A. PHYSIOLOGIC-GENETIC STUDY OF THE DIFFERENTiAL RESPONSE OF NAVY BEANS (PHASEOLUS VULGARIS L.)T0 ZINC Thesis for the Degree of Ph. D‘ MECHIGAN STATE UNIVERSITY DAViD ERNEST POLSON 1958 g I, I“ L IBP. A R y Michiga :3 Sta re 0994“] . . Unlverslty This is to certify that the thesis entitled A physiolgic-genetic study of the differential response of navy beans (Phaseolus vulgaris L.) to zinc. presented by David E . Polson has been accepted towards fulfillment of the requirements for A degree in Meme 7} ,p L7 «1/ /74'////W Major professor F—um , MRY BINDERS ' .MICIIBIII —. 1: a 7 :7 06.7 ) ABSTRACT A PHYSIOLOGIC -GENETIC STUDY OF THE DIFFERENTIAL RESPONSE OF NAVY BEANS (PHASEOLUS VULGARIS L.) TO ZINC by David Ernest Polson A differential response of navy beans (Phaseolus vulgaris L.) to Zn stress was reported in 1964. Saginaw variety was tolerant to low Zn levels, while Sanilac was extremely suscep- tible. These two varieties were employed in a genetic and physio- logic study of Zn utilization. The ultimate goal of this investigation is to determine the physiologic or anatomic mechanism(s) active in the differential reSponse, and the genetic control of this mechanism. However this particular study has only established a foundation for further investigation pertaining to the genetic control and physiologic mechanisms related to Zn utilization. Several lines of investigation have been carried out in this study: (1) a genetic study of differential sensitivity to low and high Zn in the field and greenhouse, reSpectively, as manifested by Visual symptoms and growth retardation; (2) differential response to micronutrient unbalance; (3) and physiologic studies to determine the David Ernest Polson mechanism controlling the differential response or its location in the plant. Field experiments were conducted in 1964 and 1966 with Saginaw, Sanilac, F , F , F , and backcross populations grown under 1 2 3 low Zn conditions. The degree of Zn deficiency, based on visual symp- toms, was scored from one (fully tolerant) to five (fully sensitive). Saginaw, Sanilac, F1, and F2 populations were grown at high (5.0 ppm) Zn in the greenhouse and differential tolerance was observed in the relative reduction of growth. The segregating populations were par- titioned into tolerant and sensitive phenotypes based on the perfor- mance of the parents, whose response to low Zn overlapped. Zn (50. 0, 5. 0, 0. 05, and 0.0005 ppm), Fe (75.0, 25.0, and 0.5 ppm), Cu (10.0, 2.0, and 0. 02 ppm), and Mn (50.0 and 0. 5 ppm) were utilized in the micronutrient experiments. Plants of both varieties were grown in acid washed sand and received various combinations of the above micronutrients and levels. Response to these micronutrients was determined by plant height, number of tri- foliate leaves, shoot weight, and root weight. In addition, elemental concentration in various plant tissue (leaves, petioles, stems, and roots) was determined. Reciprocal scion/ root grafts, Zn content of exudate, and radioisotope (65Zn) investigations were utilized in determining David E rnest Polson location of tolerance to high Zn mechanism, Zn uptake, and Zn dis- tribution. The data obtained from the genetic experiments indicated that tolerance to low Zn was phenotypically dominant in the F1. In addition, the results were indicative of two genes which interacted to give a 9:7 ratio in the F2. In the F3 and F1 x Sanilac population more tolerant plants than expected (based on a 9:7 ratio) were ob- served. It was concluded that the two gene model giving a 9:7 ratio was extremely close to, but perhaps not the real genetic situation. At the 5.0 ppm Zn level, it was not possible to resolve the genetic system. The F1 generation was indicative of dominance or partial dominance of the sensitive phenotype. The F2 generation implied dominance of the tolerant phenotype with a two gene model giving a 9:7 ratio. In the micronutrient interaction investigations, Saginaw was tolerant of high (5.0 ppm) Zn, while Sanilac was not. This dif- ferential response was not associated with differential uptake of Zn. Although Sanilac plants did accumulate significantly higher levels of Zn in their tissue, the concentration of Zn in the tissues of both vari- eties was high (600-800 ppm in leaves). The two varieties also differed in their susceptibility to a Cu-induced Fe deficiency. Saginaw was (again) tolerant while Sanilac was sensitive. David Ernest Polson At low Zn, the physiological studies established that Sanilac often accumulated greater quantities of Zn, but translocated smaller proportions of Zn to the tops of the plants than Saginaw. This difference in distribution may be associated with greater release of Zn into the vascular tissue as evidenced by higher concentrations of Zn in the exudate of the Saginaw variety. At the high (5.0 ppm) Zn level, reciprocal root/scion grafts of Saginaw and Sanilac established that tolerance to this level of Zn is associated with the genotype of the scion. Since differential accumulation in the leaves did not account for the response, it was postulated that there may be a difference in the distribution of extra- to intra-cellular Zn in the two varieties. Unpublished data, reported in this thesis, indicate that isolated leaf cells of Sanilac take up more Zn than Saginaw cells when the cells are obtained from plants grown at 5. 0 ppm Zn and are bathed in a high Zn solution. Three physiologic models are presented to account for the differential reSponse of the two varieties. Two models involve dif- ferential membrane or cellular properties which account for a differ- ential release of Zn into the vascular tissue and uptake into leaf cells. On the basis of these two models it was postulated that the same basic mechanism was reSponsible for the differential response of the two varieties at high and low Zn levels. The third model was based on David E rnest Polson differential stem or root binding, which was limiting at low Zn levels, and differential leaf cell permeability, which was effective at high Zn. According to this hypothesis, different mechanisms would account for the differential response at low and high Zn and these would be under different genetic control. Gene action and possible breeding programs for nutrient utilization were discussed. A PHYSIOLOGIC -GENETIC STUDY OF THE DIFFERENTIAL RESPONSE OF NAVY BEANS (PHASEOLUS VULGARIS L.) TO ZINC By David E rne st Pols on A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOC TOR OF PHILOSOPHY Department of Crop Science 1968 (3 J L}; t" . .1 0'6? ACKNOWLEDGMENTS There have been many people instruments in helping me carry out this work. Uppermost of these is my wife, Ann, and my children, Mike, Tod, and Amy, to whom I would like to sincerely express my appreciation for their support, patience, and long suffer- ing. In addition, I would like to thank my mother, who has been instrumental in giving me the desire for knowledge and in providing me with both encouragement and financial assistance when necessary. Pertaining to my academic career, Dr. M. W. Adams has provided the major guidance of my program, stimulation, and encouragement. Dr. M. J. Bukovac not only provided me with a knowledge of proper techniques but also the physical facilities. In connection with these studies, I would like to acknowledge that partial financial assistance was obtained from A. E. C. Contract #AT(11-l)- 888. Dr. F. W. Snyder assisted in preparation of some of the nutri- ent solution formulas used in the micronutrient interaction studies. Appreciation is expressed for the work of these men and others in my behalf. ii TAB LE OF CONTENTS ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION REVIEW OF LITERATURE I. II. III. IV. V. VI. VII. VIII. IX. History of Zn Nutrition Morphological Effects of Zn Deficiency Biochemical Effects of Zn Deficiency . The Occurrence of Zn Deficiency in Navy Beans (Phaseolus vulgaris) in Michigan . Conditions Affecting Zn Deficiency Critical Levels of Zn in the Soil and Plant . The Interaction of Zn with Other Micro- nutrients. Genetic Differences in Ion Accumulation or Utilization . . . A. Qualitative reSponses B. Quantitative responses . Possible Mechanism Responsible for the Observed Differential Responses . Active Uptake as a Genetically Controlled Process . METHODS OF PROCEDURE . I. II. III. IV. Low Zn Genetic Investigations High Zn Genetic Investigation . . Micronutrient Interaction Investigation Grafting Experiment iii Page ii vi 0000 10 13 15 17 20 20 25 26 32 Page V. Estimation of Zn Bound by the Stem Using Plant Exudate . . . . . . . . . . . . . . . . . 33 VI. Tracer Investigation . . . . . . . . . . . . . . 36 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 40 I. Genetic Experiments . . . . . . . . . . . . . . 40 A. Low Zn . . . . . . . . . . . . . 40 1. Field data . . . . . . 40 2. Determination of the number of genes and gene action . . . . . 47 3. Segregation within the F . 3 populat1on . . . . . . . . . . . 50 B. High Zn . . . . . . . . . . . . . . . . 53 II. Micronutrient Studies . . . . . . . . . . . . . . 56 A. Zn, Cu, and Fe . . . . . . . . . . . . . 56 B. Zn, Cu, Fe, and Mn . . . . . . . . 63 1. Differential varietal reSponse in terms of growth . . . . . . . . 63 2. Elemental concentration in plant parts of the two varieties . . . . . . . 64 III. Physiological Nature of the Differential Zn Deficiency and Toxicity Tolerance of the Saginaw and Sanilac Varieties of Navy Beans . . . 69 A. Reciprocal grafting--high Zn level . . . . 69 B. Quantity and Zn concentration of exudate . . . . . . . . . . . . . . . . 73 C. Tracer studies . . . . . . . . . 76 I. Uptake and distribution . . . . . . 77 2. Time course #1 and #2 . . . . . . 78 3. Zn concentration: Pretreat- ment and treatment . . . . . . . 84 4. Other studies . . . . . . . . . . 86 GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . 89 1. Models . . . . . . . . . . . . . . . . . . . . 96 11. Gene Action . . . . . . . . . . . . . . . . . . 104 111. Plant Breeding . . . . . . . . . . . . . . . . . 105 iv SUMMARY AND CONCLUSIONS . 1. Genetic . . A. Low Zn . . . . B. High Zn . . . . . II. Micronutrient Balance Studies--Zn, Cu, Fe, and Mn III. Physiological Studies . A. Low Zn B. High Zn IV. Discussion LIST OF REFERENCES APPENDIX Page 109 109 109 110 111 112 . 11.2 . 112 113 114 125 Table LIST OF TABLES Example of Partitioning Technique (Fictitious Data) Micronutrients and Concentrations Utilized in Micronutrient Interaction Experiment Number One . Micronutrients and Concentrations Utilized in Micronutrient Interaction Experiment Number Two Summary of 1966 Low Zn-genetic (Saginaw x Sanilac Navy Beans) Population Performances Grown in the Field (Saginaw County) Summary of 1964 Low Zn-genetic (Saginaw x Sanilac Navy Beans) Population Performances Grown in the Field (Saginaw County) Performance of Several F3 Navy Bean Lines in 1966 Selected for Zn-deficient Reaction in 1964 when Grown Under Low Zn Conditions in the Field (Saginaw County) Percent Distribution of Saginaw and Sanilac Navy Beans Grown in the Field in 1966 Under Low Zn Conditions F2 (Saginaw x Sanilac Navy Bean) Population Grown in the Field in 1966 Under Low Zn Con- ditions Adjusted to Parental Type Percent Distribution of Saginaw and Sanilac Navy Beans Grown in the Field in 1964 Under Low Zn Conditions and F2 vi Population Adjustment Page 24 28 30 41 43 46 48 48 49 Table 10. 11. 12. 13. 14. 15. 16. 17. 18. Patterns of Segregation for Reaction to Low Zn in Unselected F3 Navy Bean Families (1966) Grown in the Field Distribution of Segregating Unselected F Navy Bean Families Grown in the Field Under Low Zn (1966) 3 Summary of Saginaw x Sanilac Navy Bean Genetic Experiment in Sand Culture at High Zn in the Greenhouse . Segregation of (Saginaw x Sanilac Navy Bean) F2 Population Grown Under 5. 0 ppm Zn Level into a Saginaw and Sanilac Phenotype . Effect of Various Levels of Zn, Cu, and Fe on Growth of Saginaw and Sanilac Varieties of Navy Beans when Grown in Sand Culture in the Greenhouse Analysis of Variance of Saginaw Minus Sanilac Shoot Weights when Grown Under Different Levels of Zn, Cu, and Fe . Plant Height of Saginaw and Sanilac Navy Beans Grown in Sand Culture in the Green- house as Affected by Age and Zn Levels A Comparison of Growth Characteristics of Saginaw and Sanilac Navy Beans Grown Under Three Levels of Zn in Sand Culture in the Greenhouse Mean Accumulation (in ppm Tissue-Dry Weight) of Each Element Summed Over Other Nutrient Treatments for Each Ion Concentration, Plant Part, and Variety with Plants Grown in Sand Culture in the Greenhouse vii Page 50 51 54 55 61 62 63 64 66 Table 19. 20. 21. 22. 23. 24. 25. 26. Zn Content (ppm) of the Saginaw and Sanilac Navy Bean Leaves at Three Levels of Zn in the Nutrient Solution and at Two Levels of Fe, Mn, and Cu when Grown in Sand Culture in the Greenhouse Average Dry Weight of Grafted Plant Shoots of Saginaw and Sanilac Navy Bean Varieties Grown in a Media Containing 5.0 ppm Zn in Sand Culture in the Greenhouse Concentration of Zn and Cu in Reciprocal Root-Scion Grafted Plants of Saginaw and Sanilac Navy Beans Grown at 5.0 ppm Zn in Sand Culture in the Greenhouse The Effect of Cutting Stem at 2-3 Inches (Short) or 18-25 Inches (Long) on the Zn Content of the Exudate Obtained from Saginaw and Sanilac Navy Beans (Averaged over Six 5-hour Collection Periods) Mean Values for 65Zn Uptake and Distribution by Saginaw and Sanilac Navy Beans for 48 Hours Exposure Uptake and Distribution of 652m by Saginaw and Sanilac Navy Bean Varieties for Six Different Time Periods on a cpm Basis Ratio of 65Zn Distributed Within Plant Parts of Saginaw and Sanilac Navy Beans Averaged Over All Time Periods of Exposure 65Zn Content Expressed as cpm/g Root in Saginaw and Sanilac Navy Beans Grown Under a Normal (. 05 ppm) and High (5. 0 ppm) Level of Zn Prior to Treatment as Affected by Zn Level During Treatment (Averaged Over Plant Parts . viii Page 67 69 72 74 77 80 82 85 Table 27. 28. 29. 30. 31. The Effect of Pretreatment and Zn Concentra- tion on Total Uptake and Distribution of 652m by Saginaw and Sanilac Navy Bean Varieties The Effect of Hydrogen Ion Concentration on 65Zn Uptake by Saginaw and Sanilac Navy Beans (96 Hours) . . . . Cu and Zn Content (ppm) of Two Bean Varieties Grown in a Low Zn Soil (Saginaw County) in 1964 as Determined by Flame Spectrophotometry 6SZn Uptake in FourHours by Leaf Cells Isolated Enzymatically from Two Navy Bean Varieties Grown at Two Levels of Zn A Comparison of Several Physiological Characteristics Between the Saginaw and Sanilac Varieties of Navy Beans with ReSpect to Zn Utilization ix Page 87 88 93 95 97 Figure LIST OF FIGURE S Differential Tolerance of Saginaw and Sanilac Navy Beans Grown on a Soil (Saginaw County) Low in Zn . Navy Bean Plants Grown in Low Zn Soil (Saginaw County) Exhibiting Various Levels of Zn Deficiency Which Illustrate the "Scale" (or Given by the Numbers) Used in Estimation of Degree of Zn Deficiency Plant Arrangement During Exudate Experiment Pictures of Plexiglass Container Designed and Used for Exposing Plants to 65Zn Distribution of Saginaw x Sanilac Genetic Populations Grown in a Soil Low in‘Zn in 1966 (Saginaw County) . Distribution of Saginaw x Sanilac Genetic Population Grown in a Soil Low in Zn in 1964 (Saginaw County) . Saginaw (Left Side of Pot) and Sanilac (Right) Navy Beans Grown at 0. 05, 5. O, and 50. 0 ppm Zn in the Greenhouse Saginaw (Left Side of Pot) and Sanilac (Right) Navy Beans Grown at 10. 0 ppm Cu Plus Two Levels of Fe in the Greenhouse Scion/ Root Grafted Navy Bean Plants (Saginaw and Sanilac) Grown at 5. 0 ppm Zn in the Greenhouse Page 12 22 35 37 42 44 58 60 70 Figure 10. 11. 12. Radioautograph of Saginaw Navy Bean Plant (Sanilac Pattern the Same) Exposed to65 Zn for 288 Hours A Comparison Between Saginaw and Sanilac Navy Beans of Uptake Rate of 652n Radioautograph of Saginaw Nav Bean (Sanilac the Same) Exposed to 5Zn for 12 Hours xi Page 79 83 103 INTRODUCTION Plants selectively accumulate certain ions or groups of ions to a greater extent than others, and different species or geno- types within species differentially accumulate ions or grow under conditions of either ion deficiency or toxicity. However, the mecha- nisms reSponsible for differential ion selectivity by plants have not been identified. Varietal or species differences in ion accumulation and/ or so-called differential resistance to low or high levels of a particu- lar ion or ions provide a unique system in which to study the mecha- nism(s) involved. These systems can be studied from a physiological, anatomical, and genetic standpoint. Some of the physiologic or mor- phologic factors relating to the differential response may be identified, and then studied genetically for an understanding of the genetic con- trol of nutrient utilization in higher plants. In 1964, a Zn deficiency was reported (34) in navy beans (Phaseolus vulgaris) in Michigan. Deficiency symptoms were most severe when beans were grown on a heavy clay soil with a pH of 7. 2 to 8.0, and occurred primarily when beans followed sugarbeets in the H H crop rotation. One of the varieties, "Sanilac, was sensitive" to the Zn deficiency, while "Saginaw" was "tolerant" (33, 66). A genetic-physiologic study of these processes in beans was initiated in 1964. The primary purpose of this investigation has been to elucidate the physiological basis of the differential varietal reSponse, and determine the genetic control of this mechanism. Several lines of investigation have been followed: (1) a genetic study of the differential sensitivity to Zn deficiency as mani- fested by visual symptoms in the field; (2) a genetic study of differen- tial tolerance to high levels of Zn in the greenhouse; (3) an investiga- tion of micronutrient interaction in the two varieties; (4) and grafting, exudate, and radioisotope studies to determine the physiological mechanism(s) involved in differential tolerance to high and low levels of Zn and/or the location within the plant of this mechanism(s). The relationship of the genetic control to the physiological mechanism of nutrient utilization in navy beans will be postulated and discussed. REVIEW OF LITERATURE I. History of Zn Nutrition Thorne (98) discussed the early history of Zn in plant nutrition. He pointed out that Zn was not generally accepted as an essential nutrient until the work of Sommer (95), although evidence existed as early as 1863 that Zn was necessary for the growth of certain fungi. Research into the Spectrum of flora affected by Zn deficiency and the areas where the deficiency occurred continued in the United States until, by 1962, Zn deficiency had been reported in 30 states in a large number of crops (7, 104, 106). II. Morphological Effects of Zn Deficiency Hewitt (54) and Thorne (98), as well as others (25, 27, 49, 105), discuss the morphological effects of Zn deficiency on plants. In general they found symptoms of Zn deficiency were a general retardation of growth and loss of chlorophyll. The leaves were often smaller than normal and often developed a yellow mottling between the veins. If Zn deficiency persisted, the mottled regions coalesced to form a continuous interveinal chlorosis and eventually necrosis. These symptoms often affected the older leaves first. Many annual herbaceous crops are severely stunted partly due to lack of internode elongation. Also, they rapidly develop interveinal necrosis. Epinasty of leaves and petioles is common. Total seed yield is drastically reduced in many species due to poor growth, poor pollination and seed development, and/or delayed maturity. III. Biochemical Effects of Zn Deficiency Although Zn has been found active in several enzymatic systems, certain of the visual symptoms of Zn deficiency seem to be related to an upset auxin (indoleacetic acid) balance (54). In his review, Hewitt (54) cites the work of Skoog which indicates that between 1/10 and 1/50 of the amount of indoleacetic acid is obtained by diffusion or ether extraction from Zn-deficient stem apices or leaves of tomato than from normal tissue. In addition, auxin was inactivated by blue light more rapidly in Zn-deficient tissue than in normal tissue. Skoog and others (54) conclude that Zn is required to maintain auxin in an active condition but not for the synthesis of auxin. In contrast, Tsui (103) found that Zn-deficient tomato plants contain significantly less tryptophan than normal plants and also less indoleacetic acid. Since tryptophan is a known precursor of indoleacetic acid, he concluded that Zn was ultimately required for indoleacetic acid synthesis. Thus Zn may have a dual role, one maintaining auxin in an active condition and the other affecting its synthesis. IV. The Occurrence of Zn Deficiency in Navy Beans (Phaseolus vulgaris) in Michigan Ellis et_al. (34) indicated that Zn deficiency caused a severe reduction in yield of navy beans, when beans followed heavily P-fertilized sugarbeets in the rotation. A P-Zn interaction might have induced the Zn deficiency. Lessman (71) and Judy (65) in later work on the same problem found that P increased Zn solubility in the soil, but decreased its utilization. Lessman, utilizing nutrient solu- tions and radioactive Zn, found that washing the roots of beans prior to exposure to 65Zn increased the amount of Zn translocated to the tops of the plants. He concluded that an insoluble complex is formed - near the cell surface involving Zn and P and that washing removed this complex and allowed upward translocation of Zn. V. Conditions Affecting Zn Deficiency Reports conflict with reSpect to the effect of P on Zn availability. Two (13, 92) indicate no effect, while others (10, 11, 23, 44, 66, 110) report reduced Zn utilization in the presence of high P. Some indicate the damaging effect of P on Zn utilization to be mainly physiological and suggest a reduced translocation of Zn within the plant (8, 72, 97). This reduced translocation was thought to be due to reduced movement in the root cell (72, 97) and Zn pre- cipitation by P in the xylem tissue (8). Soil compaction and high soil moisture have also been observed to decrease Zn availability (109). Martin _et_al_. (75), in 1965, reported that raising the soil temperature from 50 to 80 F increased the availability of Zn. Contrasting results of the effect of organic matter on Zn availability have been published. Organic matter inactivated soil Zn, which may have been due to production of chelating agents such as lignin, and microorganisms which compete with the root for available Zn (21, 21). Grimes $11. (51), in 1961, reported the opposite. They found manure to be a partial replacement for Zn. The availability of Zn has been found to be affected by soil pH. In general, raising pH above 6.0 decreases Zn availability (20, 44). VI. Critical Levels of Zn in the Soil and Plant Brown and Tiffin (20) used ammoniumacetate-dithiazone to extract Zn from soils as a guide in locating geographical areas likely to be deficient. They believed 0. 