I V } ‘IHH WWNWIIHIHIIH I 106 232 IS” THE USE OF TETRAZOLIUM CHLOREDE A5 A MEANS OF DETERMINING SEED VIABILITY 3“), a w.- : m 3'.‘ ‘ ‘-‘ -.. 1m~¢ ‘4 ‘ 4 _.;';~JM3 Ah .zm Jsgwa 913 kt. 1:; 'o.~:°- 1W.“ . l 3 (in: ; 23"! 1 tn 3.! "V“. .. - .9» .‘f‘gik'i‘; cifiA‘L‘ : 4-"- ‘- 2 'V'V ““5131: "x a. ,. . If? . $45.". ‘3‘?" ~ ' a _. urg-"s & 4.! ;‘-.\}3¥'sé.’-41 a L b :2‘ r"-’c . «It u H! Ar... rah -a.‘ , ‘- ‘1— -JdLA—_ This is to certify that the thesis entitled 9W MFWW has been accepted towards fulfillment of the requirements for 777. j degree inJ___m7 Agylfilw Major professor Date Mg / 755—] i f l ._--4_J__ THE USE OF TETRAZOLIBH CHLORIDE AS A MEANS OF DE BRMIEING SEED VIABILITY By Joseph F. Delaney AN JRSTRACL Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology Year 1951 Approved tfi-HEBIS Joseph F. Delaney A recent che emical test for determining viabili'y in seeds involves the use of 2,5,5- triphenyltetrazolium,chloride. Cer- tain enzymatic systems of living tissue are capable of reduc— ing the colorless solution of s, 5,5- triphenyltetrazoliump \ chloride to a insoluble red precipitate called triphenyl forma zan. The living tissue as a result appears bright red in color. The test for viability in seeds is based on the fact that living seeds reduce the tetrazolium and become red while the dead (D eds r main unchar ed Investigators to date have varied U) (D Ho n their results. Some find the tetrazoliw test% re ed closely with germination tests especially with cereals and others have found wide variation in the comparisons of the two tests. It is the purpose of this paper to contribute further information on the apgalicability of the tetra zolium test and to point out certain difficulties involved in both the mechanics of the method and interpretation of the staining patterns result- ing in tests of certain seeds. The tetrazolium test employed in this study was a modifica- tion of the Lakon Test. (1) The seeds were soaked in water overnight and sectioned through the embryo in a manner such that the radicle, hypocotyl, and plumule regions were bisected. The half seeds were placed in a 0.5% solution of B,h,5~triphenyl- tetrazolium chloride for four hours at 30°C. At the end of the time they were removed fron the solution and all embryos exam- ined and classified according to staining. All tetrazolium tests were compared to standard germination tests. 288354 (D In six of fourteen species of seed tested the perce11t:.ge of embryos staining entirely in the ra dicle, hypocotyl, and plunule region agreed well with the percentage of germinating seeds. In three species no staining oceuured in samples show- ing low germination. The staining patterns varied in intensity and amount of stair ed a1In other species no agreement be- tween st: inh " patterns and germination could be found. Morphological eatures of certain of the seeds, such as hard seed coats and unsymmetrical embryos, Lu;de sectioning com- plicated and in some instances, impossible. Color of the embryo and dormancy of the seed also caused difficulty in comparison of the two tests. In te stirg a series of elm seed samples laving different viabilities, agreement between the tetrazolium test and germi- nation was closed in three samsles but differed widely in a fourth. The tetrazoliun test was found to be affected only by strongly acid or basic buffered solutions. A wide range of pH val‘es from 6.8 to 9.0 resulted in sinila H staining patterns. 1. Lahon G. Topographical te razoliun me hod for determin- 3 ge1m1n.ting capacity of seeds. Pl. Physiol. THE USE OF TETRAZOLIUH CHLORIDE AS A HEATS OF DETZEHINING SEED VIABILITY By Joseph F. Delaney A THQSIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in paftial fulfillment of the requirements for the degree of K no 771". O": (3 fiwC-w 1 Lu .._:l‘. l‘ w an -'_J Department of Botany and Plant Pathology ACIQIOI-TLEIE ZI‘EEITTS The author wishes to express his sincere thanks and appreciation to Dr. G. P. Steinbauer, under whose supervision this investigation was carried out, for his guidance and criticisms throughout the entire study. Acknowledgement is also made to Dr. E. E. Toole, Bureau of Plant Industry, Soils, and Agricultural Engineering, United States Depart— ment of Agriculture, for readire the manuscript and other helpful suggestions. III. IV. VI. VII. VIII. 1.7 J). o 1 ‘ "‘ T' f‘ .L"LAIT-_.- L .Ln TABLE OF COITTFZUT ITTRCWIC'I'IO" . . . . . . . . . L TIES-EU? RZLLVIEU . . . . . . A. Quick Tests in General . . S .1sz D II:1TI:ODS o o o o Pfir‘T P10 J-JL-‘ J \J 0? Ti .\;T}:1 O O O O O O O \"‘"‘| SPECIAL T EST OI? TILES n.- lg- I 1.1.