QINHERITANCE OF'NITRATE- N ACCUMUtATlON IN - LEITUCE (lACTUCA SATlVA L) . Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY ' RAMACHAND‘RA SUBRAMANYA 1977. LIBI‘ARY ” Mich}; n State UHiVLféty This is to certify that the thesis entitled .1 Nfiéz/MMKF 0/: N/M/VZ". 4/ Ana/m1 (AIM/v nv Le’rrac‘é” (lflcruCfl Sfl7/l/fl 4.) presented by 44%» rim/v2,” fad/mm»; Nyfl has been accepted towards fulfillment of the requirements for /9’{. D degree in flail/“(17016:— fl££7,( DLLL- }/’;K‘.\~_‘ .] ' Major professor Date 7751778" /7, /777 0-7639 ABSTRACT INHERITANCE OF NITRATE-N ACCUMULATION IN LETTUCE (LACTUCA SAIIVA L.) By Ramachandra Subramanya Nitrate accumulation in plants is the result of nitrate uptake in excess of its reduction. A greater capacity for nitrate reduction and subsequent assimilation in a plant is desirable to increase the yield and to breed vegetables for low nitrate content, since nitrate reduction is one of the first steps in protein synthesis. Previous work on lettuce has shown variations in nitrate accumulation in different culti- vars. Experiments reported herein with 66 cultivars showed a differen- tial nitrate accumulation. The plants were grown in sand culture using nutrient solution. The nitrate content was determined on the dry leaf tissues using a nitrate selective electrode. Two low, one medium and three high nitrate accumulators were used to study the inheritance of nitrate accumulation in lettuce. Accumulation for low levels of nitrate was found to be dominant. Genetic analyses showed that three major genes control the accumulation of nitrate in lettuce. In the three major gene systems, two genes 5 and §_determined low nitrate accumulation levels in the cultivar Valmaine. The presence of Ramachandra Subramanya both g and g dominant genes was essential for low accumulation, i.e., A and §_were complimentary to each other. The low nitrate accumulation levels in the cultivar WOnder Van Voorburg was due to a single dominant gene §_. Thus low nitrate accumu- 2 lation can occur due to the presence of both §_and g, or 92 alone or a combination of all three é:§:§2. A dominant gene §_determined the high nitrate accumulation levels in the cultivar Caravan. The gene §_was epistatic to genes A and g, but it was recessive to the dominant allelic gene 92. Thus the presence of £5 and §_in the same genotype results in low levels of accumulation of nitrate. The genotypes of the cultivars Kordaat and Valore, the high accumulators were similar. The high nitrate accumulating trend of these cultivars was determined by recessive alleles a:b:g, Thus high nitrate accumulation can occur by the presence of g_gene alone or the absence of either or both A or _I_3_ in a genotype. Accumulation of nitrate for medium levels appeared to be con- trolled by the allelic gene £2. INHERITANCE OF NITRATE-N ACCUMULATION IN LETTUCE (LACTUCA SATIVA L.) By Ramachandra Subramanya A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1977 ACKNOWLEDGEMENTS The author expresses appreciation to Drs. Shigemi Honma and Grant H. Vest for their guidance and assistance during the course of this study. Thanks are due also to Drs. W. M. Adams, L. R. Baker, R. C. Herner and D. D. warncke, who served as guidance committee members and assisted in preparing the manuscript. Special thanks to Dr. H. C. Price, who served as a guidance come mittee member and for providing the necessary instruments for nitrate determination, and to Dr. A. DeHertog for providing the laboratory facilities, where part of the research was conducted. Special thanks to Drs. W} M. Adams and J. H. Asher Jr. for advice and valuable discussions during the analyses of the data. The author thanks the students and friends for their help in many aspects of this study. Thank you Bridgette for all your help throughout the course of this study. ii TABLE OF CONTENTS Page INTRODUCTION.................................................... 1 LITERATURE REVIEW............................................... 3 MATERIALS AND METHODS.. ..... . ........... ........................ 17 RESULTS AND DISCUSSION.......................................... 27 A. Preliminary Studies............... ..... ...... ....... ...... 27 Total-N................................................ 27 Other E1ements......................................... 32 Fresh and Dry Weights.................................. 38 B. Genetic Experiments--l974................................. 41 Variations in Nitrate-N Content of Lettuce Cultivars at Different Plantings.............................. 45 Individual Crosses--1975............................... 47 Cross: Valmaine (P ) x Caravan (P3) (Low x High)...... 49 Cross: Valmaine (P ) x Kordaat (P ) (Low x High)...... 56 Cross: Valmaine (P ) x Valore (P I (Low x High)....... 59 Cross: Wonder Van Toorburg (P2) x Caravan (P3) (Low x High) 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 61 Cross: Wonder Van Voorburg (P2) x Valore (P6) (Low x High) 0 C O O O O O O O O C O O O O O O O O O O O O O I C O O O O O O O O O C O O O O O O O I O C O 63 Cross: Valmaine (P ) x Wonder Van Voorburg (P ) (Low x 1 2 LOW)..OOCCOOOCOCOOOOOOOOOOOOOOOCOOCCOOOOOCOOOOOCCOOO 65 Cross: Caravan (P3) x Ithaca (P5) (High x Medium)..... 68 SUMMARY AND CONCLUSIONS........................ ........ ......... 71 LITERATURE CITEDOOOOOOOOOOOOOOOOOOOOO0.0.0.0....OOOOOOOOOOOOOOOO 77 iii LIST OF TABLES TABLE Page 1. Lettuce cultivars screened for NO3—N accumulation in 1973... 18 2. A modified Hoagland nutrient solution used in lettuce sand cultureOOOOOOOOOOOOOOOOO... ..... 0....OOCOOOOOOOOOOOOOOOOOO.I 19 3. Nitrate recovery from added nitrate to lettuce tissue extracts as measured by a nitrate selective electrode....... 23 4. Nitrate-N (percent NO -N) content of fresh leaves from 66 lettuce cultivars grown in two nitrogen nutrient regimes for eight weeks.0..OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00.0.0... 28 5. Nitrate-N content (percent N0 -N on dry weight) of 11 lettuce cultivars grown in two nutrient N regimes and har- vested at two growth stageSOOOOOOOOOOOOOOOOOOOO0.000000CO... 3o 6. Total nitrogen (percent N) of leaf tissues of lettuce culti- vars grown in two N regimes and harvested at four and eight weekstageOOOOOOOOOOOOOOOOOOOOO...0.0...OOOOOOOOOOOOOOOOOOO. 31 7. Element analyses of eight week old leaf tissues of lettuce cultivars grown in the modified Hoagland solution (Table 2) containing standard N, K and Ca concentrations.............. 33 8. Element contents of eleven selected cultivars grown in two nutrient regimeSOOOOOOOOOOOOO0.00000000000000000000000.0.... 36 9. Mean fresh weight of leaf, root and dry weight of leaf of four week old lettuce plants grown in two nutrient regimes for the cultivars used in NO3-N study....................... 39 10. Mean nitrate-N (percent NO —N on dry weight) of ten culti— vars and their reciprocal crosses........................... 42 11. Leaf nitrate-N (percent) content of various cultivars grown in sand culture using modified Hoagland solution on four week old dry tissues for three years........................ 46 iv LIST OF TABLES-—continued TABLE 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Mean nitrate-N (percent NO -N) content (dry weight) of parents and F1 plants of cgosses used in the genetic analy- 888 Of nitrate-N Study.’.0.0000......OOOCOOOOOOCOOOOOOO0.0. Frequency distribution of Nitrate-N (percent N0 -N)/p1ant (dry weight) for the parental, F1 and F2 genera ions of crosses Of valmainexcaravanOOOO0.000000000000000000000.0. Chi-square test for goodness of fit to the postulated model for nitrate-N data in the F2 generation.................... Chi-square test for goodness of fit of the F mean to the 2 postulated mOdeIOOOOOO... ....... OOOOOOOOOCOOOIOOOOOOOO0.0.. Proposed genotypes of the lettuce parent lines used in nitrate-N StUdYooo-ooococoo-ooooooooooooooooooooooooooooooo Frequency distribution of Nitrate-N (percent NO —N)/plant (dry weight) for the parental, F and F2 generagions of crosses of Valmaine x Kordaat.............................. Frequency distribution of Nitrate—N (percent N0 -N)/p1ant (dry weight) for the parental, F and F2 generagions of crosses of Valmaine x Valore............................... Frequency distribution of Nitrate-N (percent NO -N)/p1ant (dry weight) for the parental, F and F generagions of 2 crosses of Wonder Van Voorburg x Caravan................... Frequency distribution of Nitrate-N (percent NO -N)/plant (dry weight) for the parental, F and F generagions of crosses of Wonder Van Voorburg x Valore.................... Frequency distribution of Nitrate-N (percent N0 -N)/plant (dry weight) for the parental, F and F2 generagions of crosses of Valmaine x Wonder VanIVoorburg.................. Frequency distribution of Nitrate-N (percent N0 -N)/plant (dry weight) for the parental, and F2 generations of crosses of Caravan x Ithaca................................ Genotypes and phenotypes of the F populations from various crosses Of nitrate-N accumulation StUdy ooooooooo o o o o o o o o o o 0 Mean fresh and dry weights of parents and their F popula— tion for two plantings .......... ............ ............... V Page 48 50 52 53 55 57 6O 62 64 66 69 72 76 INTRODUCTION The breeding of plants for high yields or quality requires a greater understanding of physiological mechanism. Identifiable physio- logical traits contributing to yield or quality could provide an alternate selection criteria for a variety improvement program. The determination of these traits needs to be simple and rapid if large populations are to be screened effectively. Nitrate accumulation for example would be suitable for such an investigation. Nitrate reduction is one of the first steps in the synthesis of protein in plants. In recent years there has been a growing interest in understanding genetic influences on plant nutrition. Low and high-protein cultivars of the same species have been attributed to its nitrogen nutrition (54, 70,86). Considerable work has been directed towards understanding N nutrition. The genetic control of many aspects of N nutrition is, however, missing. Lettuce (Lactuca sativa L.) and many other crop species are known to accumulate nitrates (6,19,20,23). Nitrates in the diet is undesir- able since they may lead to methemoglobinemia in humans (26,42,61,80). Nitrate under certain conditions may be reduced to nitrites in the gastro intestinal tract. These nitrites are known to interact with secondary or tertiary amines to form nitrosamines. Many of the nitros- amines are mutugenic, teratogenic, and carcinogenic to various lab animals (26); however, carcinogenic concentrations in humans are unknown and, therefore, it is important to have nitrate free food in the human diet. Cultivar difference in their capacity to accumulate NO3-N have been observed in different vegetable species (6,18,23,68). The objective of this study was to determine whether nitrate accumulation in lettuce is genetically controlled, and, if so, to learn its heritability and mode of inheritance. The pattern of inheritance would be invaluable for a plant breeder in breeding lettuce for low nitrate content. LITERATURE REVIEW Nitrate accumulation in plants has been observed for many years; however, interest in it has intensified due to an increased use of nitrogen fertilizers. The factors influencing nitrate accumulation have been studied extensively. Nitrate accumulation in plants may affect man and animals directly or indirectly since nitrate in water, food, and feeds can be microbio- logically reduced to nitrite which is 10 times as toxic as nitrates (37). Maynard‘gg'gl, (68), Viets & Hageman (92) and WOlff & wasserman (95) have recently reviewed the nitrate problem on human health and environ- ment. One of the effects of nitrite is its toxic interference of the oxygen carrying capacity of blood in both animal and humans, an effect called methemoglobinemia. Nitrates and nitrites can also reduce milk production in animals and cause reproduction problems, or even death if ingested in large quantities (37). Nitrates in forage material under damp conditions can be reduced to nitrites. Consequently, feeding of such material to livestock has been reported to be toxic (95). The release of oxides of nitrogen released from ensiled high-nitrate forage materials can be lethal to both man and animals in silage buildings with no fencing and inadequate ventilation (28,42). Cyanosis or blue babies caused by nitrates was reported as early as 1945 (25). The low stomach acidity of infants under four months of age permits the growth of microorganisms that can reduce nitrate to nitrite. Poisoning of very young infants fed with spinach high in nitrate content has been reported (80). Deaths of infants fed with water containing high nitrate have also been reported (95). In recent years, there has been an increased concern about the potential hazard of nitrosamines as toxicants formed in or through eat- ing certain foods or drugs which contain the precursor substances of nitrosamines (46,63). Nitrite reacts with secondary and tertiary amines, or quaternary ammonium compounds that occur in foods and drugs (63,95) to give nitros- amines which have been shown to be carcinogenic, teratogenic, or muta- genic to various laboratory animals (26,63). Maynard g£_§l, (68) and WOlff and wasserman (95) recently reviewed the nitrosamine situation. Nitrosamines have not been reported to occur in the commonly consumed vegetables. Although Keybets g£_§l, (57) and Heisler g£_§l, (46) could not detect any nitrosamines in spinach and beets even under conditions favorable for formation of nitrosamines other experimental results cited by Maynard £5.3l, (68) and Wolff and Wasserman (95) suggests a need for extensive research with regard to in;!i!g_synthesis of nitrosamines. Sprague (89) reported that nitrate reduction is one of the first steps in protein synthesis; therefore, the plants that have the greatest capacity to reduce nitrate will also have a better chance of more pro- tein synthesis. Differences between high and low protein cultivars of wheat species were attributed to differences in the re—export and trans- location of N (54,70,86). Croy and Hageman (29) showed a practical significance of two wheat genotypes, which differed in nitrate reductase activity and their protein yield. The capacity to reduce or assimilate nitrate varies with different crop species (75). In the same study, Olday gt 2l° (75) found the site of nitrate reduction to be concentrated in the leaf blades of cucumber (Cucumis sativus L. ), while in pea (Eisum sativum L.) nitrate reduction occurred evenly throughout the plant. The nitrate-N comprised 80% of the N present in the bleeding sap of roots of cucumber plants from which the shoots had been excised; whereas, nitrate-N constituted only 30% of the sap from pea roots, the nitrate reducing capacity of the two species were found to be different in different tissues. In cucumber only 2% of the nitrate reductase activity was in roots; whereas, 92% of the activity was found to be in the leaf blades. In contrast the enzyme activity in pea plants was 18% in the roots and 67% in the leaf blades. They concluded that, because of the greater nitrogen assimilation by pea roots, it uses NOB-N more efficiently than cucumber resulting in less nitrate and more protein in the pea. One of the major problems of high nitrates in canned vegetables is the reduced shelf-life caused by the detinning effects of tin lined cans (52,60,87). Farrow ggugl. (33) reviewed the detinning process and associated factors. High nitrate content in beets can lead to a bitter taste when cooked (79). Factors influencing nitrate accumulation in plants have been extensively studied. One of the most important factors favoring nitrate accumulation in plants is the nitrate rich environment. The availabil— ity of soil nitrate for plant uptake depends upon very complex processes. Soil nitrate can be lost by leaching, volatilization, microbial utiliza- tion, or simply be unavailable to the plant for lack of water. Nitrate, an anion, is considered to be contained entirely in the soil solution; therefore, the rate of replenishment of nitrate from other forms (organic and ammoniacal) may be more important than the amount of nitrate present at any given time (12). Increased demands on agricultural produce for higher yields, rapidly maturing vegetable crops and to maintain a bright green color and succulence especially in leafy vegetable crops like spinach, lettuce, etc., require a liberal or adequate nitrogen fertilizers in the rhizo- sphere which can result in nitrate accumulation in the plants (68,92,95). The time and absorption of nutrients by lettuce is crucial. Zink and Yamaguchi (99) reported that most of the nutrient uptake (70 percent) occurs during the period of 21 days preceding harvest, and about one- half of that is absorbed two weeks prior to harvest. Therefore, is it necessary to maintain a high nutrient level in the soil until a few days before harvest. Barker ethal, (6) showed spinach leaves contained more nitrate-N when N was applied broadcast before planting than when applied as a side dress application. This suggests that spinach accumulates nitrates when cultured for a longer period of time in nitrate rich medium. Greenhouse experiments have shown a direct relationship between time of nitrate fertilization and nitrate concentration in plants (4). Barker gt 31. (6) also showed the source and timing of N application to affect nitrate content in plants. Ammoniacal fertilizers and materials that mineralize slowly are known to reduce nitrate accumulation in plants (3,6,71) but the possibility of ammonium toxicity exists when all the N is in the ammoniacal form (65). A combination of NRA-N and NO3-N with nitrapyrin a nitrification inhibitor was reported to reduce nitrate accumulation in plants without sacrificing the yield (71). Lettuce (Lactuca sativa L.) has been reported to accumulate nitrate (23). Brown and Smith (13) reported no significant increase in nitrate content of leaf and semihead lettuce plants by increasing the fertilizer rate. Cantliffe and Phatak (23) also reported no significant increase in the plant nitrate content of four lettuce cultivars even at increased nitrogen fertilizer rate. The lack of response to additional N was attributed to the use of muck soil. Other plant nutrients have been reported to affect nitrate accumu- lation. Reports on the effect of phosphorus and potassium are varied. A decrease in the nitrate content with an increase in P application (7,39) was noted, but Barker and Maynard (4) observed no effect on nitrate accumulation in spinach when P, K, Ca, and Mg, were moderately deficient. Brown and Smith (14) also observed that insufficient P had no effect on nitrate content of vegetables. Cantliffe (17) working with beets (Beta vulgaris L.) and spinach (Spinacea oleracea L.) plants, grown at different levels of N, P, and K, observed that varying the level of P had no effect on nitrate accumulation, but plants of both species grown at high K accumulated significant amounts of nitrates. Molybdenum deficiency lead to nitrate accumulation in higher plants (47,73). Beevers and Hageman (9) in their review indicated that Molybdenum appeared to be involved in the nitrate reductase induction process and not merely activating pre-existing protein. Molybdenum is a constitutive part of nitrate reductase (1,32). Iron (1) and other plant nutrients may be involved in the reduction or accumulation of nitrate but the effect appears to be indirect (68,92). Light has long been known to influence nitrate content in plants (8,15,19). Low light intensity increased nitrate accumulation (l9). Cantliffe (19) observed increased nitrate content in spinach leaves when the light intensity was reduced from 2400 ft-c to 600 ft-c. Shade or inadequate light also resulted in high nitrate accumulation in plants (17). Photoperiod, diurnal variation, and sampling time are known to influence nitrate content in plants (41,44). Cantliffe (20) working with various radish, spinach, and snap bean cultivars harvested at 0, 6, and 12 hours after the initiation of the light period, found radish leaves and snap bean pods contain less nitrate when harvested late in the photoperiod. However, the nitrate content of radish roots and spinach leaves remained the same regardless of the harvest time. The effect of light on nitrate accumulation is indirect in that light affects nitrate reductase thus affecting the nitrate content. Beevers and Hageman (9) made a comprehensive review on the influence of light on nitrate reductase activity in higher plants. A direct and absolute requirement of photosynthetic C02 fixation for the induction of nitrate reductase in Perilla plant was reported (55). This requirement, however, does not hold true for all tissues since the enzyme can be induced without photosynthetic requirement. Thus, the effect of light appears to provide energy for synthesis of the enzyme or in some way increase and stabilize polyribosome formation (90), or increase the membrane permeability (9,11) so that nitrate transport is enhanced. Effect of light on nitrate reductase induction is also associ- ated with increased protein synthesis (9). Adequate light would also insure photosynthate for reductive energy via carbohydrates and carbo— hydrate skeletons for amino acid and nucleotide formation (59). Although Gilbert 35 al. (39) did not find a temperature effect on nitrate accumulation in various plant species, other workers have observed greater nitrate concentrations in plants grown at high tempera- ture than at lower temperatures (8,21,38). Increased nitrate content with high temperature have also been observed on barley (76), lettuce (36), pasture crops (8),zuuisorghumrsudangrass (38). Spinach cultivars grown at various temperatures and at various N levels (21) showed an increase in nitrate with an increase in temperature even at zero N treatments. The nitrogen level and higher temperatures appeared to have a synergistic effect on nitrate accumulation. Similar observations have been reported with other crops (7,21,36,38). Cantliffe (21) also indicated a deactivation of nitrate reductase activity at extreme temperatures and nitrate accumulation. Reduction of nitrate reductase activity due to heat stress was also demonstrated in barley seedlings (76). 10 All of these reports suggest that a general statement about the effect of temperature on nitrate accumulation cannot be made since the processes of absorption, translocation, and assimilation are also affected by temperature. Therefore, it is not uncommon to find contra- dicting reports of the effect of temperature on nitrate accumulation(68). water or moisture is a very important factor in nutrient uptake. Roots come in contact with more nutrient ions when growing in a moist soil than when growing in a dry soil. Ammoniacal N does not readily move but the nitrate-N an anion is considered to be contained entirely in the soil solution, which can readily move in and out of the root zone. When nitrate accumulates under low soil moisture or drought condition, high temperatures are usually associated with it. Both high temperature and moisture stress were shown to reduce or inactivate nitrate reductase activity (97). The enzyme, nitrate reductase was shown to be more sensitive to moisture stress than were certain other enzymes of the photosynthetic system (53). These results suggest that the effect of either high temperature or moisture stress is more adverse on nitrate reduction than it is on the absorption and translocation of nitrate, thus causing nitrate build—up. Herbicides have been reported to increase nitrate in crops. Cantliffe and Phatak (24) cited many reports indicating an increase in nitrate content of various crops with the use of herbicides. Beevers 23 a1. (10) found that herbicidal levels on 2,4-dichlorophenoxy-acetic acid (2,4-D) had differential effect on cucumber and corn. Nitrate reductase activity was reduced in cucumber whereas an opposite effect 11 was noted in corn. With both species the nitrate content varied inversely with nitrate reductase activity. Cantliffe and Phatak (24) did not find a dramatic increase of total-N in spinach blades and petioles, by herbicide treatments, they indicated that the increase in nitrate accumulation was the result of a decrease in some phase of nitrate reduction. Klepper (58) recently reported that the photosynthetic or triazene herbicides inhibited nitrite reductase. He found little influ- ence of these herbicides on nitrate accumulation or nitrate reductase activity. Sublethal doses of herbicides, especially triazenes have been reported to be beneficial by increasing the protein content and yield (24,49,56). An increase in nitrate reductase activity and nitrate accumulation were also observed (34,81,82,9l). Ries gt a1, (83) treated six different plant species with subherbicidal levels of simazine and found increased nitrate uptake by plants, and in some cases the plants had a high nitrate content. Cantliffe and Phatak (24) working with herbicidal levels of several herbicides found increased nitrate content in spinach petioles with many herbicides; however, with certain herbicides it appeared that the N fertilizer was more effici- ently used. Since nitrate reductase reduces nitrate, interference in the enzyme activity would result in the accumulation of nitrate in the plant. It has been shown that within the same tissue the nitrate con- tent varies inversely with the level of nitrate reductase activity (41). 