5 part per million (ppm) to be a critical level below which a Zn response might be detected. Though Steenbjerg (96) pointed out the difficulty of relat- ing concentration to elemental status within the plant, several authors have estimated critical concentration of Zn within the plant (12, 58, 79, 89, 105, 112). These estimates range from about 10 to 20 VII. The Interaction of Zn with Other Micronutrients Considerable work has been done on the interaction between the micronutrients Zn, Cu, Fe, and Mn. Under some circumstances one or more of these micronutrients have an extremely detrimental effect on the utilization of others. Fe seems most subject to altera- tion of uptake and utilization. Chapman etil. (26), working with orange trees, obtained results suggesting that Zn prevents Fe from being translocated from the exterior root cells to the vascular sys- tem. Smith and Specht (93) observed that Cu was more effective than Zn and Mn in inducing Fe chlorosis in citrus. Excesses of Cu and Zn inhibited upward transport of Fe . Lingle et_al_. (72) observed that Zn reduced the translocation of Fe to soybean tops and root uptake. Fe-deficient soybeans developed a greater capacity to take up Fe than normal plants. The relative effectiveness of several cations in inducing Fe chlorosis has been reported by Hewitt (55, 56) and Hunter and Vergnano (61). The order of severity seems to be Ni > Cu > C0 > Cr > Zn > Mo > Mn. Millikan (78) has obtained evidence of interaction between Cu and Zn in subterranean clover and alfalfa. He observed that Zn deficiency decreased the concentration of Zn within the plants and increased the copper concentration. Fuerhing (42) observed a Zn-Mn and Fe—Mn interaction, which affected the Zn concentration in corn plants. Fuering and Soofi (43) observed, in corn, that the Optimum Zn concentration for grain yield was found to be dependent upon the level of Mn. Low Mn in the leaves resulted in a low Zn requirement, while higher Mn caused a higher requirement for Zn. Thus they con- cluded that balance between the various microelements seemed to be most important. This balance seemed to be less important when the elements were in the external solution than when they had been taken up by the plant. Micronutrient unbalance seemed to affect transloca- tion more than uptake. VIII. Genetic Differences in Ion Accumulation or Utilization An early review by Collander (28) deals with preferential absorption of cations by different species of plants. He stated that plants do not absorb elements in the same proportion as they occur in nutrient solution, and that the mechanism of selective salt absorption was obscure. Working with 20 different plant species grown in several different nutrient solutions, Collander found that the concentrations of Na and Mn are about 20 to 60 times greater in the plant species of highest accumulation than the Species of lowest accumulation. The differences in the other cations studied (Li, Mg, Ca, Sr, K, Rb, Cs) were not as great but still of a magnitude two to five times as concentrated between the upper and lower species. He observed that some plant Species are invariably relatively rich in some cations and poor in others. He speculated that the root cells exert selection of cations and actively secrete them into the xylem vessels, and pointed out that "the whole problem of cation selection is an extremely intricate one. " Vose (107), in his review of varietal differences in plant nutrition, cited a report in 1863 on differential uptake of I. Subsequent reports have dealt with differential yield responses to nutrient application, differential accumulation, differential require- ments to alleviate nutrient deficiency, and differential resistances to metal toxicity (37, 45, 107). 10 A. Qualitative ReSponses A well-known example of a differential nutrient response is the requirement for Fe by two soybean lines (Hawkeye and PI- 54619-5-1). This phenomenon was first noted by Weiss (111). Effi- ciency of utilization was found to depend upon a single dominant gene. He concluded that the relatively low soluble Fe observed in the aerial plant tissue was probably due to the high pH which may have been induced by the low K content. Pope and Munger (83, 84), working with celery, found a differential susceptibility to low (0. 01 ppm) B and to low (2. 5 ppm) Mg. In both instances, efficiency was dominant and segregated 3 to 1 in the F2 population. A mutant (yellow-stripe = ysl) of corn has been found and described by Beadle (3) and Bell _et_a_l_. (5, 6) which renders the plant Fe-inefficient when homozygous. Bell et_a_l_. (5, 6) showed that the inefficiency which was due to an inability to utilize ferric Fe was restricted to the roots. Brown (14) observed differential chlorosis of an inefficient line (ySl/ysl) and an efficient line (Pa 54) on calcar- eous soils. He found that a sixfold increase in P decreased the Fe concentration in the tops of the inefficient but not the efficient line. Howell (60), working with the soybean varieties "Lincoln" and "Chief, " obtained a differential reSponse to high P. Chief 11 responded favorably to P concentrations as high as 3. 62 mM whereas Lincoln was inhibited by concentrations as low as 1. 61 mM. Foote and Howell (41), on the basis of reciprocal grafting experiments, determined that this difference was related to the genotype of the root. Tolerance was found to be due primarily to reduced P accumu- lation . Recently, differential tolerance to high Al concentrations of barley (74) and cotton (39) has been observed. Al interfered with the accumulation of K, Mg, Ca, and P in barley. It was associated with Shallow rooting in cotton. Foy and Brown (40) noted that differ- ential tolerance of the buckwheat and barley species was due to the ability to absorb and utilize P in the presence of excess Al. Two varieties of beans differentially respond to low levels of Zn encountered under field conditions in Michigan (33, 66). The Saginaw variety produced 18. 7 bushels per acre without added Zn on a calcareous soil, with a pH of 7. 8, while Sanilac produced 7. 6 bushels. The partially reduced yield of Saginaw was due mainly to delayed maturity. Sanilac, on the other hand, was extremely stunted and chlorotic (Figure 1). Saginaw was derived from crosses and backcrosses made between several navy bean types including Michelite. It has an 12 FIGURE 1. -- Differential tolerance of Saginaw and Sanilac navy beans grown on a soil (Saginaw County) low in Zn. ..- ' . -. " . o , . -. . ' . Upper picture--Differentia1 stunting, chlorosis, and necrosis of Saginaw and Sanilac navy beans. Lower picture-—Differential maturity of Saginaw and Sanilac navy beans [Sanilac (on right) normally 9-10 days earlier than Saginaw (on left) is approximately 10-14 days later] . 13 indeterminate growth habit and is generally 8-10 days later than Sani- lac in maturity (1). Sanilac has a complex genetic background based primarily on an X—Ray induced early bush mutant of Michelite (2). B. Quantitative ReSponse Although quantitative differential mineral accumulation is more difficult to study physiologically, the evidence shows real dif- ferences between and within Species. Smith (94) observed a difference in utilization of P in maize and attributed the differential response to root type. Effi- ciency was genetically dominant. A high ratio of secondary to pri- mary roots accounted for efficiency. Harvey (53) noted that differential reSponse of corn to N was controlled by a partially dominant gene system and that differen- tial growth of tomatoes induced by K was due to an incompletely domi- nant genetic system. Massey and Loeffel (76, 77) found differences ranging from 38. 4 ppm to 15. 5 ppm in Zn content of corn grain, which were attributed to higher transfer of Zn from. the ear leaf to the grain in the higher accumulating lines. Butler _e_t_a_l_. (24) found that accumu- lation of Zn in ryegrass (Lolium) was highly heritable (.401 i . 176 in the broad sense). 14 Gorsline and his associates (46, 47, 48), studying accu- mulation of a number of elements by maize, found additive gene action for ear leaf accumulation and in most cases for grain accumulation. They observed a strong relationship between Sr and Ca uptake sug- gesting that uptake of both elements is controlled by the same physio- logical mechanism. Kleese (70) utilized reciprocal grafting experiments to establish whether differential accumulation of 898r and 45Ca by soy- beans was a root or shoot phenomenon. The accumulation of both radioisotopes was controlled to a greater degree by the genotype of the stem. Rasmussen and his associates (86, 87) studied differen— tial accumulation of 89$r and 45Ca and the heritability of these phe- nomena in barley and wheat. Large genotypic differences in both species were noted. Although accumulation was higher in barley, variation was greater in wheat. Accumulation in most cases was due to additive effects and heritability in the broad sense ranged from 40 to 70 percent in wheat and 26 to 63 percent in barley. They also ob- served a close relationship between Sr and Ca accumulation. Pinkas and Smith (82) found a higher concentration of Sr in the fluid from the decapitated roots from a high accumulating variety than from a low. No differences in the morphology and internal anatomy of the two 15 varieties were observed and they concluded that Sr transport is de- pendent on metabolic activity. IX. Possible Mechanism ReSponsible for the Observed Differential Responses Vose (107) categorized factors which may be responsible for differential responses as follows: nutrient absorption (root mor- phology, exchange absorption), active uptake, translocation, and metabolism. Where the physiological mechanisms have been investi- gated, an attempt has been made to eliminate various possible mecha- nisms as causative factors in any particular instance. Smith (94) found that a high proportion of secondary to primary roots was re- sponsible for the tolerance to low P of some maize lines. Reciprocal grafting experiments have been often used to separate root and shoot as regions of control (4, 18, 41, 70). Kleese (70) found the genotype of the stem to be the major factor in deter- mining differential Sr accumulation in the tops of soybean plants. The other authors cited found the root to be the determining factor in their systems. Brown 3’21; (18), utilizing stem-root grafting, related the differential ability of root genotypes of soybeans for Fe absorption to a greater sorptive capacity and a greater tolerance to interference by competing ions. In a recent paper (22) they discuss the present 16 status of the differential iron response. The efficient genotype has a greater ability to reduce Fe+++ to Fe++ at the root. This reduction is related to the Fe stress within the plant. Thus the efficient vari- eties are better able to grow under Fe stress because the available Fe is utilized to a higher degree. Following reduction of Fe, a che- lation with citrate occurs and the Fe is transported as Fe—citrate. Thus, plants with a greater reducing capacity at the root surface also have a greater capacity to transport Fe-citrate. In contrast, Wallace El. (108) found that Fe absorption by Hawkeye (efficient), when grown in the same soil solution with PI (inefficient), was decreased. This indicated that PI exuded an inhibi- tory factor into the soil that decreased the absorption of Fe by Hawk- eye. Another possible explanation is that Fe-deficient plants might deveIOp an increased capacity to absorb and accumulate Fe in the roots (15) which would decrease the amount of Fe available to the efficient plant. Tracer experiments utilizing radioautography have been useful to determine circulation patterns and distribution of some nutrients within the plant (4, 8, 9, 63, 88). Biddulph (9) reported that Zn was precipitated along the veins of plants grown in solutions high in P. Under low P, Zn was uniformly distributed through leaves and not markedly concentrated in the veins. 17 Jacoby (63) and Cooil fig. (29), working with Na, have reported on some of the mechanisms and peculiarities of translocation. Jacoby found that radioactive Na is retained in the basal parts of the plant at low concentration. AS concentration increases, a gradual Na saturation of the stem tissue occurs and distribution becomes uniform. Cooil et a1. state that Na is transported out of the root but that this is not observed because Na is recycled down the stem and back into the root. Jyung Stil- (67, 68) have developed the technique of enzy- matic separation of cells to study cellular uptake per—s3, bypassing root absorption and translocation. Utilizing this technique they have established that Rb is actively accumulated. Fe has also been found to be actively accumulated by isolated cells (69). In addition to an isolated cell system, a cell—free system has been utilized to study the nature of Zn and Zn-containing substances within the cytoplasm (64). Zn existed in a free form and bound to a protein(s). X. Active Uptake as a Genetically Controlled Process Metabolic energy input must be involved in any scheme of genetic control of nutrient uptake and/or utilization. If the entire process were passive there would be little basis for assuming genetic control of ion utilization; with the exception of differential response 18 due to different root or vascular morphology. Brown (16) recognized this when he proposed that translocation of Fe was dependent upon oxidative phosphorylation, primarily for the production of organic acids. Although the reduction of Fe seems to be very much in- volved in the differential Fe reSponse, such a scheme cannot account for differential Zn response since Zn exists primarily in only one ionic form. The carrier hypothesis, reviewed by Hansen (52), that ions are transported across membranes via carriers, seems a plau- sible alternative. Epstein (1961) and others (36, 73) have established that K and Rb uptake by excised barley roots is mediated by the same mechanism. They have identified two mechanisms or systems of uptake, one dominant at a concentration of about . 018 mM and the other at a concentration of about 16 mM for half-maximum velocity. Na competes with the first but not the second system. Ca seems to be necessary for the selective abilities of the systems (35, 62). Luttge and Laties (73) proposed that system one (low con- centration, high affinity) Operated at the plasma membrane and sys- tem two at the tonoplast. They present their conclusion as evidence confirming the symplast theory as a means of transport of salts from the outer solution to the xylem. They differentiated between long dis- tance transport (movement into the xylem through the intercellular 19 cytoplasmic strands) via symplasmic uptake and vacuolar uptake. System one had the same physical characteristics as xylem transport which was expected if the symplasmic theory is correct. System two had different characteristics and was reSponsible for vacuolar accu- mulation. Both systems required energy (36). The work of Pardee et_al. (80, 81) is another confirma- tion of the carrier hypothesis. They have isolated and purified a pro- tein from Salmonella which might act as a carrier for S. This is the first report of the isolation of a carrier molecule. METHODS OF PROCEDURE 1. Low Zn Genetic Investigations In 1964, plants of the Saginaw and Sanilac varieties, as well as F , F 1 2, and backcross progenies from crosses between these two varieties, were grown on plots with low Zn availability located on a c00perator' s farm in Saginaw County, Michigan. This farm had been used for several years in Zn deficiency experimentation by the Soil Science and Crop Science Departments of Michigan State Univer- sity. The soil in this area is a Wisner clay loam; a calcareous, lake- bottom soil with a pH of about 7. 9. The soil has no lime requirement and the concentrations of P, K, Ca, and Mg are about 40, 330, 9200, and 600 pounds per acre, respectively. In addition, the soil was fer- tilized with 200 pounds per acre of 5-20-20 (non—Zn), plus 2% Mn. The experiment was not replicated except for parents, which were replicated twice. The various generations followed suc- cessively in a series of short 28 inch rows. The seeds were Space planted Six inches apart. The severity of Zn deficiency as indicated visually by chlorosis, necrosis, epinasty, stunting, and vigor was estimated on each plant at about six weeks of age. A scale from one 20 21 to five was used, one being normal and five extremely zinc deficient . (Figure 2). At ten weeks of age, several F plants, which received 2 scores from three to five, were transplanted in the plant science greenhouse on the Michigan State University campus at East Lansing. These plants were fertilized with Zn and eventually overcame the deficiency and set seed. In 1966, the parents, F 'S, F 's, and F 's were planted 1 2 3 on the same farm as in 1964. In addition, F3 seed obtained from the F2 plants transplanted in 1964 and selfed seed from both backcrosses in 1964 was planted. In 1964, the selfed-backcross seed was obtained only from plants possessing a fair degree of tolerance to low Zn and therefore was biased. The areaused in this experiment had previ- ously received Zn at an unknown rate during normal farming opera- tions. For this experiment, 50 pounds of P per acre was added to induce a stronger Zn deficiency. Each generation of genetic material was replicated six times. Each replicate consisted of 100 six-foot by 28 inch rows, which had been sown with 10 seeds, if available, plus two red kidney beans at each end to remove border effect. A two-foot alley was between each range. Two and one-half ranges made up one replicate. Of the 100 rows per replicate, nine were planted to each parent, three to the F1 generation, nine to the F2 generation, 10 to selected 22 FIGURE 2. -- Navy bean plants grown in low Zn soil (Saginaw County) exhibiting various levels of Zn deficiency which illustrate the "scale" (as given by the numbers) used in estimation of degree of Zn deficiency. 