- VIT-\.LIT:—r O O O O C C O O O O EFVECT OF pH 11 TJTT‘A. 011m; :33? DISCUSDIOII . Q g g Q o o o o . syn-yr li—L-‘n-L o o o o o o o o o o 0 TDI137 "H“:TF‘ J.) a1...) \JJLL--_".L LJ. 0 o o o o o o o o O O O O O O O O O O O C O 0 mac 0 O C O O O O 0 C 0 M. .Q ".1 C0 INTRODUCTION Seed investigators have long been interested in methods of measuring the germinating capacity of seeds without the necessity of a routine germination test. Though a germination test is probably the most accurate and most reliable test, it cannot be adapted to all circumstances. In many instances the time required to complete a germination test may make the re- sults worthless. This is particularly true in determining the viability of dormant seeds. Many dormant seeds require several months of after-ripening before they will germinate under the proper conditions. {any times dealers in this type of seed cannot afford to wait through months of treatments when the seed lots are available and ready for planti ". Events also arise frequently with non-dormant seed when the results of tests for viability are needed before germination ests ca be completed. The investigations of Bennett and Loomis u) and Goodsell (Bl) in determining freezing injury of seed corn are examples of such situations. As a result of such cases, many so called "rapid" methods of testing for seed viability have been devised. ZY) LITEnATURB REVISE Quick Tests in General Probably the simplest of quick tests used by seedmen is to cut Open the seed and examine it under a handlens. (19) Seeds are considered viable if the embryos are firm, plump and of good color. Abnormalities such as decayed, shriveled, rancid smelling embryos, empty or abortive seed etc. are considered non-viable. The results, however, are very gen- era and many times very erroneous. (24) Darsie, Elliot, and Pierce (7) based a viability test on the quantity of heat given off by germinating seed. They cal- culated the normal daily rise in temperature for a sample of highly viable seed. Any sample tested that had a rise in temperature lower than this normal was assumed to have a cor- responding reduction in the vigor of that sample. Waller (47) in 1901 attempted to determine viability in seeds by use of an electric current. Using an E.H.F. of 0.01 volt, he induced a current into the embryo and noted on a sensitive galvanometer the after-currents produced by the embryonic tissue. The characteristic after-currents of viable and non-viable seed were used as the basis for the test. Fisk and Hibbard (14) in their method measured the elec- trical resistance of a volume of water in which the seeds had q I been soaking for a given period of time. The dead seed n o permitted a greater outward diffusion of salts (electrolytes) than the live seed. Accordingly, a high resistance indicated high vitality and low resistance, low vitality. 1 re use of nzymatic activity for determining seed vigor (D T has been reported by many investigators. The principle of the methods was based on the assumption that the activity of the enzymatic systems was directly related to viability of the tissue. Davis (8) utilized catalase activity as the basis for his method. He determined the ratio of the cata- lase activity of soaked seeds to that of dry seeds. If this ratio was greater than 1:1, they were considered to be highly viable. If it fell below 1:1, they were considered to be low in vigor. He stated that no definite conclusion could be drawn from the activity of dry seeds alone because the D: ‘isappearance of catalase was found to lag behind the loss of viability. The soaking of the seed would rapidly dis- organize the catalase activity of dead seed and permit true estimates of activity. Davis also used the phenolase activity a similar tes . (9) Mar (:6) reported a H as the basis fo method involving the amylase activity. A method using excised embryos has shown promise as a rapid and accurate method of testing for viability. Flemion (15, 17) has reported good correlation between this method and g rmination with a wide variety of dor ant and non- Iere placed on q. dormant seed. In her method excised embryos aoist filter paper in Petri dishes. In noting their sub- sequent behavior, she could determine the viable from the 4 non-viable seeds. The viable seeds showed signs of develop- ment such as greening of the cot Mdo s, elongation of the radicle and h ypocotyl, etc. The non-viable embryos rapidly lcteriorated and became soft. If the embryos remained firm and did not show signs of development, they were classified as viable but low in vigor. As early as 1378, investigators turned to chemical tests as means of detecting viability. Dimitriewicz (10) used sul- furic acid on sectioned seed. He noted that the viable seeds turned a deep rose color in two to five minutes and non-viable seed required fifteen minutes or longer for the same reaction. Gurewitsch (22) er W-ploy d dinitrobenzene as the reagent in his method of staining viable seeds. Lesage (34) based his test on a distinct discoloeation found in non-viable seed when treated with potassium carbonate. Some of the more recent chemicals used to stain viable seed were sodium tellurate and sodium selenite. IIas gmer (a 5) reported that excised embryos stained dark indigo or black with sodium tellurate if they were viable. Browning or spot~ ting indicated weak seed and lack of color was characteristic of dead seed. Eidmann (12) using sodium selen Mt , described a viable seed as staining a dark red with a solution of the salt. Use of Tetrazolium chloride The most recent chemical test showing much promise for determining tissue viability is " he use of 2,3,S-triphenyl- tetrazolium chloride. The preparation of the chemical dates back to 189% when it was rst prep: iTEd by Peckman and Yunge.