Patent Application: US-45787695-A

Abstract:
a basal nutrient culture medium for the in vitro culture of pinus taeda , loblolly pine , is disclosed . the medium contains nitrate , ammonium , potassium , phosphorate , calcium , magnesium , sulfate , chlorine , sodium , borate , manganese , iron , zinc , copper , iodine , molybdenum oxide , cobalt , thiamine and edta .

Description:
the present invention alleviates deficiencies and problems associated with prior art culture media by providing an aqueous basal media which is reliable and efficient to use with loblolly pine tissue cultures . the invention media consists of the unique combination and concentrations of nutrients listed in table i below . table i______________________________________nutrient components and concentrations of basalnutrient mediummedium concentrationcomponent ( millimolar or mm ) ______________________________________no . sub . 3 15 - 30nh . sub . 4 6 - 15k 10 - 25h . sub . 2 po . sub . 4 1 - 2 . 3ca 2 - 7mg 1 - 9so . sub . 4 1 - 10cl . 0001 - 25na . 1 -. 3h . sub . 2 bo . sub . 4 . 05 -. 6mn . 124 -. 45fe . 05 -. 2zn . 015 -. 2cu . 0005 -. 003i . 0001 -. 001moo . sub . 4 . 00005 -. 006co . 00005 -. 006thiamine . 0002 -. 002 * edta . 05 -. 2______________________________________ * edta = ethylenediaminetetraacetic acid . the preferred combination and concentrations of nutrients for the aqueous basal media is listed in table ii below . table ii______________________________________preferred nutrient components andconcentrations of basal nutrient medium . medium concentrationcomponent ( mm ) ______________________________________no . sub . 3 16 . 9 - 24 . 9nh . sub . 4 6 . 9 - 11 . 3k 13 . 6 - 19 . 8h . sub . 2 po . sub . 4 1 . 5 - 2 . 1ca . 15 - 5 . 08mg 1 . 46 - 7 . 75so . sub . 4 1 . 68 - 8cl . 000105 - 20 . 4na . 224 -. 232h . sub . 2 bo . sub . 4 . 1 -. 5mn . 09 -. 124fe 0 . 1 - 0 . 15zn . 03 -. 15cu . 001 -. 002i . 0002 -. 0008moo . sub . 4 . 000103 -. 00517co . 000105 -. 000549thiamine . 0003 -. 00119edta . 1 - 1 . 5______________________________________ the media may include any of the additional nutrients and / or additives commonly utilized in tissue culture media , including : carbohydrates , hormones , amino acids , vitamins , activated carbon , minerals , ph buffers , gelling agents , and the like . the basal nutrient medium contains the many elements essential to plant growth at concentrations appropriate for loblolly pine . however , the basal media may be supplemented with specific growth regulators to achieve various developmental responses , including embryogenesis , adventitious and axillary shoot development , and rooting . 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 two different formulations of the invention aqueous basal nutrient media ( hereafter referred to as wv4 and wv5 basal media ) against two commonly known and used aqueous basal media formulations -- lmg 20 ( mott et al . 1987 ) and lp ( aitken - christie et al . 1987 )-- for their influences on in vitro micropropagation and shoot culturing of loblolly pine . the respective aqueous basal media formulations are listed in table iii below : table iii______________________________________basal nutrient media evaluations for in vitro micropropagationand shoot culture of loblolly pine . improved nutrient published nutrient concentrations ( mm ) concentrations ( mm ) component wv4 wv5 lmg . sub . 20 lp______________________________________no . sub . 3 24 . 9 16 . 9 1 . 0 33 . 0nh . sub . 4 6 . 9 11 . 3 -- 5 . 0l - glutamine -- -- 20 . 0 -- k 19 . 8 19 . 8 13 . 6 19 . 8po . sub . 4 2 . 0 2 . 0 2 . 5 2 . 0ca 4 . 08 4 . 08 0 . 15 5 . 08mg 7 . 75 7 . 75 7 . 75 1 . 46so . sub . 4 7 . 87 7 . 87 8 . 0 1 . 68cl 8 . 0 20 . 4 10 . 4 . 000105na 0 . 224 0 . 224 0 . 232 0 . 224h . sub . 2 bo . sub . 4 0 . 5 0 . 5 0 . 5 0 . 1mn 0 . 09 0 . 09 0 . 124 0 . 09fe 0 . 1 0 . 1 0 . 1 0 . 1zn 0 . 03 0 . 03 0 . 15 0 . 03cu 0 . 001 0 . 001 0 . 002 0 . 001i 0 . 00048 0 . 00048 0 . 025 0 . 00048moo . sub . 4 0 . 00103 0 . 