12 Other experiments (29,30,74) suggest that nitrogen fertilizer over and above that which is usually considered optimal increases the nitrate reductase level and protein content of the plant or grain. A review on the characteristics and factors affecting nitrate reductase was made, by Beevers and Hageman (9) and recently by Viets and Hageman (92). Although many endogenous compounds have been reported (35,48,64,84) to inhibit nitrate reductase or repress its synthesis resulting in nitrate accumulation in gi££g_studies, it is not certain that the same endogenous compounds are responsible for nitrate accumula- tion in 31.1-29.3 One of the major sources of nitrate intake in foods is from vegetables. Wright and Davison (96) listed Amaranthaceae, Chenopodia- ceae, Cruciferae, Compositae, Graminae, and Solanaceae as plant families which have a tendency to accumulate high levels of nitrate. Nitrate-N a natural constituent of Plants is not uniformly distributed throughout the plant, or for that matter within the edible portion (e.g., petiole versus leaf blade) (96). The nitrate content within a leaf, root, and fruit, type of vegetables has been known to vary (66). Maynard and Barker (66) stated that nitrate-N generally accumulates in older por- tions of the plant. Reproductive parts are usually low in nitrate-N, roots slightly higher while leaves even higher. The nitrate content of seeds in general is considered to be negligible. McNamara ggnal, (69) surveyed 113 seed samples representing 37 different species and found among cereals nitrate content was more in corn than either wheat or cats, but the nitrate content of corn was low. Among the major crop 13 plants tested soybean seeds contained the highest nitrate—N. The weed seeds as a group had the highest average nitrate content. Nitrate con“ tent in vegetable seeds varied with the species. Although high levels of nitrate can accumulate in leaf tissues of plants in the early vegetative growth, the nitrate content decreases in leafy and non leafy tissues as the plant matures. In mature plants, nitrate accumulates primarily in nonchlorophyllus stem and stalk struc- tures which exhibit a low capacity for nitrate assimilation (92). However, a different pattern of nitrate accumulation occurs in leafy vegetables where such as lettuce (99) and spinach (6) the nitrate generally accumulates with age. Reports of differences in nitrate accumulation among cultivars within the same species which has been recently reviewed by Maynard g£_§l, (68). Cantliffe and Phatak (23) reported cultivar differences in their capacity to accumulate nitrates in their edible parts of lettuce, radish and spinach. Minotti (72) in his initial studies with lettuces found a fourfold difference within crisphead lettuce cultivars. Continued comparisons of two cultivars Minetto and Valrio under varying growth situations showed that nitrate content varied with media, environ- ment, plant part and plant age, but the nitrate content of the cultivar Minetto was consistently higher than that of Valrio. In recent years there has been an increased interest in genetic influences on plant nutrition. Many aspects of plant nutrition are under genetic control (31). Response to N nutrition being under genetic control was first suggested by Harvey (45), when he showed that corn l4 and tomato genotypes had differential N requirements and that these differences were inherited. Savoyed leaf types of spinach were reported to accumulate more nitrate than smooth leaf type spinach (23,67). Barker gt 31. (5) con- ducted an experiment with 18 spinach cultivars which were grouped into savoy, semi-savoy, and smooth leaf types, these were grown under various N regimes. Although their results showed that savoy leaf type spinach type accumulated more nitrate than the smooth leaf type when compared as a group, there were marked differences among cultivars in nitrate accumulation within each group. A similar study (18) involving 31 spinach cultivars, including Plant Introduction accessions of various leaf types were grown under two N regimes. In this experiment leaf type was not related to nitrate accumulation and in the presence of high N fertilizer rates individual spinach lines accumulated nitrate. The author (18) suggested that the genetic variations among lines in the N assimilation pathway may account for the differential nitrate accumula— tion. Olday gtflgl, (74) explained the physiological basis for differ- ences in nitrate accumulation between the two spinach cultivars America, a high accumulator and Hybrid 424 a low accumulator. The authors ob- served, with adequate N nutrition, the nitrate content of America was about three times that of Hybrid 424. Nitrate reductase activity in the leaf blades of Hybrid 424 was significantly greater than America. The qualitative and quantitative composition of bleeding sap did not differ for the two cultivars. There was also no indication of reduced 15 nitrate uptake by Hybrid 424. The lower nitrate content of Hybrid 424 at high N nutrition was attributed to a greater nitrate reductase activity in this cultivar, especially in the leaf blades. Marked varia— tions in the levels of nitrate reductase activity was also reported with several varieties of cauliflower (9). Hageman and Flesher (40), demon— strated an inverse relationship between nitrate accumulation and nitrate reductase activities in two corn (§g§_m§y§ L.) lines. Although the genetics associated with nitrate accumulation have not been reported in the literature, there are a number of reports indi- cating genetic variations in nitrate reductase (9,92). Corn inbreds and hybrids were reported to differ widely in nitrate reductase activity and the level of activity was under genetic control (98). The heritability of nitrate reductase activity was demonstrated (85) by careful selection of corn inbreds based on enzyme assay, and appropriate inbred combina— tions, a hybrid could be obtained with a high, medium or low level of nitrate reductase activity. The inheritance of nitrate reductase activity in corn was reported to be controlled by a two gene system (93). The authors (93) concluded that both enzyme synthesis and decay are factors governing the level of nitrate reductase in corn. Efficiency of N utilization in tomatoes (Lycopersicon esculentumlu) was shown to be a highly heritable trait (77). The efficient plants under severe N stress produced more dry weight than the inefficient strains. The inheritance studies showed that relatively few genes were involved. 16 Hoener and DeTurk (51) concluded that "high protein" corn strains absorbed and assimilated more nitrate than "low protein" strains. The level of nitrate reductase was reported to be positively correlated, with soluble leaf protein (29,40,43). Grey and Hageman (36) selected wheat cultivars with high nitrate reductase activity and reported a higher grain protein in these selected lines. Others (54,70,86) have reported that the difference between high and low protein cultivars of wheat species were due to their differences in re—export and transloca- tion of N and these differences were under genetic control. Olday gt 2l° (74) working with two spinach cultivars with differ- ential nitrate reductase activity indicated that the potentially higher growth rate, higher yields, higher protein content and lower nitrate content appear to be interrelated features found in the low nitrate accumulation cultivars. Similar observations were also made by Barker e£_§l, (5) with the low nitrate accumulation spinach cultivars compared with cultivars which accumulated high nitrates. MATERIALS AND METHODS Sixty-six lettuce cultivars (Table l) were screened for NOB-N accumulation in their leaves after being grown in a modified Hoagland's (50) nutrient solution (Table 2). The plants were grown in the green- house in unground silica-sand (Wedron 4030) and watered with nutrient solution to maintain uniformity and a desired level of nutrients in each growing unit. In the preliminary screening study (1973), 236 cc paper cups (cold cups) were used to grow the plants. Holes were made in the bottom of the containers for drainage. A filter paper (Whatmanl) was placed in the bottom to prevent loss of sand through the drainage holes. Each cup contained 315 g of sand. In subsequent plantings (1974 & 1975), 473 cc styrofoam cups were used. Each cup was filled with eight g. of coarse perlite followed by 630 g. of sand. Benches on which the plants were grown were lined with black poly- ethylene and arranged so the leachate could be collected at one end of each bench for reuse. Seeds of each cultivar were sown in a flat containing a mixture of 3 perlite to l vermiculite. Seedlings were transplanted at the first true leaf stage. One day prior to transplanting each, silica-filled cup was thoroughly washed with distilled water. Two seedings were planted in each container and watered with a modified Hoagland's solution (Table 2). The following modifications were made in the nutrient solu— tion: 17 Table l: Lettuce cultivars screened for N0 -N accumulation in 1973. 18 3 Agilo Amplus Arlen Ayoncrisp Avondefiance Blondine Bourguignonne Brioso Calicel Calmar Caravan* Climax Deciso Delta Empire E 9201A Fairton Great Lakes Grand Rapids Groso Hilde Interex Ithaca* Klock KnaP Kordaat* Korrekt Kwiek Larganda Liba Magiola Marquette May Princess Montemar M.S.U. 71- I46 Portato Proeftuin's Blackpool Rapide Red Tipped Boston Regina Resistent Secura Solito Spartan Lakes Sucrine Suzan Texas 635- 1967-68 Texas 637- 1967-68 Texas 641- 1967-68 Texas 642- 1967-68 Texas 644- 1967-68 Tinto Tonika Type 57 Type 69 Valmaine 67074* Valmaine (Weslaco) Valore* Valrio Valtemp Valverde Vanmax Variety A (Weslaco) Variety B (Weslaco) Ventura Wonder Van Voorburg* Zomerkoningin * Cultivars were used as parents in the genetic analyses. Table 2: A.modi£ied Hoagland nutrient solution used in lettuce sand culture. Element concentration ppm (mg/1) in nutrient solution Stock Salt Solution of Major Elements 200 Ca 210 N 234 K 64 S 48 Mg 31 P 0.5 B 0.05 Mn* 0.05 Zn 0.02 Cu 0.01 M0 2.4 Fe IM Ca(NO3)2 4H20 IM KNO3 m Mgso4 IM KHZPO4 Stock Salt Solutions of Trace Elements H3303 MnSOAHZO ZnSO47H20 CuSOASHZO M003 Na Fe Chelate * 0.5 ppm in 1973 planting. 20 l. Elimination of chloride by substituting MnSO4°H20 for MnClz. 2. Substitution of iron chelate for iron tartrate. The pH of the final solution was 5.7. In preliminary studies plants watered with this nutrient solution showed manganese toxicity after eight weeks. Hence, in subsequent plantings Mn content was reduced from 0.5 to 0.05 ppm in the solution. The frequency of watering of the seedlings with nutrient solution was dependent on the stage of the plant growth. At each watering the sand was saturated with nutrient solution. At transplanting seedlings were watered with half the quantity of CaNO and KNO in the nutrient 3 3 solution and continued until the seedlings were established. watering with a complete nutrient solution followed. Daily watering with the nutrient solution commenced when the plants were large and required it. After daily watering began the sand was saturated weekly with distilled water to leach out salts. Nutrient solution leachate collected at the end of each bench was reused once or twice before discarding. This was the practice in the first two plantings. In preliminary screening, nutrient solution was used alternating with water. In addition to natural light plants were supplemented with fluor- escent (C.W.V.H.O.) light placed over each bench. Light meter reading at the plant level averaged 750 ft-c. The fluorescent lights were on 16 hrs. a day. The temperature in the greenhouse was set at 15°C and the auto- matic vents were set at 18°C. Fans were used to maintain uniform temperature through the greenhouse. 21 Temperatures varied from 12°C in the night to 27°C during the day from December 2, 1975 to January 2, 1976. The plants were grown in nutriculture for four weeks to obtain a sample for the NO3-N determina- tion. Harvesting was done at the beginning of a dark period. Plant tops were cut at the base just above the crown and the fresh weight of leaves were determined. All of the plants were harvested during one period of time. After the fresh leaves were weighed they were oven dried at 37.5- 43.000 and re-weighed. The dried leaves were ground in a Wiley mill to pass a 20 mesh screen. This plant tissue was analyzed for NO -N using 3 an Orion Nitrate Ion Activity Electrode (2,22). The determination of nitrate-N was similar to the method described by Baker and Smith (2), with the following modifications: 1. 0.2 g. of lettuce leaf tissue was extracted in 100 ml of 0.025}! A12(SO solution. 4)3 2. The 0.02511 A12(SO extracting solution also contained 10 ug 4)3 per m1 of NO -N and 2 ml per liter of preservative. 