptions were the untransformed roots , which appeared dim green under the fluorescence microscope , probably due to some chromophorous chemicals accumulated in roots . but the roots with gfp activity could still be identified because the green fluorescence produced by gfp protein was brighter and appeared more uniform . under the blue light produced by the fluorescence stereo microscope , red fluorescence is clearly visible in untransformed green plant tissues that are enriched with chlorophyll such as leaf and stem . in gfp - 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