Patent Application: US-45675795-A

Abstract:
this invention relates to a method for reducing contamination of in vitro cultures . in particular , this invention relates to a method for reducing contamination of in vitro cultures of woody plant mature shoot material and shoot material of outdoor origin .

Description:
sturdy branches on the donor plant are selected and treated ( either by pruning or by application of a suitable growth regulator ) at a location appropriate to stimulate the development of new shoots . if needed , a wire support ( extending about 10 cm above the pruned location ) is then attached to each branch to provide support to the isolating bag . a weather - resistant bag is then placed over each branch and corresponding wire support in order to isolate the branch . it is preferred to equip each bag with a small incision ( commonly about 7 . 0 cm in length ) to allow for the foliar application of anti - microbial agents . this opening is kept sealed using a binder clip or other suitable device except during the application of anti - microbial agents . the bottom opening of each bag is sealed around the branch using non - absorbent cotton or polyester fiber - fill and tied shut with twine or wire tie . ( while the need for incisions may be eliminated by introducing the anti - microbial agents via the bottom openings of the enclosures , it is preferred to utilize incisions for ease - of - handling purposes .) isolation in these bags is maintained during the entire time required for development of the new shoots ( usually about 2 to 10 weeks ). immediately following the treatment to stimulate new shoot development and isolation of each branch , a suitable anti - microbial agent or combination of agents ( such as broad - spectrum fungicides and / or bactericides ) is applied through the sealable opening in the bag to the treated branch enveloped by each bag ( after which the opening is again sealed ). thereafter , anti - microbial agents are periodically reapplied ( commonly about every 7 to 14 days ) to each treated branch and its accompanying developing new shoots . it is within the ability of a skilled artisan to determine the type of fungicide and / or bactericide to be applied to the enclosed branches ( as well as the schedule of applications necessary ) in order to kill contaminants on the shoots . applications of the anti - microbial agents are continued throughout the period necessary for the development of the new shoots . when the new shoots have developed to a suitable size ( a minimum of about 0 . 5 cms for ease - of - handling purposes ), the isolating bags are removed and these new shoots are excised from the donor plant . the shoots are subsequently surface - sterilized . one method of surface - sterilization is to utilize a solution of 70 % ethanol followed by submersion in a calcium hypochlorite or sodium hypochlorite solution . however , any effective method of surface - sterilization which does not result in excessive harm to the excised shoots may be utilized . after surface - sterilization , the excised shoots are cultured in vitro under aseptic conditions until harvesting of the new in vitro shoot material is desired . an alternative , preferred step would be to graft the surface - sterilized excised shoots onto surface - sterilized , seedling understocks or surface - sterilized , embryo understocks . the shoot explants ( or portions thereof ) are grafted ( using a cleft graft or other appropriate grafting methods ) under aseptic conditions onto seedling or embryo understocks of sufficient size to prevent contact between the scion and the nutrient medium . the resulting grafted explant is placed in culture , making sure that contact between the scion and the medium is avoided . the cultured explant is subsequently incubated to allow new growth to occur from the scion . when sufficient growth has occurred to allow excision of the new material , the new shoots are removed and cultured independently . this new shoot material will be virtually free of surface contaminants . bags which are suitable for use in the present method will be of sufficient size to enclose and isolate the desired area of the branch while also allowing enough room for new shoots to develop appropriately . the bag may be transparent , translucent , or opaque . the pore size of the bags should be large enough to allow sufficient gas exchange for appropriate shoot development while being small enough to hinder the entry of contaminants . materials which are suitable for use in comprising the bags include , but are not limited to , the following : paper , plastic , cloth , silk , canvas , nylon , rayon , polyester , cellophane , mylar , and combinations thereof . the donor plant &# 39 ; s environment will also effect the type of bag utilized . for example , if the donor plant is out - of - doors , the bag should be weather - resistant to prevent the necessity of replacing the enclosure part - way through the development of the new shoots . the present method is effective in controlling common external surface contaminants such as fungi , bacteria , and yeasts . anti - microbial agents which are suitable for use in the present method are those which will kill the above - noted contaminants without harming the plant . examples of such agents include , but are not limited to , the following : fungicides , bactericides , antibiotics , ethanol and other alcohols , sodium hypochlorite , calcium hypochlorite , hydrogen peroxide , mercuric chloride , and combinations thereof . agents which are suitable for use in the surface - sterilization are those which will kill the above - noted contaminants without causing excessive harm to the explant . examples of such