1.37.... . A .44. L. :19 2 v0 _ .I.IAIV . V. . (2.13.? J... .3... Kit)...» :35: 36: . a in.” . . n . MM 3..9fi\.&. .. Hy; fir surufimv . null».- . ,IIA L I: 4 it: I... x .1 1 5 1 vi) s 224. s n.» .2 i a: . 3.32:9. if. 3.: . 24.1.5. .u.fiu-I44.5\Y l. . bin-1’0...» OAKJVdvo-I. . vflnrmquar: . . rd..." 41.44.? \ .12.. I . . if. . .. 23.54.. lifuufcq )thr! 41:! ”km ILQV4‘1 .4 ‘ 2.. . . viluclh.‘ - .lY'. 22.5fI:\0 {Ed-Us”! .\ I» l.. $4¥§ru “Maggi a4...“ . 3- .. . 2.. ..\f.f.. . .Y .. .......IA_:. 2.. b4 . . . THESIS m.-. llllllllgll‘llllll.lmlll 1 9087 i LlBRARY ~ fineiilgan State Unlversity This is to certify that the thesis entitled INVESHGATION DP METHODS OF INWGOEATWG VEGETABLE. AMAKADTH (AmAaAmHus Tacoma L.) SEEDS) presented by Pmmblwe K. N2 MDKOEDA has been accepted towards fulfillment of the requirements for ' M's degree in HO'IUQJLTUQE Q4442. _ ‘V Major professor Of Date fl/H /% 07639 MS U is an Affirmative Action/Equal Opportunity Institution _._..._ __ 4 v v- ——- --.-fi-—— ”a v v ~v'v- '~ - _—~ PLACE ll RETURN BOXto mwothb checkout from your ncord. TO AVOID F INEB Mum on or baton duo duo. DATE DUE DATE DUE DATE DUE MSU I: An Affirmative WM Opportunlly Instltwon Wanna-9.: INVESTIGATION OF METHODS OF INVIGORATING VEGETABLE AMARANTH (AMARANTHUS TRICOLOR L.) SEEDS BY Phindiwe K. N. Mokoena A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1996 ABSTRACT INVESTIGATION OF METHODS OF INVIGORATING VEGETABLE AMARANTH (AMARANTHUS TRICOLOR L.) SEEDS BY Phindiwe K. N. Mokoena In humid tropics, amaranth has long been consumed as a green vegetable, not only because of its robust growth, but as a source of nutrition. One of the major constraints to amaranth cropping is seedling establishment. In this study, Amaranth tricolor L. seeds were aged and their percentage germination tested against fresh seeds. Aged seeds exhibited lower germination rates than fresh seeds. Testing moisture content (MC) for priming, presoaking seeds at 35% MC ‘was found. appropriate for' both seed. types. In. the greenhouse, seedling emergence rates were tested using different invigoration methods. Under crusted and non- crusted conditions of loam and sandy soils, priming of aged and fresh seeds at 35% IMC lgave the highest percentage emergence compared with the control, Polyethyl glycol- priming, or Solid-Matrix-Priming. ACKNOWLEDGMENTS I would like to convey my gratitude to my major Professor, Dr. John F. Kelly for his patience and support throughout my program. Even though things never seemed to work properly for me, Dr. Kelly remained supportive. His perseverance and his never-dying spirit for success served to auger my successful completion of this thesis. I also would like to thank my family, especially my mother, Ciki and. my sister Keke, who gave me special support, notably during my home-sickness moods. My friends Bernard, Thuli, Awonke, Thokoza, and all the folks in the Plant and Soil Sciences Building; thanks a lot. I would also like to thank the participants who helped bring this thesis to completion: Dr. Alan Taylor of Cornell University, Dr. Larry Copeland of the MSU Crop and Soil Science Dept., Dr. Bob Herner of Horticulture and Dr. George Axinn of Resource Development. Special thanks to my sponsors, USIA through Fulbright, and the International Center. Last but not least I would like to thank The Creator for being with me throughout my stay in the United States to acquire knowledge, which I hope, will help alleviate some of the conditions experienced by depriVed rural farmers of my country, South Africa. Hopefully, this effort will join other contributions to help correct some of the iii impoverishment introduced by the ruthless legacy of apartheid. iv LIST OF TABLES . LIST OF FIGURES . INTRODUCTION . . Chapter 1 . . . . LITERATURE REVIEW Taxonomy . . History . . Description Nutrition . Cultivation Market . . . Background of Invigorating Seeds Rationale for Invigorating Seeds Moisture Control . . . . . . . . Presoaking . Pregermination and Gel-seeding . Priming . . TABLE OF CONTENTS Solid-Matrix-Priming . . . . . . . . . . . . Seed Aging . Chapter 2 . . . . MATERIALS AND METHODS . . . . . . . Accelerated Aging Method . . . Germination Test Experiment . Initial Moisture Determination Critical Moisture Experiment . . Pregermination and Gel-seeding Experim nt . Solid-Matrix-Priming and Polyethyl Glyco -Pr Experiment . . . . . . . . . . . . . . Soil-Crusting Experiment . . . . . . . . . . Chapter 3 . . . . “BOLTS m DIBCUBBIONB O O O O O O O O O O O O O Experiment 1: Experiment 2: Experiment 3: Experiment 4: Experiment 5: CONCLUSIONS . . . LITERATURE CITED Optimum Seed Germination Test Pregermination and Gel-seeding SMP and PEG-priming . . . . . Presoaking . . . . . . . . . . Soil Crusting . . . . . . . . viii O O O O O O O O O O O O O O O H N 25 25 25 27 27 . 29 iming . 29 . 30 . 32 32 32 40 45 49 52 . 60 O 63 LIST OF TABLES Table 1. Nutritional composition of amaranth leaves and other vegetables (Grubben, 1976). . . . . . . . . . 10 Table 2. Mean germination (%) of aged and fresh A. tricolor seeds at 10, 15, 20, 25, 30, 35C and room temperature (RT) after 18 days. . . . . . . . . . . . . . . . . 32 Table 3. Mean time to 50% germination (T-50%) of aged and fresh A. tricolor seeds for 18 days at 10, 15, 20, 25, 30, 35C and room temperature (RT). . . . . . . . . . 34 Table 4. The effect of germination pattern, gel type and seed condition of A. tricolor seeds expressed as percent seedling emergence. . . . . . . . . . . . . 41 Table 5. Effects of seed treatments on mean percent emergence of aged and fresh A. tricolor seeds. . . . 42 Table 6. Effects of gel types and germination pattern on mean time to 50% germination (T-50%) (days) for aged and fresh A. tricolor seeds. . . . . . . . . . . . . 44 Table 7. Analysis of variance for mean percent emergence of Sol id-Matrix-Primed and PEG-pr imed aged and fresh A . tricolor seeds. . . . . . . . . . . . . . . . . . . 46 Table 8. Effects of priming treatment on mean percent emergence, mean days to 50% emergence (T—50) of aged and fresh A. tricolor seeds. . . . . . . . . . . . . 47 Table 9. The effect of moisture content (MC) (%) on germination of aged and fresh A. tricolor seeds when presoaked at room temperature for 7 to 10 days. . . 49 Table 10. Mean germination (%) of aged and fresh .A. tricolor seeds at 30 to 50% desired moisture content at 3OCO O O O O O O O O O O O O O O O O O O O O O O O O 50 Table 11. Mean time to 50% germination (T-50%) of aged and fresh A. tricolor seeds at 30 to 50% desired moisture content at 30C. . . . . . . . . . . . . . . . . . . 51 Table 12. Analysis of variance for aged and fresh seed types (seed), clay and sandy soil types (soil), crusted and non-crusted soil conditions (condition), administered with different treatments (trts). . . . 54 vi Table 13. Mean emergence (%) of aged and fresh A. tricolor seeds treatments under crusted and non-crusted loam and sand soil types. . . . . . . . . . . . . . . . . . . 55 Table 14. Mean emergence (%) and contrasts of control, selected treatments of presoaking, gel-seeding, SMP and PEG under crusted and non-crusted soil conditions. . 58 vii LIST OF FIGURES Figure 1. Morphology of an amaranth plant and its seed (A. tricolor L.) (Bittenbender, 1983; Grubben, 1976). . 8 Figure 2. Triphasic pattern of water uptake by germinating seeds (Bewley and Black, 1994). . . . . . . . . . . 16 Figure 3. Thermogradient tables used to germinate A. tricolor seeds at different temperature regimes. . . 26 Figure 4. Mean percent germination of aged and fresh A. tricolor seeds at 10 to 35C after 18 days. . . . . . 33 Figure 5a. Daily mean germination (%) of aged and fresh A. tricolor seeds for 18 days at 20C. . . . . . . . . . 35 Figure 5b. Daily mean germination (%) of aged and fresh A. tricolor seeds for 18 days at 25C. . . . . . . . . . 36 Figure 5c. Daily mean germination (%) of aged and fresh A. tricolor seeds at 18 days at 30C. . . . . . . . . . 37 Figure 5d. Daily mean germination (%) of aged and fresh A. tricolor seeds for 18 days for 35C. . . . . . . . . 38 Figure 5e. Daily mean germination (%) of aged and fresh A. tricolor seeds for 18 days at room temperature. . . 