(40) The compound is one of the few chemicals that produces a color in its reduced state. When reduced, the readily sol— uble 2,3 ,5 ~triphenyltetra zolium chloride forms an insoluble red precipitate called triphenylformazan. The reaction pro- ceeds as follow :71 :8 N—N" (31‘ + 211* LEI—N: ——N I H O 0 0 N N w 2,5,5-triphenyl- triphenyl formazan tetrazolium chloride Living tissue placed in a solution of 2,5,5—triphenyl- tetrazolium chloride reduces the salt and takes on a red color. This coloration is the basis of the test to di1 fer- entiate between living and non—living tissue. The complete explanation of the cause of this reduction in living cells has not een determined. However, considerable research has been reported concerning systems in the living cells that may result in this reduction. Mattson, Jenson, and Dutcher (37) hought it might be caused by a de hyer enase reIr;uiring co- q ensyme I or II. They reported tlat glucoseo de.ydr o-ena s (I) no d p. (D plus coenzyme I, in the presence of its substrate, re the salt at pH 6.3. They found that many tissues such as the f 95h? parts 0f apples, oranges, and grapes, the gill area O) of mushrooms, carrot roots, white and sweet potato, young leaves, and the stigmas and ovaries of certain pollinated flowers, readily reduced the salt. Dufrenoy and Pratt (ll) found that the sites of reduc- ing activity within the cells of sugar cane coincided with the location of oil droplets, whether in the cytoplasm nea- th plasmodesmata or between starch grains within the amylo- plasts. The treatment of the sections with reagents for phosphatase and for phosphate showed hat the sites of reduc- tion of the tetrazolium salts were also sites of intense positive reaction for phosphate ions. Kun and Abood (88) estimated the activity of succinic deiydrogenase in tissue extracts using tetrazolium chloride. Fred and Knight (20) stained Penicilium chrysggenum with tetrazolium chloride at a pH of 7.2 to 7.4. They reported tlat the reaction was retarded at lower pH and virtually stopped at pH 6.0. Potassium cyanide in M/lOO concentration inhibited the reaction. Sodium malonate, sodium azide, 3,4- dinitrophenol, sodium fluoride and iodoacetic acid merely slowed reduction at M/lOO concentrations. These chemicals are known to affect specific enzyme systems in living tissue. Jensen, Sachs, and Baldauski (26) applied tetrazolium in a test to determine the presence of certain dehydrogenases in plant tissue homogenates. They reported that many of these dehydrogenase systems requiring DPU and TPN ( di and triphos- phopyridine nucelotide } reduced the salt. They gave the redox 7 potential of tetrazolium as minus 0.08 at pH 7.0. The systems requiring DPN which they listed as reducing the salt were glucose dehydrogenase, alcohol dehydrogena e, lactic acid de- hydrogenase, malic acid dehydrogenase, beta-hydroxylbutyric dehydrogenase, 5-pnosphoglyceraldehyie dehydrogenase, and alpha-glycerophosphate dehydrogenase. Isenberg, Odland, Popp, and Jensen (25) also used tetra- solium chloride to determine the effect of maleic hydrazide on the succinate, fumarate, malate and pyruvic dehydrogenase systems in onion plants. Although the complete explanation of the reduction has yet to be determined, the literature to date ind'oates that many systems in the normal cell have the ability to reduce tetrazolium chloride. Besides enzyme studios, tetrazoli n has been used in other capacities. Kuhn and Jerchel (27) found the salt stained viable cultures of bacteria and fermenting yeasts. Uaugh.(48) tested the stem tissue of trees and shrubs. :1) us (48) demonstrated the staining of U) traus, Cheronis, and Str neoplasms in mammalian tissues. Black and Kleiner (4) studied respiration in various tissues of mice using tetrazolium, Evans and Earle (13) found that the salt aided in studying cancerous growths in humans. The chemical has also been used in a quantitative colorimetric test for reducing sugars. ($8) 03 Application to Seed Testing Probably the most important application of the use of tetrazoli‘m chloride has been in the field of seed testing. The method of testing seeds was firs t proposed by Lahen (30) in Ge many in 012. He reported a good correlation between this test and viability in testing seeds of cats, barley, wieat, rye and corn. Since his first in estigation he has 1 improved his method and has hac- it adopted by the German Seed ‘4 Testing Stations as the official method of determining the ermination capacity of cereals and ma e .