agents include , but are not limited to , the following : fungicides , bactericides , sodium hypochlorite , calcium hypochlorite , alcohols , hydrogen peroxide , mercuric chloride , various commercial sterilants , radiation , fumigants , and combinations thereof . while radiation or fumigation may be utilized for surface - sterilization , it is preferred for ease - of - handling purposes to utilize liquid , chemical sterilization agents . a skilled artisan can easily ascertain the proper strength or solution levels at which to apply the proper anti - microbial agents and / or surface - sterilization agents to achieve the desired results . the following examples are provided to further illustrate the present invention and are not to be construed as limiting the invention in any manner . the following example evaluated three procedures , both alone and in combination , for reducing contamination in cultures of mature shoots of field - grown pinus taeda l . ( see table i below ). these procedures included pre - culture isolation of mature shoots , pre - culture foliar application of fungicide to mature shoots , and in vitro grafting of mature shoots onto greenhouse - grown - seedling understocks . six - year - old trees were utilized as a source of mature material , and newly - developed shoots stimulated by pruning served as the mature shoot explants . paper drg pollination bags ( commercially available from drg packaging , toronto , ontario ) were used to isolate mature shoots during development in the field . explants were cultured on solidified nutrient medium contained in culture tubes . cultures were evaluated for fungal and bacterial contamination during the eight weeks following culture initiation . seeds of pinus taeda l . were surface - sterilized by submersion in a 3 % h 2 o 2 solution for 20 minutes . after the h 2 o 2 treatment the seeds were stratified in the dark at approximately 6 ° c . for 30 days . after stratification the seeds were surface - sterilized in a 3 % h 2 o 2 solution as before . following this treatment the seeds were planted in flats containing soilless potting medium . the flats were placed in full sun in a greenhouse , and watered with reverse osmosis ( ro ) water when necessary during germination and subsequent seedling growth . beginning five weeks after planting , the seedlings were sprayed with fungicide once every ten days . the type of fungicide employed was alternated between cleary 336wp ( commercially available from w . a . cleary chemical corporation , somerset , n . j . ; and utilized in solution at 1 / 2 tbl / gal of ro water ) and captan 50wp ( commercially available from ici americas inc ., wilmington , del . ; and utilized in solution at 2 tbl / gal of ro water ). approximately 8 - 13 weeks after planting ( when the appropriate mature shoot material was ready ), seedling epicotyls with at least 3 . 5 cm of visible stem were excised for each set of mature shoots excised at that time for treatments requiring grafting . when harvesting , the epicotyls were placed in a beaker containing ro water to prevent desiccation . each shoot was then trimmed to 3 . 5 cm by removing the shoot base , if necessary , and the shoots were placed in 500 ml wide - mouth erlenmeyer flasks . the shoots were rinsed five times in ro water , followed by a five minute wash in water containing common liquid dishwashing soap ( about 10 drops per flask ). after removing the soap residue with ro water , the shoots were washed in a 70 % ethanol solution for about 30 seconds . the shoots were then surface - sterilized for five minutes in a 20 % commercial bleach ( 1 . 05 % sodium hypochlorite ) solution ( containing four drops of tween 20 surfactant per liter ), followed by three aseptic rinsings with sterile ro water . finally , each flask of shoots was covered with a sterile petri dish lid and stored in a laminar flow hood until use . six healthy well - branched six - year - old trees , each from a different pinus taeda l . family , were selected to provide mature shoot material . twenty - four sturdy branches of the previous year &# 39 ; s growth were selected from the upper crown of each tree . ( branches chosen were those with multiple shoots from the current year &# 39 ; s growth .) each of these branches ( along with its accompanying current year &# 39 ; s growth ) were collectively referred to as a treatment branch . three treatment branches on each tree were randomly tagged for each of the eight treatments . when the seedlings for understock material were about five weeks old , all of the treatment branches selected on the six - year - old field - grown trees were pruned . when pruned , one - half of each existing branch of the current year &# 39 ; s growth was removed . for treatments requiring isolation , a wire support was attached to each treatment branch immediately after pruning with the top of the support approximately 10 cm above the top of the pruned portion . a paper drg pollination bag was then placed over each support and treated branch . the opening at the bottom of each bag was then stuffed until sealed with polyfil ( commercially available from fairfield processing corporation , danbury , conn . ); and the bags were tied shut with a twist - tie wire ( commercially available from bel - art products , pequannock , n . j .). for treatments requiring both isolation and fungicide application , a 7 . 