39 viii INTRODUCTION Poor and variable germination of amaranth (Amaranthus spp.) seeds may be associated with low vigor of the seeds. In turn, seeds with low vigor may not be able to tolerate adverse soil conditions, like crusting. Chate et a1. (1987) reported that many disappointments occur in directly drilled crops in terms of rapid unifonm emergence at a high level, because of soil crusting. In particular, Kauffman and Bass (1983) noted. that among' the: difficulties experienced. by farmers growing amaranth was crusting in fine-textured soils. Although vegetable amaranth is an important nutritional crop (Grubben, 1976), its cropping is characterized by uneven emergence or lack of uniformity in rate of emergence. In turn, overall yields are reduced, because of variability in size, quality, and harvest dates, which are important in terms of demands of the market, grading, flowering and establishing a new crop in case of sequential cropping (Matthews and Powell, 1986). This study was conducted following failure of field emergence of amaranth seedlings in a sequential cropping experiment, due to soil crusting. Since amaranth has very small seeds, soil crusting appeared to be the limiting factor for obtaining a good stand of amaranth. Even though direct-seeding offers the lowest cost method in most growing 2 situations, strict control of the soil environment is not always possible (Chate et al., 1987). Some recommendations dealing with the soil crusting problem have been reported. Rodale (1989) recommended use of double-press wheels rather than single-press wheels on crusting soils to reduce crusting, because wheels provide good soil contact on both sides of the seed but maintain loose soil directly over the seed. Seeding at higher rates per unit area and mulching of soil are also some of the recommendations for alleviating the problem. The thinning and mulching recommendations involve laborious work, which is often not desirable. Chate et al. (1987) found that it is possible to control the vigor of the seed to improve the probability of successful stand establishment. This study looked at the effect of conditioning aged and fresh amaranth seed, using different pre-plant seed enhancement treatments, on improving seed germination percentage, rate, vigor, and seedling emergence and uniformity under soil crusting conditions. Amaranth (Amaranthus tricolor L.) was chosen for this study, because of its high nutritional content, its ability to thrive in hot, humid climates, and its relatively easy cultivation; therefore, making it an important crop in the tropics, where malnutrition is prevalent. Amaranth has a short growing season of 3-6 weeks, and can be subjected to "O. 3 repeated cutting of edible tops. This attribute makes it useful in sequential intercropping, thus optimizing available land resources. However, despite all of these favorable attributes, vegetable amaranth remains an under- exploited crop, largely because of neglect by researchers and policy-makers (Makus, 1984). This thesis presents topics that deal with the manipulation of water status of seeds to improve germination and seedling emergence. The first section is devoted to determining optimum germination for the seeds. In this section, optimum temperature, optimum days for germination, and germination rate of the seeds are investigated. The second section deals with the different approaches to hydration. Included is the determination of critical moisture content for presoaked seeds, the use of gels on pregerminated seeds, and solid matrix-priming. This study was conducted to test the following hypotheses: 1. Aged and fresh amaranth seeds have different germination regimes for different temperatures. 2. Invigoration of amaranth seeds improves germination and seedling emergence. 3. Invigoration of aged seed differs from that of fresh seed. 4. In crusted soil, the extent of seed invigoration differs with the invigorating method. Chapter 1 LITERATURE REVIEW Taxonomy There is considerable confusion regarding the taxonomy and nomenclature of amaranth used as a leafy vegetable. The confusion is attributed to the characteristics often applied in determining the species, these being unstable because of ecological or genetic influences within the same species (Grubben, 1976) . Therefore, taxonomic distinction among species is difficult, and many synonyms exist (Schnetzler and Breene, 1994). The collective name 'amaranths' for vegetable amaranth is comprised of all species and cultivars of the family Amaranthaceae, principally Amaranthus cruentus L., A. dubius Mart. ex Thell., A. tricolor L. and Celosia argenea L. The grain amaranths, still cultivated in tropical mountainous regions of South. America and. Asia (A. caudatus L., A. hypochondriacus L. and A. cruentus L.) also are used as leafy vegetables. The species most often grown as vegetable amaranth is A. tricolor L. (Makus and Davis, 1984). History Historical records of amaranth show that it was consumed both as a vegetable and as a cereal grain. Grain 5 amaranth, in which the seed is consumed, originated in South and Central America and was used by the Aztecs as a staple food and for ritual ceremonies before 1519 (Early, 1994). Most varieties of vegetable amaranth, in which the leaves and succulent stems are consumed, originated in Asia (Grubben, 1976) . Amaranthaceae seeds have been found in prehistoric dwellings, even in Mediterranean Europe (Grubben, 1976). Amaranth grows naturally in open habitats as a pioneer species of riverbanks, coastal marshes, and dunes. Amaranth also spreads as a weed of artificially disturbed habitats created by human activities. In prehistoric times, wild and weedy amaranths became common potherbs throughout the tropics and subtropics. In other parts of Africa and Asia, amaranth was domesticated as a potherb (Rodale, 1989). Vegetable amaranth is now cultivated and consumed as a cooked vegetable or green in many of the countries of Africa, the Caribbean, and Asia (Singh and Whitehead, 1992). In the United States, A. retroflexus (red-root pigweed) is the common species, but it is mostly treated as a noxious ‘weed. (Rawate, 1983) because of its spiny leaf texture, making it less tender and therefore inedible. However, there have been some studies by Campbell and Abbott (1982), Abbott and Campbell (1982), Makus (1984) , Makus and Davis (1984) and Rhoden and McKelly (1995) on the feasibility of 6 growing Asian cultivars of vegetable amaranth as a summer green in the southeastern United States. Description Amaranth (Amaranthus L . ) is a herbaceous short-lived annual plant, monoecious or dioecious. The plant is upright, branching, and 0.5-2.5 m tall. Lea 7 axis or has small bundles throughout the length of the cotyledon. These provascular cells are small and elongated and have fewer reserves and more cellular organelles than the large x); 4. [mil unlh :Irhitnng (up {6 x): 5. {mil u-ilhoul up (6 I): Fig. 6. Amarzmhus tricolor L. I. branch with flnwcrt (2/3 x): 2. male fluwvr (4 x): J. [one]: flower (4 6. Irrd. Inlrml m'rw (6 x); 7. “1|er a/glumrmlu Figure 1. Morphology of an amaranth plant and its seed (A. tricolor L.) (Bittenbender, 1983; Grubben, 1976). 9 protoderm and ground meristem cells. The latter cells have more protein bodies and larger globoid crystal inclusions than the others. The perisperm is composed of starchy tissue, and its cells have thin walls and are full of angular starch grains (Coimbra and Salema, 1994). Nutrition Amaranth contributes to the diet because of its high nutritional value (Table 1). The leaves are high in protein (17.4 - 38.3%, dry matter basis); the protein comprises 4.4% sulfur-containing amino acids and approximately 5% lysine (Rhoden and McKelly, 1995) . Amaranth leaves also provide significant amounts of carbohydrates, vitamins, dietary fiber, and minerals, particularly calcium and iron. Dialyzable iron varies from 0.41 and 0.63 mg bioavailable Fe per 100 g fresh weight. In actual levels, a 100 g portion of amaranth could provide 40 to 60% of the daily Fe requirement for men and children (RDA of 1 mg/day), or 27 to 41% for women (RDA of 1.5 mg/day) (Rangarajan, 1995). Amaranth contains more fiber, niacin, and ascorbic acid than spinach, cabbage, or lettuce, on a fresh weight basis (Grubben, 1976; Abbott and Campbell, 1982), and it is similar to spinach in flavor (Makus, 1984). Used as a vegetable, amaranth has about 3 /4 as much Vitamin A as spinach, but the levels of protein, iron, and other minerals are similar. Nitrate and oxalate contents are comparable to 9 protoderm and ground meristem cells. The latter cells have more protein bodies and larger globoid crystal inclusions than the others. The perisperm is composed of starchy tissue, and its cells have thin walls and are full of angular starch grains (Coimbra and Salema, 1994). nutrition Amaranth contributes to the diet because of its high nutritional value (Table 1). The leaves are high in protein (17.4 - 38.3%, dry matter basis); the protein comprises 4.4% sulfur-containing amino acids and approximately 5% lysine (Rhoden and McKelly, 1995) . Amaranth leaves also provide significant amounts of carbohydrates, vitamins, dietary fiber, and minerals, particularly calcium and iron. Dialyzable iron varies from 0.41 and 0.63 mg bioavailable Fe per 100 g fresh weight. In actual levels, a 100 g portion of amaranth could provide 40 to 60% of the daily Fe requirement for men and children (RDA of 1 mg/day), or 27 to 