23 F plants (from 1964 transplanted F 3 plants), 21 to unselected F 2 3 plants, and 20 to each selfed-backcross generation. A single unse- lected F3 family was planted in only two replicates; thus there was a total of 63 families. Approximately six weeks after planting, readings of visual Zn deficiency symptoms were taken on an individual plant basis using the 1964 scale. The analyses of the 1964 and 1966 data were, in general, the same. Plants of both the Saginaw and Sanilac parents were found in several of the classes, making interpretation of the segregating populations difficult. To determine the number of genes and mode of action, an assumption of two phenotypes in the genetic population was made, one tolerant and one sensitive to Zn deficiency. Gradation observed within each phenotype was assumed to be due to low pene- trance and expressivity (85, 90) of the genes regulating the reSponse. The analysis of the genetic populations was based on partitioning these populations into the tolerant and sensitive phenotypes, using the non-segregating p0pulations as an estimate of the degree of pene- trance. This technique, similar to Powers' and Locke' s (85) parti- tioning technique, is illustrated in Table 1. The F2 pOpulation (partitioned into a tolerant and sensitive phenotype) then was used to estimate the number of genes and gene action. 24 In addition, the means and variances of each population were calculated. TABLE 1. -- Example of partitioning technique (fictitious data). Number of individuals in each class of Zn reaction Generation 1 2 3 4 5 Saginaw 120 80 70 30 Sanilac 10 10 100 130 50 F2 120 70 80 100 30 Percentage of parental types in each class 1 2 3 4 5 Saginaw 40.0 26.6 23.3 10.0 0 Sanilac 3.3 3.3 33.3 43.3 16.7 Saginaw+ Sanilac 43.3 29.9 56.6 53.3 16.7 Proportion Saginaw type 92.4 89.0 41.2 18.8 0 P roportion Sanilac type 7.6 11.0 58.8 81.2 100 Partitioning F2 into parental types 1 2 3 4 5 Total Total 120 70 80 100 30 400 Saginaw type 111 62 33 19 O 225 Sanilac type 9 8 47 81 30 175 25 The 1966 F3 families were split into segregating and non- segregating families on the basis of variances and means. If the vari- ance of a Saginaw-type family didn' t exceed the variance of the Saginaw parent by over 25 percent and the means were similar, it was called a non-segregating Saginaw type. The same parameters were used to identify the non-segregating Sanilac types. The segregating families were split into a 9:7 population and a 3:1 pOpulation on the basis of their segregation pattern within the five classes of Zn reaction. The two segregating populations were then partitioned into sensitive and tolerant phenotypes, using the method illustrated in Table 1. In 1964, a bias in the Saginaw readings tended to reduce the overlap between Saginaw and Sanilac. In order to correct this bias, the F population was used as an estimate of Saginaw to parti- 1 tion the F2 population. II. High Zn Genetic Investigation To gain an indication of the inheritance of tolerance to high ~Zn levels (5. 0 ppm), Saginaw and Sanilac parents, Fl' 5 and F2' 5 were planted in the greenhouse utilizing the same procedures as in the nutrient experiments (described in the next section) with respect to sand preparation and water and nutrient application. Saginaw and Sanilac varieties were planted in the same pots; the other genetic 26 material was planted two per pot, with the provision that Fl' 3 were together and also the FZ' S. Six weeks after emergence the plants were harvested, dried, and weighed. No extensive analysis was conducted on this material due to rather limited population sizes. III. Micronutrient Interaction Investigation To determine the effect on Saginaw and Sanilac of various combinations of several micronutrients at normal to extremely high concentrations, two different experiments were conducted. The first (June-August, 1965) consisted of planting Saginaw and Sanilac seed on opposite sides of one gallon glazed pots containing silica sand which had been washed three times with 0. 5 N HCl. In between each acid washing, the sand was rinsed with deionized water twice, and after the final washing the sand was rinsed four times. After planting, the sand was moistened with deionized water every day until the seedlings emerged. The plants were thinned to one of each variety per pot. At this time watering with nutrient solution was initiated. The macronutrient solution used was a slightly modified Hoagland' s (59) listed below: 27 Chemical Quantity Ca(NO3)2- 4H20 1.401 grams/liter KHZPO4 0. 1585 grams/liter KNO3 0. 5284 grams/liter MgSO4- 7H20 0. 4323 grams/liter The treatment variables were three concentrations of each of the micronutrients, Zn, Fe, and Cu, in a factorial design. The lowest concentration of each micronutrient plus the levels given for the other micronutrients, are those normally used in a Hoag- land' 5 solution (59) (Table 2). Reagent grade chemicals were used to make up all nutrient solutions. The pH of the final solution varied between 5. 3 and 5. 7. The various combinations of micronutrients gave twenty- seven different treatments. The cultures were watered three times per week (Monday, Wednesday, and Friday) with the appropriate nu- trient solution and on other days with deionized water. Nutrient solu- tion or water was added until effluent flowed from a hole in the bot- tom of the pot. Supplemental light was used in this and all following experiments (12 hour daylength). The plants were grown in an "air- conditioned" greenhouse chamber which maintained the temperature at80 Fi 5°. 28 TABLE 2. -- Micronutrients and concentrations utilized in micronutrient interaction experiment number one. . . Concentration M1cronutr1ent as . . (parts per m1111on) B H3BO3 0. 5 Mn MnClZ- 4H20 0.5 Mo HzMoO4- H20 0.01 Zn ZnSO4- 7H20 0. 05 Zn ZnSO4- 7H20 5.0 Zn ZnSO4- 7H20 50.0 Cu CuSO4- 5H20 0. 02 Cu CuSO4- 5H2O 2.0 Cu CuSO4- SHZO 10. 0 Fe Fe-Citrate 0.5 Fe Fe-Citrate 25.0 Fe Fe-Citrate 75.0 Plant height and trifoliate leaf number were recorded two weeks, four weeks, and six weeks after seedling emergence. At six weeks, the plants were removed by washing the roots out of the sand with water under pressure. root, air dried, and weighed. The plants were divided into shoot and 29 An analysis of variance of root and shoot weight, number of leaves, and plant height was made by computer. Another analysis was computed on the difference in Shoot weight of Saginaw and Sanilac. The second experiment (December, 1965-January, 1966) was conducted identically to the first except that the macronutrient solution used was slightly modified in an attempt to buffer possible pH changes within the culture during nutrient uptake. The nutrients used are listed below: Chemical Quantity Ca(NO3)2- 4H20 1. 401 grams/liter KHZPO4 0.0793 grams/liter KNO3 0.5284 grams/liter NH4H2PO4 0. 0670 grams/liter MgSO4° 7H20 0.4323 grams/liter KCl 0. 0434 grams/liter The micronutrient solution utilized Mn as an additional variable to Zn, Cu, and Fe. The micronutrients and concentrations are listed in Table 3. Six weeks after seedling emergence the number of tri- foliate leaves and plant height were recorded, the plants were then harvested as described previously, except that the roots were washed in . 5 N HC1 and rinsed in deionized water. 30 Again the plants were di- vided into roots and shoots, dried, and weighed. TABLE 3. -— Micronutrients and concentrations utilized in micronutrient interaction experiment number two . Concentration M1cronutr1ent as (parts per million) B H3BO3 0. 5 MO H2M004' H20 0. 01 Zn ZnSO4- 71120 0. 005 Zn ZnSO4« 71120 0.05 Zn ZnSO4' 7H20 5~ 0 Cu CuSO4- 5H20 0.02 Cu CuSO4' 5H20 2. 0 Mn MnClZ- 4H20 0. 5 Mn MnClzo 4H20 50. 0 Fe Fe-Citrate 0- 5 Fe Fe-Citrate 50. 0 petioles, stems, and roots. The dried plant material was further divided into leaves, These various plant parts were ground 31 in a Wiley mill (with stainless steel parts to prevent contamination) to pass through a 20-mesh screen. Samples of one-half to one g were used except when less material was available. They were dry ashed in porcelain crucibles at 500-550 C for at least six hours. The temperature was gradually raised to prevent ignition of the plant material. The ash was taken up in five ml of 2 N HCl and filtered through a Whatman No. 2 filter paper. Four rinsings of 10 ml each were made with deionized water: the crucible twice, the filter paper once, and the funnel once. The final solution volume was made up to 50 ml with deionized water. This solution was analyzed for Zn, Cu, Fe, and Mn using a Perkin-Elmer model 290 atomic absorption spectrometer. Occa- sionally, further dilution of a sample was necessary to obtain a read- ing on the scale of the machine. The reading was linear in the range used with 0 percent absorption equal to 0 ppm and 100 percent absorp- tion equal to the concentration of the highest standard solution used for each element. Values were converted to ppm/g dry weight using the following equation: percent absorption X solution volume ppm/g tissue = - 100 plant weight + ppm highest standard 32 Analysis of variance was computed on plant weights and ppm of each element for each plant part. Again the computer was utilized for the analysis. IV. Grafting Experiment To determine the portion of the plant responsible for a differential reaction of the two varieties to 5. 0 ppm of Zn, a series of grafts was made. Seeds of the two varieties were sown on opposite sides of a glazed pot containing acid-washed silica sand. The sand was moistened with deionized water. Following germination, when the primary leaves were partially expanded, grafts were made in all combinations of scion and stock with the Saginaw and Sanilac plants. Diagonal cuts were made through the hypocotyl approximately half the distance between the cotyledonary node and sand surface. A short section of sterilized nylon bristle was inserted into the region of the pith, which at this time was beginning to break down, for support of the scion. The scion of one plant was placed on the stock of another. A small piece of self-adhering rubber latex was placed tightly around the graft to prevent drying out of the graft union and to hold the graft tight. The grafted plants were watered with 5. 0 ppm Zn nutrient solution and the cultures placed in a plastic-lined clay saucer con- taining about 1/2 inch of the same nutrient solution on the bottom. A 33 large plastic bag was placed over the culture and saucer so that it was airtight and supported above the plants by stakes and served as an effi- cient moist chamber. After five days the plants were taken out of the chamber and the watering schedule of nutrient solution (containing 5.0 ppm Zn) three days per week and deionized water on the alternate days was initiated. Approximately 30 grafts were made in each pos- sible combination (Saginaw/ Saginaw, Saginaw/Sanilac, Sanilac/ Saginaw, Sanilac/Sanilac). Graft success was about 50 percent... Photo- graphs were taken when the plants were approximately one month old. At two months the plants were harvested by washing the roots out of the sand, washing the roots with . 2 N HCl, segmenting at the graft union, drying, and weighing. Later the dried plant material was ground, ashed, taken up in acid, and analyzed for Zn and Cu using the techniques previously listed. V. Estimation Of Zn Bound by the Stem Using Plant Exudate TO determine whether the stems of the Saginaw and Sanilac navy beans bound Zn to the same extent, Zn content of exudate was determined. Seeds Of the two varieties were sown on Opposite sides Of glazed pots. Following emergence, the number Of plants was thinned to two of each variety per pot, and the irrigation procedure, described previously in the nutrient interaction experiment, was 34 initiated. The pots were divided into three groups, which received a different level of Zn in the nutrient solution. These levels were 5. 0, 0.05, and 0 ppm (the zero Zn level did receive Zn as a con- taminant as the plants didn't develop deficiency). At two months of age, eight cultures from each level Of Zn (designated pretreatment) were selected from the groups on the basis Of plant uniformity. These plants were watered for three days with deionized water to flush out . Zn. The three pretreatment groups were divided into two groups Of four pots each. All leaves were removed from the plant by cutting at the base of the petiole. The cut surface was covered with vaseline to prevent exudation. Immediately after initiation Of the treatment, two levels of Zn (5. 0 and 0. 5 ppm), one plant of each variety was cut Off about three inches from the sand surface. The other plant Of each variety was cut Off 20-26 inches from the sand surface. The lower cutting usually left two to three nodes, while the higher left seven to eight. The stems were bent over and the cut ends placed into small (5 ml) plastic vials (Figure 3). Exudate was collected continuously for six periods of five hours each, starting at eight A. M. , a new vial being placed under the end of each stem at the beginning of each 5 hour period. 35 FIGURE 3. -- Plant arrangement during exudate experiment. long stem ll/ sand surface short stem > collection vial ————— elastic band glazed pot with hole at bottom 2 plants on left = Saginaw O 2 plants on right = Sanilac 36 The weight and Zn concentration Of each sample of exudate was determined. The exudate was directly aSpirated into the spec- trometer. Analysis Of variance was computed on exudate weight, Zn concentration, and total Zn exuded. VI. Tracer Investigation--Whole Plant All tracer studies on the whole plant followed a similar pattern Of preparation. Plants were grown in silica sand until the primary leaves were approximately 3/ 4 expanded (about 12 days from sowing). The roots were then washed out Of the sand, rinsed Off, and the plants transferred to a special plexiglass container de- signed for isotope-containing nutrient experiments (Figure 4). Generally the containers were filled with Hoagland' s nutrient solution up to about 1 inch from the top (1500 ml). Aeration was continuous. Supplemental light was given by fluorescent lamps (1200 foot candles) on a 12 hour daylength cycle. Following a 12 hour acclimation period (unless otherwise specified), 65Zn was added and the plants allowed tO take up Zn for a period of time. Specific details concerning treatment variables and uptake period will be given for each experiment in the results section. After the uptake period, the plants were removed from the 37 FIGURE 4. -- Pictures of plexiglass container designed and used for exposing plants to Zn. (Outside dimensions = 18%" x 2%" x 4%") 38 solution and the roots were blotted, washed in dilute HCl, blotted, rinsed in H20 four minutes, and blotted. At this point, the plants were either pressed in order to make radioautographs or sectioned for counting. Pressing was done by placing the plants between two pieces Of heavy blotter paper, usually with the primary leaves and roots severed to prevent recycling during the initial part of the pressing Operation. The blotter paper with the plant in between was placed between two pieces of composition board. Several of these units (plant, paper, and boards) were stacked with a lead weight on tOp and placed in an oven until dry (2-4 days). The plants were re- moved from the blotter paper with the aid Of a long-bladed spatula, mounted on a clean piece of blotter paper using rubber cement and covered with Saran wrap. Each plant and a sheet of Kodak Blue Brand Medical X-Ray film was placed between two composition boards in a dark room. These units were stacked, covered with aweight, and placed in a box for the duration Of the exposure period, which was from 60 to 96 hours in various experiments. The films were then developed. The plants were divided into leaves, petioles (in most cases), upper stem, lower stem (from the cotyledonary node down), and root for determination of 65Zn content. These plant parts were placed in small wax—coated medicine cups and dried. Following 39 drying, the plant material was pressed into the bottom Of the cup using a cork stopper covered with aluminum foil mounted on a glass rod. This was done in order to Obtain uniform geometry of plant material for isotope content determination. The material was as- sayed for 65Zn using a Geiger-Mueller Tube detector attached to a Tracerlab Versa/matic II scaler. The analysis Of these data was generally on a counts per minute basis instead of total Zn because a relative comparison between the two varieties with reSpect to uptake and distribution was all that was desired. RESU LTS AND D ISCUSSION 1. Genetic Experiments A. Low Zn 1. Field Data. -— The genetic populations were scored from 1 to 5 in both 1964 and 1966. In both years, the Zn deficiency readings were taken at about six weeks after emergence. Some Of the plants initially scored as deficient often outgrew the deficiency. Eventually these plants appeared normal, but they matured late. The genetic data Obtained from 1964 and 1966 are sum- marized in Tables 4 and 5. The distribution of the various genetic populations Of the two years can be seen in Figures 5 and 6. The severity of the Zn deficiency was not equal for the two years. With the exception Of the Saginaw parent, all Of the populations had a higher mean (greater degree Of Zn deficiency symptom) in 1964 than in 1966. In 1966, plants of the Saginaw variety were scored from 1 to 4 while those of Sanilac were scored in all classes. This distri- bution was probably due to the variability of the soil in the experi- mental plots and the threshold level of Zn differentiating sufficiency 40 41 TABLE 4. -- Summary Of 1966 low Zn—genetic (Saginaw x Sanilac navy beans) population performances grown in the field (Saginaw County). Class of Zn reaction Generation Total éVI::n Variance 1 2 3 4 5 C e Saginaw # 229 116 34 3 382 1.51 .476 % 60 30 9 1 0 Sanilac # 14 19 76 278 56 443 3.77 .706 % 3 4 17 63 13 F1 # 122 19 10 151 1.26 .326 % 81 12 7 F2 # 179 98 127 83 26 513 2.37 1.563 ”/o 35 19 25 16 5 F3 (selected) # 16 14 48 116 91 285 3.88 1.180 % 5 5 17 41 32 F:3 (unselected) # 357 222 171 172 41 963 2.29 1.562 % 37 23 18 18 4 F1 x Sag (selfed) # 442 252 192 125 18 1029 2.05 1.254 "/0 43 24 19 23 2 F1 x San (selfed) # 368 212 235 161 12 988 2.23 1.32 % 37 21 25 16 1 42 FIGURE 5. -- Distribution of Saginaw x Sanilac genetic populations grown in a soil low in Zn in 1966 (Saginaw County). 100 -. j 75 ._ Saginaw ‘1 50 _ ' 4 25 — .1 0 4 Sanilac l 1 T _— a 100 — l _ U .21 50 — H O ES 25 - F .4 \/\ .5 0 I F I F I 2 I I I I I C! .2 E 100 - — g 75 _ F3 (selected) _ £14 50 - - “3 25 a - 5c: 0 -_//\. F3 (unselected) \\—\ Pllll jljlj 100 q 1 75 _ F x Saginaw . 50 .4 (selfed) 4 25 ._ F1 x Sanilac _ (selfed) 0 I I I I I I I I I I l 2 3 45 1 2 3 4 5 Class Of Zn Deficiency Reaction 43 TABLE 5. -- Summary of 1964 low Zn-genetic (Saginaw x Sanilac navy beans) pOpulation performances grown in the field (Saginaw County). Class of Zn reaction Generation Total éVIean Variance 1 2 3 4 5 core Saginaw # 49 18 67 1. 