00103 0 . 00517 0 . 00103co 0 . 000105 0 . 000105 0 . 000549 0 . 000105thiamine 0 . 00119 0 . 00119 0 . 0003 0 . 00119myo - inositol 5 . 55 5 . 55 0 . 55 5 . 55edta 0 . 111 0 . 111 0 . 111 0 . 111______________________________________ in this example , the basal nutrient media were prepared with the addition of a similar quantity of sucrose ( 20 g / l ), activated carbon ( 5 g / l ), and gelling agent ( 8 g / l agar ). plant growth regulators were not included in any of the media . seedling shoot sections were cultured on each of the media , and subcultured to fresh media at eight - week intervals . new axillary shoots were excised upon reaching at least 2 . 0 cm in length , and cultured independently . approximately six months after initiating the original cultures , a sample of axillary shoots from each medium and family were evaluated for rooting under greenhouse conditions . specifically , in this example seedlings from each of eight loblolly pine families were grown under greenhouse conditions for ten weeks . epicotyls from the ten - week - old seedlings of each family were excised , and the stems were shortened to 3 . 0 cm by removing the shoot base . these shoots were washed for five minutes with liquid dishwashing soap , and then surface sterilized for 30 seconds in a 70 % ethanol solution followed by five minutes in a 20 % solution of household bleach ( 1 . 05 % sodium hypochlorite ) containing four drops per liter tween 20 surfactant . after the surface sterilization treatment , the shoots were rinsed three times with sterile water and stored in a laminar flow hood until use . under aseptic conditions , the shoot apex of each epicotyl was removed by severing the stem at the point where the most distal needles come together and the stem becomes visible . each decapitated shoot was then trimmed to approximately 2 . 5 cm of visible stem by removing the base of the shoot damaged during surface sterilization . each prepared 2 . 5 cm shoot section was then placed in culture by inserting the base approximately 1 . 0 cm into the nutrient medium contained in test tubes . for each family , one explant per tube and 45 tubes per test medium were initiated . the cultures were then randomized and incubated for eight weeks at 25 ° c ., under a 16 hr / day photoperiod of approximately 60 ue . sup .. s - 1 . m - 2 light from vita - lite fluorescent bulbs . at eight - week intervals , all axillary shoots with at least 2 . 0 cm of visible stem were excised from the original explants and other resulting shoot sections . the excised shoots were divided into 2 . 0 cm sections and the resulting shoots and shoot sections were cultured in test tubes containing fresh test medium as before . cultures were incubated as before . after 24 weeks from when the original epicotyls were cultured , the number of axillary shoots with at least 2 . 0 cm of visible stem and an intact apex was recorded for each genotype . data were also collected on the number of shoots with fascicular needles , the number of shoots with terminal bud development , and the amount of shoot growth occurring over an eight - week period . at 24 weeks from initiation , after recording the above data , a random sample of axillary shoots from each medium was selected for each family to evaluate the rooting potential of the shoots . the shoots were shortened to 2 . 0 cm by removing the shoot bases and the basal 0 . 5 cm of each shoot was immediately dipped in a 1000 mg / liter indole - butyric acid solution prepared with 50 % ethanol . the base of each shoot was then inserted 1 . 0 cm deep into a 1 : 1 ( by volume ) perlite : vermiculite mix contained in flats . the shoots were incubated in a greenhouse for 10 weeks under intermittent mist and approximately 50 % shade . the rooting medium temperature was targeted at 27 ° c . at the end of 10 weeks , the number rooted shoots was recorded . a sample of the rooted shoots originating from each in vitro nutrient medium was planted in pots and grown for several months under greenhouse conditions . the evaluation results are listed in table iv below . table iv______________________________________evaluation of basal nutrients for in vitro micropropagationand shoot culture of loblolly pine . means were calculated across eight families . improved published media media wv4 wv5 lmg . sub . 