41% for women (RDA of 1.5 mg/day) (Rangarajan, 1995). Amaranth contains more fiber, niacin, and ascorbic acid than spinach, cabbage, or lettuce, on a fresh weight basis (Grubben, 1976; Abbott and Campbell, 1982), and it is similar to spinach in flavor (Makus, 1984). Used as a vegetable, amaranth has about 3 /4 as much Vitamin A as spinach, but the levels of protein, iron, and other minerals are similar. Nitrate and oxalate contents are comparable to 10 those of other leafy garden vegetables (Abbott and Campbell, 1982). Amaranth greens can be prepared as a fresh salad or steamed, boiled, stir-fried, sauteed, baked, or pickled. Table 1. Nutritional composition of amaranth leaves and other vegetables (Grubben, 1976). Values for 100 g of dry matter Prot- CHO Fi- Cal- Fe Nia- Vit . Calo- Crop eins 9 her cium mg cin C iea mg mg mg mg mg kcal Amaranthus 33 33 10 1,667 27 10 667 320 app. Brassica 21 57 11 571 7 4 571 329 app. Lactuca 23 50 8 583 17 7 250 317 sativa Spinacia 25 6 - 1,625 37 7 313 162 oleracea Cultivation Unlike most plants used for greens, amaranth grows best in hot, humid climates, or during summer months in temperate climates (Makus and Davis, 1984). Amaranth grows rapidly and succulently at day temperatures above 30C and night temperatures above 20C. Optimum vegetative development and market quality generally require large quantities of water. Watering regimes depend on soil type and the stage of growth, but irrigation should occur above the Pf=4.2 wilting point, to avoid yield reductions (Rodale, 11 1989). Amaranth grows best in loose, fertile, well drained soil (Makus, 1990) . Since amaranth seeds are small, crop establishment needs special attention. Rodale (1989) warned that due to the small seed size, increased risk of poor plant establishment due to soil crusting occurs frequently. Amaranth can be seeded directly at a depth of 0.5 cm or transplanted at 21 days or 7-cm length. Shallow planting depth is important to maximize emergence (Rodale, 1989). Spacing can range from 10-40 cm (Mortley et al., 1992) depending on the type of harvest to be followed. Plants are harvestable 3-9 weeks after transplanting by clipping them 20-25 cm above the soil line, and can be allowed to ratoon (growth of repeated harvests of the same plant) (Makus and Davis, 1984). Harvesting also occurs by clear-cutting (uprooting) and reseeding (Campbell and Abbott, 1982). Amaranth also can be grown in mixed cropping systems, and because of its vigorous growth, it is useful in 'intensive multiple cropping systems' (Grubben, 1976). Market In general, little has been done in terms of research on the cultivation, varietal improvement, or market potential of the crop, especially for developing countries (Makus, 1990) . It has been documented (Grubben, 1976; Grubben, 1979; Oke, 1980) that in most tropical areas the crop is used in small-scale gardens for home consumption and 12 markets; therefore, it forms an important part of food security. There has been a potential acceptance of amaranth use as a mid-summer vegetable crop in the United States (Makus, 1992; Rhoden. and. McKelly, 1995), therefore its consumption and market potential are expected to increase in the future (Campbell and Abbott, 1982). Background of Invigorating Seeds Seed viability and low vigor affect the performance (i.e. percentage and rate of emergence) of seeds, which ultimately result in low yields. The slow rate of emergence associated. with. low-vigor’ seed. results. in. small. plants. Vigor also is associated with seed size; large seeds emerge faster small seeds (TeKrony and Egli, 1991). Invigoration of seed involves primarily the use of presowing techniques for the improvement of seed germination, emergence, and stand establishment, which are essential for early and maximum yields. The aspect of invigoration addressed in this research is the enhancement of physiological and. biochemical events in seeds during controlled hydration, and suspension of germination by low water potential of the imbibing medium (seed conditioning), either liquid or moist solid (Khan, 1992). 13 Rationale for Invigorating Seeds The topic of seed hydration and invigoration has been reviewed by Khan (1992). The rationale for invigorating seeds is to mobilize the seeds' resources and to augment them with external resources to maximize improvement of stand establishment and yield (Khan, 1992). Physiological seed treatments used in this study to improve or enhance seed performance based on seed hydration include presoaking, pregermination, and matriconditioning. Prolonged seed hydration, especially at low water potential, influences the rapidity, synchrony, and the percentage of seeds that germinate (Khan, 1992). As a result, rate and uniformity of seedling emergence is improved. Vigorous plant stand is influenced by the interaction of seed quality, seed-soil water relations, and growing media environment (Taylor et al., 1992). Various environmental stresses, such as soil crusting encountered after sowing, may decrease or prevent seedling establishment (Taylor et al., 1992). Seeds which are too small or which have low germination and seedling vigor are limited first by the percent viability. Invigorating seed may be used to upgrade seed quality before sowing, by eliminating non-viable or poor quality seeds. Bradford (1986) reviewed various methods of osmoconditioning to improve seed quality and vigor. Seed l4 enhancement techniques include seed hydration, biological seed treatments, and seed coatings. Treatments which include seed hydration under controlled conditions before sowing have attracted interest over the years. Two techniques, namely, fluid drilling and seed priming have been studied for the improvement of the rate of germination (Taylor et al., 1992). Moisture Control Moisture control is a seed prehydration treatment in which the amount of water absorbed by the seed is controlled precisely to increase percentage and rate of germination and increase the uniformity of stand establishment (Bewley and Black, 1994). Seed moisture content plays a major role in the life cycle of seeds (Taylor et al., 1992). Moisture content is associated with seed development, maturation, harvest, and storage. Water uptake, or imbibition in seeds, is the first key step toward germination; therefore, water is essential to change the status of seed development from quiescence to active growth (Taylor et al., 1992). The environmental forces that determine the rate of water imbibition by seeds are complex, but the ability to imbibe water is dependent on cell water potential and is a result of cell wall matric forces, cell osmotic concentration, and cell turgor pressure 15 (Copeland and McDonald, 1995; Vertucci, 1989; Bradford, 1986). Germination of seeds is characterized by three phases (Figure 2) (Copeland and McDonald, 1995; Bewley and Black, 1994). The triphasic water uptake of seeds can be explained on the basis of water relations. In their resting stage, dry seeds are characterized by low moisture (5-15%) and relatively inactive metabolism (Copeland and McDonald, 1995; Bewley and Black, 1994). At this stage seeds have a lower water potential (-100 Mpa) than that of the surrounding moist substrate. Thus, a large water potential gradient initially exists between the seed and high water potential medium such as solution or water (Taylor et al., 1992, Vertucci, 1989). In this phase, imbibition is crucial in initiating seed germination. The events include controlled physical and physiological processes which direct flow of water into the dry seed and result in a large increase in seed moisture. 16 Phase 1 Phase 2 Phase 3 H < > _._- (increase in fresh weight) Visible ge£Lination starts here Water uptake 0 Time Figure 2. Triphasic pattern of water uptake by germinating seeds (Bewley and Black, 1994). Phase two is the lag phase of water uptake; it occurs before the onset of visible germination or radicle emergence. In this phase, the seed's water potential plays a minor role. The osmotic potential and pressure potential create a static relationship between seed moisture and water uptake. During this phase, major metabolic events take place in preparation for radicle emergence (Bewley and Black, 1994) . Enzymes are activated to break down stored tissue, to aid in the transfer of nutrients from storage areas in the cotyledons or endosperm to the growing points, l7 and to trigger chemical reactions that use breakdown products for the synthesis of new materials (Copeland and McDonald, 1995). In phase three, another large increase in water uptake results, marking the transition from seed to seedling by the protrusion of the radicle. Cell elongation results in additional water uptake by the seedlings (Taylor et al., 1992) . Water uptake is influenced by decreases in water potential, resulting from production of low-molecular- weight, osmotically active substances resulting from the postgerminative hydrolysis of stored reserves (Bewley and Black, 1994). The water potential differential between the environment and the seed establishes the gradient, but not the rate of imbibition. The rate of imbibition is influenced by the permeability of the seed to water; therefore, imbibition rate can be regulated by the available water in the environment to the seeds. Components that influence the hydration rate are properties of the seed and the environment in which it is situated. Such components include seed morphology, structure, composition, moisture content, and temperature (Taylor et al. , 1992; Bewley and Black, 1994). Inhibition is accompanied by gains in weight and ‘volume. However, the jpercent increase in. weight is not necessarily proportional to the percent increase in volume. 