3 The use of 0.2 g. of plant material was necessary since the tissues had high nitrate contents. Preservative was used to prevent bio- logical changes in nitrate concentrations. The extracting solution containing 2 ml per liter of preservative was reported, not to inter- fere with the nitrate determination (2). The nitrate determination in this study lasted 10-14 days and no changes either in the extracting solution or in the standard solutions could be detected. The nitrate 22 concentrations were determined on a pH meter with expanded millivolt scale. The extracting solution with the plant material in suspension was read directly without the filtration step. N03-N was read as ppm and recorded as percent NO -N on a dry weight basis. 3 To assess the efficiency of nitrate recovery and the sensitivity of the nitrate ion activity electrode, nitrate varying from 10 to 100 ppm was added to the lettuce extract and the amount recovered was ascertained (Table 3). Recovery was nearly complete; these results are in agreement with that of Paul and Carlson (78) and Cantliffe g}; 31. (22). The preliminary screening was designed to observe: l) Cultivar differences in free NOB-N in lettuce leaves, 2) effect of age on NO3-N accumulation, and 3) effect of NOB-N concentration in the media on the nitrate accumulation in lettuce leaves. Sixteen plants of each cultivar were watered with nutrient solution containing two levels of N (210 and 420 ppm N). There was also an increase in Ca and K in the latter solu- tion, since nitrogen was applied as salts of Ca and K. Design of the experiment was a randomized block with 2 replications, 66 cultivars, 2 harvests, and 2 nitrate, Ca and K levels. After growing the plants for 4 weeks, one—half of the plants (4 plants) from each nutrient level per replication were harvested. The fresh weight of leaves and roots and the dry weight on the leaves were recorded. The remaining half of the plants were harvested at the end of 8 weeks. On these 8 week old plants a representative sample from each cultivar per treatment was used for NOB—N determination. The re- mainder of the plant material was used for the determination of other elements. 23 Table 3: Nitrate recovery from added nitrate to lettuce tissue extracts as measured by a nitrate selective electrode. Added Percent N03-N (ppm) Expected Found Difference Recovery 0 63 63 -- -- 10 73 72 -1 99 20 83 80 —3 96 40 103 100 -3 97 60 123 124 +1 101 100 163 161 -2 99 24 The analysis of fresh tissue showed differences among cultivars in nitrate concentration. Eleven cultivars with the largest differences [Low (L), Medium (M), and High (H)] were selected for additional studies. Total N and NOB-N determinations, based on dry weight, were made for the 11 cultivars on 4 and 8-week old plants grown at both nutrient levels. The total N was determined by Kjeldahl method. Tissue analyses for the other elements were made on 8 week old plants for all cultivars grown with 210 ppm N and for the selected 11 cultivars grown at 420 ppm. On the other elements K was determined by Flame Photometer, all others by Spectroscopy. Based on the nitrate content of dry tissues, 11 cultivars were selected and all possible crosses and their reciprocals were made. Since lettuce is a cleistogamous plant and the stigma is pollinated prior to flower opening, it was necessary to carry a dominant marker gene in the pollen parent in order to distinguish the crosses from the selfs. The markers were seed coat color, leaf color and the presence of anthocyanin. The four main lettuce types cos or romaine, butterhead, crisphead, and leaf types were included in this study and also served as markers. Six plants of each parent and 3 to 12 F plants (including 1 reciprocals) were grown in 1974. Six F1 plants from each cross were not obtained due to the difficulty encountered in making crosses with certain cultivars. A completely randomized design was used. Modified Tukey's test (88) was used for mean comparisons. 25 For this study the modified Hoaglands‘s nutrient solution contain— ing the standard nutrient concentration (Table 2) was used. The plants were grown for 35 days prior to leaf sampling. Fresh and dry weights and nitrate-N contents of the dry tissues were determined. The parental and F1 plants that were sampled for nitrate-N were allowed to flower for hybridization or to obtain F seeds. 2 In 1975, parental F1 and F2 populations were grown. The leachate of the nutrient solution was not reused in this study. The number of plants varied from 10-20 for parents, 9-20 for F s, and 50-200 for F l 2 populations for each cross. The F2 populations were randomized on the greenhouse benches. The parental and F populations were interspersed 1 among the F2 populations to obtain the environmental effects. All calculations regarding the means, variances, and standard errors were obtained on the individual data rather than from the con- densed frequency table. The population means were compared by the use of a "t" test. The ranges of the parental populations and F1 distribu- tion overlap and the absence of back crosses did not allow for the partitioning procedures as described by Burton (16) and Leonard ggual. (62) to obtain a quantitative estimate of the number of gene controlling the trait. Therefore, the procedure of using the mean of recessive parent and the arithmetic mean of the two parents was used to separate the low and high nitrate accumulating phenotypes. In the use of the recessive parent mean as part of class separa- tion, individuals greater than the mean of recessive parent (high accumu- lator) in the F2 population were classified as high accumulators; 26 therefore, an equal number below this mean were also classified as high accumulators. The total number of plants in the F2 less the number of high accumulators gave the number of low accumulators or the second phenotypic class. . In a second procedure, the arithmetic mean of the two parents was used as the dividing point between the low and high accumulator classes. This partitioning procedure gave results consistent with the previous procedure in all but one of the crosses (Wonder Van Voorburg x Caravan). In this cross a weighting was used to compensate for the parental over- lap. The weighting procedure consisted of 100 minus the percent overlap of recessive class below the arithmetic mean of the two parents/100 (Rb)(N) where Ro is the expected ratio of recessive plants based on the use of mean of recessive parent value, and N is the total number of F 2 plants. The total number of plants in the F less the number of high 2 accumulators gave the number of low accumulators. Calculation of in- dividuals fer the low and high accumulator classes were determined from the original data. Chi-square tests were used to compare the observed and the theo- retical ratios. RESULTS AND DISCUSSION A. Preliminary Studies The preliminary observations on the fresh tissue showed cultivar differences in nitrate content in both nutrient regimes (Table 4). Nitrate content on the dry tissues of the selected 11 cultivars also showed cultivar differences (Table 5). The cultivar differences noted in this study are in agreement with earlier reports that lettuce culti— vars differ in their capacity to accumulate nitrate (23,68,72). Increased nitrate-N content in leaves of plants grown in higher nitrate regime compared with lower nitrate regime was observed (Tables 4 and 5). This finding supports earlier reports indicating that the available nitrate-N was one of the factors in nitrate accumulation (68,92,95). These results suggested that growing the plants for four weeks using modified (standard nutrient level) Hoaglands's nutrient solution was adequate for the inheritance of nitrate accumulation study. Total-N Total-N in the leaf tissue was ascertained to understand the rela- tionship between nitrate-N and total-N. The total—N content of four week old leaf tissues of the two nutrient regimes did not differ, but the total-N content of the eight week old tissues from plants grown at high nutrient regime was greater than those grown in the standard nutrient regime (P = 0.05, Table 6). 27 Table 4: Nitrate-N (percent NO -N) content of fresh leaves from 66 lettuce cultivars grown in two nitrogen nutrient regimes for eight weeks. Nutrient Regime* Cultivar l 2 Agilo 0.035 0.054 Amplus 0.050 0.068 Arlon 0.027 0.066 Ayoncrisp 0.034 0.053 Avondefiance 0.024 0.074 Blondine 0.029 0.070 Bourguignonne 0.028 0.066 Brioso 0.043 0.070 Calicel 0.026 0.064 Calmar 0.032 0.056 Caravan 0.052 0.084 Climax 0.034 0.050 Deciso 0.029 0.051 Delta 0.034 0.057 Empire 0.042 0.050 E 9201A 0.028 0.044 Fairton 0.026 0.054 Great Lakes 0.031 0.086 Grand Rapids 0.028 0.055 Groso 0.027 0.066 Hilde 0.026 0.068 Interex 0.030 0.072 Ithaca 0.052 0.079 Kloek 0.032 0.066 Knap 0.042 0.079 Kordaat 0.055 0.063 Korrekt 0.039 0.074 Kwiek 0.032 0.066 Larganda 0.034 0.053 Liba 0.048 0.066 Magiola 0.048 0.060 Marquette 0.056 0.067 May Princess 0.038 0.068 Montemar 0.028 0.050 M.S.U. 71-146 0.042 0.066 Portato 0.024 0.040 Proeftuin's Blackpool 0.028 0.047 Rapide 0.030 0.090 Red Tipped Boston 0.029 0.061 continued Table 4—-continued 29 Nutrient Regime* Cultivar 1 2 Regina 0.029 0.095 Resistent 0.028 0.075 Secura 0.020 0.058 Solito 0.034 0.083 Spartan Lakes 0.037 0.060 Sucrine 0.029 0.086 Suzan 0.026 0.051 Texas 635- 1967—68 0.034 0.078 Texas 637- 1967-68 0.026 0.054 Texas 641- 1967-68 0.032 0.060 Texas 642- 1967-68 0.025 0.047 Texas 644- 1967-68 0.024 0.048 Tinto 0.029 0.068 Tonika 0.030 0.068 Type 57 0.016 0.054 Type 69 0.030 0.045 Valmaine 67074 0.014 0.058 Valmaine (Weslaco) 0.017 0.051 Valore 0.043 0.066 Valrio 0.036 0.056 Valtemp 0.025 0.060 Valverde 0.039 0.048 Vanmax 0.027 0.056 Variety A (Weslaco) 0.022 0.074 Variety B (Weslaco) 0.022 0.051 Ventura 0.034 0.050 WOnder Van Voorburg 0.016 0.034 Zomerkoningin 0.028 0.068 Mean 0.032 0.062** *1 Plants grown in modified Hoagland solution containing standard nutrient concentration (Table 2). 2 Plants grown in modified Hoagland solution containing 2 Ca(NO3)24 H20 + 2 (KN03). *1: Means compared by a t-test (P = 0.01). .Hw>oa Na uamuwmenwwm oozma .Hm>ma NH anemones unmoemeawem«g .oafimou unmanned w afimufiz momma “Emcze....mm.o..£..A .Amo.o u my .a.m.g ha menace eflauws eoaumummmm ammzw m:N A mozvmo N + A mozsvw sues aoauaaom eamsmmom emwmweoaium maawmu unmauuaz .aoauaaom meoawmom vowmwvoalnfl daemon uaowuunzN 30 mmN.H ««mme.o mem.H mamnm.a nom.o «soon.o some: one.o oom.o Na.o nmmm.o e~.o nmoe.o wussuoo> am» “muses omH.H oao.o ~.H soma.a ~¢.o oeoa.o moum>am> nmm.a mwm.o m~.a 6mm.a o~.o mvmm.o muoam> mme.o Hm.o wa.o ommm.o ea.o omwa.o oomamozumeamsam> onn.o mm~.o mw.o ammo.o q~.o mmm.o «sonouoqamsam> coo.a om~.o m.H mq.a me.o mmsm.o enumeha owH.H mmm.o m~.H oeo.a m¢.o euem.o momma enuummm ooq.a mmw.o ou.a vooH.H o~.o memo.H nauseous: mam.H nmm.o me.a meem.a wo.o mmH.H momnuH oo~.H oqo.o mN.H vonH.H oo.o oomo.o uxmuHQM oom.H mmm.o m~.H wnw.a om.o meo.H someuom mmm.a mom.o Ho.a oom.a eq.o rwae.a am>umo N new a as w x33 4 as w use a umpauaso 05H on aeofiuusn use some Hamuo>o ww< om< Noamuww uaowuuaz .mowoum museum can no wommo>uwm new museums z museums: can ca sebum mum>fiuaao oosuuwa HH mo Aunwwma hum do 2! oz udoouoav assumes Zloumuuwz um oHAMH 31 Table 6: Total nitrogen (percent N) of leaf tissues of lettuce culti- vars grown in two N regimes and harvested at four and eight week stage. Growth Stage 4 week old 8 week old Nitrate Cultivar 1z 2 l 2 Content Caravan 4.67 4.26 3.12 4.54 High (H) Kordaat 4.7 5.38 3.74 4.42 H Korrekt - - 4.25 5.14 Med. M Ithaca - 4.65 3.92 4.56 H Marquette 4.86 4.98 3.67 4.49 H Spartan Lakes 4.76 4.27 3.94 4.52 M Type - 57 - - 3.98 5.78 H Valmaine - 67-74 4.25 4.29 3.72 4.3 Low L Valmaine-Weslaco 4.1 4.53 3.32 4.02 L Valore - - 4.46 . 5.11 H Valverde - — 3.88 4.56 H wonder Van Voorberg 4.54 4.88 4.63 5.38 L Mean 4.55nS 4.66 3.89* 4.74 * Mean values within a growth stage compared by a t-test (P = 0.05). 21. Plants grown in modified Hoagland's nutrient solution (Table 2). 2. Plants grown in modified Hoagland's nutrient solution with 2(KN03) + 2 Ca(N03)24H20. -Not enough tissue for analysis. n8Not significant at 5% level. 