20 lp______________________________________ % explants surviving to produce axil . 77 82 36 28shootsavg . axil . shoot growth over 8 - week 37 38 17 39period ( mm )% rooted axil . shoots 15 13 6 15avg . length of primary needles ( mm ) 39 41 26 33 % axil . shoots with fascicular needles 20 23 85 31 % axil . shoots with terminal resting 2 5 64 0bud______________________________________ several important aspects of in vitro shoot culture -- including survival , shoot production , shoot : growth , and rooting potential -- were improved for loblolly pine when the different invention basal nutrient media were utilized . improved rooting , as well as improved shoot growth , were partially a result of the enhanced juvenility that occurred in the shoots grown on these novel media formulations . evidence of this desirable juvenile condition lies in the juvenile morphology exhibited by these shoots . as in most pine species , this juvenile morphology was characterized by longer primary needles , a less frequent occurrence of fascicular needles , and a near absence of resting , terminal buds . the data in table iv clearly indicate that the use of the invention aqueous basal nutrient media achieved overall superior results in explant survival , shoot production , shoot growth , and rooting in the micropropagating and culturing of shoots of loblolly pine when compared to the commonly - used , published media . healthy , rooted loblolly pine plants resulted from the use of this invention . the following example evaluated an invention aqueous basal nutrient medium ( wv5 basal media ) against two commonly - used , published basal media formulations -- sh ( schenk and hildebrandt 1972 ) and dcr ( becwar et al . 1990 )-- for their influences on the initiation of somatic embryogenesis in loblolly pine . the somatic embryogenesis method followed in this example is taught in commonly assigned u . s . pat . no . 5 , 413 , 930 ( which is hereby incorporated by reference ). the respective aqueous basal media formulations are listed in table v below : table v______________________________________media evaluation for somatic embryogenesis in loblolly pine improved publishedmedium media ( conc / mm ) media ( conc / mm ) component wv5 sh dcr______________________________________nh . sub . 4 11 . 3 2 . 6 5 . 0no . sub . 3 16 . 9 24 . 7 5 . 0k 19 . 8 24 . 8 4 . 7h . sub . 2 po . sub . 4 2 . 0 2 . 61 1 . 3ca 4 . 08 1 . 37 3 . 0mg 7 . 75 1 . 63 1 . 5so . sub . 4 7 . 87 1 . 74 1 . 08cl 20 . 40 2 . 74 1 . 2na 0 . 224 0 . 12 0 . 22h . sub . 2 bo . sub . 4 0 . 5 0 . 081 0 . 1mn 0 . 09 0 . 059 0 . 132fe 0 . 1 0 . 054 0 . 1zn 0 . 03 0 . 0035 0 . 03cu 0 . 001 0 . 0008 0 . 001i 0 . 00048 0 . 00602 0 . 005moo . sub . 4 0 . 00103 0 . 00041 0 . 001co 0 . 000105 0 . 000422 0 . 00011ni -- -- 0 . 00011glycine -- -- 0 . 0267nicotinic -- 0 . 04065 0 . 00406acidpyridoxine -- 0 . 00243 0 . 00243thiamine 0 . 00119 0 . 01484 0 . 00297edta 0 . 111 0 . 059 0 . 111______________________________________ in this example , the nutrient media for embryogenic tissue initiation and proliferation were prepared with the addition of a similar quantity of sucrose ( 30 g / l ), myo - inositol ( 500 mg / l ), 2 , 4 - dichlorophenoxyacetic acid ( 3 mg / l ), n 6 - benzyladenine ( 0 . 5 mg / l ), casein hydrolysate ( 500 mg / l ), and gelrite ( 1 . 25 g / l ). ( gelrite is a gellan gum manufactured by merck , inc .) the nutrient medium for embryo development consisted of the wv5 basal nutrients ( table v ) with the addition of sucrose ( 30 g / l ); myo - inositol ( 100 mg / l ); maltose ( 60 g / l ); polyethylene glycol 4000 ( 60 g / l ); and gelrite ( 2 . 