18 Water volume does not displace its weight directly, because water affects the degree of swelling of seed polymers (Taylor et al., 1992). Presoaking Presoaking involves moisture control of hydrated seeds, which is enough to elevate their moisture content until the phase of imbibition or until moisture level is sufficient to initiate the early events of germination, but not sufficient to permit radicle protrusion (Figure 2) (Copeland and McDonald, 1995; Taylor et al., 1992; Tarquis and Bradford, 1992). The aim, therefore, is to find the percentage moisture at which the soaked seed will not germinate, but which will stimulate more rapid germination when taken out and placed under optimum germination conditions. Presoaking is a seed hydration approach involving soaking of seeds in a limited amount of water prior to planting, and to enhance germination. and. seedling' growth. by' controlling' the imbibition conditions and reducing the vagaries of adverse soil conditions (Bradford, 1986). Presoaking as a means of improving germination has been known for years. Studies that confirm the effectiveness of presoaking have been reviewed by Khan (1992). Because the amount of water absorbed by the seed is controlled precisely to ensure that germination does not occur, presoaking is a simple method of accelerating the l9 germination of seed. This gives the same results as other methods, such a osmotic priming or solid-matrix-priming, since physiological mechanisms which result in greater seedling performance are considered the same (Copeland and McDonald, 1995; Khan, 1992). It also is considered a 'user- friendly' technology which can be adapted by the farmer (A.G. Taylor, Personal Communication). Pregermination and Gel-seeding Pregermination, or chitting, ipertains to the seed hydration approach in which seeds are soaked under optimal hydration and temperature conditions to a point at which the radicle is just visible (Khan, 1992). Before planting, the pregerminated seeds are mixed with gels (fluid drilling). On other occasions, primed and pregerminated seeds are sown with gels. Seeds also can be germinated in gels and then sown (flow sowing). Seeds also may be allowed to imbibe (primed), but not to germinate, and then sown with gels. The methodology employed in fluid drilling or gel seeding has been reviewed by Gray (1981), Taylor and Harman (1990) and Khan (1992). Examples of gels include sodium alginate formulations, hydrolyzed starch-polyacrylonitiles and synthetic clays. Gels also can be used as a means of introducing hormones, starter fertilizers, or pesticides close to the seeds and developing seedlings (Gray, 1981). 20 Pregermination (gel-seeding) has proved effective for small, slow-germinating vegetable seeds such as celery, parsnip and carrot (Gray, 1981). The major advantage of the method is earlier, higher, and more uniform and predictable emergence, giving earlier growth, higher yields, and more uniform crop maturity. Permitting planting in cool soils and incorporation of nutrients and pesticides in gels are also advantages. The major disadvantage is the susceptibility of seeds to damage during handling, and loss of viability during storage of germinated seeds (Khan, 1992). Another technical, logistic, and economic disadvantage of using fluid-drilling, especially for commercial purposes, is the requirement of specialized planting equipment or modification of existing equipment to sow pregerminated seeds (Taylor and Harman, 1990). Priming Seed priming or osmoconditioning is a seed-hydration approach in which seeds are hydrated in a low—potential solution (Khan, 1992) . Seeds are soaked in an aerated osmoticum of low water potential so that their moisture content is increased and pregermination metabolic activity is initiated, but the moisture level remains below that needed to initiate germination (Bewley and Black, 1994) . The topic of priming was reviewed thoroughly by Bradford (1986) and Khan (1992). 21 In priming, physiological and biochemical events in seeds are enhanced during the suspension of germination by low osmotic potential and negligible matric potential of the imbibing medium. Salts or non-penetrating organic solutes in a liquid medium or matrix solution are used to establish equilibrium water potential between seed and the osmotic medium needed for conditioning. Priming is effective in improving germination and seedling establishment, reduction of germination time, and synchronization of germination. Taylor et a1. (1992) and Pill et al. (1994) found that primed seeds germinated more rapidly than non-primed seeds, especially under adverse field soil conditions. As a result, plants have more vigor, which leads to an advantage over weeds. Other field studies (Matthews and Powell, 1986) showed that poor seedling emergence is not associated with failure to germinate, but with low vigor, resulting in the failure to complete the post-germination, pre-emergence stage of growth. Therefore, vigor of sown seeds is the major physiological constraint on the field performance of most vegetable seeds (Matthews and Powell, 1986). Chate et a1. (1987), however, found that even though germinated seeds could establish higher stands compared with non-germinated seeds in adverse conditions, soil contact with seed is still important. There is controversy as to whether priming invigorates low-vigor seeds more than it does high-vigor seeds. Clarke and James 22 (1991) found priming of little benefit to low-vigor leek seeds. With amaranth seeds, Pill et al. (1994) found that the invigorating effect of priming was more pronounced for low-vigor than for high-vigor seeds. Copeland and McDonald (1995) concluded that priming seems to be successful with small-seeded crops, but less so with large-seeded crops. Although the benefit of solutions such as polyethylene glycol (PEG), NaCl, glycerol, and mannitol; help to supply seeds with nitrogen and other essential nutrients for protein synthesis during germination, their disadvantage is their occasional toxicity to the germinating seedling (Copeland and McDonald, 1995), or in the case of PEG, low oxygen solubility at higher concentrations. As a result, PEG, when used as an osmoticum, often requires aeration to ensure an adequate supply of oxygen to seeds. In addition, it is difficult to treat large quantities of seeds commercially using PEG. Solid-Matrix-Priming Solid-Matrix-Priming (SMP) or matriconditioning refers to seed conditioning by osmotic and/or matric component of the solid matrix water potential. It is another approach to controlled seed hydration, using solid carriers with low matric potentials (Copeland. and. McDonald, 1995). Khan (1992) reviewed work done on the use of solid carriers to 23 shorten time of germination and to improve the rate and synchrony of germination. Seed-conditioning materials should have a high osmotic solute content or a high hydrophilic surface (Khan, 1992). Examples of solid carriers include natural substances such as vermiculite, peat moss and sand; or commercial substances such as diatomaceous silica products and vermiculite. Other substances include leonardite shale, bituminous soft coal, and expanded calcined clay (Khan, 1992; Copeland and McDonald, 1995). Bead Aging Seed aging usually is caused by a number of factors. One could be the loss of viability because of seed storage conditions (i.e. Relative Humidity and temperature). The other could be the natural low viability of a seed lot (low vigor lots), or it could be the natural deterioration of vigor in mature seeds (physiological aging), or the physical damage which causes structural damage (i.e. seed coat) (Matthews and Powell, 1986). In experiments conducted using aged seeds, artificial deteriorating methods are used to speed the aging process. However, there has been growing discussion in the literature (Nath et al., 1991) about the assumptions that such