32 The total-N of four week old tissues appeared similar at both nutrient regimes, suggesting the plants at this stage had sufficient N in both nutrient regimes, since the uptake of N was similar. The greater total-N in eight week old leaf tissues from plants grown in the higher nutrient regime appeared to be due to the greater N availability in the media. In the high nutrient regime, nitrate was 26.5% of the total N and at the standard nutrient regime it was 17.6% of the total N. This finding is analogous to an earlier report (99), where lettuce continued to accumulate nitrate with age. This finding also suggests that the available nitrate-N is one of the factors in nitrate-N accumulation. The total-N in the lettuce leaf tissues of subsequent plantings were not determined since both low and high accumulators of nitrate—N appeared to be similar in their total-N at four week stage. The total- N of eight week old leaf tissues appear to differ among cultivars but did not suggest a relationship between the nitrate accumulation and total-N content. Other Elements Other elements were determined on the eight week old leaf tissues for each cultivar grown in the standard nutrient regime to learn if the low and high nitrate accumulation would also differ in other elements (Table 7). Element content of eight-week old leaf tissues on 11 culti- vars grown at the two nutrient levels are shown in Table 8. The leaf tissues from the high nutrient regime was greater than those of standard nutrient regime in K, but was lower in P and Mg and B (P = 0.05). 33 00=d0uaoo 0.000 0.44 0.00 0.00 0.000 0.400 00.0 40.0 00.0 40.0 00.0 xmo0M .40 0.00 0.44 0.40 4.00 0.000 0.000 00.0 00.0 00.0 00.0 00.0 moanuH .00 0.000 0.00 0.00 0.40 0.000 0.000 40.0 00.0 00.0 04.0 40.0 wouumue0 .00 0.000 0.00 0.00 0.00 0.000 0.000 00.0 00.0 00.0 04.0 00.4 m000m .00 0.000 0.00 0.00 0.00 0.000 0.00 00.0 00.0 00.0 04.0 00.0 omouu .00 0.000 0.04 0.04 0.00 0.000 0.00 00.0 00.0 00.0 04.0 00.0 m000m0 cameo .00 0.00 0.00 0.00 0.40 0.000 0.00 00.0 00.0 00.0 00.0 40.4 mmxmA amouo .00 0.000 0.00 0.00 0.00 0.400 0.00 00.0 40.0 00.0 00.0 04.4 souu0mm .00 0.00 0.00 0.00 0.00 0.000 0.00 00.0 40.0 40.0 04.0 00.0 «0000 m .00 0.40 0.04 0.40 0.00 0.000 0.00 00.0 00.0 00.0 00.0 00.4 «H0050 .00 0.000 0.44 0.40 0.00 0.000 0.400 00.0 04.0 00.0 04.0 00.0 mu0m0 .40 0.000 0.44 0.00 0.00 0.400 0.000 00.0 00.0 00.0 04.0 40.0 000000 .00 ,0.000 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«000 000 0003 unw0o mo 00000msm usuam0m an QHAQH 34 Umflflflufiou 0.00 0.00 0.00 4.0 0.000 0.00 04.0 40.0 00.0 00.0 00.0 00:00 040 04000 .00 0.00 0.44 4.04 0.40 0.000 0.00 00.0 40.0 00.0 04.0 40.0 00:00 040 04040 .04 0.000 0.00 0.00 0.40 0.000 0.000 00.0 04.0 00.0 04.0 04.0 00:00 000 04000 .04 0.00 0.04 0.00 0.00 0.000 0.000 00.0 00.0 40.0 00.0 40.0 00:00 000 00000 .04 0.00 0.04 0.00 0.00 0.000 0.00 00.0 00.0 00.0 00.0 00.0 04440 .04 0.00 0.00 0.04 4.00 0.000 0.000 00.0 40.0 00.0 00.0 00.4 0000050 .04 0.00 0.00 0.04 0.00 0.000 0.00 40.0 00.0 00.0 00.0 00.4 04040 amuumam .44 0.000 0.40 0.40 0.40 0.000 0.000 40.0 04.0 00.0 04.0 00.0 ou00o0 .04 0.00 0.04 0.04 4.00 0.000 0.00 00.0 00.0 00.0 00.0 00.4 «H0000 .04 0.000 0.44 0.00 0.40 0.000 0.00 00.0 00.0 00.0 00.0 00.0 pamumHmmm .04 0.000 0.04 0.04 0.00 0.000 0.00 00.0 00.0 00.0 40.0 00.0 ma0000 .04 0.00 0.00 4.00 4.00 0.000 0.00 00.0 00.0 00.0 00.0 00.4 000000 040000 000 .00 0.000 0.44 0.04 0.00 0.000 0.00 00.0 40.0 00.0 00.0 00.4 400040 .00 0.40 0.04 0.00 0.00 0.000 0.00 00.0 00.0 00.0 00.0 00.0 000000400 m.a0:uwmoum .00 0.00 0.00 0.00 0.00 0.000 0.00 00.0 40.0 00.0 40.0 00.4 cumunom .00 0.00 0.04 0.00 0.00 0.000 0.00 00.0 00.0 00.0 00.0 00.4 040-00 .0.0.zfl .00 0.40 0.00 0.40 4.00 0.000 0.40 00.0 00.0 40.0 00.0 00.4 “damage: .40 0.000 0.04 0.04 0.00 0.000 0.00 00.0 00.0 00.0 04.0 40.0 0000:000 042 .00 0.000 0.00 0.00 0.00 0.000 0.00 00.0 00.0 00.0 00.0 00.4 muums0umz .00 0.000 0.00 0.00 0.40 0.000 0.000 00.0 00.0 00.0 04.0 40.0 m0o00umz .00 0.000 0.44 0.00 0.00 0.000 0.000 00.0 04.0 00.0 00.0 40.0 4000 .00 0.000 0.00 0.00 0.00 0.000 0.000 00.0 04.0 00.0 04.0 00.0 40:40u40 .00 0.040 0.40 0.00 0.00 0.000 0.000 00.0 00.0 40.0 00.0 40.0 00030 .00 0.000 0.00 0.00 0.00 0.000 0.000 00.0 00.0 00.0 00.0 00.0 uxauuom .00 0.000 0.00 0.00 0.00 0.000 0.000 00.0 04.0 00.0 04.0 00.0 ummwuqm .00 0.000 0.04 0.00 0.00 0.000 0.400 00.0 00.0 00.0 00.0 40.0 04am .00 800 N 0033030 . oz 04 a0 0 :0 mm as 02 40 «z 0 M veneHusooII0 00000 35 0.00 0.00 0.00 0.00 o.~¢~ 0.000 m0.o Hw.N 00.0 00.0 40.0 GHwawnoxuoaom .00 0.000 0.00 0.0m 0.00 o.0~N m.HOH 0m.o m¢.N 00.0 00.o m.m wuanuoo> am> umvaoz .00 o.~m0 0.00 0.00 0.0N c.0mm o.oma 00.0 00.~ 00.0 H0.o 0.0 muaudm> .00 0.00N 0.00 0.00 0.0m 0.00N 0.000 00.o 0N.N 00.0 0m.o 0.0 000000030 m 00¢0u~> .00 0.000 0.00 n.04 n.0N o.¢HN 0.00 00.0 0o.N 00.0 om.o om.m 000000030 < 000000> .m0 0.00 o.0N o.qm «.m0 o.0mH 0.00 M0.o 00.0 00.0 00.0 00.0 xmacm> .N0 c.0wm c.40 0.00 0.00 o.0NN 0.00 00.0 H0.~ 00.0 Hm.o 00.0 mvum>am> .00 o.0oa c.0m N.00 0.NN o.mm~ 0.000 00.0 N0.N M0.o mm.o 0N.0 mamuam> .00 0.00m o.mq 0.00 0.00 o.HHN 0.000 00.0 ON.N mm.o om.o om.m oauam> .mm o.~m0 0.00 0.00 0.00 o.m~m o.0NH 00.0 00.N 00.0 00.0 00.0 unoam> .00 0.000 0.00 0.0m 0.00 0.000 0.00 00.0 00.0 00.0 mN.o 40.0 000000030 maHmaHm> .00 0.00 0.0m 0.0m «.ma 0.0HN o.N0 00.0 00.0 om.o 0N.o No.0 40000 mcwmaam> .00 0.000 0.00 0.mm 0.0N c.0mm 0.000 00.0 00.0 N0.o 00.0 00.0 00 mama .mm c.000 o.00 H.m0 o.om c.00N 0.000 ow.o 00.0 00.0 mm.o om.m 0m 0009 .00 0.000 c.00 0.00 0.0N o.~om 0.00 00.0 Nm.~ mm.o 0m.o om.m 030609 .mm 0.00 0.00 0.00 0.00 0.0mm o.m0 ~0.o Ho.N m0.o 00.0 om.m ouQHH .Nm o.NNH 0.00 0.00 0.0N 0.0mm 0.00 00.0 mm.~ 00.o 0m.o No.0 00:00 000 00x09 .00 590 N um>HuH=u .oz 00 a0 0 =0 00 50 000 00 «z 0 V0 vmnafiuaooii0 manma 36 wonawuaoo 0.000020.04«00.m4mZm~.o~mzo.H4Nmzo.0m «00.omZm~.N M0.o «0m.o «00.0 mp 0 0.000 o.0m 0.00 0.00 o.0N~ 0.000 00.o 04.0 00.0 04.0 om.m wuonuoo> nm> 000003 m 0.000 0.44 mua4 0.00 0.000 0.00 00.0 00.N 00.0 Hm.o 00.4 mvum>am> m 0.0m0 0.00 4.00 0.00 o.m~m o.0~0 00.0 00.0 40.0 mm.o 00.0 muoam> 0 0.000 o.04 0.0m 0.00 0.040 0.00 00.0 00.0 00.0 mN.o 40.4 oumamwzlma00300> 0 o.00 0.0m 0.4m «.mH 0.000 0.00 00.0 00.0 om.o 00.0 00.0 40000 ma0maam> m 0.400 0.00 0.m0 0.0m o.o0N 0.000 00.0 40.N 00.0 ~m.o om.m 0m mama z 0.00 0.0m 0.04 N.m0 o.m- 04mm 40.0 0~.N 00.0 mm.o 00.4 00300 «muummm m 0.000 c.0m 0.0m N.m0 0.000 o.m0 00.0 um.~ 00.0 m~.o 00.4 muumzvumz 2 0.000 0.00 0.00 0.00 0.00m o.~00 00.0 m~.~ «0.0 00.0 00.0 uxmuuom m 0.000 0.00 ~.o0 0.00 0.000 0.000 00.0 «4.0 00.0 04.0 00.0 ummwuom m 0.00 o.44 0.4m 4.00 o.00~ 0.000 00.0 00.N 00.0 00.0 00.0 000500 m o.om 0.0m 0.00 0.00 0.000 0.00 00.0 00.0 04.0 00.0 40.4 am>mumo uaouaoo 800 N um>0udso 83002 00 :0 0 =0 00 a: 02 00 02 0 0 .mco0umuuamocoo 00 0am M .2 0000a0uw wcwawmuaoo aowuaaom camawmom vmwmwvoa a0 aaouw madman .0 .mma0wmu unm0uusa oBu a0 aBOuw mum>0uaso wouumamm aw>me mo muamucoo anBmHm “0 manna 37 .00>00 00 00 00000000000 002 m2 .Amo.o u my uw0ulu 0 an 00000500 0E50oo 0 003003 00:00> 0002 0 0.00 0.04 0m.m~ 00.00 0.000 o.~0 00.0 0o.N m0.o 00.0 00.0 MW 0 o.m0 0.04 0.00 0.00 0.400 0.00 40.0 00.0 mm.o om.o 00.0 000nuoo> 00> 000003 m 0.00 0.0m 0.0m 0.00 0.000 o.m0 40.0 00.0 ~4.o 00.o 00.0 0000>00> m 0.40 0.0m 0.00 4.00 0.000 o.m0 00.0 00.0 m0.o 00.0 mm.0 0no00> 0 0.00 0.04 0.40 9.00 0.000 o.m0 04.0 00.0 0m.o m0.o 00.0 oo00m0310000fi00> 0 0.00 o.- 0.0m 0.00 0.00 o.m4 M4.o 00.0 04.0 00.0 04.0 40000 0000B00> m 0.00 0.04 0.4m 0.00 0.000 o.m0 00.0 mm.N 00.0 mm.o om.0 0m 0009 z o.00 0.44 m.om o.00 0.400 0.00 o0.o NM.N 00.0 00.0 00.0 00000 0000000 m 0.000 0.0m 0.00 0.00 0.000 0.00 00.0 00.0 00.0 00.o 00.0 000030002 2 0.40 0.44 0.0m 0.00 0.000 o.m0 40.0 mm.~ 40.0 mN.o 00.0 ux0uuom m 0.40 0.04 N.Nm 0.00 0.000 0.00 mm.o 00.0 N0.o 00.0 00.0 000000M m o.m0~ 0.04 m.om 0.00 o.m- o.00 00.0 mm.~ 00.0 m~.o 00.0 000000 m 0.000 0.04 N.~m 0.00 0.000 o.mm 00.0 0N.N 00.0 00.0 04.0 c0>0u0o 0000000 I Baa N u0>0u0do 0u0uu0z 0< 0N 0 so 0m 02 w: 00 02 m M .MOZMN 000 o m4NAmozv0oN w00000uaoo 00000000 00000003 00000006 00 0300» 00000m .N 00:00uaoollw 00000 38 Ca and other micro nutrients of leaf tissues were similar for both nutrient regimes. The higher K in leaf tissues of plants grown at the high nutrient regime was probably due to the greater availability of K in the media. Calcium contents were similar in leaf tissues at both nutrient regimes although the Ca content was twice as high in the high nutrient regime. A decrease in P content with increased N was also noted with beet leaf blades and spinach leaves (17). A decrease in Mg content of tomato shoot tissue was also reported by Wilcox gt a}, (94) when the plants were grown with nitrate-N in the media for seven days as compared with three day treatment. Micro elements content, except B, were similar in leaf tissues from both nutrient regimes; this was probably due to similar concentra— tions in both nutrient solutions. The other elements were not determined in the subsequent plantings, since the preliminary observations did not show a marked difference in the elements between the low and high nitrate accumulating cultivars as a group. Fresh and Dry Weights Fresh and dry weights of leaves and fresh weight of roots were determined on the four week old plants of various cultivars grown at both nutrient regimes (Table 9). Analysis of variance of the fresh weight of leaves, suggested cultivar differences in each nutrient regime. The fresh weight of roots 39 Table 9: Mean fresh weight of leaf, root and dry weight of leaf of four week old lettuce plants grown in two nutrient regimes for the cultivars used in N03-N study. Plant Part 'Eresh ' Dry Leafy Root Leaf Nutrient Regimez Cultivar 1 2 l 2 1 2 Agilo 20.95 22.79 7.775 8.675 0.9988 1.1425 Amplus 29.00 24.62 9.100 6.865 1.3275 1.2525 Arlon 24.96 15.86 6.925 5.065 1.3650 0.9825 Avoncrisp 25.95 16.37 8.690 5.025 1.0988 0.9100 Avondefiance 19.71 11.12 6.305 3.205 0.8225 0.4575 Blondine 24.59 21.13 7.190 5.780 1.1725 1.1063 Bourguignonne 21.29 20.83 6.450 6.930 1.1050 1.0210 Brioso 20.34 20.93 6.100 5.580 0.9188 0.9688 Calicel 22.73 16.08 4.705 4.950 1.2304 1.2350 Calmar 25.55 12.73 3.990 3.300 1.0775 0.7867 Caravan 27.42 26.17 6.615 6.400 1.2013 1.1875 Climax 22.70 24.73 5.265 5.415 1.0800 1.2938 Deciso 28.38 18.92 6.355 5.030 1.2475 1.0435 Delta 23.08 17.78 8.250 6.050 1.2555 1.1025 Empire 23.38 13.90 4.515 3.300 1.2325 0.8300 E 9201 A 11.64 9.48 4.725 3.365 0.6863 0.5142 Fairton 21.57 20.59 4.425 5.315 1.0388 1.0925 Great Lakes 21.79 24.10 3.155 4.380 1.0963 1.4113 Grand Rapids 30.12 23.20 7.290 5.850 1.3900 1.2113 Groso 22.40 17.03 5.515 6.275 1.1713 0.9988 Hilde 26.73 24.88 7.975 8.005 1.5363 1.6125 Interex 23.55 23.40 6.725 7.000 1.3913 1.3475 Ithaca 22.36 27.93 3.815 4.900 1.0450 1.3913 Kloek 26.37 22.70 5.775 6.140 1.4113 1.2938 Knap 23.82 32.08 4.915 8.230 1.2513 1.7838 Kordaat 22.67 25.79 5.325 6.405 1.2325 1.3113 Korrekt 22.22 29.43 6.100 8.840 1.3555 1.5625 Kwiek 26.22 23.63 6.655 7.465 1.4713 1.4588 Larganda 26.48 24.79 6.230 7.525 1.7635 1.7325 Liba 21.12 18.68 5.590 4.675 0.9550 1.0975 Magiola 24.48 21.55 4.230 6.825 1.3525 1.2813 Marquette 26.47 21.48 4.100 4.740 1.2913 1.2050 May Princess 21.80 24.52 6.865 8.050 1.0290 1.2888 Montemar 13.99 11.72 2.950 4.415 0.7163 0.6400 M.S.U. 71-146 20.14 20.79 3.780 4.325 1.0575 1.0825 Portato 21.07 16.76 6.465 6.390 1.1588 1.2713 Proeftuin's Blackpool 21.28 20.36 7.515 7.250 1.2621 1.1788 continued Table 9--continued 40 Plant Part Fresh Dry Leafy Root Leaf Nutrient Regimez Cultivar l 2 1 2 1 2 Rapide 20.75 17.62 4.205 3.765 1.0863 1.0463 Red Tipped Boston 19.04 15.07 5.315 3.690 1.0713 0.9613 Regina 20.58 18.03 6.100 6.150 1.2488 1.2238 Resistent 21.19 17.99 5.415 5.555 1.2350 1.1000 Secura 21.15 17.62 5.640 5.600 1.1225 1.0450 Solito 21.12 22.88 5.750 6.625 1.1663 1.4813 Spartan Lakes 17.72 22.54 4.005 5.355 1.9725 1.3625 Sucrine 17.37 21.30 5.015 7.680 1.1000 1.4500 Suzan 19.50 20.53 5.540 6.940 1.1875 1.4700 Texas 635-1967-68 22.67 . 21.70 6.525 6.015 1.3325 1.2675 Texas 637-1967-68 20.13 20.90 6.015 7.415 1.1113 1.4763 Texas 641-1967-68 15.27 22.84 4.380 6.950 0.8525 1.4125 Texas 642-1967-68 23.25 14.58 7.300 5.430 1.4325 1.1400 Texas 644-1967-68 22.57 18.54 6.490 5.840 1.2625 1.3363 Tinto 20.59 20.78 5.505 5.950 1.0338 1.2025 Tonika 25.09 22.73 7.460 4.240 1.3629 1.2263 Type 57 24.23 21.88 6.350 5.775 1.2488 1.1913 Type 69 21.72 21.25 6.375 4.890 1.2325 1.1163 Valmaine 67074 21.03 22.62 6.780 7.500 1.3700 1.6025 Valmaine (weslaco) 25.53 19.27 6.630 6.985 1.6038 1.6013 Valore 22.38 25.94 5.125 6.675 0.9800 1.3838 Valrio 18.17 15.80 5.530 2.625 0.9650 0.5950 Valtemp 25.12 14.27 8.440 5.025 1.4050 1.0225 Valverde 23.88 21.34 4.830 5.980 1.2489 1.0388 Vanmax 19.96 12.14 3.017 4.430 1.0350 0.6975 Variety A (weslaco) 18.73 18.13 5.340 6.565 1.1388 1.1563 Variety B (weslaco) 20.54 17.50 6.500 4.940 1.1088 0.8325 Ventura 17.40 19.37 5.790 5.275 0.9200 1.0850 WOnder Van Voorburg 19.44 17.41 5.725 5.680 1.1238 1.2125 Zomerkonigin 22.78 21.45 7.055 7.115 1.1825 1.3775 Mean 22.20 20.17 5.858 5.829 1.1923 1.1823 yMean separation LSD 0.05 = 3.79. 21. Plants were grown using modified Hoagland solution (Table 2). 2. Plants were grown using modified Hoagland solution containing 2 Ca (N03)24H20 + 2 KNO 3. 