27 . 197 % 73 27 Sanilac # 15 24 31 70 4.23 .609 % 22 34 44 F1 # 20 32 9 61 1.82 .450 % 33 52 15 F2 # 55 154 63 64 78 414 2.89 1.804 % 13 38 15 15 19 BC Saginaw # 28 25 1 54 1.5 .283 % 52 46 2 BC Sanilac # 4 21 12 2 9 48 2.81 1.553 % 8 44 25 4 19 and deficiency within the plots. Other factors that could have con- tributed to the Observed variability in degree Of Zn deficiency symp- toms are slight differences in soil moisture, soil temperature, and elemental concentration (particularly P). Environmental factors 44 FIGURE 6. -- Distribution of Saginaw x Sanilac genetic population grown in a soil low in Zn in 1964 (Saginaw County). 100- _ 75_ Saginaw _ 50— \ _ 8 25.— Sanilac - / g 0 U l I I I I I 7 I I I '5 «3100— H F “I 5 75a 1 _ g 50-} _ 3 25—4 1.72., / \ g. 0 I I I I l 7 I I I I 0.. “5100.. _ s" 75 .. F1 xSaginaw _ 50-1 _ 25 _ F1 x Sanilac_ O I I I 1 r I l 1 2 3 4 5 1 2 3 4 5 Class Of Zn Deficiency Reaction 45 apparently act in such a manner that plants of identical genotype can shift to one side or the other of the Zn threshold level. Thus at least in this work, the genes involved in low Zn tolerance can be regarded as having relatively low penetrance (85, 90). In 1966, 10 percent of the Saginaw population was scored in classes 3 and 4 and 25 percent Of the Sanilac was scored in classes 1, 2, and 3, which emphasizes the low degree of penetrance of the genes for tolerance and sensitivity, respectively. The F population 1 in both years was similar to the Saginaw population in Zn deficiency symptoms. In 1964, the mean score Of the F population was 1. 82 1 as compared to 1. 27 for Saginaw and 4. 23 for Sanilac. In 1966, the F1 scored 1. 26 as compared to 1. 51 for Saginaw and 3. 77 for Sanilac. These data suggest essentially complete dominance for the gene(s) imparting tolerance to low Zn. The means Of F2 and F3 populations were intermediate between the parents, although somewhat closer to Saginaw. The backcross to Saginaw exhibited full low-Zn tolerance whereas the backcross. to Sanilac was intermediate. Selfed material obtained from the 1964 Saginaw backcross population was not as tolerant to low-Zn conditions as the original backcross grown in 1964 (E = 2. 05 vs. 1. 51). This indicated that genes for sensitivity were ex- posed in the backcross self population. If the trait were governed 46 by multiple factors, it is doubtful that a generation of selfing would expose sufficient numbers of genes conferring sensitivity to account for the loss Of tolerance Observed in this population. The F population Obtained from F 3 plants selected in 2 the field in 1964 for inefficient Zn utilization also indicated a small number of genes. These F2 plants, after six weeks to two months in the greenhouse with nutrient fertilization, set seed which gave rise to a "selected" F3 population. This pOpulation in 1966 had a mean value of 3. 88 compared to 3. 77 for the Sanilac parent (Table 6). TABLE 6. -- Performance Of several F3 navy bean lines in 1966 selected for Zn-deficient reaction in 1964 when grown under low Zn conditions in the field (Saginaw County). Family 339161: aClass of Zn reaction Tetal 8122:: 1 2 3 4 5 34 14C 8b 6 12 10 3 39 2. 85 37 5 3 2 12 14 3 34 3. 35 39 4 2 2 5 21 11 41 3. 90 32 4 2 7 25 9 43 3. 95 38 5 3 1 4 14 14 36 3. 97 33 3 1 6 10 8 25 4. 00 35 5 1 15 21 37 4. 49 36 5 1 7 22 30 4. 70 a1--highly tolerant; 5-—highly sensitive. bNumber of individuals scored in class. COriginally scored 4 but later scored 1. 47 This indicated that true-breeding fully sensitive plants could be selected in the F2 population. 2. Determination of the Number of Genes and Gene Action. -- Gene number refers to the number of segregational units, while gene action is used in the classical Mendelian sense and refers to dominance, additivity, gene interaction, etc. Though it is difficult to draw a firm conclusion with regard to a specific number of phenotypic classes present, the as- sumption of two seems to be the most conservative. The variability Of the two varieties was used to estimate the number of individuals of the segregating populations in each class of Zn reaction which were phenotypically tolerant or sensitive. This "partitioning" method aided in splitting the F and other segregating populations 2 into tolerant and sensitive phenotypes. The probability that a plant in a given class is tolerant was computed. In 1964, the F1 was used as an estimate Of the response of tolerant phenotypes because there was no overlap in scoring of Saginaw and Sanilac parents. Scoring Of both varieties, but eSpecially Saginaw, was unconsciously biased for either a low or high rating in 1964. Using the F it was possible 1’ to partition class 3 into tolerant and sensitive phenotypes. The results of the partitioning are shown in Tables 7 and 8 for 1966 and Table 9 for 1964. These results indicated that two 48 TABLE 7. -- Percent distribution of Saginaw and Sanilac navy beans grown in the field in 1966 under low Zn conditions. Class Of Zn reaction Variety Number 1 2 3 4 5 Saginaw # 229 116 34 3 382 % 59.94 30.36 8.90 .78 Sanilac # 14 19 76 278 56 443 % 3.16 4.29 17.15 62.75 12.64 Total % 63. 10 34.65 26.05 63.53 12.64 % Saginaw 94. 99 87. 62 34.17 1. 23 0 TABLE 8. -- F2 (Saginaw x Sanilac navy bean) population grown in the field in 1966 under low Zn conditions adjusted to parental type. Class of Zn reaction 1 2 3 4 5 % Saginaw 94. 99 87.62 34.17 1. 23 0 F2 Field Data Total 179 98 127 83 26 Saginaw Type 170 86 43 1 Sanilac Type 9 12 84 82 26 Observed Expected (adj. ) (9:7) Total Saginaw Type 300 289 Total Sanilac Type 213 224 x2 prob. = .5 - . 3 49 TABLE 9. -- Percent distribution of Saginaw and Sanilac navy beans grown in the field in 1964 under low Zn conditions and F pOpulation adjustment. 2 Class Of Zn reaction Variety Number 1 2 3 4 5 Saginaw # 49 18 67 % 73. 17 26.87 F1 # 20 32 9 61 % 32.78 52.46 14.75 Sanilac # 15 24 31 70 % 21.43 34.29 44.29 Total % 32.78 52.46 36.18 34.29 44.29 % Saginaw (using F1) 100 100 40. 77 O 0 Observed Expected (adj. ) (9:7) F2 Total Saginaw Type 235 233 Total Sanilac Type 179 181 genes are acting in a complementary manner tO give a 9:7 ratio in the F2 generation. A chi-square test indicated that the 9:7 ratio was compatible with the data. In 1964, the deviation of the Observed from the expected 9:7 ratio was so small that a chi-square probability was not calculated. 50 3. Segregation Within the F3 Population. -- If a ratio of nine tolerant to seven sensitive in the F population is assumed, it 2 should be possible to predict the performance Of various F3 families. One half of the F families should segregate while the other half 3 should not segregate into tolerant and sensitive phenotypes. One half Of the segregating families Should segregate three tolerant to one sensitive and the other half nine to seven. Seven-eighths Of the non- segregating families should be sensitive and the remaining tolerant. The performance of the F population in 1966 is given in 3 Table 10. A given family was placed in a particular category by TABLE 10. -- Patterns Of segregation for reaction to low Zn in unselected F3 navy bean families (1966) grown in the field. Segregating Nonsegregating Expected 31. 5 31. 5 Observed 34 2 9 Ratio- -TOlerant/ Sensitive Tolerant Sensitive 9:7 3: 1 Expected 17 17 4 28 Observed 15 19 17 12 comparing the mean and variance Of a family with that of the Saginaw and Sanilac parental populations. The partitioning values for the 51 families segregating 3:1 and 9:7 into tolerant and sensitive pheno- types are given in Table 11. TABLE 11. -- Distribution Of segregating unselected F3 navy bean families grown in the field under low Zn (1966). Zn reaction class Totals Ratio 1 2 3 4 5 Expected Observed 9:7 Tolerant 48 51 18 6 -- 126 123 Sensitive 3 7 36 44 11 98 101 Total 224 224 3:1 Tolerant 123 66 19 -- -- 209 208 Sensitive 7 9 36 15 70 71 Total 279 279 When one compares the number of Observed to expected F families in each category (Table 10), the observed ratio of segre- 3 gating tO nonsegregating families closely approximates the expected. In addition, the number Of families segregating 3:1 and 9:7 is close to the expected. However, there is a large discrepancy between the nonsegregating families which were tolerant and sensitive when com- pared to the expected number. There are many more tolerant than 52 sensitive plants rather than the expected reverse based on the 9:7 genetic model. From these data, it appears that although there is some question regarding the absolute validity Of the 9:7 ratio in F2, there is also evidence supporting this interpretation. Data in Table 11 indicate how closely the partitioning method transforms the Observed numbers of individuals in segregating F families regarded as toler- 3 ant and sensitive to the expected numbers of each. The distribution Of the 1966 Sanilac F1 backcross selfed pOpulation very closely approaches that of the F population. If one 2 considers the source of this backcross self population, further evi— dence is Obtained for the 9:7 hypothesis. This population was biased due to natural Selection for Zn tolerance because only tolerant plants Of the 1964 F1 x Sanilac would have set seed. The expected genotypes Of this backcross are Aa Bb, Aa bb, aa Rb, and aa bb, assuming Saginaw (tolerant) to be AA BB and Sanilac (sensitive) to be aa bb. Only plants Of the Aa Bb genotype would have been tolerant and would have set seed. This genotype is the same as that Of the F1 from which the F2 generation was produced. In effect, the selfed backcross to Sanilac population was an F population. 2 From these Observations, although a two gene model may be valid, these genes may not be acting strictly in a complementary 53 fashion to give a 9:7 ratio. There are several indications that the genesjinvolved are few in number. The ease with which plants were selected in the F2 population which were true breeding for low Zn sensitivity provides evidence favoring a small number of genes (2-3). Also, the segregating populations are not distributed continuously and normally as would be expected if the trait were strictly polygenic without dominance. Instead, these populations are somewhat discon- tinuous and skewed toward tolerance of the low Zn condition. This indicates a low gene number with dominance controlling the trait. There appears to be no way of resolving this question with the data presently available. Advanced generation backcrossing and intercrossing Of advanced generation families which are homo- zygous and give Specific reactions to low Zn conditions would provide more information. This information, in addition to data from segre- gation patterns of the existing advanced generations, should make it possible to answer the question of gene number and the mode Of their action in the Mendelian sense. B. High Zn A summary of genetic data Obtained from several popula- tions of a Saginaw x Sanilac cross grown in nutrient sand culture with 5.0 ppm Zn is presented in Table 12. The three different lots pre- sented were grown at two different times. Lot one responded with 54 somewhat greater growth Of Saginaw than lots two and three, neces- sitating a splitting Of the lots into two groups. In both instances, the populations were Split into four categories on the basis Of shoot weights (Table 12). TABLE 12. -- Summary of Saginaw x Sanilac navy bean genetic experiment in sand culture at high Zn in the greenhouse. Dry weight (g) Of plant tops Lot 1 _. 0-2 2-5 5-8 8 - Saginaw 1 1 4 Sanilac 5 F1 3 1 F2 12 12 5 1 Lots 2 and 3 0-2 2-4 4-6 6 - Saginaw 3 5 4 Sanilac 12 F1 2 1 1 F2 41 37 11 3 Because of the relatively small pOpulation it is difficult to make an estimate Of gene number and action. However, sensitivity to the high level appears to be dominant because the performance Of 55 the F1 generation, with a few exceptions, approximates that of Sanilac. Plants of the Sanilac variety fall only in the lowest weight range and Saginaw in the higher three ranges. If the F2 population is Split into two groups on the basis of the parental performance, the values Obtained are extremely close to a 9:7 ratio Of tolerant to sensitive (Table 13). The expected values are 68 to 54. Thus a con- flict appears between the F1 and F2 data. The response Of the Sanilac phenotype under high Zn is partially to completely manifested in the F1, while in the F2 the ratio of tolerant to sensitive plants suggests recessiveness Of the Sanilac phenotype. TABLE 13. -- Segregation of (Saginaw x Sanilac navy bean) F population grown under 5. 0 ppm Zn level into a Saginaw and Sanilac phenotype. (Sanilac) (Saginaw) Total number Of F2 plants 0_2 grams < 2 grams Lot 1 12 18 Lots 2 and 3 41 51 Total 53 69 A possible root and/or shoot interaction between plants within the same pot due to differential accumulation of Zn and other ions may account for this discrepancy. However, this is only specu- lation. 56 Additive gene action may be an alternative to the 9:7 ratio. The F1 generation would be somewhat intermediate between the Sagi- naw and Sanilac parent with regard to growth at the high (5. 0 ppm) level of Zn. The F2 generation would also be somewhat intermediate. Both of these conditions appear to be fulfilled if the data are indica- tive of the real situation. With the present data, there is no possibility of distinguish— ing which of the various alternatives might be correct. The determina- tion Of the true genetic action is Of importance as the genetic model limits the nature of the hypothetical physiological model. If gene num- ber and action are the same at the low and high level Of Zn, a common mechanism may be postulated. If they are not the same, a common mechanism, at least in its entirety, cannot be involved. Greater amounts Of data from F2 and backcross populations and an experimental design removing the possible competition effect but still measuring the pot to pot variability would help clarify the genetic picture with regard to the control system Of high Zn tolerance. II. Micronutrient Studies A. Zn, Cu, and Fe The purpose Of this and the following experiment was to determine the effect of high concentrations of various micronutrients 57 on growth and ion accumulation of the Saginaw and Sanilac varieties of navy beans. By increasing the concentrations Of certain micro- nutrients, deficiencies in other micronutrients might be induced and the two varieties might react differently to this unbalance. This was initially suggested by the chance Observation of a differential nutrient chlorosis induced by nutrient solutions containing especially high Zn concentrations, as well as high contents of other elements. Saginaw appeared normal, while Sanilac was chlorotic under these conditions. The effect of some of the combinations and concentrations of Zn, Cu, and Fe on the appearance Of the two varieties can be seen in Figure 7. Photographs A, B, C, and D illustrate the effect Of the three levels of Zn. At the normal level (A) both varieties grew well and at the 50. 0 ppm level (D) both were extremely stunted. However, at the 5.0 ppm level Of Zn, Saginaw grew well while Sanilac was stunted. This effect is Observed in B and C, which were photographed at three weeks and six weeks after emergence, respectively. The differential stunting was observed at all levels Of the other micro- nutrients except 10. 0 ppm Cu. Figure 8 illustrates the response due to interaction of high Cu and Fe on the two varieties. In photograph A (10. 0 ppm Cu) Sanilac was chlorotic while Saginaw was normal. However, the appli- cation Of high Fe (75 ppm) overcame the chlorosis as evidenced in B. 58 Avocomnofio swam among 095: qN 805 o .m -u .m :N San no .0 -134 69.658me 93 E GN Ema o .om 6:6 .o .m .8 .o 3.6 638m 6:86 .25: 25.3 66:63 new :8 do 635 as: 3659.8 -- s. 5505 59 an 866 o .8 -- .O Amocownofio swam meOB xfimv GN Ema o .m 11.0 66:53.80 .. .s HMDUE 60 6m 85593--..m 658811.614 .omsoncomwhm 9: 5 oh mo mHO>OH 043 mafia :0 Sam o .8 pm :Bozw wcwon b3: AEMEV owficmm cum fioa mo ogm to: Emcfimww I- .w HMDUHE 61 This effect was Observed at the two lower concentrations of Zn but not at 50. 0 ppm. The differential growth in terms Of shoot weight for the two varieties at the various levels Of each micronutrient can be seen in Table 14. The analysis of variance data are given in Table 15. TABLE 14. -- Effect Of various levels of Zn, Cu, and Fe on growth of Saginaw and Sanilac varieties of navy beans when grown in sand culture in the greenhouse. Averagea weight (g) of shoot Nutrient Level Saginaw Sanilac Difference Zn 0.05 ppm 3.9 3.2 0 7 5. 0 ppm 3. 8 1.4 2.4 50.0 ppm 0.3 0.3 0.0 Cu 0.02 ppm 3.8 2.0 1.8 2.0 ppm 3.8 2.0 1.3 10.0 ppm 1.0 0.8 0.2 Fe 0.5 ppm 2.7 1.6 1.1 25.0 ppm 3.5 1.7 1.8 75.0 ppm 2,0 1.6 0.4 aAverage of three replications and nine treatments. The analysis Of variance of the raw data is given in Appendix Table 1. Although tabular data will not be shown, root weight, trifoliate leaf number, and plant height responded similarly to shoots to high Zn. 62 These data establish the fact that Saginaw is capable Of withstanding much higher levels of Zn than Sanilac (Table 14). TABLE 15. --Analysis of variance of Saginaw minus Sanilac shoot weights when grown under different levels of Zn, Cu, and Fe. 85:22:12? d. f. M. S. F value Replications 2 1. 965 Treatment 26 8. 846 6.156** Zn 2 42. 85 29. 819M< Cu 2 16.255 11. 312** Fe 2 13.235 9. 210** Zn x Cu 4 10. 498 7. 305** Zn x Fe 4 3. 388 2. 358* Cu x Fe 4 3. 388 2. 358* anCuxFe 8 2.026 1.410 Error 52 1. 437 Total 80 >I<—-Significant at Q. .05 **—-Significant at C1. . 0125 The reason for the small difference between the two varieties at either 50. 0 ppm Zn or 10. 0 ppm Cu is that at these con- centrations neither variety grows well. The effect of 5. 0 ppm of Zn in the nutrient solution was Observed as early as two weeks after emergence of the seedlings (Table 16). 63 TABLE 16. -- Plant height of Saginaw and Sanilac navy beans grown in sand culture in the greenhouse as affected by age and Zn levels. Zn level Age Saginaw Sanilac % San. /Sag. O. 05 Two Weeks 11. 7 15.1 129 5.0 9. 8 7. 5 77 50.0 2. 7 2. 8 104 0.05 Four Weeks 23.6 18.3 76 5.0 24. 1 10.0 41 50.0 2. 7 2. 8 104 0.05 Six Weeks 36.3 20.0 55 5.0 36. 9 11. 3 31 50.0 2. 7 2. 8 104 B. Zn, Cu, Fe, and Mn 1. Differential Varietal Response in Terms Of Growth. -- The micronutrients and concentrations (ppm) involved in this experi- ment were: Zn--0. 005, 0. 05, and 5. 0; Cu--0. 02 and 2. 0; Fe--0. 5 and 50. 0; and Mn--O. 5 and 50. 0. In general, the results of the pre- vious experiment were confirmed, although the magnitude of the differences between the two varieties at 5. 0 ppm Zn was slightly less (Table 17). In addition to the effect Of Fe and Cu, Mn tended to de- crease the magnitude Of the difference in growth between the two varieties. The overall analysis Of variance data for trifoliate leaf number, height, and plant weights are in Table 2 Of the Appendix. 64 TABLE 17. -- A comparison Of growth characteristics of Saginaw and Sanilac navy beans grown under three levels of Zn in sand culture in the greenhouse. Growth . . . . ‘70 Zn level Characteristic Probab111ty Sanilac Saginaw San. /Sag. 0.005 21.1 37.9 55.7 0. 05 Plant in <. 05 16.9 38.9 43. 4 5.0 n' 4.7 14.3 32.9 0. 005 Trifoliat 12.9 15.9 84. 3 0.05 L af Noe <.01 10.2 15.3 66.7 5. 0 e ' 7. 8 14. 3 54.5 0. 005 3. 87 4.29 90. 2 0.05 Sh°‘(’t)Wt' 0.066 3.68 3.62 101.6 5.0 g 0 75 1.59 47 2 0. 005 2.17 2. 52 86.1 0.05 R00: Y” 11.2 2.13 2.10 101.4 5.0 g 0 32 0.75 42.7 2. Elemental Concentration in Plant Parts of the Two Varieties. -- The concentration of Zn, Cu, Fe, and Mn in the leaves, petioles, stems, and roots averaged for the two varieties is given in Table 3 Of the Appendix. Portions of these data were lost and esti- mates were made by comparing the ratios Of the elemental content of Saginaw to Sanilac for the particular plant part in the other repli- cates. All summary and analysis of variance tables were made using these estimates. At the 5. 