aging- accelerating methods cause similar. damage to that which would occur in natural aging. Nath et a1. (1991) argued 24‘ that seed detoriation was a matrix of interrelated events, each susceptible to different environmental constraints, instead of a continuum of damage, resulting first in a loss of vigor and ultimately in loss of viability. Chapter 2 MATERIALS AND METHODS Commercial greenleaf vegetable amaranth (Amaranthus tricolor L.) seeds (Johnny's Select Seed, Albion, Maine) with a 76% labeled germination, were used to evaluate preplant seed treatments for seedling establishment. All seeds were treated with 20 ppm of Captan to prevent fungal growth during the aging process. To produce aged seeds with lower vigor than fresh seeds, the method of accelerated aging was used (Copeland and McDonald, 1995). Accelerated Aging Method Fresh amaranth seeds 'were aged. by“ placing' a small amount of seeds on a seed rack inside a plastic dish with a tight-fitting lid in an oven of 41C and 100% RH for three days. Germination Test Experiment Optimum germination rates of aged and fresh seeds were tested in a laboratory, using a thermogradient table with five temperature ranges (10, 15, 20, 25, 30 and 35C)(Figure 3). .A room temperature (25Ci2) control also was included. Two 25 26 M\ 2““ mmumu Figure 3. Thermogradient tables used to germinate A. tricolor seeds at different temperature regimes. 27 thermogradient tables with identical temperature ranges were used as two replications. Fifteen seeds of either fresh or aged seeds were placed on a layer of germination paper moistened with distilled water at each of the different temperatures. More distilled water was applied daily to maintain moisture of the seeds. Germination (visible radicles) of the seeds was counted daily for eighteen days. The germination percentage and days to 50% (T-50) radicle emergence were calculated and analyzed using ANOVA and mean separation by F-test and LSD (P=0.05). Initial Moisture Determination The moisture contents (MC, fresh weight basis) of 1.5 g aged and fresh seeds were determined by the oven-drying method (3 h at 130C) . Following oven-drying, seeds were allowed to cool for 1 h in a desiccator before reweighing. The loss in weight was calculated. as percentage moisture content (Grabe, 1989) . The procedure was repeated three times, and the average was used as the initial moisture content of the seeds. Critical Moisture Experiment The required moisture content for priming aged and fresh seeds was determined using an algebraic formula which increases the seed moisture content to a desired level. 28 Knowing the initial moisture content of a known quantity of seed (Initial Moisture Determination Experiment), the critical moisture can be determined by the following formula: Critical moisture, fw basis = [(Desired % moisture content - Initial % moisture content) x (Initial weight of seed)]/(100 - Desired % moisture content) (A.G. Taylor, Personal Communication). The critical moisture content value (f.w. basis) was converted into the amount of water to be mixed with the initial weight of the seed in order to increase the seed moisture content to a desired level. Desired moisture levels used ranged from 30 to 100% in 5% increments. For each desired moisture level, the procedure was repeated twice. The desired moisture level that gave the least germination rate was used as that critical moisture level that will allow seeds to imbibe water, but prevent radicle emergence (i.e. primed seeds). The seed and water mixture was held in air-tight glass tubes for seven to ten days. Then the seeds were dried for 12 h at room temperature and germinated under their optimal germination conditions (Germination Test Experiment), and their germination rates were recorded. 29 Pregermination and Gel-seeding Experiment Fresh and aged seeds were germinated under optimum conditions (Germination Test Experiment) until radicle emergence. Pregerminated and non-pregerminated seeds were mixed with four gels; Natrosol 250 (hydroxyethyl cellulose) (Hercules Inc., Wilmington, Del.), Laponite 445 (magnesium silicate) (Laporte, Hackensack, N.J.), Liqua-gel (polyacrylamide) (Miller Chem. & Fert. Corp., Hanover, Pa.) and Agri-gel/Viterra hydrogel (water-soluble polymer) (Nepara Chem. Co. , Harriman, N.Y.) . Controls of aged and fresh seeds without treatments also were included. Seedling emergence of gel-seeds and seeds without gels was investigated in the greenhouse. Five seeds per pot were planted 6 mm deep in a greenhouse medium (Baccto, Houston, Tex.), in pots of 10 cm diameter which randomly allocated in three blocks. Plants were watered as needed, and seedling growth was recorded daily for 3-4 weeks. Days to 50% (T-50) emergence and total percent emergence were analyzed by ANOVA using MSTAT (MSTAT, East Lansing, Mich.). solid-Matrix-Priming and Polyethyl Glycol-Priming Experiment A sample of 0.5 g seeds was mixed with 10 ml distilled water and 10 g Celite and Aqua-mend or 50 g of silica sand. Celite (Aldrich. Chem. Co., Inc., Milwaukee, Wis.) is a diatomaceous silica protective carrier, silica sand (Fisher 30 Scientific Co., Fair Lawn, N.J.) is a sand product, and AquaMend (Hydration Technology Corp., Santa Monica, Calif.) is a potassium polymer. The seed-protective carrier mixture was held at room temperature for seven to ten days. Greenhouse seedling growth of the Solid-Matrix-Primed (SMP) seeds was compared with growth of control seedlings and Polyethyl Glycol (PEG)-primed seedlings. The PEG—primed seedlings were obtained by mixing a 25 ml solution of PEG (270 g.L“) providing -1.25 Mpa osmotic potential, with 0.5 g seeds for seven to ten days. After PEG-priming, the seeds were rinsed in distilled water while stirring for one ‘minute. The seeds were drained and air-dried at room temperature for 1 h. The greenhouse seedling growth investigation methods and materials were the same as those in previous experiments. Boil-Crusting Experiment To test the best seed conditioning method under adverse conditions, all of the above treatments were tested under soil-crusting conditions in the greenhouse. The only exception was pregerminated seeds (Pregermination and Gel- seeding Experiment) because seeds (especially aged seeds) took long and variable time to germinate, causing large variation of radicle length. Crusted and non-crusted soils of sandy (Marlette fine sandy loam) and loam (Glassoboric Hapludalf mixed mesic) 31 soil types (Michigan State University Horticulture Teaching and Research Center, East Lansing, _Mich.) were used. The soil was steamed to eliminate weed seeds and finely seived. Crusted soil was obtained by irrigating soil (131 ml.sec“) at 0.5 m height. Seeds were planted in 10-cm diameter pots with holes underneath so that daily watering could be performed in trays, without interfering with the crusted or non-crusted top soil. Seedling growth was monitored using the same methods and materials as in other greenhouse experiments. Chapter 3 RESULTS AND DISCUSSIONS Experiment 1: Optimum Seed Germination Test Fresh seeds exhibited mean germination of 78-80% at 20, 25, 30C and room temperature (25Ci2) (RT) (Figure 4 & Table 2) . There were no significant differences in germination response (P=0.05) at 20, 25, 30C, and RT. Aged seeds showed optimum mean germination of 52% at 30C (Table 2). Temperatures of 10 and 15C resulted in 0% germination for both fresh and aged seeds, as did 20 and 35C for aged seeds (Table 2, Figures 5a & 5d). Table 2. Mean germination (%) of aged and fresh A. tricolor seeds at 10, 15, 20, 25, 30, 35C and room temperature (RT) after 18 days. Temperature 10 15 20 25 30 35 RT Mean Germination (%) Aged 0a# 0a 0a 28b 52c 0a 14d Fresh 0a 0a 78b 79b 80b 59c 79b F Mean separation using LSD within horizontal rows by F test, P=0.05. 32 33 100 + Aged seed + Fresh seed Mean Germination (%) Temperature (C) Figure 4. Mean percent germination of aged and fresh A. tricolor seeds at 10 to 35C after 18 days. Aged seeds showed low percentage germination compared to fresh seeds (Figure 4). At very low (10, 15 and 20C) or high (35C) temperatures, aged seeds did not germinate (Table 2, Figure 5a & 4d). Fresh seeds exhibited a wide range of temperatures (20, 25, 30C and RT) for optimum percent germination; only 10 and 15C temperatures had the lowest percentage germination (0%). The shortest times for seeds to germinate were two and four days for fresh and aged seeds, respectively, both at 30C (Figure 5c). For aged seeds, the shortest average time 34 to T-50 was 7.5 days at 30C (Table 3). The highest T-50 for fresh seeds occurred at room temperature (Table 3 & Figure 5e), whereas RT and 25C (Figure 5b & 5c) resulted in the highest T-50 for aged seeds. Fresh seeds exhibited a wide range of temperatures (20, 25, 30 .and 35C) for low T-50, whereas aged seed germination was confined to one specific temperature (30C) for rapid germination (Table 3) . Even though RT (Table 2) had significantly similar percentage germination (P=0.05) as other temperatures (20, 25, 30, 35C) for fresh seeds, it took longer for the seeds to germinate (Table 3). Part of the reason for this difference could be that the environment at RT was not controlled as strictly as in the thermogradient tables; e.g. light conditions. Table 3. Mean time to 50% germination (T-50%) of aged and fresh A. tricolor seeds for 18 days at 10, 15, 20, 25, 30, 35C and room temperature (RT). Temperature 10 15 20 25 30 35 RT T-50% (days) Aged - a* - a - a 13.0c ‘7.5b '- a 14.50 Fresh - a - a 5.0b 4.0b 4.0b 4.0b 9.0c *Mean separation using LSD at P=0.05. 