41 and dry weight of leaves for the two nutrient regimes was similar. The fresh leaf weight from the standard nutrient regime was greater (P = 0.05) than those grown from a higher nutrient regime (Table 9). Preliminary observations on the nitrate-N content and yield (fresh weight) for the various cultivars failed to show a relationship between these two variables at four weeks of age. B. Genetic Experiments--1974 Based on the results of preliminary screening, ten lettuce culti- vars were selected for further studies based on their low (L), medium (M), and high (H) nitrate content. The mean nitrate-N contents of these cultivars and their reciprocal crosses are summarized in Table 10. Although the results from the preliminary studies suggested that cultivars may be classified as low, intermediate and high nitrate-N accumulators (Tables 4 and 5) in 1974, the medium and high accumulators could not be separated from each other. The nitrate-N content of Valmaine (L) and Wonder Van Voorburg (L) were lower than Caravan (H), Kordaat (H), Ithaca (H), Marquette (H), Korrekt (H), Valore (H), Valverde (H) and Type-57 (H) (Table 10) in 1974 as in the previous year. The nitrate-N contents of F1 plants from low x high nitrate crosses were not significantly different from the low accumulator parent which suggests a dominance for low nitrate accumulation. Nitrate-N content of F1 plants from low x low crosses was low and did not differ significantly from the parents. Results from crosses involving only high nitrate accumulators were variable. Table 10: Mean nitrate-N (percent N0 -N on dry weight) of ten cultivars and their reciprocal crosses. No. of Mean Nitrate Generation Plants Nitrate-Ny 32 content Valmaine 67074 Pl 4 1.0494 0.0631 L Valmaine Weslaco P2 6 0.7733 0.0233 L WOnder Van Voorburg P3 6 1.1567 0.0396 L Caravan P4 6 1.6867 0.1742 H Kordaat P5 4 1.9181 0.0978 H Ithaca P6 4 1.9663 0.2181 H Marquette P7 3 1.7442 0.1890 H Korrekt P8 4 1.6765 0.0075 H Valore P9 6 1.7133 0.0886 H Valverde P10 4 1.8250 0.0081 H Type-57 P11 4 1.8844 0.0762 H Cross P1 Ple3 7 0.6370 0.0296 LxL P3xP1 8 0.6635 0.0052 LxL P4xP1 4 1.0906 0.0135 HxL Ple5 4 0.9506 0.3130 LxH PsxP1 8 0.9763 0.033 HxL Ple6 4 0.9370 0.0022 LxH P6xP1 3 0.9225 0.0515 HxL Ple7 4 1.0850 0.0578 LxH Ple8 4 0.9700 0.0076 LxH P8xP1 4 1.0500 0.0430 HxL Ple11 4 0.9900 0.0587 LxH Plle1 4 0.8500 0.0296 HxL continued Table 10--continued 43 No. of 2 Generation Cross Plants N03-N 3 Cross F1 szP3 6 0.6100 0.0084 LxL P3xP2 6 0.5967 0.0109 LxL PéxP2 3 1.1333 0.0702 HxL PZxP5 4 0.9050 0.0111 LxH PSxP2 4 0.8009 0.0003 HxL szP6 4 0.9788 0.0958 LxH P6xP2 4 0.8748 0.0785 HxL szP7 4 1.1019 0.0282 LXH szP8 4 0.8965 0.0100 LxH P8xP2 4 0.9841 0.1044 HxL szP9 6 0.7567 0.0036 LxH P9xP2 5 0.7520 0.0091 HxL P4xP3 6 0.9100 0.0150 HxL P3xP4 2 0.8913 0.0330 LxH P3xP6 4 (0.8044 0.0016 LxH P6xP3 4 0.8880 0.0242 HxL P3xP9 4 1.300 0.0355 LxH ngP3 4 1.2850 0.0404 HxL P4xP5 4 1.1583 0.0790 HxH P4XP6 4 2.2678 0.1045 HxH P5XP7 4 1.2330 0.0039 HxH P7xP5 4 1.235 0.0490 HxH PSxP8 4 2.238 0.1997 HxH P8xP5 4 2.1675 0.0970 HxH P6xP7 4 1.7131 0.1331 HxH P7xP6 4 1.6718 0.1175 HxH continued 44 Table 10--continued No. of 2 Generation Cross Plants NOB-N 3 Cross F1 p9xP5 4 1.6718 0.1829 HxH P9xP8 4 1.9650 0.0596 HxH Plong 4 1.1781 0.0200 HxH yMean comparison, modified Tukey's method. To compare two means differing in their sample size use lower 'n' value, for example P = 1.0494, n = 4 and P2 = 0.7733, n = 6. The minimum significant value (n =4 ) = 0.4831. The two P and P means do not differ by this value, therefore, these means are not significantly different at the 5% level. minimum significant difference = 0.4831 0.3945 0.34164 0.20596 0.27896 0.2583 wow-waD ll 45 The F1 generation of three of eight such crosses [Caravan (P4) x Kordaat (P5), Kordaat (PS) X Marquette (P7), and Valverde (P10) x Valore (P9) Table 5] showed a lower nitrate-N content than either parent. Low accu- mulation trait of the F1 generation resulting from two high accumulator parents suggests that the F1 plant contained two or more dominant alleles, each locus probably controlled an enzymatic step essential for low nitrate accumulation. The nitrate-N contents of five other high x high F plants re- 1 mained high and were similar to their parents (Table 10). Nitrate-N contents of reciprocal crosses were not significantly different. Variations in Nitrate-N Content of Lettuce Cultivars at Different Plantingg The nitrate content of parental lines which appeared to be medium or high accumulators was variable from year to year. For example, cultivar Korrekt, Type-57, Valore and Valverde appeared to be medium or mediumrhigh accumulators in 1973, but all were found to be high accumu- lators in 1974 (Table 11). Cultivar Ithaca which appeared to be a high accumulator in the 1974 and 1974 planting, was found to have medium levels of nitrate in 1975. Based on this information it was assumed that the medium and high accumulators were probably the same and the inconsistency observed was due to the sensitive nature of the trait. The difference between the cultivars designated low and high was con- sistent for the three years of testing. The mean nitrate content was lowest in 1973 and highest in 1975, this was due to the increased frequency of watering the plants with nutrient solution. Table 11: Leaf nitrate-N (percent) content of various cultivars grown in sand culture using modified Hoagland solution on four week old dry tissues for three years. Cultivar 1973 1974 1975 Caravan 1.49 1.6867 2.0233 Ithaca 1.19 1.9663 1.6075 Kordaat 1.01 1.9181 2.2700 Korrekt 0.63 1.6765 — Marquette 1.09 1.7442 - Type-57 0.97 1.8844 — Valmaine-Weslaco 0.48 0.7733 1.2525 Valmaine-67074 0.35 1.0494 - Valore 0.95 1.7133 2.0525 Valverde 0.96 1.8250 - Wonder Van Voorburg 0.46 1.1567 1.4038 Mean 0.871 1.581 1.768 47 Individual Crosses--1975 Nitrate in plant tissue is always in a dynamic state since it represents the difference between absorption and nitrate reduction (assimilation). Nitrate absorbed can also move into or out of a parti- cular plant part. Hence, one factor may alter tissue nitrate concen- trations by affecting any one or all the processes of absorption, assimilation, and translocation (68). For example, factors such as nitrate availability (68,92,95), light (9,68), shade (l7), temperature, and other factors (9,68,92,95) have been known to influence the tissue nitrate-N content. To avoid the diurnal fluctuations in tissue nitrate levels, all the lettuce plants were harvested at one time under dark conditions. Thus the nitrate measured was the excess unreduced portion at that time. Although every effort was taken to obtain a uniform environment, the parental lines showed a wide range in their nitrate content. The mean nitrate-N contents of parents and F1 plants are shown in Table 12. The low nitrate accumulator lines used were Valmaine and Wonder Van Voorburg. The high accumulator parents were Caravan, Kordaat and Valore (Table 12). The cultivar Ithaca which was used in one cross was a medium accumulator in 1975 but was high in 1974 and 1973 (Tablell). The nitrate-N content of F1 populations of crosses low x low and low x high were low accumulators. The reciprocal crosses did not differ significantly and therefore were pooled. 48 Table 12: Mean nitrate-N (percent N0 -N) content (dry weight) of parents and F plants of crosses used in the genetic analyses of nitrate-N study. Parent Lines Percent N03—N 2 P1 Valmaine 1.2525 3 P2 Wonder Van Voorburg 1.4038 ab P3 Caravan 2.0233 c P4 Kordaat 2.2700 c P5 Ithaca 1.6075 b P6 Valore 2.0525 c F Population -1 plxpzy 1.0355 Ple3 1.5475 13le4 1.2292 Ple6 1.1825 P2xP3 1.4275 szpé 1.4675 zMean separation within the column (t-test 5% level). yReciprocal crosses included. 49 Cross: Valmaine (P1) x Caravan (P3)(Low x High) In order to obtain the best estimate of the gene number involved in the crosses, two partitionint procedures were considered to separate the phenotypes. The first procedure consisted of using the recessive parental mean. Plants in classes greater than the recessive parental mean in the F2 population were classified as high accumulators, and an equal number of plants lower than this mean was also classified as high accumulators. The total number of plants in the F2 less the number of high accumulators gave the number of low accumulators or the second phenotype. In the second method the arithmetic mean of the two parents were used as the dividing point between the low and high accumulator classes. The population means were compared by a t-test at P = 0.05. The P value of Chi-square tests indicate that the calculated value is sig- nificant at the first P value but not at the second. For example, in the cross Valmaine (L) x Caravan (H), the division of phenotypes sug- gested a segregation ratio of 9 Low:55 High. The calculated X2 value for goodness of fit to the proposed 9:55 ratio was 0.6644 with a P value of 0.5-0.25. The Chi-square 0.6644 was significant at the 50% level but not at the 25% level. Therefore, the proposed ratio of 9:55 was accepted on the basis of lack of significance at 25% level. Frequency distributions of nitrate-N content for Valmaine (P1) Caravan (P3), and their F1 and F2 populations are summarized in TabLe13. The nitrate content of Caravan was greater than that of Valmaine and the F generation (P = 0.05). The mean of the F 1 population (1.5475) 1 0 5 Amo.o u mv ummulu m mp aabHoo aHnuHs GOHumummwm comma oomH.o Hsoma.H m m s OH mH He on wH OH HH N com NH OHHo.o mmhqm.H H s H - H OH Hm MHmo.o nmmuo.~ N I q H q H m mH cm>mumu mHmo.o mmNnN.H H N m o m q H om demaHm> mm Munoz o.m m.~ o.~ q.~ N.N o.~ w.H o.H ¢.H N.H o.H w.o musmHm mo .oz mmMHo mo uHaHH Home: .nm>mumm x oaHmaHm> mo mmouo mo mGOHumuoaow mm was Hm .Hmuaouwa mnu How Auanoz xuvv udMHm\AZI oz uaoonmav Zloumuqu mo GOHuanHHuwHw moamndoum umH oHan .MH mHan 51 approximated the arithmetic mean of the two parents (1.6379). These two values were not significantly different from each other. The F1 mean was not significantly different from Valmaine, the low accumulator, suggesting dominance toward low nitrate accumulation. The F2 population mean was greater (P = 0.05) than the low accumu- lator parent Valmaine and the F but not significantly different from 1 the high accumulator parent Caravan. The parental types were recovered in the F2 population and a majority of the segregating population fell in the high accumulator range (Table 13). Using the recessive parental mean to separate the two classes (low and high) the number of plants which fell beyond (greater) the mean (2.0233) in the F population was calculated and multiplied by 2 2 to obtain the total number of high accumulators (49+20+6+8+5)2 = 176 (Table 13). The number of low accumulators is calculated by subtracting the high accumulators from the total F population (200-176 = 24), thus 2 giving a ratio of 24 low:176 high accumulators. Since the arithmetic mean of the two parents (1.6379) approximates the F1 mean (1.5475) and the arithmetic mean of the parents was greater (P = 0.05) than the low accumulator parent. The dividing point between the low and high accumulators appears to be between the 1.4 and 1.6 class (Table 13). The observed 23 low:177 high is similar to the previous partitioning (24:176) procedure. This observed phenotypic ratio suggests a 9:55 model. The X2 value gave an acceptable fit to the model (P = 0.5-0.25. Table 14). The observed F mean, 1.9364, 2 approximates the expected F2 mean of 1.9149 for a 9:55 model (P = 0.990— 0.975, Table 15). 52 N .dOHuanHuumHv m no momma GOHumHnmom N m we waHQOHuHuummm .m 30H mnu uoa wan Hm>mH m sts wSu um unmoHMHame mosHm> anpmumHsono may» .mnHm> wouanmzx .muamuma 03u mo some oHuwanuHHm wsu no momma aOHumHaaom Nm wcHsOHuHuumm .N .osHm> some Hmuaoumm m>Hmmwomu mzu_so momma QOHumHnmom Nm maHaOHuHuumm .HN I H5125 nu HHmH 36 2.3.3.3 EH28 .. 93% x .1me 82”: x 92,33... wusnuoo> am> umvsoz II m.otmn.o II NNN.o Nnnm NNuwNH II ONHowH 30H x 30H x msHmaHm>m mHOHm> x mN.o1m.o H.01mN.o mmq.o Non.H Hum omuomH ocuqu Nqume :me x 30H wussuoo> om> Howaoz am>mumo x H.o1mN. mn.0la.o mNo.N oomo.o Hum omuomH acquooH wquNmH anm N 30H wusnuoo> am> Houses mn.otm.o m.01mm.o mmqo.o moo.o mum m.mmum.NHH mmuHHH meNHH stm x 30H muon> x oaHmaHm> mn.ola.o mo.onmn.o mmqo.o omQN.o mum m.mwum.NHH awHHHH «wnoHH anm x 30H ummvqu N mVHing—HE? mN.OIm.o mN.OIm.o owmo.H qqoo.o mmum NnHHmN NNHHMN omanN anm x 30H sm>mumo x oaHmaHm> N H N H Hmwoz anmHBOH anHmHBOH HameHBOH GOHuMHaaauo< mmouu 1 Na 3835 . 