0 ppm Zn level no values are given for 65 petioles and stems because Of the stunting Of Sanilac. The analyses Of variance on these data are given in Appendix Table 4. Roots accumulated much higher concentrations Of the four elements than did the other plant parts, particularly at the higher concentrations Of micronutrients. Leaves generally accumulated higher concentrations than petioles and stems, which accumulated about the same levels (Table 18). Data in Table 19 Shows two effects Of Fe, Cu, and Mn on Zn content Of the leaves of the two varieties. First, Zn content Of the leaves is decreased significantly by increasing levels Of other micronutrients only when the nutrient solution is high in Zn. Second, relatively lower concentrations Of Zn are found in the leaves Of Sagi- naw than Sanilac, in most cases and particularly, at the high level of Zn. It appears that Saginaw may have a greater ability to maintain an internally balanced concentration of Zn with external fluctuations of other micronutrients. Saginaw also appears to have a more restricted movement Of Zn into the leaves or less total uptake of Zn at the high Zn concentration in the nutrient solution. Differential accumulation does not appear to account for the differential tolerance Of the two varieties because both accumulate high levels Of Zn. Therefore, either the two varieties differ in the 66 wmm m3 3. com Nam mmH mm Em C .m we mm 3. mmfi m: mm 8 mm: No .0 :0 mac wmm 2: gm vow mvm om 4mm om mm mu. 30 S; S 3 34 >2 m .o :2 mug mm: 13.: www 9.65 mg am now om >3... 3. mg NHN 161v mm 52 wow m .o oh so: I: 1: EL. pm“: I: I: mum o.m m3 m3 em 2: SH 2: 2: cm mo .o v: m: cm m: o3 o: E: m: moo .o 2N aoom 83m 303mm “mod poom 53m 3030mm mood 333 nofiflom “dogmam 335:: 5 Omficmm Bmfimmm . . cowumpuooocoo .Omzoncoonw on» 5 0.33:0 wcmm 5 GBOpw manna 53> 19395? was 33.3 Ema .Gofimficoocoo so“ some .HOm mucosa—moi acorn—5G .550 ao>o poEESm «nofiofio some Ho 353.95 40:76:33 Ema :3 2533383005 982 In .3 mimja. 67 distribution Of intra- and extra-cellular Zn, or they have physiologi- cal processes within the cell Or cell organelles which differ in their ability to withstand high levels of Zn. TABLE 19. -- Zn content (ppm) of the Saginaw and Sanilac navy bean leaves at three levels of Zn in the nutrient solution and at two levels of Fe, Mn, and Cu when grown in sand culture in the greenhouse. . Concentration Zn in nutrient solution Element Coniggtniation Variety 0. 005 O. 05 5. 0 Fe 0. 5 Saginaw 145 88 791 Sanilac 123 109 812 5. 0 Saginaw 85 92 556 Sanilac 109 93 741 Mna 0. 5 Saginaw 135 91 660 Sanilac 119 88 929 5. O Saginaw 95 89 687 Sanilac 1 13 113 624 Cu 0. 5 Saginaw 102 91 803 Sanilac 129 1 18 907 5. 0 Saginaw 129 89 544 Sanilac 102 84 646 aVarietal differences statistically significant. Zn may act two ways at the toxic level, either poisoning some Of the physiological processes within the cell or interfering 68 with the utilization Of other micronutrients. Several authors have pointed out the interference Of Zn with the uptake and distribution Of other micronutrients, particularly Fe (26, 61, 72, 93, 110). In most cases, Zn and also Cu act to induce severe Fe chlorosis. In the work reported here, high Fe (25 and 75 ppm) overcame the detrimental effects Of Zn and Cu to a moderate extent. However, since increasing the concentration Of other micronutrients in the nutrient solutions did not completely overcome the effects Of high Zn (5. 0 ppm) or high Cu (10. 0 ppm), it is apparent that either some other nutritional factor was out of balance or the high level of these cations was toxic pg _S__e_. It seems probable that there exists a cation level within plants of these two varieties below which certain cations induce deficiencies Of other cations. Above this level, these particular cations would be toxic in and of themselves. The fact that Saginaw seems to be able to grow with relatively unbalanced nutrient solutions suggests that this variety can regulate, to some extent, the internal physiological nutritional status regardless Of external nutrient concentration. Sanilac lacks this capacity. In addition, both varieties are affected equally when the cations reach toxic levels. From these data and other Observations the dividing line between ion interference and toxicity per ES seems to be around 5. 0 ppm Zn. 69 III. Physiological Nature Of the Differential Zn Deficiency and Toxicity Tolerance Of the Saginaw and Sanilac Varieties Of Navy Beans A. Reciprocal Grafting--High Zn Level To determine whether the roots or shoots were the plant parts active in determining tolerance to high levels Of Zn, grafts in all possible combinations of Saginaw and Sanilac shoots and roots were made. The reciprocal grafting data Show that the genotype Of the scion determines tolerance to high Zn (Figure 9, Table 20). This TABLE 20. —- Average dry weight of grafted plant shoots Of Saginaw and Sanilac navy bean varieties grown in a media containing 5. 0 ppm Zn in sand culture in the greenhouse. San. /San. San. /Sag. Sag. /San. Sag. /Sag. Average dry weight (g) 2.3 2.5 15.8 18.5 Total number of plants 6 11 5 4 does not imply that tolerance to low Zn is also determined by the genotype Of the scion. At present, the relationship between the physiological mechanism(s) involved in differential tolerance at the two levels Of Zn has not been established. 70 .mmsoncmmnw 05. E QN Sam o .m «a Esoam Aomdcmm paw Bacfimmmv manna :32 firms podmam pooiqoflom .. .m mmDOHm 71 .UODGSCOU I: .m mmDUHm 72 The difference in tolerance to high Zn is not due to dif- ferential Zn accumulation. Although Sanilac had more Zn in the shoots than Saginaw, both varieties accumulated a high level (Table 21). TABLE 21. -- Concentration Of Zn and Cu in reciprocal root-scion grafted plants of Saginaw and Sanilac navy beans grown at 5. 0 ppm Zn in sand culture in the greenhouse. Plant Zn b Cu b Graft part (ppm) '70 (ppm) "/0 San. /San. Leaves 818 19 22 22 (ma Stems 1148 27 33 33 Roots 2312 54 44 45 Total 4278 99 San. /Sag. Leaves 803 17 32 25 (10) Stems 841 18 35 27 Roots 3038 65 62 48 Total 4682 129 Sag. /San. Leaves 653 13 23 28 (7) Stems 573 11 18 22 Roots 3909 76 42 51 Total 5135 83 Sag. /Sag. Leaves 595 17 24 41 (4) Stems 405 12 12 20 Roots 2424 71 23 39 Total 3424 59 aNumber Of plants . bPercent derived from elemental concentration in each plant part given in the body of the table and not total accumulation and distribution. 73 Thus, some factor other than differential content of Zn in the shoots must determine tolerance or sensitivity to high Zn. Nevertheless, this factor(s) will have to be located in the shoot. B. Quantity and Zn Concentration of Exudate The intent of this experiment was to determine whether or not the stems of Saginaw and Sanilac retained Zn to the same degree. If the stem retains Zn throughout its length, exudate collected from a short stem and a long stem should have different amounts of Zn. If Saginaw and Sanilac stems differ in their capacity to retain Zn, this should be detected when comparing exudate from plants with short (3-4 inches) stems and long (20-30 inches) stems. In most cases, it was possible to collect sufficient exudate for analysis within the five hours. However, in a number of instances this was not possible. In a few cases, this was due to in- advertently kinking the stem while bending it for exudate collection. Occasionally exudate leaked from the cut surface where the petiole had been removed from the stem even though the surface had been smeared with vaseline. In a few pots, none Of the plants within the pot produced sufficient exudate for analysis. Estimates of missing values were made by averaging replicates for that time period; and the other time period samples for the particular plant. 74 The values for exudate weight, Zn concentration, and total Zn are given in Table 5 of the Appendix. Variety and cutting height were significantly different for all three variables. The specific effect of cutting height on the two varieties when summing over all other effects is given in Table 22. TABLE 22. -- The effect of cutting stem at 2-3 inches (short) or 18-25 inches (long) on the Zn content Of the exudate Obtained from Saginaw and Sanilac navy beans (averaged over six 5-hour collection periods). . Stem Weight of Concentration Total Zn Variety length exudate Of Zn of exudate pm x g (9) ppm p Saginaw Short 1. 35 0. 71 1. 20 Saginaw Long 1. 23 0. 58 0. 85 Sanilac Short 0. 73 0. 45 0.49 Sanilac Long 0. 35 0.29 0. 10 Weight of exudate and‘Zn concentration of this exudate were greater in Saginaw than Sanilac at either stem length. The dif- ference in exudate weights suggests that either Saginaw has a larger 75 or more efficient root system which is capable Of taking up greater quantities of water, or the xylem system of Saginaw has a greater capacity for movement of sap. There may also be a difference in osmotic pressure within the stelar tissue of plants of the two vari- eties. Due to the limited nature Of the data, it is not possible to decide whether the root or stem is more important in determining the difference in exudate weight. The volume Of exudate delivered at a specific length may be a very complex phenomenon, affected by total cross-sectional area, the diameter Of the individual vessels (which would affect the total amount Of wall area and thus the amount Of resistance to sap flow), and flow rate. The contribution these various factors make in determining the total volume of exudate in the two varieties is un- known. However, the difference in volume or weight of exudate Obtained from the short stems and the long stems may be largely due to a difference in total cross-sectional area Of the xylem at high versus low cutting height. The difference in Zn concentration of Saginaw and Sanilac exudate indicates that either Saginaw has greater ability to absorb Zn from the medium or a greater capacity to translocate Zn from the root system into the vascular system. 76 The stems, apparently, retain Zn to some degree since the Zn content Of the exudate decreased with increased stem length. The fact that there is a greater proportional reduction in the concen- tration of Zn in Sanilac exudate than in Saginaw suggests that there is a greater capacity for retention in stems Of Sanilac. This difference may be due to the greater Zn concentra- tion in the sap Of Saginaw than Sanilac. If a much higher amount Of Zn is transferred into the vascular system Of Saginaw, stem reten- tion would be nearer full capacity than that of Sanilac and proportional binding would be less. In this case, the absolute binding capacity Of the stem might have been the same in the two varieties, but the dif- ference in Zn transferred into the vascular tissue could give the im- pression of differential binding. The effect Of pretreatment and treatment on the two varieties is given in Table 6 of the Appendix. Pretreatment had the more significant effect on the Zn content and exudate weight of the two varieties. Increasing pretreatment and treatment Zn levels increased Zn concentration and exudate weight. C. Tracer Studies The primary goal Of the tracer experiments was to deter- mine if there were any detectable differences between the Saginaw 77 and Sanilac navy bean varieties with respect to uptake and distribu- tion Of 652m For this reason, most of the studies were conducted from the standpoint of a comparison of Saginaw with Sanilac. These studies, in general, will be reported as a series of small experiments. 1. Uptake and Distribution. -- Plants were grown, as out- lined in the methods section, and then placed into nutrient solution containing 6E’Zn for 48 hours. The plants were removed and washed, and then dissected into leaves, upper stems, lower stems, and roots. There were four plants Of each variety. A summary Of the data is given in Table 23. TABLE 23. -- Mean values for 652n uptake and distribution by Saginaw and Sanilac navy beans for 48 hours exposure. Saginaw Sanilac Plant part cpm % of total cpm % Of total Leaves 100 38 48 27 Upper stem 17 6 20 11 Lower stem 27 10 28 15 Roots 119 45 86 47 Total 263 182 Varieties, plant parts, and their interaction were statisti- cally significant. There was a real difference between the two vari? eties with regard to total uptake and distribution Of 65Zn on a cpm 78 basis. Saginaw took up more Zn (263 vs. 182 cpm) and translocated higher percentages of the Zn into the leaves (38 vs. 27%) than Sanilac. Differences existed between the two varieties when the cpm data were adjusted for tissue weight, but were not significant. When adjusted for weight of root only, Saginaw was far superior to Sanilac with respect to uptake and translocation Of 65Zn into the leaves. 2. Time Course #1 and #2. -- The previous experiment indicated that there was a difference in the distribution Of 652n in plants of the two varieties at the time the experiment was terminated. The time-course experiments were designed to establish the rate Of uptake and distribution over a time period. In the initial experiment, four plants of each variety were removed from the 652m nutrient solution at 6, 12, 24, 48, 96, and 192 hours. Plants were removed at 48, 192, and 288 hours in the second experiment. In addition, the petioles were analyzed sepa- rately from the leaf blade and radioautographs were made Of both varieties at all three time periods in the second experiment. These radioautographs indicated that there were no strik- ing qualitative differences in distribution pattern of Zn between the two varieties, although Saginaw did appear to have more 652n in the smaller veins. The radioautographs also indicated a higher concen— tration Of 652n in the pulvinus regions Of the petioles (Figure 10). 79 FIGURE 10. -- Radioautograph of Sagiigasw navy bean plant (Sanilac ' pattern the same) exposed to Zn for 288 hours. The region of heavy labeling could have been due to greater deposi- tion of isotope or possibly due to the configuration of the xylem ves- sels in this region. According to Doutt (32), the vascular tissues are compressed in this region. Such a compression Of label-containing tissue could give the appearance of greater concentration of label. The data for the initial experiment are summarized in Table 24. These data confirm that the Saginaw variety trans- locates a higher proportion of 65Zn into the leaves than does TABLE 24. --Uptake and distribution Of 65 80 Zn byr Saginaw and Sanilac navy bean varieties for six different time periods on a cpm basis. Saginaw Sanilac Time (hours) Plant part % % Cpm Distribution Cpm Distribution 6 Leaves 64 6 30 3 Upper stem 22 2 21 2 Lower stem 38 4 30 3 Root 910 88 824 91 Total 1034 905 12 Leaves 283 16 149 13 Upper stem 51 3 31 3 Lower stem 80 5 57 4 Root 1326 76 912 80 Total 1740 912 24 Leaves 353 21 327 18 Upper stem 49 3 61 3 Lower stem 80 5 101 5 Root 1233 72 1354 73 Total 1715 1843 48 Leaves 490 29 650 22 Upper stem 70 4 95 3 Lower stem 99 6 103 4 Root 1019 61 2086 71 Total 1678 2934 81 TABLE 24. -- Continued. Saginaw Sanilac Time (hours) Plant part C m % C % p Distribution pm Distribution 96 Leaves 1210 24 1689 21 Upper stem 133 3 215 3 Lower stem 202 4 298 4 Roots ' 3452 69 5814 72 Total 4997 8016 192 Leaves 5666 32 5381 26 Upper stem 460 3 550 3 Lower stem 455 3 547 3 Roots 10919 62 14413 69 Total 17500 20891 Sanilac. The difference in distribution of Zn between the two varieties becomes more apparent when one compares the ratios Of 652n con- tained within the various plant parts Of the two varieties. The ratios in Table 25 denote the greater capacity of Saginaw to translocate 65Zn into the leaves. From these ratios, it appears that the greater proportion of 65Zn contained in the leaves Of Saginaw results from a greater release or movement Of Zn out of the roots and stems than in the case Of Sanilac. 82 TABLE 25. -- Ratio Of 652n distributed within plant parts of Saginaw and Sanilac navy beans averaged over all time periods of exposure. Saginaw Sanilac cpm cpm/g tissue cpm cpm/g tissue Leaves/Stems + Roots .29 . 15 . 22 . 13 Leaves/Stems 3. 13 .50 2. 64 .48 Leaves + Stems/Roots .42 . 64 .32 .54 As Opposed to the initial radioisotope study, in both time- course experiments, Sanilac absorbed more Zn than Saginaw. This is illustrated for time-course experiment 1 in Figure 11. The expo- sure time in the initial experiment, however, was only for about 48 hours. As can be seen in Figure 11, Saginaw takes up 65Zn faster initially for about 24 hours then there is a lag period for another 24 hours, while Sanilac takes up 652n at a slower rate, initially, but does not have a lag period. Apparently, the plants in the initial experiment were removed prior to the shift in the uptake rates Of the two varieties. From 96 hours to 192 hours the rate Of uptake in the two varieties is essentially the same. In general, the data presented here have been from the first time course experiment, which ran from 6 to 192 hours. The data from the second, which ran for 48 to 288 hours, have not been 83 FIGURE 11. -— A comparison between Saginaw and Sanilac navy beans of uptake rate Of 65Zn. 20000 - 16000 - 12000 -. Sanilac Saginaw cpm / plant 8000 -I 4000 - I ,I I I l 0 40 80 120 160 200 Time (hours) 84 presented, but the patterns of distribution and uptake in the two vari- eties are essentially the same. 3. Zn Concentration: Pretreatment and Treatment. -- Two experiments were conducted to determine whether there is a differ- ential effect of concentration of Zn upon Zn uptake in the two varieties. In both experiments, plants were grown in sand culture until 12 days Old. In the first experiment, 0. 05 ppm Zn was used during this 12 day period. In the second, 5. 0 ppm Zn was included. At the end Of this period, the plants were removed and placed in nutrient solutions containing 0. 0005, 0. 05, and 5. 0 ppm Zn for 24 hours. 65Zn was added to each nutrient solution at an equivalent dilution of radioactive Zn to cold Zn. A significant varietal response was Obtained only when radioisotope activity was expressed on a root weight basis. In this instance, the concentration by variety interaction was significant. The effect Of Zn concentration on the two varieties can be seen in Table 26. Greater differences may not have appeared due to the short exposure time to 65Zn. The difference in radioisotope activity of the plant mate- rial at the two pretreatment levels is partially due to differences in the amount of 652n label added. Though there may be differences in the amount Of 652n taken up at the two pretreatment levels, these 85 differences cannot be detected due to the differences in radioisotope activity Of the treating solutions. Comparisons between varieties and treatment levels within a specific pretreatment are valid, how- ever. TABLE 26. -— 65Zn content expressed as cpm/g root in Saginaw and Sanilac navy beans grown under a normal (. 05 ppm) and high (5. 0 ppm) level of Zn prior to treatment as affected by Zn level during treatment (averaged over plant parts). Concentration cpm/g root weight of Zn ppm Saginaw Sanilac Normal pretreatment 0. 0005 328 352 O. 05 296 299 5. 0 2011 2646 High pretreatment 0. 0005 195 201 O. 05 146 270 5. 0 200 633 In the high Zn treatment, uptake of 65Zn was significantly greater in Sanilac than Saginaw at both pretreatment levels. Pre- treatment with high Zn magnified this difference, which indicates that high Zn pretreatment in some manner preconditioned plants of the Sanilac variety so that these plants absorbed greater quantities of Zn. 86 There were relatively small differences between the dis- tribution of Zn in the two varieties; however, Saginaw usually trans- located equal or higher proportions of 652m into the leaves (Table 27). The increase in percent of Zn accumulated in the leaves with increas- ing Zn concentration in the nutrient solution may indicate a certain retention capacity in the roots and stems which becomes more and more saturated as Zn absorption by the roots increases. AS root and stem retention capacity are approached, higher levels of Zn are trans- located tO the top. Although Saginaw and Sanilac differ little in percent dis- tribution of 65Zn, total Zn accumulated in the plant parts differs markedly due to the greater uptake of Zn by Sanilac. 