35 00 0'1 + Aged seed + Fresh seed a: o: 0: <3 N O -L U" Mean germlnatlon (96) _a C Tlme (days) Figure 5a. Daily mean germination (%) of aged and fresh A. tricolor seeds for 18 days at 20C. 36 -°- Aged seed 30 .......................................................... + Fresh seed N 01 p-———-——— —---—— ——---_-———--—--—-—--_-----—--—-—————_---—-----——---_--—-—---——-——- N 0 Mean germlnatlon (96) a _L O e e 1011 1’213134131'61'713 Tlme (days) Figure 5b. Daily mean germination (%) of aged and fresh A. tricolor seeds for 18 days at 25C. 37 50 + Aged seed + Fresh seed 40 --------------------------------------- E 8 30 F“? ....................................... _______________ .5 E o O) 20 ................................................................................. 5 d) :2 10 5 9 1b 171 15131713151318 Tlme (days) Figure 5c. Daily mean germination (%) of aged and fresh A. tricolor seeds at 18 days at 30C. 38 30 + Aged seed 25 K ________________________________________ + Fresh seed 3 ‘:20 --------------------------------------------------------------------------------- 0 "Ta" C E§15+ -------------------------------------------------------------------------------- 5 0) § 10 ....................................................................... IE 8 e 13 1’1 1’2 1’3 1‘4 1'5 16 1Y7 118 Tlme (days) Figure 5d. Daily mean germination (%) of aged and fresh A. tricolor seeds for 18 days for 35¢. 39 + Aged seed 30 ----------------------------------------- + Fresh seed ________________________________________________________________________________ M 01 p—o—-———_----—--—-—_________-_——--—_--_-___ --- -_—_._____—_—___.—-____________-——.—_-— N O _a 01 p_____—_—_---—_———-——---—__——-_——------—- —_-— —--.—---------——-—--——-—-_——_——--—_-- _a o p-—————________-__—___ ___ ______—___——-— —————_ —-———————_—_-_--—---_-----_-—-——___ Mean germlnatlon (%) /. ' . ’ 0" ' ' ' ' ' 4"” Y 4% 1r 3 'r 'r i 1 2 3 4 5 6 7 8 9101112131415161718 Tlme (days) Figure 5e. Daily mean germination (%) of aged and fresh A. tricolor seeds for 18 days at room temperature. This experiment supports one of the original hypotheses; that aged seeds' germination rate is much lower and slower than that of fresh seeds. It also showed the wide temperature germination ability of fresh seeds compared with aged seeds. These results were similar to those found by Clarke and James (1991) and Powell et al. (1991) in their study of germination performance of leek and brassica seeds, respectively. Aged seeds' germination rates were slower, and the time required for germination was greater than for 40 fresh seeds. The reason for the slower germination is due to reduced seed vigor. Reduced vigor involves the accumulation of degenerative changes until eventually the ability to germinate is lost (Naylor and Gurmu, 1990). Experiment 2: Pregermination and Gel-seeding There was a significant interaction (Table 4) between seed type, germination pattern and gel type on mean percent emergence. Overall, fresh seeds which were non- pregerminated, with all four gels; or pregerminated without a gel, significantly had better percentage emergence than all aged seeds (Table 5). 41 Table 4. The effect of germination pattern, gel type and seed condition of A. tricolor seeds expressed as percent seedling emergence. Source of variance DF S.S. M.S. F Blocks 2 280 140 Seed type 1 16667 16667 37.31* Error (a) 2 893 447 Germination 1 1307 1307 7.0 ns Seed type X Germination 1 4507 4507 24.14** Error (b) 4 747 187 Gel type 4 4587 1127 6.5** Seed type x Gel type 4 2400 600 3.46** Germination X Gel type 4 14426 3607 20.81** Seed X Germination X Gel 4 3360 840 4.85** Error © 32 5547 173 Total 59 54640 m ns, *, **:N0nsignificant or significant at P=0.05 or 0.01 respectively. ©or fresh seeds, there was a significant difference in percentage emergence between germination pattern and gel types. Non-pregerminated fresh seeds with Laponite, Natrosol, Agri-gel; or pregerminated fresh seeds without a gel, emerged significantly better than all other fresh seeds treatments. With all four gels, non-pregerminated fresh seeds gels gave significantly higher percentage emergence than pregerminated seeds with the four gels. When no gel was included, pregerminated fresh seeds gave significantly 42 higher percentage emergence than non-pregerminated seeds (Table 5). For aged seeds, no significant difference in percentage emergence occurred with pregerminated or non-pregerminated seeds with all four gels; without a gel, pregerminated seeds obtained higher percentage emergence than non-pregerminated seeds and all other treatments with gels (Table 5). Table 5. Effects of seed treatments on mean percent emergence of aged and fresh A. tricolor seeds. Seed Type Aged Fresh Gel Type Germination Pattern Emergence (%) Laponite No Pregermination 6.67cd 86.67a# Pregermination 6.67cd 26.67bc Liqua-gel No Pregermination - d 20.67bcd Pregermination 6.67cd 6.67cd Natrosol No Pregermination 13.33cd 66.67a Pregermination 6.67cd 13.33cd Agri-gel No Pregermination 6.67cd 83.33a Pregermination 6.67cd 20.00bcd No gel No Pregermination - d 20.00bcd Pregermination 40.00b 80.00a ' Mean separation using LSD P=0.05 For fresh seeds, no significant difference in days to T-50 occurred, either with gel type and/or germination pattern. For aged seeds, non-pregerminated seeds with 43 Laponite and Agri-gel, and pregerminated seeds without a gel; had a significantly lower T-50 than all other treatments (Table 6). The highest percentage emergence as well as the shortest time to T-50 for fresh seeds were; non- pregerminated seeds with Laponite or Agri-gel gels, or pregerminated seeds without a gel. The best percentage emergence treatment in aged seeds, giving the shortest time to T-50 was pregermination without a gel. The significant response of non-pregerminated fresh seeds to gels might be supported by the claims that the role of gels is to provide water for growth of germinated or dry seeds (Gray, 1981). Therefore, Laponite and Agri-gel gels were the best in providing water for the growth of non- pregerminated seeds. 44 Table 6. Effects of gel types and germination pattern on mean time to 50% germination (T-50%) (days) for aged and fresh A. tricolor seeds. =LC-Iel Type l-E:;mination Pattern If Aged Fresh T-50% (days) Laponite No Pregermination 10.0c 5.3af Pregermination 15.0d 7.0ab Liqua-gel No Pregermination - e 8.5b Pregermination 15.0d 9.0bc Natrosol No Pregermination 10.0c 7.0ab Pregermination 11.0c 7.0ab Agri-gel No Pregermination 9.0bc 6.0a Pregermination 15.0d 9.0bc No gel No Pregermination - e 7.0ab Pregermination 7.0ab 7.0ab ' Mean separation using LSD P=0.05 In contrast. with. results found. in 'this experiment, studies by Odell et al. (1992), Pill (1986) and Ghate and Phatak (1982) on different crops found that pregerminated seeds with gels hastened seedling emergence and improved seedling emergence. The probable explanation for such differences may be the vulnerability of pregerminated seeds to physical damage during handling, and the variability of germination (even at optimal germination conditions) which force storage of seeds, resulting in reduced viability Another reason for variations in crop (Gray, 1981). 45 response to fluid-drilling noted by Pill (1986); was the variability of radicle length at the time of sowing a batch of seeds with variable germination rates. Taylor and Harman (1990) offered another reason for variation of pregerminated seeds. They suggested that reduction in plant stands of pregerminated seeds might be due to desiccation intolerance, and therefore death of seeds because of insufficient moisture for continued growth. Following that reasoning, with results obtained in this experiment, the possible explanation might be the inability of the gels to supply sufficient moisture to the pregerminated seeds to continue growth, or the failure of gels in protecting the vulnerable radicle. Another possibility for low performance of pregerminated seeds could be the desiccation intolerance of already germinated seeds during the germination process. As a solution to the variability of pregerminated seeds, Pill (1986) suggested seed osmoconditioning before pregermination. Experiment 3: SMP and PEG-priming Since there were significant interactions among priming methods and seed type (Table 7), discussion for main effects was ignored. Emergence of fresh seeds did not significantly differ as a result of the type of priming method used. Within aged seeds, priming with PEG and Celite resulted in the best percentage seed emergence (Table 8). 