2:32qu N .aowumuoaow m 0:» SH muwp ZlmumuuHa How Hmwoa wmumHSumom 0£u ou uHm mo mmmcmoow Mom umou muddvmlwno "qH mHnMH 53 Table 15: Chi-square test for goodness of fit of the F2 mean to the postulated model. Cross Model OBS EXP X2 P Valmaine x Caravan 9:55 1.9364 1.9149 0.0002 0.990-0.975 Valmaine x Kordaat 9:7 1.7868 1.6977 0.0047 0.975-0.900 Valmaine x Valore 9:7 1.6352 1.6025 0.0007 0.990-0.975 wonder Van Voorburg x 3:1 1.5822 1.620 0.0009 0.990-0.975 Caravan WOnder Van Voorburg x 3:1 1.5836 1.566 0.0002 0.990-0.975 Valore Caravan x Ithaca 6:10 1.8445 1.7364 0.0119 0.950-0.900 54 Based on the F2 progeny of the cross Valmaine x Caravan, a 3~major— gene system is suggested for the inheritance of low and high nitrate—N accumulation in this cross. The F1 mean of low x high.was low, suggest— ing a dominance toward low accumulation. Apparently, both parents may have contributed dominant gene(s) towards the observed F1 phenotype. The F2 distribution shows a unimodal distribution and is skewed towards high accumulation. The F2 and P3 population means were not significantly different from each other, and a major portion of the F population fell 2 in the high accumulator range suggesting that a dominant gene was con- tributed by the high accumulator parent. Based on the ratios observed in this cross (Table 14) the proposed genotype of Valmaine is AA BB cc and that of Caravan is aa bb CC (Table 16) where the presence of Aland B genes are necessary to bring about low nitrate accumulation, and g_is the dominant gene controlling high nitrate accumulation. The F1 genotype is Aa Bb Cc and was classified as a low accumu— lator, but the observed ratio of 9 low; 55 high in the F population 2 suggests that the genotypes having ArB-C, A-b-C, a-B-C, a—b-C, Arb-c, a-B-c and a-b-c are high accumulators; while, the genotype having ArB-c were classified as low accumulators. Although the F generation 1 was classified as a low accumulator, the F distribution shows a large 1 portion (80%) of its population in the parental overlap (Table 13). The F1 mean and the arithmetic mean of the low parents were not sig- nificantly different from each other. The F1 mean 1.5475 was greater than the P1 (L) mean (1.2525) at 10% level but not at 5% level. The F1 plants also showed heterosis in fresh and dry weights (see Table 24, page 76). 55 Table 16: Proposed genotypes of the lettuce parent lines used in nitrate-N study. Nitrate-N Cultivar Accumulation Genotype Valmaine P1 Low AA BB cc WOnder Van Voorburg P2 Low aa bb CZCZ Caravan P3 High aa bb CC Kordaat P4 High aa bb cc Ithaca P5 Medium aa B2B2 cc Valore P High aa bb cc 6 f.'.' -. -- .‘qu 56 All of the observations suggest that the observed low accumulator phenotype of the F1 generation probably was due to hybrid vigor and/or due to a dilution effect. The observed 9L:55H in the F2 population suggests that the presence of §_gene controlling high accumulation, is epistatic to the A and B alleles. The genes A and _B are complimentary to each other which results in low accumulation. The contribution of the recessive allele g_was not detected because of the F population 2 size. Cross: Valmaine (P1) x Kordaat (P4) (Low x High) Frequency distributions for nitrate-N content of Valmaine (P1), Kordaat (P4), and their F1 and F2 populations are summarized in Table 17. The nitrate content of Kordaat was greater than that of Valmaine and the F1 generation (P - 0.05). The means for the F1 and Valmaine were not significantly different from each other suggesting dominance for low accumulation. The F2 population mean was greater than Valmaine (1) and the F1 mean, but less than Kordaat (H) in its nitrate content (P - 0.05). Using the recessive parental mean to separate phenotypic classes of high and low, the number of plants which fell beyond the mean of Kordaat (2.27) in the segregating population was multiplied by 2 to obtain the number of high accumulators 42 x 2 = 84. The number of low accumulators was 200 - 84 - 116. This observed ratio of 116 L:84 H suggested a 9:7 model and gave a good fit to the model (P - 0.75-0.5, Table 14). Thus, a two major gene system controlling nitrate accumulation was suggested. .Amo.o u my ammulu m mp HESHoo angHB GOHumummmw comma mem~.o owowH.H H w w mH NH AN QH mm 0H «N m H com a Ammo.o m~a-.H m N m H a HH 7 .5 «mmH.o HooHN.~ m H N H H H mH “ensues aHmo.o mmNm~.H H N m o m H H om maHmaHm> Hm News: o.m m.~ o.~ H.H H.~ o.N w.H o.H H.H H.H o.H m.o muamHm Ho .oz mmmHo Ho uHaHH Hume: . .ummvuo x oonaHm> mo mmouo mo m:0Humumcow Nm vow Hm .Hmuaoumm mnu How AuanoB muvv uQMHm\A21 oz unmouomv Zloumuqu mo sOHuSAHHumHv mucosvmnm "HH mHHmH 58 The ratio also suggested that low nitrate accumulation was the result of an epistatic effect. Similarly, ratios were obtained by using arithmetic mean of the two parents (1.7613) as the dividing point between the lows and highs. The number of plants in the F2 population below the arithmetic mean of the two parents (low accumulators) was 26+33+l9+24+8+l = 111 and the number of high accumulators was 25+22+19+8+7 = 89 (Table 17). This observed 111 low:89 high suggested a segregation ratio of 9:7 and gave a good fit to the model (P = 0.90-0.75, Table 14). The observed F2 mean of 1.7868 approximated the expected F2 mean 1.6977 for a 9:7 model (P = 0.975—0.90, Table 15). A two major gene system is suggested based on the following observations: the F1 plant was completely dominant for low accumula- tion, which was contributed by Valmaine, whose genotype was designated as AA BB cc; based on the observed F ratio the proposed genotype of 2 Kordaat is aa bb cc (Table 16); the genotype of the F was Aa Bb cc, 1 and the observed F ratio suggested that the presence of both the A_and 2 §_a11eles are necessary to bring about low nitrate accumulation; the genotypes having A:b_or §:§_and 3:2 were high nitrate accumulators while the A:B_genotypes were low accumulators; the genes A_and B are complimentary to each other; both Valmaine and Kordaat were homozygous for the recessive c gene; therefore, the presence of a third gene was not evident. 59 Cross: Valmaine (P%) x Valore (P6)T(Low x High) Frequency distribution of nitrate-N content of Valmaine (P1), Valore (P6) F1 and F2 populations are summarized in Table 18. The mean nitrate content of Valore was greater than that of Valmaine and the F1 population (P = 0.05). The F1 and P1 population means were not signifi- cantly different from each other (Table 18), suggesting dominance for low accumulation of nitrate-N. The nitrate content of the F2 population mean was greater than that of Valmaine (L) and the F (P = 0.05), but 1 less than Valore (H) (P = 0.05). The partitioning procedure using the recessive parent (Valore) mean (2.0525) to calculate the number of high accumulator was 44 x 2==88 and the number of low accumulators was 200 - 88 = 112. Thus an ob- served ratio of 112 low:88 high suggested a segregation ratio of 9:7. These data gave a good fit to the model (P = 0.95-0.9, Table 14), based on a 2 major gene system with epistasis. Similar results were obtained by using the arithmetic mean of the two parents (1.6525) as the dividing point between lows and highs. The number of low accumulators was 34+36+22+8+1 = 111 and the high accumulators was 41+14+29+6+7+2 = 89 (Table 18). Thus, the observed 111 low:89 high also gave a good fit to the 9:7 model (P = 0.9-0.75, Table 14). The results of this cross were similar to the previous cross, Valmaine x Kordaat. The genotype of Valore (high accumulator) is designated as ea bb cc and for Valmaine AA BB cc (Table 16). The observed F mean, 1.6352 approximates the expected F mean 1.6025 for a 2 2 9:7 model (P = 0.990-0.975, Table 15). 60 .Amo.o u mv umwulu m up aasHoo aHsuHs aOHumumqom ammzN .voHoom mama mmmwouo HmuoumHumM% GHHH.o ommme.H H u a 0 am «H He «H on «N w H com me Hoao.o mmmeH m H m H HH HHH wOMH.o nmNmo.N H H c m N m H mH mHOHm> ¢Hmo.o mmNmN.H . H N m o m H H 0N osHmaHm> Nm Names o.m m.N o.N q.N N.N o.N m.H o.H q.H N.H o.H m.o muaMHm «0 .oz mmMHo mo uHaHH Home: .muon x oonaHm> mo mwouo mo msOHumumsow Nm was Hm .Hmuoouma msu How AuanoB zuvv ustn\AZI oz unmouomv Zlmumuqu mo aOHHanuuva moaosvoum HwH mHan 61 Cross: Wonder Van Voorburgf(Pn) x Caravan (Pa) (Low x High) (- J— Frequency distribution of nitrate-N content of Wonder Van Voorburg (P2), Caravan (P3), and their F1 and F2 populations are summarized in Table 19. The mean nitrate-N content of Caravan (H) was greater than that of WOnder Van Voorburg (L) and F1 (P = 0.05). The F1 and P1 popu- lation means were not significantly different from each other (Table 19) suggesting complete dominance for low accumulation of nitrate-N. The population means of the P F1, and F generations were not 2’ 2 significantly different from one another, but their nitrate content was lower than the P3 parent mean (P = 0.05). Using the recessive parental mean to separate the lows and highs, the number of plants in the F2 distribution beyond P mean (2.0233) were calculated from the original 3 or raw data and multiplied by 2 (24 x 2 = 48). The number of low accumulators was 200 - 48 = 152. Thus, the observed ratio of 152 low: 48 high suggested a segregation ratio of 3:1 and gave a good fit to the model (P = 0.9-0.75, Table 14) which suggested a single major gene differentiated between these two parents for accumulation of nitrate. Partitioning the F population into low and high classes, by 2 using the arithmetic mean of the two parents, 1.71, a slightly different ratio was observed. This was perhaps due to a 20% overlap of the parental distributions. Therefore, the classes were given weighted values. The weighting procedure consisted of 100 minus the percent overlap of the recessive class below the arithmetic mean of the two parents, divided by 100 (R0) (N); where RO is the expected ratio of recessive plants based on the mean of recessive parent value and N is 62 .Amo.o n my ummulu m zn aEDHoo cHnuHS GOHumumamm cmmzu HHOH.O mNNOm.H H q HH HN OH OH OH HN O N OOH NH NOOO.O mmHNH.H H O s H OH Hm OHOO.O HOONO.H N n q H O H m mH am>mumo wuonuoo> HOOH.O mOOOHH H u H u m H H H OH am> “meaos m Nunez O.H O.H H.~ N.N O.H O.H O.H H.H N.H O.H O.O muaaHm N we .oz wmmHo mo uHaHH Home: .am>mumo x myopuoo am> nouaoz mo mmouo mo maOHuwuoaow Nm was Hm .Hmusoumo map How Austma mumv waMHm\AZI oz Howouomv Zloumuqu mo oOHuonHuumHv zoooavoum "OH mHOma 63 the total number of F2 plants. The number of low accumulators was 200 - 40 - 160. Thus the observed ratio 160 low:40 high suggested a segregation ratio of 3:1 and gave an acceptable fit to the model (P = 0.25-0.1, Table 14) which again suggests a single major gene. Based on the segregation in the F population of this cross and 2 the information obtained from the cross Valmaine (L) x Caravan (H), the proposed genotype for Wonder Von Voorburg is aa bb C C and for Caravan 2-2 (P3) aa bb CC (Table 16). The allele 92 is dominant over g_where the presence of 92 results in low nitrate accumulation. The F1 genotype aa bb C g_was dominant for low accumulation of nitrate-N. The expected F2 mean (1.620) approximates the observed F model (P = 0.990-0.975, Table 15). 2 mean (1.5822) for a 3:1 Cross: WOnder Van Voorburgj(Pfl) x Valore (P,) (Low x High) A T) Frequency distribution of nitrate-N content of parents, F1, and F2 populations are summarized in Table 20. The nitrate content of Valore (P6) was higher than the means of Wonder Van Voorburg and F1 population (P = 0.05). The F1 and F2 means were not significantly dif- ferent from each other suggesting a complete dominance for low accumula- tion of nitrate-N. The P2, F1 and F2 population means did not significantly differ from one another and were lower than the P6 mean (P = 0.05, Table 20). Using the recessive parent mean to separate the classes the number of plants in the F population beyond the recessive parent mean (2.0525) 2 was calculated 21 x 2 = 42. The number of low accumulators was 200 - 42 = 158. The observed 158 low:42 high suggested a segregation 64 .Amo.o u mv ammulu m up nasHou aquHB cOHumummom ammzN mowo.o mommm.H H H 0 MH wH ow no me «H H OON Nm OHOO.O mmHOq.H H H O O H OH Hm mOMH.o nmNmo.N H H w m N m H mH MU.HOHSH wuapuoo> NomH.o mmmqu H I H I m N H N 0H om> umwooz Nm Nunez w.N o.N q.N N.N o.N w.H o.H q.H N.H O.H m.o musMHm mo .oz mmmHo mo uHaHH Homo: N H .oHOHm> x wusnuoo .Hmusmumm on» How Auanms zuvv unon\AzI 00 am> wovaoz mo mwouo mo maoHumuoaom m was m z unmouoav zIoumuqu mo =0Hu=nHuumHo hooosvoum uON mHan 65 ratio of 3:1 and gave an acceptable fit to the model (P = 0.25-0.l, Table 14). Partitioning the F population into low and high phenotypes using 2 the arithmetic mean of two parents (1.73), the observed ratio of 154 low:46 high determined from the raw data suggested a segregation ratio of 3:1 and gave an acceptable fit to the model (P = 0.5=0.25, Table 14). Nitrate accumulation in this cross appears to be controlled by one major gene with dominance for low nitrate accumulation. The observed F mean (1.5836) approximated the expected F mean (1.566) for 2 2 a 3:1 model (P = 0.990-0.975, Table 15). The P2 genotype, as assigned in the previous cross Wonder Van Voorburg X Caravan, was aa bb C292. The genotype proposed for Valore in the cross Valmaine (P1) x Valone(P6) was aa bb cc (Table 16). The F genotype in this cross is aa bb C c. 1 2-' The presence of 92, which is a dominant gene, was responsible for the low accumulation of nitrate-N and the absence of it resulted in high nitrate-N accumulation. Cross: Valmaine (P1) x Wonder Van Voorburg (P6) (Low x Low) Frequency distribution of nitrate-N content of Valmaine (P1), WOnder Van Voorburg (P2) and their F1 and F2 populations are summarized in Table 21. The mean of the parental and F were not significantly 1 different from one another. The mean of parental and F I 2 populations were not significantly different; however, the F population mean was 2 greater than the mean of the F generation (P = 0.05, Table 21). 