5 cm slot was cut in the center of the top edge of the bag to allow a small spray nozzle to enter the bag and spray the shoots . to close and seal this small hole , the top of the bag was folded over ( folding away from the clear window side of the bag ) and clamped shut with a binder clip ( commercially available from officemate international corporation , carteret , n . j .). isolation in these bags was maintained during the entire time required for development of the new shoots . for treatments requiring fungicide application , the terminal ends of the treatment branches were sprayed with fungicide after pruning and bagging were completed . fungicide applications were subsequently applied once every ten days , alternating between solutions of cleary 3336wp ( 1 / 2 tbl / gal ) and captan 50wp ( 2 tbl / gal ). treatment branches were sprayed to cover all of the current year &# 39 ; s growth until run - off occurred . applications continued throughout the entire time required for development of the new shoots . after the treatment branches had been prepared , the shoot tips of all of the remaining branches on the tree were pruned off , including the main leader , to reduce the inhibitive effects that apical dominance might have on new shoot development . when the majority of the newly developed mature shoots for each treatment on each tree were between 1 to 4 cm in length ( approximately five to eight weeks after pruning ), the shoots were removed from the trees by carefully twisting them off at the point of attachment to the pruned branch . collectively , up to 20 mature shoots were removed from the three treatment branches in each treatment on each tree . the mature shoots from each treatment and each tree were kept separate and placed in labeled 125 ml flasks containing ro water . in the laboratory these shoots were washed and surface - sterilized ( as previously described for the seedling material ). following sterilization , each flask was covered with a sterile petri dish lid and stored in a laminar flow hood until use . following surface - sterilization , mature shoots cultured directly or grafted were handled differently . for shoots in treatments not requiring in vitro grafting , each mature shoot was placed on a sterile petri dish and the basal section of stem damaged by the sterilization process was removed using a # 10 scalpel blade . for shoots in the treatments requiring in vitro grafting , each mature shoot was grafted onto a seedling understock as described below . with the aid of a dissecting microscope , mature shoots were grafted onto seedling understocks using a cleft graft . the grafting took place under aseptic conditions just prior to culturing . following the surface - sterilization of both seedlings and mature shoots , each 3 . 5 cm seedling shoot was individually removed from a flask and placed on a sterile petri dish . using a # 10 scalpel blade , a 0 . 5 cm section of stem was removed from each end of the shoot . at the apical end of the shoot section , a 0 . 5 cm longitudinal cut was made through the stem . at this time a mature shoot from a separate flask was removed and placed on the same sterile petri dish . ( from this point on , care was taken not to touch the basal end of the seedling understock with either the mature shoot or any instrument that came in contact with the mature shoot ). using a # 10 scalpel blade , the mature shoot was shortened to 1 . 0 cm of visible stem and then a longitudinal wedge was cut in the base of the shoot , making one cut on each of the two opposite sides of the stem to create a wedge of approximately 30 ° to 40 °. holding the seedling understock and the mature scion in separate pairs of forceps , the basal wedge of the mature shoot was inserted into the longitudinal cut at the apical end of the seedling shoot section , thus forming the cleft graft union . in joining the scion with the understock , the surface of the scion was lined up with the outer surface of the understock on at least one side of the graft union to ensure cambial contact . care was taken to make sure that the scion and understock fit snug together and that no gaps existed in the graft union . following preparation of grafted and ungrafted shoots , the resulting explants were carefully placed in 25 × 150 mm culture tubes , with the basal 0 . 5 cm of each explant inserted into a gel - solidified nutrient medium ( dispensed in 20 ml portions ). the culture tubes were closed using magenta 2 - way caps ( commercially available from magenta corporation , chicago , ill .) and sealed with nescofilm ( commercially available from karlan research products corporation , santa rosa , calif .) to prevent exterior contaminants from entering . the cultures were incubated in a culture room for eight weeks at 25 ° c ., under a 16 hour photoperiod of approximately 60 μe . sup .. s - 1 . m - 2 light ( vitalite fluorescent bulbs commercially available from duro - test corporation , fairfield , n . j .). at one week intervals , beginning seven days after initiation of each treatment , the presence or absence , and type of contamination was recorded for each culture by visual observation . the different treatments are noted in table i below , while the results of the treatments are listed in table ii . table i______________________________________eight treatments examining the effects of the presence orabsence of three procedures on reducing contamination incultures of mature loblolly pine shoots . treatment # isolation fungicide grafted______________________________________ 1 * 0 0 02 + 0 03 0 + 04 0 0 + 5 + + 06 + 0 + 7 0 + + 8 + + + ______________________________________ * treatment 1 is the control . 