46 Priming fresh seeds with silica sand and Celite gave the shortest time to T-50, whereas priming with PEG gave the longest time to T-50. Combining the highest percentage emergence with the shortest time to T-50, Celite and PEG gave the best responses for aged seeds (Table 8) . If a choice between priming with PEG and Celite was given, Celite would be more favorable, because it is easier to handle than PEG (Taylor and Harman, 1990). Table 7. Analysis of variance for mean percent emergence of Solid-Matrix-Primed and PEG-primed aged and fresh A. tricolor seeds. Source of variance D.F. 8.8. M.S. r Block 3 110 36 Seed type 1 15210 15210 51.00** Error (a) 3 830 277 Priming method 4 2460 615 2.65 ns Seed type X Priming 4. 4940 1235 5.33** Error (b) 24 5560 . 232 Total 39 29110 ns, **: No significance and significance P=0.05, respectively. 47 Table 8. Effects of priming treatment on mean percent emergence, mean days to 50% emergence (T-50) of aged and fresh A. tricolor seeds. Seed Type Aged Fresh Priming Emergence T-50% Emergence T-50% ”“1”“ (’5) (days) (*1 (681!!!) Control 49.00abc* 15.008 53.00a 4.503b Celite 53.00a 9.00d 51.006b 3.008 Silica 30.26bc 7.00cd 71.00a 3.30a sand AquaMend 28.720 9.00d 52.506 5.80bC PEG 57.50a 8.30d ’ 60.00a 9.00d * Mean separation by LSD at P=0.05. In their priming experiment with leek seeds, Clarke and James (1991) noticed the same trend as in this experiment; that, even though on the overall, priming can increase germination performance of aged seeds, their viability will still be lower than that of fresh seeds. The authors cited part of the reason for this as that aging the seeds had some effect on the nucleic acids of the whole seeds prior to germination. Even though subsequent priming increased germination percent, this was not enough to replace all the loss of nucleic acids. In conclusion to their study, Clarke and James (1991) suggested that the relationship between priming and aging was not simple, and therefore proposed that as seeds move down a viability curve, the beneficial effects of priming are reduced. 48 Performance of PEG-primed aged and fresh seeds was the same (Table 8). In their experiment, Pill and Evans (1994) found that PEG—priming invigorated mechanically harvested (low-vigor) amaranth seeds more than hand-harvested (high- vigor) seeds. They also showed that mechanically injured seeds can activate their nucleic acids during the priming process, whereas aged seeds cannot. In their review on seed vigor, Matthews and Powell (1986) suggested that in controlling field conditions such as crusting, the economic benefits of presowing treatments such as priming and pregermination can be seriously undermined when seeds are of low-vigor. They argued that such expensive treatments can be of little benefit to low- vigor seed. They suggested that the major benefits of the many seed treatments lie in the indirect influence they have in ensuring that only highly germinable, vigorous seed lots are given expensive treatments. This leads to the conclusion that, in this experiment, even though overall response to priming was higher for aged than fresh seeds, priming fresh seeds would be more economical than priming aged seeds. Supporting this conclusion, are the arguments noted from prior statements that, the overall percentage emergence of primed aged seeds would not surpass that of fresh seeds due to viability problems. 49 Experiment 4: Presoaking Using the gravimetric technique (see Materials and Methods: Initial Moisture Determination) in determining the initial moisture content (fresh weight basis), the average was 13% for both aged and fresh seeds. The critical moisture content to prime seeds are shown in Table 9. However, the critical seed moisture content selected should prevent germination, because seeds become desiccation-intolerant after visible germination (A.G. Taylor, Personal Communication). Based on the results obtained, seeds started to germinate at 40% MC, therefore, 35% desired moisture content would be selected as the critical moisture content to prime seeds. Table 9. The effect of moisture content (MC) (%) on germination of aged and fresh A. tricolor seeds when presoaked at room temperature for 7 to 10 days. Desired MC (%) 30 35 40 45 50 55 60 65 75 99 Germination (%) Aged O 0 1 3 5 50 70 80 85 90 o o 3 7 4o 67 82 85 90 96 Table 10 showed that the mean percentage germination for 30, 35 and 40% desired MC was not significantly different in aged versus fresh seeds. 50% desired MC had 50 significantly the highest mean germination, but since 5% of the seeds (Table 9) had already germinated, these MCs would be undesirable for priming seeds. With fresh seeds, 30 and 35% desired MC gave the least mean percentage germination; whereas 40, 45 and 50% desired MC gave 59.5, 68.0 and 84.9% mean germination, respectively (Table 10). Even though 40, 45 and 50% desired MC gave higher percentage germination than 30 and 35% desired MC, they cannot be used as critical moisture contents, because the seeds were already visibly germinating (Table 9). Table 10. Mean germination (%) of aged and fresh A. tricolor seeds at 30 to 50% desired moisture content at 30C. Desired Moisture Content (%) 3O 35 40 45 50 Mean Germination (%) Aged 49.900 51.450 52.65C 58.50b 66.50a* Fresh 50.009 51.00d 59.50C 68.00b 84.90a *Mean separation-using LSD within horizontal rows by F test, P=0.05. Although mean percentage germination of 30 and 35% desired moisture contents did not surpass that of control aged and fresh seeds at 30C (Table 2 & 10), time to 50% germination was reduced (Table 3 & 11) by two days for aged seeds and one day for fresh seeds. In the case of aged seeds, these results were similar to those found by Nath et al. (1991). In their experiment with wheat seeds, seed 51 hydration treatments applied after aging did not allow recovery of germination percentage. The authors attributed their findings to the fact that aged seeds might have lost viability prior to treatment application. The hydration treatments did, however, result in marginal decrease in T-SO of the remaining viable seeds, as well as T-50 of fresh or non-aged seeds. Contradicting the results found in this experiment and those found by Nath et al. (1991) are reports by Tarquis and Bradford (1992) quoting prehydration studies which improved the vigor of aged seeds. One factor which could be attributed to these contradictions is that duration and temperature of the presoaking or prehydration treatments differed in each of the experiments. Another factor could probably be the difference in crop species that were under investigation. Table 11. Mean time to 50% germination (T-50%) of aged and fresh A. tricolor seeds at 30 to 50% desired moisture content at 30C. :— Desired Moisture Content (%) 30 35 40 45 50 T-50% (days) Aged 6 6 6 4 4 .EFESh_ 3 3 3 2 2 52 Although the initial moisture content (f .w.) of aged and fresh seeds was the same (13%), fresh seeds seemed to imbibe faster than aged seeds, since the latter obtained higher percentage germination rates (especially at 45 & 50% desired MC) than the former (Table 9). The reason for this scenario is that seeds (aged or fresh) will have the same moisture content as long as they equilibrate to the same Relative Humidity (A.G. Taylor, Personal Communication). Vertucci (1989) used seeds ,of different chemical composition but identical water potentials. It was found that the permeability of the seeds' tissue to water was more important than initial moisture potentials in determining imbibition rates. In this experiment, the aged seeds' coats appeared to be less permeable than those of fresh seeds. Nonetheless, Vertucci (1989) concluded that seed permeability was a complex function of seed morphology, structure, composition, moisture, and temperature that could not be elucidated easily. Experiment 5: Soil Crusting There was a significant interaction between seed type, soil type, soil condition, and treatments1 (Table 12). 1 Treatments = Seed Desired Moisture Contents (30, 35, 40, 45, 50, 55 & 60%), Non-pregerminated gel-seeding (Laponite, Liqua-gel, Natrosol, Agri-gel & AquaMend gels), Solid Matrix Priming (Celite & Silica sand), Polyethylene glycol Priming, and Control. 