4. Other Studies. -- Several experiments were conducted on micronutrient interaction, hydrogen ion concentration and the effect Of root removal. The micronutrient interaction studies were inconclusive, but offered further evidence that Sanilac absorbs greater quantities Of Zn than Saginaw, yet transfers a smaller proportion Of this Zn to the shoots Of the plants. The hydrogen ion experiment also indicated that Sanilac absorbs greater quantities of Zn than Saginaw. However, increasing amounts of 65Zn were accumulated with increasing hydrogen ion con- centration in both varieties (Table 28). 87 2 on 3 mm 2 83 8.3258 a 2 S S on was nos .o 5898 2..- 2 mm 2 am 32 68:88 ON cm 3 mm mm Ea no .o 5653.6. S 3 2 2 am .88 oaanam am 2 3 2 on 83 o .m o .m aafiwam. mm 2 mm 2 m: on: 3:98 2 on 2 mm S SS mooo .o safiwam mm 2 cm 2 2 SE oflanam S .3 S 5 mm 3.3 no .o eafimam S on. 2 S K SSH oflanam 2 am 2 2 mm $2: o .m mo .o encamnm 83m 83m Ema Sam «oom .8304 yoga 3030mm mo>mod poo.“ coo: won . dog «on on D w\8ao u “a .H a “E a m 53.55 333: HSOH . . 28358536 “coonom 3.9.3 :N Ho>3 GN . dosh??? coon 49m: Omfinmm paw kmfimwm 43 :Nmm mo :ofiusfifimwp can 333: ~33 no :ofiwfiqoocoo :N pom unofiuwofioua mo Soto meat I- Sm mqm<fi 88 TABLE 28. -- The effggt of hydrogen ion concentration on Zn uptake by Saginaw and Sanilac navy beans (96 hours). cpm/g tissue pH Saginaw Sanilac 5. 0 4788 5256 6.0 2326 3956 7. 0 2822 3943 8. 0 370 424 The results of the root removal experiment were also inconclusive, but indicated the trend Of greater translocation of Zn in Saginaw but less total uptake. GENERAL DISCUSSION A genetic study was conducted at low Zn levels based on the frequency of appearance and severity Of Zn deficiency symptoms. A limited genetic study was conducted at toxic or high levels of Zn. Information Obtained on the basis of visual symptoms may not accu- rately reflect the fundamental genetic control reSponsible for the differential appearance of those symptoms because symptoms pe_r_ _s_e_ are not primary events and may be affected by the ontological processes leading to the deficiency lesion, as well as by other char- acteristics such as maturity, drought tolerance, etc. , which are also under genetic control. Nevertheless, the initial genetic infor- mation on differential Zn utilization should be useful in developing more Specific experiments. HOpefully, when the physiological mechanism responsible for the differential Zn reSponse of Saginaw and Sanilac plants has been determined, a genetic study can be con- ducted in detail at that level. Such an investigation should confirm or reject-the conclusions Of this study which were based on the appearance or lack of visual symptoms Of Zn deficiency or toxicity. 89 90 It is clear from the literature (83, 84, 107, 111) that all the genetic work on differential varietal reSponse to nutrients has been based on appearance of gross visual symptoms. In fact, the only differential response in which very much is known with reSpect to mechanism is with soybeans and Fe (14). Information regarding the mechanism is relatively recent, yet the genetic study by Weiss (111) was conducted in 1943 on the differential ability Of this PI selec- tion and the variety Hawkeye to grow under Fe stress as indicated by visual symptoms. Although no attempt has been made to correlate the genetic information gained in 1943 with recent physiological infor- mation, studies with isolines reveal that the genetic situation at the physiological level may be more complex than the early conclusions indicated (22). The genetic investigations reported in this thesis have proven fruitful, though inconclusive. The "partition" method used to estimate the degree Of overlap between phenotypes has proven valuable in trying to interpret the data. Although the specific details Of the genetic model cannot be defined until more data become available, the information presently at hand indicates that very few gene pairs (2-3) regulate the differential response to low Zn. The two-gene, complementary factor model appears tO be a 91 close approximation to the real genetic situation. Future data should clarify this situation. Because a few genes appear to be involved in determining tolerance to low Zn, they should have major effects on the mechanism responsible. For this reason, the physiological differences between tolerant and sensitive plants should be rather discrete (as Opposed to the behavior under polygenic control). Due to these discrete differ- ences, the identification of the mechanism(s) responsible for the dif- ferential sensitivity should be possible, though perhaps difficult. A genetic study of this mechanism(s) also should be possible. Initially when Sanilac was Observed to develop chlorosis in a particular nutrient solution while Saginaw did not, the question was raised concerning the generality of the mechanism responsible for the differential tolerance of Saginaw and Sanilac under low Zn. DO these varieties differ only in degree Of utilization of Zn or is the control mechanism(s) concerned with the utilization of other elements also? Two micronutrient experiments indicated that Saginaw has a greater ability than Sanilac to withstand extreme micronutrient unbal— ance (Figures 7 and 8). This suggested that a common physiological or anatomical mechanism(s) is responsible for the differential response of Saginaw and Sanilac plants under various micronutrient stress conditions. 92 The genetic investigation of the differential response to high Zn, as exhibited by stunting, was undertaken to determine whether the same physiological mechanism(s) is Operative both at low and high Zn levels. If the same genetic model were applicable in both instances, this would constitute supporting evidence (though not proof) that a common mechanism is involved. Since inconclusive genetic data were Obtained, it was not possible to answer this ques- tion (page 55). Even though the genetic models may be different at low and high levels of Zn, this does not imply that the mechanisms reSponsible for the differential response in the two varieties are not related. A mechanism at one level of Zn could be a component of a more complex mechanism which Operates at the other level. This question cannot be answered until the mechanism responsible for the differential reSponse of the two varieties at both levels of Zn is determined. Although the specific mechanism(s) has not been identified, some of the characteristics of this mechanism(s) have been established. The grafting experiment established that Shoots of the plants deter- mine the tolerance Of Saginaw to high Zn conditions (Figure 9). Grafting experiments at low Zn levels have not been successful. One of the most persistent differences between the two varieties was the differential distribution of Zn in them. Saginaw 93 generally had a higher proportion of Zn in the shoots Of the plants at low or normal Zn levels. This was first Observed with limited data (unpublished) Obtained from the two varieties grown on a low Zn soil in 1964. A summary of the data for Cu and Zn content is given in Table 29. Generally, total content of Zn or Cu was similar, but the shoots of Saginaw contained a higher percentage than Sanilac. TABLE 29. -- Cu and Zn content (ppm) of two bean varieties grown in a low Zn soil (Saginaw County) in 1964 as determined by flame spectrophotometry. Saginaw Sanilac Roots Leaves Roots Leaves -Zn +Zn -Zn +Zn -Zn +Zn -Zn +Zn Zn —913 24 41 41 26 44 24 28 \ V J \ W _/ \ V _/ \ V j ' 37 82 70 52 \ V #1 W V 119 122 93923.2 12 70.9 63.4 94.2 63.6 35.0 10.2 V—‘W \ V j \ v j \ V J 35.2 134. 3 158.2 45. 2 \ V— J ¥ V _/ 169. 5 203.4 A stem exudate study (Table 22) established that Saginaw transfers greater quantities Of sap which contain higher concentrations 94 of Zn than Sanilac. This difference in sap volume possibly could account for the greater movement of Zn into the leaves Of Saginaw plants if Zn moved by a mass flow mechanism which is a function of sap-flow rate (4). Almost all of the radioisotope work has indicated that a higher percentage of radioactive Zn is transferred into the shoots of Saginaw than Sanilac. Time-course experiments indicated that Saginaw had a higher proportion of 652n in the leaves than Sanilac (Table 24). The major proportion Of this difference appeared to be due to greater tranSport out Of the root, and tO a slight degree, less binding of Zn by the stems of Saginaw (Table 25). It is apparent that Saginaw definitely translocates more of the Zn taken up by the root into the aerial portion of the plant than Sanilac does. This difference in translocation of Zn may be related to the tolerance Of Saginaw to low Zn conditions. While Saginaw generally translocated a higher proportion of Zn to the leaves than Sanilac, there is ample evidence that Sanilac under some conditions took up more Zn than Saginaw (Table 24). When the Zn in the nutrient solution is increased, Sanilac takes up a greater quantity of Zn (Table 28). The difference in uptake between the varieties is magnified by growing the plants at initially high Zn concentrations (5. Oppm), (Table 27). This indicates that the sensitivity of Sanilac at the 5. 0 ppm level of Zn may be due to a much greater 95 uptake Of Zn by the roots. Although reciprocal grafts indicated that the Shoots of the plants determine tolerance or sensitivity to high Zn, mineral analysis of grafted plants indicated that root uptake may be affected by the upper portion Of the plant (Table 21). The difference in tolerance of the two varieties to high Zn levels could also be due to a difference in the ratio Of extracellular to intracellular Zn within the leaves. 65Zn uptake studies (unpublished) utilizing isolated leaf cells (67) have suggested that there may be a Significant difference between membrane properties within leaf cells of the two varieties. Table 30 illustrates the greatly enhanced level of Zn uptake by cells of Sanilac from plants grown at 5. 0 ppm Zn for one month. TABLE 30. -- 65Zn uptake in four hours by leaf cells isolated enzymatically from two navy bean varieties grown at two levels of Zn. Variet Zn level mM Zn/mg y (ppm) cells (dry weight) . a San1lac 0. 05 0. 57 Sanilac 5. 0 9. 94 Saginaw 0. 05 0. 64 Saginaw 5 . 0 0. 69 aAverage of four replications. 96 Little physiological work has been done with these two varieties grown at low or deficient levels of Zn due to the difficulty of eliminating Zn contamination with the present facilities. Never- theless, uptake and distribution of Zn by Saginaw and Sanilac plants grown at normal (. 05 ppm) and high (5. 0 ppm) Zn concentrations dif- fer markedly. Also, there may be a great difference in distribution at low levels of Zn. A summary of the physiological differences in Zn utiliza- tion between Saginaw and Sanilac is given in Table 31. Although some of the differences between the two varieties noted appeared small, they could be significant because most of the experiments were conducted over a relatively short period Of time. In the field or greenhouse, small differences between the two varieties may be greatly magnified with increasing time. 1. Models These differences can be used as an aid in postulating physiological models which could account for the differential tolerance Of Saginaw and Sanilac at the two levels of Zn. Either a single mecha- nism or possibly different mechanisms may be responsible for toler- ance at low and high Zn levels. Models for both possibilities will be proposed. 97 on 6389 .a 28E Hm .mH £3 3an. mm 3an mm 3an 2 8:9,”... mm .5 .mm .3 wins pm 6 N 3an on 3an am .pm .mm 3an ofifimoom maoonm SSE 30H 33 SSE 83 @3339: «on mo>m3 5 mmoH H.233 muoosm 5.3 SE 5.3 .533 33 UGHO TH ammh 3:82 5 OAOE 3980 EN 5.3 fits hzmwooamov madmafiaoaxo wcfimmpw pow Eoflfifionofia E cowfimoafioo amazoaofim qoflmfiqooqoo 35355 08304, 3839mm 39$: 38.6: E :N mo oxmpaD mamas :N am“: no.“ Ema .HO wcfiqofiwpaooopm . mfimbofl an 5.3 an 39% .832ch an 56 562559555 mocoamfiom Owflcmm BmEmmm c030 mom 039530 «swab .GOESNSSS SN 3 aooamop £33 mcmoo. b5: mo won—039’ Omfidwm paw Bmcwmmm of :ooafioo. mofimflgownwno HwmeoHofimmna Hanosbm mo comfiamaaoo «<4 I- .3 mamdwrfi 98 MODEL 1. -- Differential Membrane Permeability. Conditions: 1. Same mechanism for resistance of Saginaw at high and low levels of Zn. 2. Same genetic control. Characteristics: 1. Greater membrane permeability in Sanilac going from outside to inside (plasma-lemma and tonoplast) throughout the whole plant. 2. Less leakage from membranes of Sanilac (inside to outside). 3. NO difference in anatomy or morphology of the two varieties. Consequences Of Model: Property Saginaw Sanilac 1. Vascular and cytoplasmic accumulation low high 2. Total Zn uptake lower higher 3. Release of Zn into stelar tissue from symplasm (30, 73) high low 4. Water movement into xylem (if membrane properties Similar for other ions as for Zn) high low End Result: 1. At low Zn levels Sanilac has the capacity of taking up more Zn but transports much less to the top of the plant because a lower percentage of Zn is released into the xylem due to greater cellular accumu- lation Of Zn in the roots and possibly stem. Under low levels of Zn availability, Zn uptake is low in both genotypes, but the plants of the Saginaw variety have the capacity to mobilize more Zn to the portions Of the plant where there is a requirement for this ion. 2. At high Zn levels Much greater uptake of Zn and greater cellular accumulation by Sanilac. Due to the high ex- ternal concentration, relatively high levels of Zn are tranSported via the xylem. Due to the more limited permeability of the cells of the Saginaw variety, toxic levels of Zn do not build up within the cells of Saginaw, but do in Sanilac. 99 MODEL 11. -- Changing Membrane Permeability. Conditions: 1. Same mechanism acting differently at low and high levels of Zn. 2. Same genetic control. Characteristics: 1. Membrane of Saginaw moderately permeable and relatively con- stant over a broad range of Zn levels. 2. The membranes Of Sanilac have limited permeability at low levels of Zn. As Zn concentration increases, membrane permeability increases greatly. Consequences: Property Saginaw Sanilac 1. Total Zn uptake fluctuates little greatly 2. Cellular accumulation Of Zn fluctuates little greatly End Result: 1. At low Zn levels Saginaw plants probably take up greater quan- tities of Zn due to greater permeability of cortical cell membranes at low Zn concentra- tions. This uptake would be into the cytoplasm and movement of greater quantities Of Zn into the stelar tissue would occur. Thus there results the greater ability Of Saginaw to mobi- lize Zn into plant parts under conditions Of Zn stress. 2. At high Zn levels Zn uptake and movement into the shoots Of the plants is much greater in Sanilac than Saginaw due to increased permeability of membranes within the root cells. In addition, much higher levels of Zn accumulate intracellularly in the leaves of Sanilac than in Saginaw due to the greater permeability Of the membranes in the leaf cells. Higher levels Of Zn build up inside the cells Of Sanilac than in Saginaw due to greater uptake by cells Of the roots and leaves. 100 MODEL III. -- Translocation and Membrane Permeability. Conditions: 1. Two different mechanisms involved at low and high levels of Zn. 2. Two different genetic control systems. Characteristics: 1. Limited capacity of Sanilac to transfer Zn to Shoots Of the plants. 2. Limited membrane transport Of leaf cells of Saginaw. Consequences: Property Saginaw Sanilac 1 . Total Zn uptake adequate adequate 2. Zn transfer adequate inadequate at low con- centrations 3. Leaf cell accumulation of Zn adequate, not but restricted restricted at high Zn concen- trations End Result: 1. At low Zn levels Both varieties take up sufficient Zn, but due to the limited capacity of Sanilac to tranSport Zn, only Saginaw grows at a normal or near normal level. Sanilac plants suffer from Zn deficiency. 2. At high Zn levels Although Sanilac plants restrict Zn transport to a certain extent, toxic levels of Zn still reach the shoots Of the plants. Due to high permeability Of leaf cells of Sanilac, toxic levels of Zn accumulate. Saginaw plants re- strict the accumulation Of Zn in the leaf cells and are not affected to the same degree as Sanilac plants by the high level of Zn. 101 Three models are suggested by the data Obtained in these studies. There may be other models which are closer to the actual mechanism(s). However, a discussion Of the models presented may be useful in more clearly defining the problem and in leading to more critical experiments. The Observation that Saginaw transfers higher percentages of Zn to the shoot is in agreement with Model I. In addition, greater membrane permeability of Sanilac may account for the greater uptake of Zn that was Observed in the radioactive and mineral analysis work. If there is less leaka‘gefrom the symplasm, there would be lower concentrations Of Zn in the exudate of Sanilac (as Observed). These factors would all impart differential tolerance to low Zn conditions. The model suggests that high Zn tolerance is located in the shoot (as was indicated by the grafting experiments). It also indicates the possibility of high Zn accumulation by Sanilac plants, as external concentration is increased, due to the higher membrane permeability. This was Observed in the radioisotope studies (Tables 26 and 27). Model II is based on the assumption that membrane per- meability to Zn in Sanilac increases with increasing external Zn con- centration. This could be accounted for by changes in membrane permeability to Zn per se or to increased numbers of carrier 102 sites or an increased rate Of turnover Of the carrier-Zn com- plex. Zn uptake by Saginaw does not exhibit fluctuation in uptake and accumulation Of Zn with external Zn concentration, at least not to the same extent as Sanilac- If overall permeability of the cells increases in Sanilac with increasing concentrations of Zn or, perhaps, other ions, Sani- lac may be subject tO micronutrient unbalances while Saginaw is not (Table 29). Sanilac would also be susceptible to high external levels Of Zn which may result in accumulation of very toxic concentrations of Zn in Sanilac cells but not Saginaw (Table 30). The third model is based on the premise that there is a specific mechanism responsible for the differential reaction Of the two varieties at low levels of Zn and another mechanism at high levels of Zn and that these two mechanisms are under different genetic con- trol. The characteristics of this model include limited transport of Zn ions to the tops of Sanilac plants. Such a limited transport could be due to slower movement Of sap due to less cross-sectional area of xylem, more restricted end walls in tracheids and vessels, greater restriction to movement at nodal areas, and perhaps other factors which provide greater opportunity for immobilization Of Zn in root and stem tissue. Plants treated with a low amount Of radioactivity 103 for 12 hours revealed that nodes do constitute a barrier to xylem transport of Zn (Figure 12). In addition to or instead of restricted FIGURE 12. -- Radioautograph Of Saginaw navy bean (Sanilac the same) exposed to 65Zn for 12 hours. l L movement of sap, root and/or stem cells of Sanilac may also pos- sess greater binding capacity. Either possibility would decrease net movement of Zn to the shoots of Sanilac plants and could be respon- sible for the differential reaction of Saginaw and Sanilac at low Zn. At high Zn levels, leaf cells of Saginaw either do not accumulate toxic levels of Zn or these cells are capable Of carrying out normal metabolic functions in the presence of higher Zn levels than cells of Sanilac. 