0 = procedure not applied , while + = procedure applied . table ii______________________________________results from evaluation of isolation ( i ), fungicide application ( f ), and in vitro grafting ( g ) for controlling in vitro contamination . % of treatment # of contaminatedtreatment description cultures cultures______________________________________1 control 120 972 i 106 923 f 120 814 g 120 945 i + f 74 536 i + g 103 917 f + g 120 678 i + f + g 85 52______________________________________ individual procedures ( treatments 2 - 4 ) resulted in a small reduction ( 3 - 16 %) in overall contamination ( table ii above ). these individual procedures , when utilized alone or when combined with another procedure , had a profound effect on the proportion of contaminations involving specific contaminants . both isolation and fungicide application significantly reduced fungal contaminations , while in vitro grafting was effective in reducing bacterial contaminations . combinations of procedures ( treatments 5 - 8 ) significantly reduced overall culture contaminations compared with cultures of untreated control shoots ( table ii ). the combination including both isolation and fungicide resulted in a reduction in overall contamination from 97 % seen in the untreated control shoots , down to 53 %. the combination of all three procedures resulted in greatest reduction in overall contamination , down to 52 % compared with 97 % seen in the untreated control shoots . the following example evaluated two combinations of procedures for reducing contamination in cultures of mature shoots of field - grown pinus taeda l . ( see table iii below ). these procedures included pre - culture isolation of mature shoots , pre - culture foliar application of fungicide to mature shoots , and in vitro grafting of mature shoots onto greenhouse - grown seedling understocks . nine - and ten - year - old trees were utilized as a source of mature material , and newly - developed shoots stimulated by pruning served as the mature shoot explants . non - woven polyester duraweld pollination bags ( commercially available from pbs international , scarborough , north yorkshire , united kingdom ) were used to isolate mature shoots during development in the field . explants were cultured on solidified nutrient medium contained in culture tubes . cultures were evaluated for fungal and bacterial contamination during the eight weeks following culture initiation . seeds of pinus taeda l . were surface - sterilized by submerging in a 3 % h 2 o 2 solution for 20 minutes . after the h 2 o 2 treatment , the seeds were stratified in the dark at approximately 6 ° c . for 30 days . after stratification , the seeds were surface - sterilized in 3 % h 2 o 2 as before . following this treatment the seeds were planted in flats containing soilless potting medium . the flats were placed in full sun in a greenhouse , and watered with reverse osmosis ( ro ) water when necessary during germination and subsequent seedling growth . beginning five weeks after planting , the seedlings were sprayed with fungicide once every ten days , alternating between solutions of cleary 3336wp ( 1 / 2 tbl / gal ) and captan 50wp ( 2 tbl / gal ). approximately 8 - 13 weeks after planting ( when the appropriate mature shoot material was ready ), seedling epicotyls with at least 3 . 5 cm of visible stem were excised for each set of mature shoots excised at that time for the treatment requiring grafting . when harvesting , the epicotyls were placed in a beaker containing ro water to prevent desiccation . each shoot was then trimmed to 3 . 5 cm by removing the shoot base ( where necessary ) prior to being placed in 500 ml wide - mouth erlenmeyer flasks . the shoots were rinsed five times in ro water , followed by a five minute wash in a aqueous solution of liquid dishwashing soap ( 10 drops per flask ). after removing the soap residue with ro water , the shoots were washed in 70 percent ethanol for 30 seconds . following this , the shoots were surface - sterilized for five minutes in a 20 % solution of commercial bleach ( 1 . 05 % sodium hypochlorite ) containing four drops tween 20 surfactant per liter , and then aseptically rinsed three times with sterile ro water . each flask of shoots was then covered with a sterile petri dish lid and stored in a laminar flow hood until use . several healthy , well - branched trees , representing four genotypes which were nine to ten years of age were selected to provide mature shoot material . as many as eight sturdy branches of the previous year &# 39 ; s growth were selected from the middle to upper crown of each tree to serve as treatment branches . when the seedlings for understock material were five weeks old , all of the treatment branches selected on the mature , field - grown trees were pruned . each treatment branch was pruned back to the previous year &# 39 ; s growth . for treatments requiring isolation , a wire support was attached to each treatment branch immediately after pruning , with the top of the support approximately 10 cm above the top of the pruned portion . a non - woven polyester duraweld pollination bag was then placed over each support and treated branch . the opening at the bottom of each bag was then stuffed using polyfil , and the bags were tied shut with a twist - tie wire . for treatments requiring both isolation and fungicide application , a 7 . 