(:;) 0‘} Lakon used a 1 percent solution of 8,3,5—triyhenyltetra- urv zolium chloride buffered between pH 6 and 7. he irst soaked H, o.— the grains of wheat, rye and barley in water overnight ( Sin to eighteen heirs . The embryos were then excised from the starchy endosperm and pericarp an d placed in the tetrazolium solution, making certain t1 at all the ervrbryos were completely submerged. They were kept thus at room temperature for seven or eight hours. At the end of this time the embryos were removed and mm mined for staining. He classified as viable only those embryos that had stained red in the regions of the plumule and adjacent tissue bearing root primordia. In testing corn, Lakon modified his technique. He bisected instead of excising the embryo. He also clas s1 fie ed as viable all those corn embryos that had stained either completely or .0 .L at least in the region of the shoot, including initi;ls c L1 the secondary radicles and the sou.te llum. he stated that J 8.5 long as the corn embryo could produce good secoa3.ary roots it would produce a normal seedling. Porter (42) in his review of seed technology considered the tetrazolium test as being superior to the other rapid methods and having consider: ble practical application for a quick evaluation of certain kinds of seed, especiall" of the Gramine ae. Porter, Durrell, and 30mm (41) tested various samples of seeds of several mem ers of the grass family, seeds of three leguminous species, and seeds of buckwheat and cotton. In a number of instances good correlation was obtained between the staining test and germination test. They encountered difficulty with hard seed in cotton. They also reported that it was impossible to section regularly through the epicoty or plumule region of the Legumes. This difficulty ullifiea the test in determining the so called "baldhead" injury in this group. Cot crell (6) reported close cor relation bet en the tetra- zolium test and gew a-ina ion tests of barley, wheat, peas, and vetches. She pointed out that tr e test was lin lited by the size of the seed being tested. Seeds of species such as mustard, turnip, etc. would not allow detailed examination, especial].v in the case of embryos that were ex remely small in the parsnip or fo ded as in the mustard. Sheul (44) reported that the test was reliable for fresh seed but that with older seed (viability less than 60 percent) it was much less accurate. The resulting coloration ranged 10 from a carmir e to a light pink in the viable seeds of barley and oats. He could not find a correlation between the light staining and slowness of germination. Goodsell (Bl) employed the tetrazolium test in making early appraisal of minimal freezing lamage to seed corn. He found that the test could be used directly if the moisture content of the grains was less than 49 perc nt when the freez- .1. ing occurred regar'less of the amount 0 '“F If the P5 Pb H m (D N '4 d- O 1 moisture content was higher, the grains had to be dried prior to testing in order that the injured kernels might lose their ability to reduce the tetrazolium solution. He concluded that the delay in breakdown of the enzyme systems was responsible "1 'for the reduction in the grains n mvi Ligh moisture content. Bennett and Loomis (5) also reported difficulty in apply- ing this test to freshly frozen seed corn. They stated that the dead kernels slowly lost their ability to reduce tetrazolium but tha‘ after several days or weeks the Lakon test could be applied. Flemion and Poole (18) tested a wide variety of dormant and non—dormant seed with the tetrazolium.method, using their excised embryo method as a standard. They reported a signif- icant correlation between the two methods but found large and frequent deviations in individual tests. The results of the previous investigations in determir— ing seed viability with tetrazolium chloride have varied. Some reported good corfelation with germination tests; others 11 have found a wide diversity of staining patterns that could not be correlated with germination. It i the purpose of this U1 paper to contribute further information on the applicability of the tetrazolium test and to point out certain difficulties involved in both the mechanics of the method and the int (1) I’— pretation of the staining patterns resulting in tests 0 H) certain seeds. O {u MTER ALB AED ME HODS 7 The majority of the seeds used in this study was gathered in the vicinity of1‘ Iichiga State College, East Lansiis, Michigan. The collected seed was dried at room r temperature and stored in sealed containers in the labora- Both.germination and tetrazolium tests were carried out on all lots of seed. The germination test was used as a standard in evaluating the