104 Although there may be other factors involved in the dif- ferential reaction of the two varieties, differential root uptake seems to be eliminated. Sanilac takes up nearly as much or more Zn than Saginaw at normal levels, and the genotype Of the shoots has been shown to be the determining factor at toxic levels of Zn (Table 31). Differential rates of movement within the symplasm have not been discussed, but could possibly account for the greater accumulation of Zn by the roots Of the Sanilac variety. Also, differential metabolism and differential accumulation of Zn within the cell organelles could be involved. In addition, the properties of the sap of the two varieties may be altered by the differential production Of organic acids (19, 57). In this instance, there may be great differences between the two varieties in solubility of Zn in the sap at various levels of exter- nal Zn. None Of these have been investigated in this study. 11. Gene Action Models have been presented which explain the behavior of the two varieties Of beans at low and high Zn concentrations. Re- gardless Of the physiological and genetic models which ultimately prove correct, the question of the nature Of the genetic control must be raised. It has been concluded from this investigation that at least two gene pairs interact to impart the tolerance to low Zn levels 105 Observed in Saginaw plants. These genes either govern critical enzymatic steps in a common biosynthetic pathway leading to an end product responsible for low Zn tolerance or produce different poly- peptide chains which combine tO form an active enzyme which directly or indirectly confers tolerance. Another alternative is that the vari- ous genes act to produce different non-related enzymes. In this instance, the interaction Of phenotypic characters which result from separate genes is necessary for tolerance. The first two possibilities involve biochemical interaction to produce a tolerant phenotype, while the last alternative involves "gene-product" interaction to produce the tolerant phenotype. The mode of action of the genes in the first instance wOuld give either tolerant or sensitive plants. However, if both genes are responsible for separate phenotypic characters, (as in the final alternative above), each or one of the character(s) may be responsible for a small amount of tolerance to low Zn. Under conditions of slight deficiency, these single genes for tolerance may be expressed. This may be the source Of some Of the confusion with the 1966 genetic data which were Obtained when Zn deficiency conditions were not very severe. III. Plant B reeding The first prerequisite in any plant breeding program is the establishment Of precise, realizable goals. With respect to 106 utilization of nutrients, goals have not been well established. Indeed, there has been little work on which characteristics are or are not desirable. Perhaps this is due to a lack Of knowledge concerning the complexities Of nutrient utilization. To oversimplify the total con- cept, nutrient utilization can be thought of as consisting Of uptake, transport, and intracellular utilization. Ideally these three compo- nents can be brought under genetic control and manipulated SO that plants may have an ability to withstand micronutrient unbalances at toxic and deficient ends of the scale. At the same time, these plants should have the ability to utilize efficiently higher quantities of nutri- ent to produce greater yields than they are now able to do. Simply put, the goals the plant breeder should establish are (1) a general adaptability with a high buffer capacity and (2) a specific ability to utilize high levels Of nutrients when such are available. It may not be possible to develop varieties that meet both Of these Specifications. Overall tolerance to micronutrient balance implies that a plant is able to maintain a favorable internal ionic re- lationship when the external relationship is unfavorable. Thus higher levels Of nutrients and other ions do not affect these plants. For this reason, plants may not be able to utilize high levels of fertilizers. The plant breeder may have to decide whether he desires a variety with a wide range of adaptability to nutrient levels or whether he wants a variety capable Of utilizing high levels of fertilizer. 107 With respect to a breeding program for Zn utilization, decisions regarding goals will have to depend upon the nature Of the tolerance of Saginaw to low and high Zn. Certainly tolerance to low Zn levels, and ability to maintain balance in the presence of external ion unbalance, would seem to be desirable. However, the ability to withstand high levels of Zn may not be desirable. Judy §_t_al_. (66) have shown that Sanilac responds to additional Zn fertilization (in the intermediate range), as indicated by seed yield to a greater extent than Sagi- naw. Sucha reSponse may be related to the sensitivity of this variety to the toxic-high level of Zn. If this were the case, such a sensitivity would be desirable because toxic levels Of Zn are rarely encountered in the field and the plants could utilize higher levels Of Zn fertilization for higher production. Saginaw, on the other hand, due to the mecha- nism that conditions its ability to withstand extreme levels of Zn, is limited in its ability to utilize moderately higher levels of Zn. The actual mechanism involved will be of significance in the formulation Of the goals Of the program. If there is only one mechanism at both Zn levels, with the same genetic control, the de- cision will have to be made whether to breed for tolerance to low Zn or for greater yield reSponse to high Zn fertilization. If there are two different mechanisms, it will be possible to Obtain plants with low Zn tolerance and response to high Zn 108 fertilization. It will be necessary to select for recombinant plants having the tolerance to low Zn of Saginaw and the sensitivity to high Zn of Sanilac. SUMMARY AND CONCLUSIONS The differential ability Of the Saginaw and Sanilac navy beans to withstand Zn stress was investigated by genetic, micro- nutrient balance, and physiologic experimentation. The conclusions from these investigations are summarized in the following paragraphs. 1. Genetic A. Low Zn Populations of Fl' 3, F 's, F 's, and backcrosses Of 2 3 Saginaw x Sanilac when grown on low Zn soil (Saginaw County) in 1964 and 1966 responded differentially to Zn stress. Individual plants were scored on a 1 to 5 scale on the basis Of visual Zn deficiency symptoms. Due to overlap of Saginaw and Sanilac populations in several classes, the segregating generations were partitioned into tolerant and sensi- tive genotypes based on the performance Of Saginaw and Sanilac, respectively. The F1 generation indicated that tolerance tO low Zn was phenotypically dominant. Tolerance appeared to be mediated by two genes which gave a ratio of nine tolerant to seven sensitive in the F2 109 110 generation. The F3 generation was not entirely in agreement with the 9:7 ratio as there were more tolerant families than predicted. The backcross to Sanilac population was not conclusive, and more tolerant plants than expected were Observed. It was concluded that the number Of genes determining tolerance to low Zn is low, probably two. These genes apparently interact to give a phenotypic ratio of 9:7 (tolerant to sensitive) in the F2 generation. However, this ratio may not be inviolate. These genes may govern the synthesis of various product combinations which impart various degrees of tolerance to low Zn. Depending upon available Zn and the threshold level within the plant, these combina- tions may interact to produce an array Of phenotypic ratios. The 9:7 ratio may be an average Of this array. The F population may have a 3 different threshold level due to a different level of background homo- zngSity. B. High Zn In addition to differential tolerance Of Saginaw and Sanilac navy beans to low Zn levels, a differential response to 5. 0 ppm Zn (100 X normal) was Observed in the greenhouse. It was not possible to establish the number Of genes or gene action for this trait. The F1 generation indicated that sensitivity was dominant or that there 111 was no dominance. The F2 population distribution was characteristic of dominance for tolerance, or possibly indicated partial dominance. A ratio of 9 tolerant to 7 sensitive plants was observed in the F2 which, if true, may indicate that tolerance to low and high Zn levels is mediated by the same genetic control system. II. Micronutrient Balance Studies—-Zn, Cu, Fe, and Mn The effect of various levels Of Zn, Cu, Fe, and Mn upon the growth of Saginaw and Sanilac navy beans in sand culture in the greenhouse was studied. In addition, the accumulation Of these four elements in plant parts was determined. Zn at 5. 0 ppm caused marked stunting of Sanilac, whereas growth Of Saginaw was little affected. This differential growth did not seem to be related to differential uptake since both varieties accumulated high levels Of Zn. Cu induced a differential Fe-chlorosis which could be corrected by adding Fe. Sanilac was more sensitive to Cu in this respect than Saginaw. It was postulated that the effect of low and high Zn and high Cu may be due to the same physiological or ana- tomical mechanism. 112 III. Physiological Studies A. Low Zn Exudate experiments established that Saginaw transferred greater volumes of plant sap during a 30-hour period than Sanilac. In addition, the Zn concentration in the exudate was approximately three times greater in Saginaw. Whether this difference in Zn con- centration was due to greater Zn uptake by Saginaw roots or to greater transfer Of Zn into the vascular tissue is not known. Radioisotope studies established that Sanilac plants normally take up higher levels of Zn but transfer small proportions Of this Zn to the top of the plant. This indicates that the high con- centration of Zn in the exudate Of Saginaw is due to greater release Of Zn into the vascular-tissue. B. High Zn Reciprocal scion/root grafts Of Saginaw and Sanilac grown in high Zn established that tolerance to this level of Zn is mediated by the genotype of the scion. Differential accumulation did not account for the differential tolerance of low Zn. Unpublished data, reported in the discussion, indicated that tolerance at high Zn may be due to a difference in the distribution Of extra- and intra-cellular Zn in the 113 two varieties. Isolated leaf cells of Sanilac took up much more Zn at high external concentrations than did Saginaw. IV. Discussion Three models were presented as possible mechanisms for the differential tolerance of the two varieties of navy beans. The first two models involved differential membrane or cellular proper- I ties which account for differential release of Zn into the vascular tis- I; sue and also accumulation Of different levels of Zn within the cell. On “— the basis of these two models it was postulated that the same basic mechanism was reSponsible for the observed reSponses at high and low Zn. The third model was characterized by differential stem and/ or root binding which limited the availability of Zn to Sanilac at low external concentrations. At high Zn levels, the cells of Sanilac were postulated as being more permeable than Saginaw and thus more liable to accumulate toxic levels of Zn while the cells Of Saginaw are not. Gene action and possible breeding programs for nutrient utilization were discussed . 10. LIST OF REFERENCES Adams, M. W. 1967. Personal communication. Anderson, Axel L., E. E. Down, and Gerald Whitford. 1960. The Sanilac pea bean: Its history, development and characteristics. Mich. Agr. Exp. Sta. Quart. Bull. 43: 214-236. I I Beadle, G. W. 1929. Yellow stripe: A factor for chlorOphyll i deficiency in maize located in the Pr pr chromosomes. ,__ Amer. Naturalist. 63: 189-192. Bell, C. W., and O. Biddulph. 1963. TranSport Of calcium. Exchange versus mass flow. Plant Physiol. 38: 610-614. Bell, W. D., L. Bogorad, and W. J. McIllrath. 1962. Yellow- stripe phenotype inmaize: 1. Botan. Gazette 124: 1-8. Bell, W. C., L. Bogorad, and W. J. McIllrath. 1958. Response of the yellow stripe maize mutant (ysl) to ferrous and ferric ion. Botan. Gazette 120:36-39. Berger, Kermit C. 1962. Micronutrient deficiencies in the U. S. Jour. of Agric. and Food Chem. 10: 178-181. Biddulph, O. 1953. ‘ Translocation of radioactive mineral nutri- ents in plants. Kansas Agr. Exp. Sta. Rpt. 4: 48-58. Biddulph, O., Susann Biddulph, R. Cory, and H. Koontz. 1958. Circulation patterns for phosphorus , sulfer, and calcium in the bean plant. Plant Physiol. 33:293-299. Bingham, Frank T. , and James P. Martin. 1956. Effects of soil phosphorus on growth and minor element nutrition Of citrus. Soil Sci. Soc. Amer. Proc. 20: 382-385. 114 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 115 Bingham, F. T., and M. J. Garber. 1960. Solubility and availability of micronutrients in relation to phosphorus fertilization. Soil Sci. Soc. Amer. Proc. 24: 209-213. Boawn, Louis C. 1965. Zinc concentration in hOp leaves in relation to adequate and inadequate levels of zinc nutri- tion. Agron. Jour. 57: 523-524. Boawn, Louis C. , Frank G. Viets, Jr. , and Carl L. Crawford. 1954. Effect of phosphate fertilizer on zinc nutrition of field beans. Soil Sci. 78: 1-7. Brown, J. C. 1967. Differential uptake Of Fe and Ca by two corn genotypes. Soil Sci. 103: 331-338. Brown, J. C. 1966. Fe and Ca uptake as related-to root-sap and stem exudate citrate in soybeans. Physiol. Plantarum. 19: 968-976. Brown, J. C. 1963. Iron chlorosis in soybeans as related to the genotype Of root stock; 4. Sorption and translocation as 2 separate phases of iron nutrition. Soil Sci. 96: 387-394. Brown, J. C., R. S. Holmes, and L. O. Tiffin. 1961. Iron chlorosis in soybeans as related to the genotype of root stalk: 3. Chlorosis susceptibility and reductive capacity at the root. Soil Sci. 91: 127-132. Brown, J. C., R. S. Holmes, and L. O. Tiffin. 1958. Iron chlorosis in soybeans as related to the genotype of root- stock. Soil Sci. 86: 75-82. Brown, J. C., and L. O. Tiffin. 1965. Iron stress as related to the iron and citrate occurring in the stem exudate. Plant Physiol. 40: 395-400. Brown, J. C., and L. O. Tiffin. 1962. Zinc deficiency and iron chlorosis dependent on the plant species and nutrient ele- ments in Tulare Clay. Agron. Jour. 54: 356-358. :- I-mwxrixn am tour" I. I'm“ m ‘1 7 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 116 Brown, J. C., L. O. Tiffin, and R. S. Holmes. 1960. Compe- tition between chelating agents and roots as factor affect- ing absorption of iron and other ions by plant species. Plant Physiol. 35: 878-886. Brown, J. C., C. R. Weber, and B. E. Caldwell. 1967. Effi- cient and inefficient use of iron by two soybean genotypes and their isolines. Agron. Jour. 59: 459-462. r___ Burleson, C. A., A. D. Dacus, and C. J. Gerard. 1961. The Sf effect Of phosphorus fertilization on the zinc nutrition Of “.7 several irrigated crops. Soil Sci. Soc. Amer. Proc. 25: 365-368. Butler, G. W., P. C- Barclay, and A. C. Glenday. 1962. Genetic and environmental differences in the mineral composition of ryegrass herbage. Plant and Soil. 16: 214-228. Camp, A. F. 1945. Zinc as a nutrient in plant growth. Soil Sci. 60: 157-164. Chapman, H. D., G. F. Liebig, Jr., and A. P. Vanselow. 1939. Some nutritional relationships as revealed by a study of mineral deficiency and excess symptoms on citrus. Soil Sci. Soc. Amer. Proc. 4: 196-200. Chesters, C. G. C., and G. N. Robinson. 1951. The role of zinc in plant metabolism. Biol. Rev. 26: 239-252. Collander, Runar. 1941. Selective absorption of cations by higher plants. Plant Physiol. 16: 691-720. COOil, Bruce J. , Rollo K. Le Da Fuente, and Ramon S. De La Pena. 1965. Absorption and transport of sodium and potassium in squash. Plant Physiol. 40: 625-632. Crafts, A. S. 1961. Translocation in plants. Holt, Rinehart, and Winston. New York. DeRemer, E. Dale, and R. L. Smith. 1964. A preliminary study on the nature Of a zinc deficiency in field beans as determined by radioactive zinc. Agron. Jour. 56: 67-70. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 117 Doutt, Margaret T. 1932. Anatomy of Phaseolus vulgaris L. var. Black Valentine. Technical Bulletin NO. 128, Michigan State College, Agricultural Experiment Station. Ellis, Boyd G. 1965. ReSponse and susceptibility: In "Zinc Deficiency-~a symposium." Crops and Soils. 17: 10-13. Ellis, Roscoe, Jr., J. F. Davis, and D. L. Thurlow. 1964. Zinc availability in calcareous Michigan soils as influ- enced by phosphorus levels and temperature. Soil Sci. 28: 83-86. Epstein, E. 1961. The essential role of calcium in selective cation transport by plant cells. Plant physiol. 36: 437- 444. Epstein, Emanuel, D. W. Rains, and O. E. Elzam. 1963. Resolution of dual mechanisms of potassium absorption by barley roots. Proc. Natl. Acad. Sci. 49: 684-692. Epstein, Emanuel, and R. L. Jefferies. 1964. The genetic basis Of selective ion tranSport in plants. Ann. Rev. Plant Physiol. 15: 169-184. Findenegg, G. , and E. Broda. 1965. Mechanisms Of uptake of trace elements by plant roots. Nature. 208: 196-197. Foy, C. D., W. H. Armiger, A. L. Fleming, and C. F. Lewis. 1967. Differential tolerance Of cotton varieties to an acid soil highin exchangeable aluminum. Agron. J our. 59: 415-418. Foy, C. D., and J. C. Brown. 1964. Toxic factors in acid soils: II. Differential aluminum tolerance in plant species. Soil Sci. Soc. Amer. Proc. 28: 27-32. Foote, Beverly D., and Robert W. Howell. 1964. Phosphorus tolerance and sensitivity of soybeans as related to uptake and translocation. Plant Physiol. 40: 610-613. Fuerhing, H. D. 1960. Interrelationships of the trace elements zinc, boron, iron, manganese, and copper on the growth and composition of corn. Ph.D. Thesis, University Of Nebraska. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 118 Fuering, H. O., and G. S. Soofi. 1964. Nutrition of corn ona calcareous soil: II. Effect of Zn on the yields of grain and stover in relation to other micronutrients. Soil Sci. 28: 79-82. Giordano, P. M., J. J. Mortnedt, and R. I. Papendich. 1966. ReSponse of corn (Zea mays L. ) to zinc, as affected by placement and nitrogen source. Soil Sci. Soc. Amer. Proc. 30: 767-770. Gerloff, G. C. 1963. Comparative mineral nutrition of plants. Ann. Rev. Plant Physiol. 14: 107-124. Gorsline, G. W., J. W. Ragland, and W. I. Thomas. 1961. Evidence for inheritance Of differential accumulation of calcium, magnesium, and potassium by maize. Crop Sci. 1: 155-156. Gorsline, G. W., W. 1. Thomas, and D. E. Baker. 1964. Inheritance of P, K, Mg, Cu, B, Zn, Mn, Al, and Fe concentration by corn (Zea mays L. ) leaves and grain. CrOp Sci. 4: 207-210.“ Gorsline, G. W., W. 1. Thomas, D. E. Baker, and J. L. Rag- land. 1964. Relationships of strontium-calcium accu- mulation within corn. Crop Sci. 4: 154-156. Gouch, Hugh G. 1957. Mineral nutrition Of plants. Ann. Rev. Plant Physiol. 8: 31-64. Grossenbacher, K. A. 1939. Autonomic cycle of rate Of exuda- tion of plants. Amer. Jour. Botany. 26: 107-109. Grimes, D. L., L. C. Boawn, C. W. Carlson, and F. G. Viets, Jr. 1961. Zinc deficiency of corn and potatoes as related to soil and plant analysis. Agron. Jour. 53: 68-71. Hanson, J. B. 1967. Roots-—Selecters Of plant nutrients. Plant Food Review 13: 8-10. > Harvey, Paul H. 1939. Hereditary variation in plant nutrition. Genetics 24: 437-461. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 119 Hewitt, E. J. 1963. The essential nutrient elements: Require- ments and interactions in plants. In F. C. Steward, Ed. Plant Physiology--A Treatise. Vol. III. Inorganic Nutri- tion Of Plants. Pp. 137-360. Academic Press. New York. Hewitt, E. J. 1951. The role of mineral elements in plant nutri- tion. Ann. Rev. Plant Physiol. 2: 25-52. Hewitt, E. J. 1948. Relation of manganese and some other metals to the iron status Of plants. Nature 161: 489- 490. Hiatt, A. J. 1967. Relationship of cell sap pH to organic acid change during ion uptake. Plant Physiol. 42: 294-2 98. Hiatt, A. J., and H. F. Massey. 