0 g / l ). megagametophytes from immature loblolly pine seeds were cultured on each of the media , and subcultured to fresh media at 7 and 10 weeks after culture initiation . proliferating embryogenic tissue was removed from the original explants at week seven and cultured independently . after ten weeks , samples of proliferating tissue from several families initiated on the wv5 basal medium were transferred to embryo - development medium to assess the embyogenicity of these cultures . specifically , in this example immature cones containing seeds from six loblolly pine families were collected mid - july in ravenal , s . c . after collection , the cones were stored at 4 ° c . in the dark for approximately five weeks . seeds from several cones in each family were removed and pooled for each of the six families , and stored overnight on moist filter paper in the refrigerator . seeds were then surface - sterilized in 10 % household bleach ( 0 . 53 % sodium hypochlorite ) for 15 minutes , followed by three , 5 - minute rinses in sterile water . following sterilization , the seed coat , nucellus , and any other exterior tissues were aseptically removed from the megagametophytes . the megagametophyte explants were then placed horizontally on the three media contained in petri dishes ( table 4 ). ninety - six explants were cultured on each medium ( 8 explants per petri dish ). the petri dishes were then sealed with parafilm and incubated in the dark at 23 ° c . for seven weeks . at the end of four and seven weeks , data were collected on the extrusion of proliferating embryogenic tissue from the micropylar end of the megagametophytes . at seven weeks , this proliferating embryogenic tissue was then transferred to fresh medium , identical to the initial culture . these new cultures were then incubated as described above for an additional three weeks . at ten weeks from culture initiation , data on the number of embryogenic cell lines continuing to proliferate were recorded for each medium . a sample of genotypes proliferating on wv5 medium was transferred to the embryo - development medium to assess the embryogenicity of these cultures . tissue samples of these genotypes were transferred to fresh embryo - development medium every three weeks . after nine weeks on embryo - development medium , data on the number of cotyledonary stage , embryos were collected for each of the genotypes sampled . table vi______________________________________percentage of initially cultured genotypes withproliferating embryogenic tissue ( means calculated across six families ) improved media published mediatime in culture wv5 sh dcr______________________________________4 weeks 46 34 337 weeks 30 16 1310 weeks 21 10 7______________________________________ the results in table vi clearly indicate that the frequency of extrusion and proliferation of embryogenic tissue was improved for loblolly pine when the invention aqueous basal nutrient medium was utilized . although the percentage of cultures extruded on wv5 medium declined somewhat over a ten - week period , the percentage of proliferating cultures remained higher than that of dcr and sh medium at ten weeks . of the sample of genotypes transferred to embryo - development medium , 89 % produced mature , cotyledonary stage somatic embryos . among these responding genotypes , the number cotyledonary stage somatic embryos produced ranged from 1 to 485 . the use of an invention basal nutrient medium resulted in a more successful initiation and proliferation of embryogenic tissue than that provided by commonly - used , published media for somatic embryogenesis of loblolly pine . healthy , cotyledonary somatic embryos resulted from the use of this invention . 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 . abdullah , a . a ., m . m . yeoman , and j . grace . 1985 . in vitro adventitious shoot formation from embryonic and cotyledonary tissues of pinus brutia ten . plant cell , tissue and organ culture 5 : 35 - 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