results of the tetr zolium test. As nearly all of the seeds used in this study had some type of dormancy, various treatments to overc01ne this dormanc cy were necessary before germination could take place. A sum- mary of these treatments along with the refere11ces to the recommended treatments is given in Table I. ”fter the treatments used to break dormancy, the seeds to be germinated were placed in moist paper toweling and placed in a germinating chamber at a temperature alternating between 25°C for sixte can hours and 3000 for eight hours. The tetrazolium test was a modification of the method used by Lakon. (32) The tetrazolium chloride we .5 dissolved istilled water and made up to a 0.5 percent solution. Po gm n Since this chemical is light sensitive, the solution was stored in the dark. The seeds to be tested with the tet razolium solution were in moist paper toweling and allowed to absorb 91 first place water overnight. They were then carefully sectioned with a V sharp razor blade through the embryo. Tne cut was made to pass through the radicle, ypocoty , plumule, and cotyledons. The first three regions were those considered b Lalzon as important in estimating viability. Cottrell (6) reported that the cotyledons did not necessarily have to be stained entirely. She classified the seeds viable as long as some staining de- velOped in the cotyledons along with complete staining of the other regions of the embry However, not all the seeds in this investigation could be sectioned in a manner such that the cut surface passed through.all regions of the embryo. The folded cotyledons in hear and the offset radicle in Ailanthus, for ex nple, make ‘L I sectioning difficult. Plate A shows she ones of the various seeds studied and the dotted line indicates the plane of sec- tioning which proved most effective for the es t. Due to the small size of the seed, a disecting microsc0pe was emplogm in some cases to insure the cutting of the essential parts. As the seeds were bisected, one half of each see~ was placed in distilled water in a vial until one hunlred seeds were cut. The water was then poured off and the seeds covered with a 0.5 percent solution of tetrazolium chloride The vials containing the seed were placed in a 5000 chamber eeds were re- U) for four hours. At the end of this time the 1 moved and the embryos ex nined for staining. TEELTMEETS USED TO 0V3? TEEEI f}g-fi kl;..J T'N‘.r‘\"' —’JL'.L4L$ 1-4 H'\ xr .L Reference for :{ind Of Seed Treatment to breari treatl‘lcnt Dormane; recommended Acer negundo L. Acer platanoide L. Ailanthusg landulosa Desf. 5etula Dpapyrife ra II arsh Cata lpa Speciosa Harder Crataegus spp. Fraxinus ,ennsy — anica Harsh Gleditsia triecanthos L. Haclur pomifera R- f.) Schneie Pinus de11siflora Sieb a Zucc Plata I1“ 5-3 occidentalis L. Robin's. pseudcaeacia L. Thuja occidentalis L. Tilia americana L. moist paper toweling 5 C., 2 months moist pr; per toweling 5 C., 5 months moist paper towelin3. 5C. 60 day's moist paper toweling 5 C., 51/2 months moist paper towelirb 5 C., 2 months 5 hrs. sulfuric acid 5 hrs. washed in r 1nin3 wate er, (fir- 5 weeks at 55 . 5 weeks at 5 C. in moist peat r1oist paper toce ling 5 C., 5 :onths 1 hr s111furic acid, 10 mir . washing in running water moist p aper towelin; 5 C., short period 1H0 ist pa per towelin3 5 C., +5 1.1.011t118 moist paper toweling 5 C., 2 months 1 hr. sulfuric acid, 10 min. washing in running water moist paper toweling 5 C., 2 months extract seed from 20 min. in sulfuric acid, 2 hr. washing running water in V pod, 1 ‘vvo 1'_ CflafiwlCn 1- '3 .3 1r Chateau. U0 :0 Do A. (19 Chadwick ( 5 For.Res.Dig. (ZS Flemion U. s. D. ,1. (l: Chadwick ( 5) Barton ( l) Tartor ( 2) 0 Plate A. Sketches showing internal seeds tested. The dotted line sectioning for the Tetrazolium A. Acer nevundo, 51; B. Acer platanoides, H ()1 indicates the plane of Test: 41; C. Ailanthus glandulosa, 1X; C. Betula alba, 6X; 5. Catalpa speciosa, 1.51; F. Crataegus spp., GI; G. Fraxinus pennsylvanica, O "27' 1X; H. Gleditsia triacanthos, 5X; I. Maclura pomifera,5n; r ’1' J. Pinus densiflora, 71; n. L. Robinia pseudoecacia, E. Tilia americana, 101; Drawings 1115 M. Thuja occidentalis, 5n; '5'...