1 66 .Amo.o u mv umoulu m up naoHoo anuHs aOHumummmm smoZN .ooHoom mums mommouo HmoouoHommm OHHH .O HHKOHH H H O H OH, HO OH mm H H OOH .H OHHH .O .138 .H H m H m H OH .HHrH wuonuoo> BOH .O emOOOHH H I H I m N H H OH 5; 33oz OHOOO HHHNmNH H N O O m H H OH mfimaHHO Hm N982 O.H H.H H.H O.H O.H O.H H.H N.H O.H O.O 35.: mo .oz mmmHo mo uHaHH Home: .wuspuoo> oo> Hovao x maHmaHm> mo mmouo mo waOHumuwoow Nm com Hm .HMuowume mzu How AuanoB zuvv quHm\AzI oz unmouomv zloumuqu mo QOHuanuumHv monosvoum "HN mHnma 67 Observation of the parental F1 and F2 distributions suggests that the parents may be of the same genotype. Based on the results of crosses of Valmaine (L) and Wonder Van Voorburg (L) with the high accumulator parents (Caravan and Valore) however, it was demonstrated that Valmaine and Wonder Van Voorburg are genetically different. Partitioning was based on the F1 and F2 distributions. The F2 distribution showed that a major portion of the F population centered 2 around the class 1.4 (Table 21). The original data showed that the maximum number of plants fell between the classes 1.25 and 1.4. The mean of these two classes is 1.33 which is also the arithmetic mean of the two parents (1.3269). The number of individuals in the F2 popula- tion dropped in the 2.0 to 2.6 classes. The 2.0 class approximates the mean of the three high accumulators (Caravan + Kordaat + Valore/3 = 2.1153). In order to partition the low and high accumulators, the number of plants below the arithmetic mean of the two parents (1.3269) were calculated to obtain the low accumulators (89 x 2 = 178). Twenty- two (200 - 178) individuals were classified as high accumulators. The observed 178 low:22 high gave an acdeptable fit to the model of 57L:7H (P - 0.75-0.5, Table 14). This ratio suggests that three major genes make up the parents Valmaine (P1) and Wonder Van Voorburg (P2). The proposed genotype of P1 is AA BB cc and that of P is aa bb C C , 2 ---—2—2 Table 16. The F1 genotype is Aa Bb C22_and is a low nitrate-accumulator. In the F2 population the following genotype A-B-Cz, A—B-c, Arb-CZ, a-B—Cz, and a-b-C2 would be low accumulators while the genotypes Aeb-c, 68 a-B—c, and a-b-c would be high accumulators. The recombination of the genes yielded a few high accumulators from this low x low cross. This would only be possible based on the proposed genotypes of Valmaine (AA BB cc) and Wonder Van Voorburg (aa bb C292). Cross: Caravan (Pd) x Ithaca (PE) (High x Medium) J J Frequency distributions for the nitrate-N content of Caravan (H), Ithaca (M) and their F populations are summarized in Table 22. The 2 mean nitrate content of Caravan was greater than that of Ithaca (P = 0.05, Table 22). This cross was made to obtain information as to whether high accumulators would recombine and produce segregants that were higher nitrate accumulators than the parents. Initially this cross was planned as a high x high; however, the parent Ithaca in the 1975 planting was classified as a medium accumulator. The F2 distribution suggests that the segregation for nitrate-N fell within the parental distributions. The majority of the segregants were scattered about the mean of Caravan (H). It appears that the genotype of Ithaca (M) was different from that of Caravan. The mean of the F2 population (1.8445) approximates the arithmetic mean of the two parents (1.8154), which was not significantly different from either parent. The absence of the F1 generation in this planting made it difficult to identify the dominance direction. Using the parental arithmetic mean, the division of the medium and high phenotype were identified. The observed 23 medium:27 high suggests a two—major 69 .umoqu m mo astoo aHnuHS GOHumumomm ammzN OOHHO AHHHHHHH H H H H HH O H OH N.H mmoo.o pnNoo.H N m N m 0H mumnuH MHmo.o mMMNoN N I H H H H m mH om>mumo Nm Nammz woN ©0N *VON N.N OQN ”0H 00H QoH N.H O.H woo mug-Hm mo .oz mmmHo mo uHfiHH Home: .momnuH x am>mumo mo mmouo mo maOHumuooow Nm wow .HMuaonm mnu How AuanmB muov momHm\AzImoz unmouomv zloumuqu mo coHuvnHHumHv mooosvmum “NN mHan 70 gene difference between the parents. This observed classes also sug- gested a ratio of 6M:10H and gave an acceptable fit to this model (P = 0.25-0.10, Table 14). The proposed segregation ratio is based on the genotype of Caravan as ea bb CC (Table 16), and that of Ithaca to be aa BZBACC. The F1 genotype would be aa sz Cc and a high accumulator phenotype. The ratio of 51131011 is based on the genotypes of medium to be aa B2_B-2 cc, aa B213”2 CC, aa B.B_ Cc and aa B b cc, while the genotype 2 2 having aa Bab CC, aa Bab Cc, and aa bb cc would be high accumulators. (- l. The observed F mean (1.8445) approximates the expected F mean (1.7634) 2 for a 6:10 model (P = 0.95-0.90, Table 15). 2 SUMMARY AND CONCLUSIONS Data from the segregation of F2 population from crosses, involving Valmaine (L), Wonder Van Voorburg (L), Caravan (H), Kordaat (H), and Valore (H), suggest that three major genes determine the inheritance of nitrate accumulation in lettuce. In the three major gene systems the two genes, A and B, appear to determine the inheritance of nitrate accumulation in the crosses of Valmaine (L) x Kordaat (H) and Valmaine (L) x Valore (H) (Table 23). In these crosses, the mean nitrate content of the F population approxi- 1 mate the mean for Valmaine (L). The F2 population showed a ratio of 9L:7H suggesting two major genes with epistasis. The presence of both Azand B alleles was necessary to give Valmaine (L) phenotypes, i.e., A and _B_ were complementary to each other. The presence of a third major gene, 9, was demonstrated from the cross Valmaine (L) x Caravan (H) (Table 23). In this cross, the F1 appeared partially dominant towards low accumulation with the F genera- 2 tion demonstrating a segregation ratio of 9L:55H. This suggested a third major gene may be involved in controlling nitrate accumulation. Based on crosses involving Valmaine (L), the genotype of Valmaine (L) is designated as AA BB cc and the genotype of Caravan (H) as aa bb CC based on the cross Valmaine x Caravan. Gene g_from Caravan (H) controls high nitrate accumulation. The §_alle1e is dominant over g_and the 71 72 Table 23: Genotypes and phenotypes of the F populations from various crosses of nitrate-N accumulation study. Genotypes Cross Model Low Medium High Valmaine x Caravan (High) 9L:55H A—B-c -- - -C Arb-c a-B-c a-b-c Valmaine (L) x Kordaat (H) 9L:7H A-B-c -— A-b-c a-B-c a-b-c Valmaine (L) x Valore (H) 9L;7H A-B-c -- Arb-c a-B-c a-b—c wonder Van Voorburg (L) x 3L:1H a-b-C2 -- a—b-C Caravan (H) a-b-CZC WOnder Van Voorburg (L) x 3L:1H a-b-C2 -- a-b-c Valore (H) Valmaine (L) x 57L:7H a-b-C2 —- A-b-c WOnder Van Voorburg (L) A—B-c a-B-c a-b-c Caravan (H) x Ithaca (M) 6H:10M -- aa—Bsz-cc aa-sz-CC aa-Bsz-Cc aa-BZb-Cc aa—Bsz-CC aa-bb-cc 73 presence of 9.18 epistatic to A and §_genes. The low nitrate accumula- tion genotypes in this cross were A—B—c and the high accumulator types were - - C, Arb-c, a-B-c, and a-b-c. The presence of an allelic gene, £2, controlling nitrate accumula- tion for low levels was demonstrated in the cross wonder Van Voorburg (L) x Caravan (H) (Table 23). In this cross, the F appeared dominant 1 toward low accumulation and the F population segregated 3L:1H. The 2 genotype of Caravan (L) was designated as ea bb CC in the cross Valmine (L) x Caravan (H). The designated genotype for Wonder Van Voorburg (L) is aa bb C29_. The allelic geneg2 is dominant over g, Thus two dominant allelic genes are identified at the {g} locus with the direc- tion of dominance 92> Q> _c_, where £2 controls low nitrate accumulation and is dominant over the g_and g_a11e1es. The allelic gene 9 controls high nitrate accumulation and is dominant over 3 whereas the recessive allele, 9, did not appear to have an influence on either low or high accumulations of nitrate-N. The presence of gene 92 was also noted in the cross Wonder Van Voorburg (L) x Valore (H). The presence of A, B, and g2 genes were also demonstrated in the cross Valmaine (L) x Wonder Van Voorburg (L). The segregation of high accumulators in this Low x Low cross would only be possible from the proposed genotype of Valmaine (AA BB cc) and WOnder Van Voorburg (aa bb C292). The recessive genes 2, h, and 2. appear to contribute little to the nitrate accumulation. The high accu- mulation with §:h:g_genotype may be due to the absence of both A-B_or Q2 genes, rather than the presence of the recessive genes. 74 The low nitrate accumulation was dominant over high nitrate accumu- lation as noted in the F1 generation of all L x H crosses (Table 12). Low nitrate accumulation may occur in three ways, first low nitrate accumulation may be the result of both genes A_and‘B_which are compli— mentary to each other as noted in crosses involving Valmaine (L). Alternatively, low nitrate accumulation may be controlled by a single dominant gene‘gL2 as noted in crosses involving Wonder Van Voorburg (L). Moreover a combination of‘A.'--‘_l§.and_(;2 as noted in the cross of Valmaine (L) x WOnder Van Voorburg (L) may also yield low nitrate accumulators (Table 23). High nitrate accumulation may be due to the absence of geneslé and §.in a genotype, as demonstrated from crosses involving Valmaine (L). Otherwise, it may be due to the presence of a dominant gene §_as noted in the cross between Valmaine (L) x Caravan (H). The presence of gene §_(high accumulator) from Caravan resulted in high nitrate accumulation even in the presence of genes A and B'suggesting that gene §_is epistatic to genes A and B (Table 23). The gene B2 determined a medium level of nitrate accumulation in the parent Ithaca as noted in the cross Ithaca (M) x Caravan (H) (Table 23). Preliminary studies on total-N of the parental lines at the four week old stage suggested that the difference in nitrate accumulation between low and high accumulators was due to the greater nitrate reduc— tion capabilities of low accumulators rather than a greater nitrogen (nitrate) uptake by the high accumulators. 75 Although the preliminary observations suggested cultivar differ- ence in the other elements, a comparison of each of these elements within and between the low and high nitrate accumulator did not show a marked variation in their content. Probably the two variables are not related. Preliminary studies on the fresh and dry weight of parent lines did not suggest a relationship between the nitrate content and yield (fresh or dry weight) at the four week stage. However, the F genera- 1 tion between a low and a high accumulator were higher in yield than one or both parents (Table 24), probably due to hybrid vigor. The fixing of low nitrate content along with higher yields in the segregating populations is worthy of further investigation. In order to better understand the mechanism of nitrate accumula— tion in lettuce, a knowledge of nitrate reductase activity and total nitrate uptake by the low and high nitrate accumulators of lettuce is essential. 76 Table 24: mean fresh and dry weights of parents and their F1 popula- tion for two plantings. Dgcember 1975 January 1976 Fresh wt Dry wt Fresh wt Dry wt P1 35.1608 1.76658 35.1628 2.68598 92 32.315a 1.6100a 33.797a 2.2578b F1 49.193b 2.7901b 40.089b 2.9574a 35.16a 1.7665a 35.162a 2.68593 38.283 1.8240a 40.570b 2.8622a 42.51b 2.1760b 41.609b 3.1673b 35.16a 1.7665a 35.162a 2.6859a 40.113a 1.48538 36.1293 1.9266b 52.789b 2.6078b 40.117a 2.9878a P1 35.16a 1.76658 35.1628 2.6859a 96 43.368 1.8780a 38.197a 2.2014b F1 56.115b 2.8723b 43.618b 3.0468a 32.315a 1.6100a 33.797a 2.2578a 38.28a 1.824a 40.570b 2.8622b 55.34b 2.615b 46.389b 3.4286c 32.315a 1.61008 33.7973 - 2.2578a 43.36b 1.8780ab 39.197b 2.2014a F1 48.905b 2.2990b 42.938b 2.5453a P1 Valmaine P2 WOnder Van Voorberg P3 Caravan P4 Kordaat P5 Valore zMean separation using a "t" test (P = 0.05) within a column and within a cross. LITERATURE CITED 6. 10. 11. LITERATURE CITED Anacker, W. F., and V. Stoy. 1958. Proteincromatographe und Calciumphosphate. I. Reiningunng von Nitrate aus Weisen Blattern. Biochem. Z. 330:141-159. 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