1958. Zinc levels in relation to zinc content and growth Of corn. Agron. Jour. 50: 22-24. Hoagland, D. R., and D. I. Arnon. 1950. The water-culture method for growing plants without soil. Calif. Agr. Exp. Sta. Bull. 347. Howell, R. W. 1954. PhOSphorous nitrition of soybeans. Plant Physiol. 29: 477-483. Hunter, J. G., and O. Vergnano. 1953. Trace element toxic- ities in oat plants. Ann. Appl. Biol. 40: 761-777. Jacobsen, Louis, Raymond J. Hannapel, David P. Moore, and Michail Schaedle. 1961. Calcium influence on ion ab- sorption. Plant Physiol. 39: 58-61. Jacoby, B. 1964. Function of bean roots and stems in sodium retention. Plant Physiol. 39: 445-449. Johnson, W. J., and W. G. Schrenk. 1964. Nature of zinc containing substances in the alfalfa plant cell. Jour. of Agric. and Food Chem. 12: 210. Judy, W. H. 1967. Zinc availability from SOil applied zinc sulfate and zinc EDTA. Ph. D. Thesis, Michigan State University. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 120 Judy, W., J. Melton, G. Lessman, B. Ellis, and J. Davis. Field and laboratory studies with zinc fertilization of pea beans, corn, and sugar beets in 1964. M.S.U. EXpt. Sta. Res. Rpt. 33. Farm Science. Jyung, W. H., Sylvan H. Wittwer, and Martin J. Bukovac. 1965. Ion uptake by cells enzymatically isolated from green tobacco leaves. Plant Physiol. 40: 410-414. Jyung, W. H., S. H. Wittwer, and M. J. Bukovac. 1965. Ion uptake and protein synthesis in enzymatically isolated plant cells. Nature 205: 921-922. Kannan, Sheshadri. 1966. Mechanisms Of foliar absorption in higher plants with special reference to iron. Ph.D. Thesis, Michigan State University. Kleese, R. A. 1967. Relative importance of stem and root in determining genetypic differences in Sr-89 and Ca-45 accumulation in soybeans (Glycine max L. ). Crop Sci. 7:53-55. Lessman, Gary Max. 1967. Zinc-phosphorus interactions in Phaseolus vulgaris. Ph. D. Thesis, Michigan State Uni- versity. Lingle, John C., Lee O. Tiffin, and John C. Brown. 1963. Iron-uptake-tranSport of soybeans as influenced by other cations. Plant Physiol. 38: 71-76. Luttge, Ulrich, and George G. Laties. 1966. Dual mechanism of ion absorption in relation to long distance transport in plants. Plant Physiol. 41: 1531-1539. MacLeOd, L. B., and L. P. Jackson. 1967. Aluminum toler- ance of two barley varieties in nutrient solution, peat, and soil culture. Agron. Jour. 59: 359-363. Martin, W. E., J. G McLean, and James Quick. 1965. Effect of temperature on the occurrence of phosphorus induced zinc deficiency. Soil Sci. Soc. Amer. Proc. 29: 411- 413. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 121 Massey, H. F. , and Frank A Loeffel. 1967. Factors in inter- strain variation in zinc content of maize (Zea mays L. ) kernels. Agron. Jour. 59: 214-217. Massey, H. F., and Frank H. Loeffel. 1966. Variation in zinc content of grain from inbred lines Of corn. Agron. J our. 58: 143-144. Millikan, C. R. 1953. Relative effects Of zinc and copper defi- ciencies on lucerne and subterranean clover. Aust. Jour. Biol. Sci. 6: 164-177. Nelson, Lyle E. 1956. Response of soybeans grown in the green- house tO zinc applied to a black belt soil. Soil Sci. 82: 271-274. Pardee, A. B. 1966. Purification and properties of a sulfate- binding protein from Salmonella typhimurium. J our. Biol. Chem. 241: 5886. Pardee, A. B., L. S. Prestidge, M. B. Whipple, and J. Drey- fuss. 1966. A binding site for sulfate and its relation to sulfate transport into Salmonella typhimurium. J our. Biol. Chem. 241: 3962. Pinkas, Leonard L. H., and Lawrence H. Smith. 1966. Physio- logical basis of differential strontium accumulation in two barley genotypes. Plant Physiol. 41: 1471-1475. Pope, Daniel T. , and Henry M. Munger. 1953. Heredity and nutrition in relation to magnesium-deficiency chlorosis in celery. Proc. Amer. Soc. Hort. Sci. 61: 472-480. Pope, Daniel T. , and Henry M. Munger. 1953. The inheritance Of susceptibility to boron deficiency in celery. Proc. Amer. Soc. Hort. Sci. 61: 481-486. Powers, LeRoy, and L. F. Locke. 1950. Partitioning method of genetic analysis applied to quantitative characters of tomato crosses. U. S. D. A. Technical Bulletin NO. 998. Rasmussen, D. C., L. H. Smith, and R. A. Kleese. 1964. Inheritance Of SR89 accumulation in wheat and barley. Crop Sci. 4: 586-589. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 122 Rasmussen, D. C., L. H. Smith, and W. M. Meyers. 1963. Effect Of genotype on accumulation Of Sr in barley and wheat. Crop Sci. 3: 34-37. Rediske, J. H., and O. Biddulph. 1953. The absorption and translocation Of iron. Plant Physiol. 28: 576-593. Rosell, Ramon A. , and Albert Ulrich. 1964. Critical zinc con- 1"“ centration and leaf minerals of sugar beet plants. Soil Sci. 97: 152-167. Sang, James H. 1964. Penetrance, expressivity, and thresh- olds. Jour. Hered. 49: 143-150. Schmid, Walter E. , and Gerald C. Gerloff. 1961. A naturally occurring chelate of iron in xylem exudate. Plant - -' Physiol. 36: 226-231. Seatz, L. F., A. J. Steiger, and J. C. Kramer. 1959. CrOp response to zinc fertilization as influenced by lime and phosphorus applications. Agron. Jour. 51: 457-459. Smith, Paul F. , and Alston W. Specht. 1953. Heavy metal nutrition and iron chlorosis of citrus seedlings. Plant Physiol. 28: 371-382. Smith, Stuart N. 1934. Response of inbred lives and crosses in maize to variation of nitrogen and phosphorus supplied as nutrients. Jour. Am. Soc. Agron. 26: 785-804. Sommer, Anna L. 1928. Further evidence Of the essential nature of zinc for the growth Of higher green plants. Plant Physiol. 3: 217-221. Steenbjerg, F. 1951. Yield curves and chemical plant analyses. Plant and Soil 3: 97-109. Stukenholtz, D. D., R. J. Olsen, Gerald Gogan, and R. A. Olson. 1966. On the mechanism of phosphorus zinc interaction in corn nutrition. Soil Sci. Soc. Amer. Proc. 30: 759- 763. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 123 Thorne, Wynne. 1957. Zinc deficiency and its control. Ad- vances in Agron. IX: 31-65. Tiffin, L. O. 1963. Plant acids and iron translocation. Plant Physiol. 38: p. ix. Tiffin, L. O. 1966. Iron translocation. 1. Plant culture, exudate sampling, iron-citrate analysis. Plant Physiol. 41: 510-514. Tiffin, L. O., and J. C. Brown. 1962. Iron chelates in soy- bean exudate. Science 135: 311-313. Tiffin, L. O., J. C. Brown, and R. S. Holmes. 1960. Chelat- ing agent and plant nutrient interactions affecting the iron nutrition of soybeans. Soil Sci. Soc. Am. Proc. 24: 120-123. Tsui, Cheng. 1948. The role of zinc in auxin synthesis in the tomato plant. Amer. Jour. Botany 35: 172-179. Viets, F. G. , Jr. 1961. Zinc deficiency Of field and vegetable crops in the western U. S. U. S. D. A. Leaflet 495. Viets, F. G., Jr., L. C. Boawn, and C. L. Crawford. 1954. Zinc content of bean plants in relation to deficiency symptoms and yield. Plant Physiol. 29: 76-79. Viets, F. G., Jr., L. C. Boawn, and C. L. Crawford. 1954. Zinc content and deficiency symptoms of 26 crops grown on a Zn-deficient soil. Soil Sci. 78: 305-316. Vose, P. B. 1963. Varietal differences in plant nutrition. Herbage Abstracts 33: 1-13. Wallace, A., R. A. Jeffreys, and V. Q. Hale. 1962. Differen- tial ability of two soybean varieties to take up Fe59 from soil added to a nutrient solution. Soil Sci. 94: 111-114. Ward, R. C., E. J. Langin, R. A. Olsen, and D. D. Stucken- holtz. 1963. Factors responsible for poor response Of corn and grain sorghum to phosphorus fertilization: III. Effect of soil compaction and moisture levels and other properties on phosphorus zinc relations. Soil Sci. Soc. Amer. Proc. 27: 326-330. 110. 111. 112. 124 Watanabe, F. S.,7 W. L. Lindsay, and S. R. Olsen. 1965. Nutrient balance involving phosphorus, iron, and zinc. Soil Sci. Soc. Amer. Proc. 29: 562-565. Weiss, Martin G. 1943. Inheritance of physiology Of efficiency in iron utilization in soybeans. Genetics. 28: 253-268. Wood, J. G., and Pamela M. Sibly. 1950. The distribution of zinc in oat plants. Australian Jour. Sci. Res. B3: 14- 27. fluid"! mm” .wxzn: Ind-H I _ . F V I in- IlIE 5. ..IIEI¢II:I .4: APPENDIX 126 TABLE 1. -- Analysis of variance for plant height, number of trifoliate leaves, and plant weight for Sanilac and Saginaw navy beans in micronutrient interaction experiment number one. Source Degrees Mean squares Of of variance freedom Plant height No. trif. Zn 2 14457. 5946** 2636. 8580** Cu 2 4931.2613*=i~' 411. 5617** Zn x Cu 4 1949. 6440 130. 8641** Fe 2 536.2119 111.7839** Zn x Fe 4 197.5390 56. 2901* Cu x Fe 4 127.3076 6.6234 Zn x Cu x Fe 8 153.2597 13.6712 Error A 54 341.1872 17.1378 Variety (Var) 1 5373.3662** 300.2551===* Var X Zn 2 1998.5205=1<* 88. 4711** Var x Cu 2 1479. 6687** 45. 9032** Var x Zn x Cu 4 672. 2304707: 26. 6748** Var x Fe 2 435.5699** 18.3971* Vaern x Fe 4 107.0390 14.1131* Vaerux Fe 4 82.3631 9.0637 Var x Zn x Cu x Fe 8 124. 4758* 9. 7659*< Date 2 4069.088 ** 593228351: Date x Var 2 2074. 7551** 36. 0020* Date x Zn x Var 4 534. 4465** 12. 3477* Date x Cu x Var 4 309.1779** 5. 2576 Date x Zn x Cu x Var 8 96.1934 1.9032 DatexFexVar 4 41.4773 2.5051 Date x Zn x Fe x Var 8 13.8029 0.9758 Date x Cu x Fe x Var 8 11.5853 1.7042 Date xanCu x Fe xVar 16 10.8253 1.1262 Error B 322 58.7189 4. 9095 Total 485 <.01 Level Of Significance: ** " .01 - .05 127 TABLE 1. -- Continued. Source Degrees Mean squares of Of variance freedom Weight tops Weight roots Zn 2 147. 5604** 40. 0385** Cu 2 62. 4965** 8. 2790** Zn x Cu 4 17. 2563*”? 2. 6503*"-< Fe 2 7.3119 1.6605 Zn x Fe 4 1. 6900 0.4804 Cu x Fe 4 2.3339 0.2762 Zn x Cu x Fe 8 1. 2065 0.1216 Error A 54 2.9127 0.6298 Variety (Vyal‘) 1 43. 867240;: 15. 3704*::: Var x Zn 2 21. 9579** 6, 305444: Var x Cu 2 7, 345144 1. 8819** Var x Zn x Cu 4 5. 3759203 1, 4424** Var x Fe 2 5. 9335** 1. 6278* Var xan Fe 4 1.5570 0.7153 Var x Cu x Fe 4 1 6748 0.4287 Var x Zn x Cu x Fe 8 0 9545 0.4989 Error B 54 0.7688 0.3519 Total 161 Level Of Significance: 53* : <. 01 Z" .01 — .05 128 TABLE 2(a). -- Analysis of variance of plant height. number of trifoliate leaves. and plant weights for Sanilac and Saginaw navy beans in micronutrient interaction experiment number two. Mean squares Source Degrees varliznce fregdom 13:13 [:35 No. trif. Height Zn 2 18.3792“ 46.6669Ml 135.0069* 5922.2500“ Fe 1 5.6406 0.6869 8.0277 1.7777 Zn 1: Fe 2 7. 2552‘ 0. 9552 24. 4652 212.6944 Mn 1 4.8767 4.2025' 144.0000* 720.0277' anMn 2 0.5134 1.6608 1.2708 152.1111 Fe x Mn 1 0.1167 0. 2025 16.0000 9. 0000 Zn x Fe x Mn 2 2.4529 3.10087 8.3125 40.0833 Cu 1 7.7006‘ 1.0000 0.6944 240.2500 Zn x Cu 2 4.8939 1.6758 1.5486 540.7500 FexCu 1 4.5156 1.5211 1.3611 136.1111 Zn x Fe x (‘u 2 2. 8952 1.4044 28.4236 168. 6944 Mn 1:01 1 1.0850 0.1111 93.4444’ 261.3611 Zn x Mn x Cu 2 0.1384 0.1169 6. 7569 36.1944 Fe x Mn xCu 4.4450 1.3611 106.7777‘ 113.7777 anFe an xCu 2 10.78884 4.4744* 80.2152' 954.1111“ ErrorA 48 2.7484 1.2115 32.4305 241.0763 Variety (Var) 1 5. 8000* 2. 3002 841. 0000’” 9409. 0000‘” Var xZn 2 2.4575' 0.6786 36.2708** 460.3333’? Vaere 1 0.2100 1.8225 11.1111 0.6944 Var x Zn x Fe 2 0.2046 0.8258 0. 9236 17.6944 Var x Mn 1 0.7656 0.1736 90. 2500MI 693. 4444* Vaerann 1 0.1814 0.7436 11.3125 25.8611 Var x Fe x Mn 1 1.1917 1.1736 30. 2500* 14.6944 Var x Zn x Fe x Mn 2 0. 3904 0.3586 20. 2708* 218. 3611 Var x Cu 1 0.4556 0.3600 1.0000 2.7777 Var xZn x Cu 2 0.6231 0.8933 7.8958 136.6944 Var x Fe x Cu 1 0.0000 0.5877 0.4444 156.2500 Var x Zn x Fe x Cu 2 3. 6354* 0.0719 2.1319 171.0000 Var x Mn x Cu 1 0. 6267 0.1877 72. 2500'" 2. 7777 Var x Zn x Mn x Cu 2 2. 0867' 0.4477 3. 9375 108.6944 Var x Fe x Mn x Cu 1 0.5017 0.0044 30. 2500* 96.6944 Var x Zn x Fe x Mn x Cu 2 0. 5154 0.2002 5.1458 173.6944 Error B 48 0. 8540 0.8789 6.2638 102.8541 Total 143 Level of Significance: ' - .05 - .10 129 'I‘AHlJ-Z 2(1)). -- Analysis of variance of elemental accumulation in various plant parts of Saginaw and Sanilac navy beans in micronutrient interaction experiment number two. Mean squares Source Degrees of of Leaves variance freedom Mn Cu Zn Fe Zn 2 12498.4 189821.7** 6146996.2** 1248595.6** Fe 1 3383.3 724. 5 153860.0** 1218417207"? Zn x Fe 2 19479. 6 22038. 5* 72532. 6* 894994. 8** Mn 1 74020271“l 58282.05”?t 91657.5it 110833.5 Zn x Mn 2 8132.8 5517.8 74993.1?” 210419.9 Fe x Mn 1 11772. 2 4879.3 57081.1' 16362. 6 Zn x Fe x Mn 2 59286. 8' 8380.1 38994.1 240298. 0 Cu 1 72720.1' 34689. 0* 310156.1=3=" 647086.1' Zn x Cu 2 16965.4 5502.2 253412.547 1248194 Fe x Cu 1 367. 3 98648. 3‘” 17800.0 647086.1 an Fe :4 Cu 2 11629.0 8670.7 32179.9 153493.3 MnxCu 1 22801.0 20952.5 12155.0 11502.5 Zn x Mn x Cu '2 915.4 4005.3 15420.4 1518338 Fe x Mn x Cu 1 3344. 6 30537. 5* 10353.0 164497. 8 Zn x Fe 1: Mn x Cu 2 903. 4 6195.5 22000.0 216571. 0 Error A 46 (48) 21945. 4 5494.1 19755. 4 170200. 3 Variety (Var) 1 59211.1“! 29498. 0’” 52326. 5* 363910. 5* Zn x Var 2 22396. 5' 1432. 5 38340.1* 385419. 3* Fex Var 1 2224.6 5.8 36131.6' 325185.0* Zn x Fe x Var 2 744.0 2199. 5 26251.7' 210972. 5' Mn x Var 1 25706. 7' 2508. 3 73125.1' 50887. 8 Zn x Mn x Var 2 28110. 0* 3700.0 132095. 7““ 145619. 8 Fe x Mn x Var 1 633.3 3.0 28420.3' 8265.8 Zn x Fe x Mn x Var 2 6862. 2 2965. 7 39954. 5* 42871. 5 Cu x Var l 37636.0* 5890.5' 7906.1 101.6 Zn x Cu xVar 2 240.2 469.0 1988.3 11149.2 Fe x Cu x Var 1 2970.2 1813.3 91556.6** 14460.0 Zn x Fe x Cu x Var 2 44659. 0’” 5925. 3‘ 146338. 3““ 37545. 7 Mn x Cu x Var 1 28561.0’ 1437.6 112616.1** 9168.0 Zn x Mn x Cu x Var 2 5961.0 2369.1 49748. 4* 26670.0 Fe x Mn x Cu x Var 1 6058.0 39.0 166. 8 885.0 Zn x Fe x Mn x Cu x Var 2 18589.1' 15360.2 3130. 3‘ 9943.1 Error B 39 (48) 771. 8 2452.1 11148. 6 16637.1 Total 143 130 'l':\l51.l'2 2(1)). —— Continual, Menu squares Source Degrees of of l’etiole variance freedom Mn Cu Zn Fe Zn 1 356.5 22.0 270.0 1007 5 Fe 1 2330.5 104.2 32230.0” 44850 3* Zn x Fe 1 546.2 84.4 5355.1 437.8 Mn 1 62883. 8** 425.0 894. 3 75.3 Zn x Mn 1 882.0 1944.0 19866. 3-’=* 7. 6 Fe an 1 0.5 2.0 6256.5' 4718.0 anFean 1 6.5 8.2 5535.8 110.5 Cu 1 3444. 0 2948.2 3965. 5 356.5 anCu 1 16971.7 287.0 1625.3 65.0 Fe x (‘u 1 90.1 1040. 2 7021. 3' 225.1 Zn x Fe x Cu 1 3589.341 852. 0 189. 8 8456 3* MnxCu 1 263.3 2301.0 364.3 23.0 Zn x Mn x Cu 1 55.5 988. 2 27034. 65* 2007 5 Fe x Mn x Cu 1 3516. 3* 9720.4>?‘* 4718. 0 102.1 anFean xCu 1 297.5 204.2 635.5 5937.7’ ErrorA 28 (32) 694.1 1197.1 2113.9 1850.8 Variety (Var) l 635. 5’ 18760.0 “ 9420. 8 13324. 5** Zn x Var I 1 1035. 0~»== 8140.2! 250.3 8493. 8* FexVar 1 137.8 18.4 404.3 2871.1' Zn x Fe x Var 1 94.0 2. 7 4360 'i' 2330. 5’ Mn x Var 1 656. 3 28.2 16354 3 14. 3 Zn x Mn xVar 1 1225.5“ 805.0 364.3 918.8 Fe x MnxVar 1 128.3 150.0 1239.8 1512.1 Zn x Fe x Mn x Var 1 380.0 100.0 3255.0 3.0 (.‘u x Var 1 7193.35”? 2795.0’ 123.8 7543.8* Zn xCu xVar 1 250.3 2.7 1020.5 1725.5 Fe xCu xVar 1 1113.8‘ 2262.0 263.3 106.3 Zn x Fe x Cu x \-'ar 1 1725. 5' 3850. 7 3231 8‘ 6032. 5* Mn x Cu x Var 1 2593. 8* 2. 7 9821. 3” 446. 3 Zn x Mn x Cu x Var l 4. 6 40.0 742. 6 2849. 3' Fe x Mn x Cu x Var 1 3026. 3* 10922. 7 r 82.5 356. 5 Zr x Fe x Mn x Cu x Var 1 0. 8 5673. 4' 1020. 5 1560.1 Error 8 16 (32) 395.6 1470.7 1681.4 1200.6 Total 75 (95) 131 TABLE 2(b). -- Continued. Mean squares Source Degrees of of Stem variance freedom Mn Cu Zn Fe Zn 1 16907.0’ 656.3 2242.7 8.2 Fe 1 27608.1* 1155.1 23688.2 178365.0** Zn x Fe 1 18040.2' 17631.3 8740.2 170.7 Mn 1 833282.7** 3687.8 2.0 3725.0 an Mn 1 5460.2 28119.3 4959.4 13.5 Fe x Mn 1 15657. 0' 22478. 8 1365.0 10542. 0* Zn x Fe x Mn 1 6176.0 106. 3 3528. 4 1633. 5 Cu 1 1426.0 80562.1* 7848.2 13395.4* Zn x Cu 1 0.1 1708. 6 522. 7 54.0 Fe x Cu 1 4320.2 15.8 228.2 459.4 Zn x Fe x Cu 1 20533. 5* 27169.0 13160. 2 1504.2 Mn x Cu 1 2053.5 1418.3 1751.0 2882.0 Zn x Mn x Cu 1 3952. 7 26433. 8 3105. 4 1014.0 Fe x Mn x Cu 1 16801. 0' 38841. 3 570. 4 1650. 0 anFe x MnxCu 1 6305.0 14.3 630.4 216.0 ErrorA 31 (32) 4755.0 13868.0 9188.6 1958.8 Variety (Var) 1 4482.7* 4121. 3 112.7 486.0 Zn x Var 1 18040.2** 0.5 368.2 247.0 Fe xVar 1 165.4 61.8 150.0 2016.7 an Fe xVar 1 1335.0 2552.3 5642.7 408.4 Mn x Var 1 2970. 4* 304. 6 247. 0 216. 0 Zn x Mn x Var 1 15352. 0“ 8683. 0* 828. 4 247. 0 Fe anxVar 1 73.5 4523.8 135.4 1504.2 anFeanxVar 1 2773.5 1811.2 22.0 1305.4 Cu x Var 1 9922. 7M 4360. 5 11528. 2' 170. 7 Zn x Cu x Var 1 3901. 5"l 3914. 3 600. 0 35. 0 Fe x Cu x Var 1 12650. 0Ml 834. 3 793. 5 24. 0 Zn x Fe x Cu x Var 1 1552. O 2490. 8 3504. 2 30. 4 Mn x Cu 1: Var 1 12285. 4'” 5843. 8 3151.0 66. 7 Zn x Mn x Cu x Var 1 3337. 0* 2156. 5 672. 0 1785.4 Fe x Mn 3: Cu 2: Var 1 7210. 6M 3372. 5 6240. 4 368. 2 anF" x MnxCuxVar 1 1472.7 1211.3 8400.0 315.4 Error B 22 (32) 669. 3 2614. 6 3007. 7 1130. 8 132 TABLE 2(1)). -- Continued. Mean squares Source Degrees of of Roots Variance freedom Mn C11 Zn FL- Zn 2 122707. 8 420406. 5"” 41734489. 6’"? 56371364. 0"“?- Fc 1 396900. 0* 926245. 8** 2021610. 07” 562603493. 3'1m Zn x Fe 2 217915.1-‘r‘ 51384.0 1222872.6** 49513629.0=7=* Mn 1 11697540.0** 49173.0 26082.2 5961736.1 Zn x Mn 2 110579.13 5028.8 62117.2 40069400 Fe x Mn 1 432087.1* 29212. 5 4692. 2 5223510. 2 Zn x Fe x Mn 2 207675.1’k 35375.0 20330. 3 4317006. 8 Cu 1 3778815.1* 6116141.1** 4331254. 6** 24479405. 4* Zn x Cu 2 157265.6' 53094.2 3366445.8** 5073306.?) Fe x Cu 1 59780. 2 750966. 6*"! 705880. 0‘ 23430442. 2* an Fe xCu 2 384208.1' 11530.8 677811.6' 34735308 Mnx Cu 1 289802.7' 11718.0 14440.0 8635761.7 an MnxCu 2 159101.2' 862.8 23287.4 4967644.? Fe x Mn x Cu 1 27390. 2 4726. 5 84584.0 6327740. 2 Zn x Fe x Mn x Cu 2 399982.651“? 5835. 8 167626. 7 6302985.1 ErrorA 45 (48) 61480.0 38216.3 2201589 555942.5 Variety (Var) 1 22750.6 2747. 5 135702. 2... 2473280. 4* Zn x Var 2 5478. 4 1436.1 47435. 2 1180397. 5 Fe x Var 1 58081.0’ 7965.5 113232.2’» 2221590.2* Zn X Fe x Var 2 20928. 8 26916.1' 13397-‘«1.7*’OK 1102600. 6 Mn x Var l 24701.3 1362.8 24596.6 3681281.7’7‘ Zn x Mn x Var 2 9344.1 1368. 2 4921. 6 5373564.6’1‘* Fe x Mn xVar 1 38155.1' 11253.6 10990.0 3896018.0** Zn x Fe x Mn 11' Var 2 30831. 0' 32916. 2' 7000.1 5073985, l’MK Cu xVar 1 1653.7 7906.1 80561.3' 5642208.4*"= anCuxVar 2 10773.8 4956.9 1057.0 5120117.7’v‘* Fe x CuxVar 1 11628.0 25095.8 107693.3* 5442111.3** an FexCuxYar 2 32991.7' 3881.3 21781.6 4475331.7Ml Mn xCu x Var 1 940.4 7014.0 88903.3' 4410000.0** an Mn xCu xVar 2 7717.8 463.9 32413.5 1958872.5* Fe x Mn x Cu x Var l 13884.6 3595. 8‘ 19090.0 3823980. 2* Zn x Fe x Mn x Cu x Var 2 33224.2' 45194. 5' 4096.3 2151202.1* Error B 35 (48) 14218.6 1346034 23926.3 493004.11 Total 143 133 :UuUEEu—Zdhnh .cNuN m ) A. pm; :3 mmom. 3.: NN— mg mg. 5.x... «1-2-2.; U «.N 3: Nun «m2: mom: N: mam mvm NNN voo (3-2-" flUNE MN mwv S wNmm cNNH wN~ m: can Nwm v {u .noT— NU NN om: Nm mvow 2;: mm“ N: Em. m5. woof“ .73.; ~U_EN& "N mmm nae m3. «$2 :3 36¢ cc.— mwm v .73; .7“ NU ON mmN ~ov~ mam hmmN cm «Na «.3 can voo .72; .u~ ~UNE mu 3:. mm flan Nvm~ S; :3 E: awe «14.4.; NU w— hmd an arm mmwN I: mNN NmN mm: voo.-~.-~.ufi dUHSZANN : won «3. manN wo— vm «On 9: on mm 0: pm“ HN— wMN mam :w «w ov-oo~-oo~-~ NU m: mm 8h ~va $3 Na «5 N~N wN~ rm mo. c: vx X: :6 Now 2: v fooToo—L HUNE 2 EN mm mNmN Nm Np vc :2 mm wv E. h: an 2: N: wmm mp 372-08; NU «— ww mm mem m2 2. mm NE m: mm 3.. 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N: 5: ,3. «N m2 2: mm: «.2 me v: 70072:; ~U~2~L~Nm fl :U :2 oh cN 5U :2 wk :N :U :2 mm :N :U 52 oh :N oEN .3500 3 03¢; a $853.95. uocz Emum 3:333 .254 CCCNQEQUCOU acmEumeF “CUSZLUQXU cowwuvaufi: acof—HDCOLU—E n: .95 p.353: mason %>mc Ho magma “cm—Q mzoEm> 5 3:68.20 No .8538 you mtma E cosmbcmucoo 692214-- .m MJQ3 onus Ho>o~ oEu $9.2 8.3m 5293/ 230.“. . oEN Ho EMEB «cofipmwuh «:oEammboum .395: “sesame: can «cofiuwoboua ufin @3223 um weapon .30: on a uw>o nose: m> coon ham: Bmcqmwm cum omzcmm 503 6300200 memo—63 no QEN ~30..— vcm :ofiwuucoocoo on: .oumvzxw “0 EMS? -- .m Mdméd. 136 TABLE 6. -- The effect of pretreatment and treatment on exudate weighfl‘and Zn concentration of Saginaw and Sanilac navy beans. Zn level Exudate (ppm) Weight (g) Concentration (ppm) Pretreatment Treatment Saginaw Sanilac Saginaw Sanilac 0.00 0.5 1.27 0.33 0.44 0.19 0.00 5.0 0.91 0.40 0.23 0.14 0.05 0.5 1.41 0.66 0.34 0.14 0.05 5.0 1.72 0.34 0.92 0.28 5.00 0.5 1.57 1.01 0.88 0.67 5.00 5.0 0.86 0.51 0.98 0.82