- Platinus occidentalis, on; -c u: 0. Ulmus americana, 7X. reproduced from reference 19. Plate A RESULTS OF TESTS A summary of the percentages of viability determined by germination and the tetrazolium test is given in Table II. The percent germination in all cases was be s d on the number of seedlings showing g od development and normal in every respect. Those seeds that were hard, insect injured, dis- 9.. eased, decayed, or empty were classified as non-germ _inate . With the seeds that were sectioned and soaked in tetra— zolium solution, a wide range of staining patterns resulted. Some stained a de 5p red throughout; others stained only partially and still others produced no stain at all. In order to reduce the number of types of staining patterns, arbitrary groups were set up based on the percentage of sta n appearing in the entire region of the radicle, hypo cotyl, and plum Those that were entirely stained were again subdivided into heavy, medium and light staining. Table III shows the percentage of seeds classed in the various groups. To arrive at the percents of viability with the tetra- zolium test, the staining groups were compared with the per- cents of actual germination. The comparison indicated in a general way that only those embryos staining entirely would be viable. Using this criteria for classification, six of the fourteen species of seeds compared f1vorably with germi— nation. Those species were Acer negundo, Ailanthus glawndulo a, 18 Framinusgpennsylvanica, Gleditsia triacanthos, Haclura_pomifera, and Pinus densiflora. Two other species, Acer platanoides and Catalpa speciosa, were classified slightly different to bring them in agreement with the percent germination. The slightly stained embryos of A. platanoides were not counted in the total and the three percent of the embrvos of Catalpa speciosa that were placed in three-fourth stained were counted in the total viable. Explanation of these variations in classification will be given under the detailed description of the individual species. As shown in Table II, comparisons using the described classification were not favorable in the remaining six species. Wide variations in the two tests a1 re apparent. In order to 0 point out these ind1viduel variations and characteristic fea- tures of the seed that may cause the variation, each specie TABLE I I COM ’LIJOI OF TH' MT5N PTICTXT VIw'E IL TI AS :JTTTLIF D BI' GIRLI"LTIOJ AITD TIT? LCLIUL TLSTS Viability " Germination Test Tetrazoiiun test mind of Seed Ho. of Keen No. of Jean Seed Percent Seed Percent Acer negundo 400 57 400 51 Acer platen01iCe 400 59 200 65 Aile nthu -s glendulosa 400 96 £00 97 Betula alba 400 6 400 O Catnip: speeioee 400 06 £00 9? Crataegus spp. 400 3 [CO 4: Fraxinus pennsylvanic: 400 41 £00 40 G1 dit ia trimeantnos CUO as 200 92 Maclura pomifera 300 100 200 09 Pinus densiflora 400 81 £00 75 Platanus occidentalis 400 E 400 5 Robinia pseudoaencia 400 43 400 CO 53 4 O O Thuja occidentalis :OO Tili O emericana 4 1‘3 2 HH .38. «a .38. 5 3333“. HH 9:3 .3 H38. 5 82.33 cozy. NH N H c on «advanced «Haas on 44 udaducocdooo ensue S H m a om flogmogoa 33an dv ad N H H m naadpcovdooo usauamam ON a N H a m nN H¢ auoauducou madam H mm ouomwlcm shadows 4 N N No nonpcdouauu «anoavoao cm 3 .2 a m o m mm 83.71932“ 833nm n4 m m m mm .mmu odwmmaano m an _ H mm unoaoono mmadnmo do a «pad «Hanan m on $ 301233 3583 2 m H can R NH noBofipfim .33. we H a On occdwoc noo< I vegan: 38 53» 353» 353.. 353a .5qu 53% has: 5&5 3.5 802: v.8: oz ,4} «\H in 3:33 flapoaaoo 2:5 .3 22 Bums SquoadeMH 2H ZH¢Hm QB GZHQmooo< QMHmHmm Plate h I). spond to staining patterns shown in " : ZN}- : ' ' '- ,' c_, __ is. ‘ '1' ‘4” .77- wg'I—rym'fiflt‘ 1:" .1 - .'l“};l:.~?.':rn - ‘1‘;".:', :3 ‘5 .' p Plate B. Patterns of stain ng in the embryos of Ulnus americana. Intensity of stinplin: corresnonds to the intensity of the stain. A. M x no" stain P. No stain 10X normal. .M EFFECT OF pH 03 TJT ‘ ACLIUI TEST Studies were made of the effect of pH on the staining patterns of seeds in the tetrazolium test. Previous investi- gators have buffered their solurions at various pH values. Lahon (3.3) used a solution buff red at pH 6 to 7. Goodsell (El) employed a solution as high as pH 13.21. Bennett and Loonis ( 3) reported that corn embryos stain ell between pH 6. 8 and 8.0 but tended to stain onl" faintlyr at lower pH values. To study the effect of pH on the degree of sta ining, various buffered solutions of tetra zolium chloride were made up using Coleman Buffer Tablets. The range of pH .as frori . fl é F to 11.5 with increments of 0.5. Seeds of Linum usitatissimum (Flax) and Helianthus mnius (sunflower) were utilized for this study. The seeds were sectioneC and placed in distilled water in Petri dishes. In order that all groups might receive t’ a '1 t {.2'1 1 '1‘ 7 a or) LP" '20 'L‘I‘QTT I rang ~ t 1. .."*c. 1 l'fip,“.;‘ a £1“! Sw11-e r{ -Jk~ til eJ-lb, llu Qvflaylo Ca") b .LVJ Hr;-L 4 Cd VIC—L v 9 Low *Jd on‘v 3 m eed. l1; U) to a dish until all sixteen dishes had one half nrocess was then repeated until all dishes had fifty half seeds. The water was rapidly poured off and the val iou3 buff- ered solutions of te trs zoliun chlori iCe were poured in, cover- 113 tle seefs. The dishes were then transferred to a COACCCn temperature germinator and m inte ined at thirty degrees centi- grade in the dark for four hours. At the end of this time they were “GHOVGQ 3nd the embryos exanined for staining. A $nnnry of given in Table V. The results snowed that a uni red staining occurred over a wide range of pH values from pH 8.5 to 9.0. The lower pH values -esulted in a lighter