5 cm slot was cut in the center of the top edge of the bag to allow a small spray nozzle to enter the bag and spray the shoots . to close and seal this small hole , the top of the bag was folded over ( folding away from the clear window side of the bag ) and clamped shut with a binder clip . isolation in these bags was maintained during the entire time required for development of the new shoots . for treatments requiring fungicide application , the terminal ends of the treatment branches were sprayed with fungicide after pruning and bagging were completed . fungicide applications were then subsequently made once every two weeks , alternating between solutions of cleary 3336wp ( 1 / 2 tbl / gal ) and captan 50wp ( 2 tbl / gal ). treatment branches were sprayed to cover the pruned site and all newly developed shoots until run - off occurred . applications continued throughout the entire time required for development of the new shoots . when the majority of the newly developed mature shoots for each treatment on each tree were between 1 to 4 cm in length ( approximately five to eight weeks after pruning ), they were removed from the trees by carefully twisting them off at the point of attachment to the pruned branch . the mature shoots from each treatment and each genotype were kept separate and placed in labeled 125 ml flasks containing ro water . in the laboratory , these shoots were washed and surface - sterilized via the procedure described above for the seedling material . following sterilization , each flask was covered with a sterile petri dish lid and stored in a laminar flow hood until use . following surface - sterilization , mature shoots cultured directly or grafted were handled differently . for shoots in treatments not requiring in vitro grafting , each mature shoot was placed on a sterile petri dish and the basal section of stem damaged by the sterilization process was removed using a # 10 scalpel blade . for shoots in the treatments requiring in vitro grafting , each mature shoot was grafted onto a seedling understock as described below . with the aid of a dissecting microscope , mature shoots were grafted onto seedling understocks using a cleft graft . the grafting took place under aseptic conditions just prior to culturing . following the surface - sterilization of both seedlings and mature shoots , each 3 . 5 cm seedling shoot was individually removed from a flask and placed on a sterile petri dish . using a # 10 scalpel blade , a 0 . 5 cm section of stem was removed from each end of the shoot . at the apical end of the shoot section , a 0 . 5 cm longitudinal cut was made through the stem . at this time a mature shoot from a separate flask was removed and placed on the same sterile petri dish . ( from this point on , care was taken not to touch the basal end of the seedling understock with either the mature shoot or any instrument that came in contact with the mature shoot ). using a # 10 scalpel blade , the mature shoot was shortened to 1 . 0 cm of visible stem and then a longitudinal wedge was cut in the base of the shoot , making one cut on each of the two opposite sides of the stem to create a wedge of approximately 30 ° to 40 °. holding the seedling understock and the mature scion in separate pairs of forceps , the basal wedge of the mature shoot was inserted into the longitudinal cut at the apical end of the seedling shoot section , thus forming the cleft graft union . in joining the scion with the understock , the surface of the scion was lined up with the outer surface of the understock on at least one side of the graft union to ensure cambial contact . care was taken to make sure that the scion and understock fit snug together and that no gaps existed in the graft union . following preparation of grafted and ungrafted shoots , the resulting explants were carefully placed in 25 × 150 mm culture tubes , with the basal 0 . 5 cm of each explant inserted into a gel - solidified nutrient medium ( dispensed in 20 ml portions ). the culture tubes were closed using magenta 2 - way caps and sealed with nescofilm to prevent exterior contaminants from entering . the cultures were incubated in a culture room for eight weeks at 25 ° c ., under a 16 hr photoperiod of approximately 60 μe . sup .. s - 1 . m - 2 light ( vita - lite fluorescent bulbs ). at one week intervals , beginning seven days after initiation of each treatment , the presence or absence , and type of contamination was recorded for each culture by visual observation . the different treatments are noted in table iii below , while the results of the treatments are listed in table iv . table iii______________________________________three treatments examining the effectiveness of combiningvarious procedures on reducing contamination in culturesof mature loblolly pine shoots . treatment # isolation fungicide grafted______________________________________ 1 * 0 0 02 + + 03 + + + ______________________________________ * treatment 1 is the control . 0 = procedure not applied , while + = procedure applied . table iv______________________________________results from the evaluation of various combinations of isolation ( i ), fungicide application ( f ), and in vitro grafting ( g ) forcontrolling in vitro contamination . # of treatment # of contaminatedtreatment description cultures cultures______________________________________1 control 30 1002 i + f 28 933 i + f + g 30 33______________________________________ in this example , the combination of shoot isolation and fungicide application reduced the incidence of culture contamination from 100 % seen in the untreated control shoots down to 93 % ( see table iv ). however , the combination of all three procedures , isolation , fungicide application , and in vitro grafting , resulted in a reduction in contamination from 100 % seen with the untreated control shoots , down to 33 % ( table 4 ). the procedures of isolation , fungicide application , and in vitro grafting , when utilized alone , provided marked reduction in contamination with cultures of mature shoots of loblolly pine . generally , combinations of two of these procedures provided a more substantial reduction in overall contamination . however , the greatest and most significant reduction in culture contamination was consistently achieved by combining all three procedures . 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 . aitken - 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