stain d O O hardly any staiuiii took place. At the higher (’1 pH range, the solution itself tended to turn a yellowish orange color. The staining in this upper pH range was verv intense however. Even the decayed tissue turned a yellow color. With these two Species, apparently only the strongly acid or strongly basic solutions interfere with the staining. ‘J TALL? V t SThII:I:K-} I133 QB- LDC) OPS FLrLJ": 1:31] “TLOTIJQ VaEEes Flax Sunflower ll.5 solution i1 slightLr yellow— solution slightly yellow orange in color decayed areas yellow ll.0 very deep red in live very deep red tissue 10.5 solution slightlyy ellow solution slightly yellow 10.0 tissue deep red, solution same as Flax clear 9.5 deepening of red aior same as Flax 9.0 deep red in living tissue, staining becoming deeper dead areas yellow than lower pH values 8.5 bright red slightly darker red than pH C.0 8.0 " " bright red 7.5 n h n n 7.0 n n n n 6 . 5 n n n n 6.0 slight degr e lighter H H 5.5 lighter than pH 8.0 " " 5.0 light pink slight degree lighter 4.5 faint pink lig Mat r th n pH 5. 4.0 very faint or none noticeably less app ox— 1 T , -C I imately 1/; that of en 7.0 The e"periments repor ed in this paper were carried out with the intention of de erm hing patterns of Staining that would yield a reliable estimate of viability. The results of these experiments have failed to show any general type of staining that could be used. With six of the fo wteen species in the assorted list of seeds and three degrees of viability of the elm seed, the staining of the entire region of the ypocotyl, plumule, and radicle either light or dark could be used in good agreument with results of germination tests. With the remaining species, variations of this type of stain- ing had to be used as an index of viability. For instance with Acer platanoides, the seeds thought to be lightly stained had to be eliminated to bring the estimate of viability in Vaccord with germination. No adjustment could be made in the elm sample stored at 35 percent relative humidity. With this sample the embryos that stained entirely far outnumbered the seeds actually ge-m1nat1n Similar y with the samples of Betula alba, Platanus (DJ H re U) H :5 o o 9 U) H :5 Q) d- H. O :5 o *5 U} (—1- {D I.) 5 l occide ntalis, and Thuja occid nnta ing patte us could bring the estimate of viability to cor- respond with germination. The two species, Crataegus sun. and Tilia aneric :1r_:, sh wed wide variation in compa Hi ons of the two tests. 1hey the (:1 test such as {1) illustrate a difficult problem in conpa: ing the tetrazolium test to the staniard gem 1:1ina ion test. Both of these species of seed are known to be extremely dormant. (15, 2 They reguire not only scarification with sulfuric acid but long periods of after—ripening before germination can take place. The reported germination of these species in this paper was probably far below-their potential g (v F' 1"; F’ f p f3 C: O 5.): g.) 0 T1 false illustrate the usefulness of the tetrazolium tes , if proper y interpreted, in investigations of dormancy in seeds. The germination test would seem b0 indicate that seeds of these species were almost worthless and yet the tetrazolium test if reliable Shows them to be potentially hi5 h in vi" ability. As shown earlier, many investisators have reported good orrelation be tween the sults of germination and tetrazolium test for viability with seeds of cereals. The fault in apply- ing this test to other types of seeds such as those used in this study may be in the standard of comparison utilized. Flemion (13) used her so called "rapid" method of testing seeds in which she extracted the embryos and noted their be- havior on moist blotters. This method has not been used widely due probably to the iculty of extracting the embryos. Many difficu ties were encountered using the standard germi— nation in this study as a standard of comparison. host of the seeds tested had some d-gree 01 dormancy. Whether the treatments completely overcame dormancy is uncertain. In the merican and Cratae c: e of Tilia EL 0) r‘. .13 :1) Tus see. it is very do abtful. ( \5‘) o-—“' ning and the ir te rfe rence U1 U) H. D: 0) d. :3‘ (D <1 :3 "S p. 9.) d- P. O :3 S. U) (’1' 91 H. of dormancy, other difficulties were encountere The proper sectioning of the seed prior to treatment with tetrezolium solution constituted a problem. Porter et_§l. (él) found that seeds of Legumes could not be sect1 one d rc*ulurl thro :h the plunule and consequently the injury kn en es "beldheeds" could not be detected. Tl e "beldheed" condition in Legumes clessif ies bfle seed abnormal according to the Official Rules of Seed Tcs ting. (£5) The seeds of ”“011ra and 11111, for example, as shown in Plate A offered a problem in sectioning all regions of the embry The size of the seed as pointed out by Cottrell (8) would probably limit the type of seLds to be tested by tet rezolium chloride. She doubted if seeds the Size of parsnip or the f old ed conoition in the smell muste rd seeds could be prOperly f“ .1 O (I) tionel and observed for ste ini e in all essential regions. nerd seeds U) uch as were present in the Robinia ssxmples in this study present a problem. They sometimes remain in the "herdseed" condition for many weeks before they become per- meable and swell. The sectioning ope r1 tion she tered the dry embryos. Some correction factor will h1ve to be introduced to compensate for these dry herd seeds. Another problem noted by Flemion (18) and also noted in this study was the factor of color in the embryo. Most em- bryos tend to be ifi ite or c1eem colored and the red staining of the tetr zoliu11. chloride produces a good contrast. But in samples such as Ace platanoides, the 5re en color of the embryo interfers with the red staini1c, especially when the staining is light. Jevertheless, the tetrazolium method even in its present state has many advantages over the other rapid methods of est- ing seed. It has been shown to indicate a fair evidence of viability. This plus the fact that it can easily be carried out, makes tle test of 5rea t valw to nurs erymcn and dea ers in all types of seec. Tne test can be performed over a wide rarse of pH values at ordinary room tempe erature. It dose not re iuire elaborate e iui ment.15ht, however, rocs inter with the test. But before this method can be used with accuracy in iled SbUdiGS must be {3 tests of all ty pes of seed, more det made on the seeds that are low in vigor. Tes enzymatic aetivity, such as the tetra; ium test, all have the inherent problem of these 10*! vigor seeds. In considering he tetrazolium.method, Jenson.et_gl. (13) felt that the lack of stain with the tetrazolium does indica.te a loss in via- tive stain does not necessarily indi- (I) H0 bility but t1at th— 0 po cate viability. It has been shown in the review of literature that many systems in the living cell reduce the salt. It ray be possible, especially in lightly stained seeds, h;t the reduction is the result of an enzymatic system yet to be in- n an embryo that, for all practical purposes,‘can [—1. CU iva ted Q) ead. p, be con51ide red rJ. 0‘» 0 SUMMARY The Lakon Test for seed via ility using 8,5,5-triphenyl— tetrazolium chloride was performed on a variety of sec— tioned seeds. The resulting staining patterns were com- {1. pare to standard germination tests. In six of the fourteen species of seed tested the per- -‘ centage of embryos staining entirely in the radicle, lync- cotyl, and plumule region agreed well with the percentage . § of gerainating seeds. In three species no staining occurred in samples showing lOW'germination. The stainird patterns varied in intensity and amount of stained area. In other speies no agreement between staiaila patterns and germ- ination could be found. The morphological features of certain of the seeds such as hard seed coats and unsymetrical embryos, made section— ing complicated and in some instances, impossible. Color of embryo and dormancy of the seed also caused difficulty in comparison of the two tests. In testing a series of elm seed samples havin5 different viabilities, agreement between the tetrazolium test and germination was close in three samples but differed widely in a fourth. The tetrazolium test was found to be affected only by I‘ll A “ELFB' m 1' 'I ‘ ".~'. Barton, L. Hastening the germination of some Coniferous seeds- Amer- Jour. Bot. 7.33—115. 1930 Dormancy in Tilia eeds. Contri. Boyce Thompson Inst. 6:39-89. 198d Bennett, N. and Loomi is, E. E. Tetrasolium chloride as a test reagent for freeziig injury of seed corn. Pl. Physiol. M .153-171. 1049 Black, I. M. and Kleiner, I. S. The L89 tet1 azolium chloride for the s ' ti issue slices. Science 110:33 f tiipheny — re 31*“ ire tion of pagat on by se Cn-dJ1cL, L. C. 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The use of perforated celeo— phane for the growth of cells in tissue culture. Jour. hation. Cancer Inst. 8lee-llQ. 19%? 14. 15. 1‘3. 17. (“"1 N) 01 C) Fisk,(}. L. and Bibbard, R. P. 1 method for determining seed viability by electrical conductivity measuremen 3. Papers Mich. Acad. Sci. Arts and Lett. 5:95-108. 1928 Flemion, F. Rapid method of determining the viability of dormant seeds. Contri. Boyce Thompson Inst. ’5"? ‘7': 0'20 9 :Q‘Ug’kud . lJuU Breaking the dormancy of seeds of Crataegus species. Contri. Boyce Thompson Inst. 9:409—435. 1958 Excised embryo method as a rapid method for determining the germinative capacity of ormant seed. d Contri. Boyce Thompson Inst.' 15:229-241. 948 and Poole, E. Seed viability test with 2,5,5- tripEEnyltetrazolium.chloride. Contri. Boyce Thompson Inst. 15:24t-b58. l9é8 Forest Service. F I U. 8 Ag r iculture , W C n "r a A Dbl-It. l d: ’C d manual 0 b. Dept. . Pu S (O 0 r9 Fred, R. B. and {n'sh', S. G. 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