53 Compared with the control, some seed-invigorating treatments of aged seed in crusted loam soil gave higher significant mean percentage emergence (Table 13) . Those that did were, 35, 50, 60% desired MC, non-pregermination seeds in Laponite and Agri-gel gels, SMP seeds with silica sand, and PEG-primed seeds. With. fresh. seeds 'under' crusted. conditions on loam, treatments that produced significantly higher mean percentage emergence than the control were; 30, 35, 40 and 45% desired MC, non-pregerminated seeds in Liqua-gel, Natrosol, Agri-gel and AquaMend gels, and SMP seeds with Celite. With aged seeds under crusted sandy soil, the control treatment's mean percentage seedling emergence was surpassed by the following treatments; 35 and 60% desired MC, non- pregerminated seeds with Laponite, Agri-gel and AquaMend gels, and SMP seeds with silica sand. 54 Table 12. Analysis of variance for aged and fresh seed types (seed), clay and sandy soil types (soil), crusted and non-crusted soil conditions (condition), administered with different treatments (trts). SOURCE OF VARIANCE DF 8.8. M.S. r Blocks 1 76.6 76.6 Seed 1 8326.6 8326.6 44.0” Error a 1 189.1 189.1 Soil 1 27639.1 27639.1 242.3“ Seed X Soil 1 1.6 1.6 0.01” Error b 2 228.1 114.1 Condition 1 4726.6 4726.6 22.4“ Seed X Condition 1 1314.6 1314.6 6.23“ Soil x Condition 1 2139.6 2139.6 10.1‘ Seed x Soil x Condition 1 4064.1 4064.1 19.3‘ Error c 4 843.8 210.9 Trts 15 53198.4 3546.6 17.9” Seed x Trts 15 30098.4 2006.6 10.2“ Soil x Trts 15 11685.9 779.1 3.95“ Seed x Soil x Trts 15 10623.4 708.2 3.59“ Condition x Trts 15 18398.4 1226.6 6.22“ Seed x Condition x Trts 15 14510.9 967.4 4.91“ Soil x Condition x Trts 15 15585.9 1039.1 5.27“ Seed x Soil x Condition 15 14360.9 957.4 4.86“ X Trts Error d 120 23662.5 197.2 Total 255 241673.4 ns, ""No significance, significance at 5% and 1% , respectively. 55 mooH mooa oomom oomom oomom _oooov Hroom Cooom one Aeoom ooooe memos ounce moooe oomom rooom oomom team modem oomom 66008 86808 rooom moooo ounce nooom oueaoo ounce mooo 8080» 88008 moooe mOOH memos emoom ocozmdca Cooom mooo woos oomom mood woos mooom mooH Hooueuod oomom mooo woos memos dooom modem emoom Heoom Homouumz Heoom mooo ounce oomom moooe mooov memos reoom Hooumoved oomom ooooe memos woos rooom woos eeoom moooe ooecodmd oomom oomom ooooe oowoe egos ounce Aeoom memos 02 wow omom mom: rooom 830m one... 3.63 ES 33 02 won dream mOOH ounce Heoom ounce Cooom rooom woven 02 won moooe rooom so endow oomom Eros ounce endow oz emu omom mooH dream moooe “room endow ounce Heoom or you done» 8808 modem rooom mace woos oomom canoe or eon rooom 803 82:. 898m 83 6868 862. roeom 02 won .w. OOEOOAOEH and: DECEMMOHH roofin— coo<_ 863m 0064 £896— ooo<_ soon: 0084 Odom H Econ ucmm _ EMS oounououcoz tonnage nous: mucoEumouu mooom Mowoowhu .moohu aeom comm use good commouoncoc 0cm poundeo .< romeo one doom mo Awe oucoouofio coo: . ma OHQMH 56 With fresh seeds under crusted sand, treatments 30, 35, 45 and 55% desired MC, non-pregerminated seeds with Agri-gel and PEG-primed seeds; produced significantly higher mean emergence than the control. With aged seeds under non-crusted loam soil conditions, mean percentage emergence for the control was significantly lower than for seeds primed at 40% desired MC, non- pregerminated seeds in Laponite, Liqua-gel, Agri-gel and AquaMend gels, SMP seeds with Celite and PEG-primed seeds. Mean percentage emergence of 35 and 60% desired MC, non-pregerminated seeds in Natrosol, Agri-gel and AquaMend gels, SMP seeds with Celite, and PEG-primed seeds with fresh seeds under non-crusted clay soil conditions was significantly higher than that of the control. With aged seeds under non-crusted sandy soil conditions, all seed-invigorating treatments except priming at 45% desired MC produced significantly higher mean percentage emergence than the control. Comparing mean percentage emergence of the control with all other seed-invigorating treatments with fresh seeds under non-crusted sandy soil conditions; 35, 40, 45, 55 and 60% desired MC, non-pregerminated seeds in Laponite and Natrosol gels and PEG-primed seeds, significantly surpassed the control (Table 13). Due to the large number of treatments, and the significant four-way interaction, data were difficult to 57 interpret (Tables 12 & 13). To facilitate simpler presentation of data while testing the hypothesis that in crusted soil, the extent of seed invigoration differs with the invigoration method, promising treatments (from Table 13) were contrasted with each other or with a control under crusted and non-crusted soil conditions. The selected major treatments (priming by presoaking at 35% desired MC, gel- seeding non-pregerminated seeds with Agri-gel, SMP with Celite and silica sand, PEG-priming and control), were contrasted using SAS (SAS Institute, Inc., Cary, NC). Contrasting treatments in crusted soil, there were significant differences (P=0.05) for; 35% MC vs. Agri-gel, 35% MC vs. control, Agri-gel vs. control, Celite plus sand vs. control and PEG vs. control (Table 14). Contrasting treatments in non-crusted soil conditions, there were no significant differences between 35% MC and Agri-gel, but there were significant differences in percentage emergence between 35% MC and control, Agri-gel and control, Celite plus sand and control, and PEG and control (Table 14). 58 Table 14. Mean emergence (%) and contrasts of control, selected treatments of presoaking, gel-seeding, SMP and PEG under crusted and non-crusted soil conditions. Soil Condition Treatment Crusted Non-crusted Mean Emergence (%) 35% MC 75.0 75.0 Agri-gel 95.0 75.0 Celite 57.5 45.0 Silica Sand 67.5 37.5 PEG 67.5 70.0 Control 25.0 25.0 Contrasts 35% MC vs Agri-gel ** ns 35% MC vs control ** ** Agri-gel vs control ** ** Celite + Sand vs control ** ' ** PEG vs control ** ** **,ns: Significance by F-test at P=0.05 or non-significance, respectively. Finally, the hypothesis was found true in that, in crusted soil, all invigorating methods differed in extent of invigoration. All selected treatments performed better than the control under both crusted and non-crusted soil conditions. Furthermore, all selected treatments resulted in equal or better percentage emergence under crusted than non-crusted soil conditions except for PEG-priming treatment (Table 14) . These results also prove the hypothesis that 59 invigoration of amaranth seeds improves germination and seedling emergence. Under crusted conditions, using Agri-gel for seeding non-pregerminated seeds was a better invigorating method than 35% MC, but under non-crusted conditions, both treatments performed the same (Table 14). Therefore, under crusted conditions, using Agri-gel to seed non-pregerminated seeds would be preferred over 35% MC. Comparing priming techniques in crusted soil, presoaking at 35% MC showed better potential for improving percentage emergence of aged and fresh seeds than did PEG-priming or SMP. CONCLUSIONS Makus ( 1984) stated that future commercialization of amaranth as a green vegetable depended upon solving problems associated with seedling establishment. The hypothesis that invigoration of seeds can improve emergence of seeds has been proven correct. Furthermore, aged seeds, with lower germination rates than fresh seeds at 20, 25, 30, 35C and room temperature; exhibited different emergence rates than fresh seeds for different invigoration methods. In gel- seeding, non-pregerminated fresh seeds emerged better than aged seeds. After PEG-priming and SMP, aged seeds responded better to priming than fresh seeds, but the overall percentage emergence of aged seeds .was lower than that of fresh seeds. When presoaking was used for priming, aged and fresh seeds did not differ in percentage germination (in the lab), except at higher than 45% desired moisture contents. In sub-optimal conditions (soil crusting) of loam and sandy soil types, this study showed that priming aged and fresh seeds by presoaking them in 35% desired moisture content (f.w.) can increase their vigor over control seeds, PEG-primed or Solid-Matrix-Primed seeds. Priming or invigoration method to be commercialized depends on the development of a reliable, economical, environmentally friendly, large-scale aerobic method that suppresses pathogen growth on the seed (Parera and Cantliffe, 1994; 60 61 Taylor and Harman, 1990) . The presoaking method for seed invigoration is less expensive than SMP or PEG-priming, and, therefore, could be affordable even for small-holder farmers of lesser developed. countries“ .Based on the preceding reasons and from results of this study, it would be recommended that the presoaking method (at 35% desired MC) be used to invigorate seeds under crusting conditions. For instance, 1.5 g seeds would be primed by soaking in 0.5 ml of distilled water at room temperature for 7 to 10 days. Further research. is needed for the success of the presoaking priming method. Since invigorating methods are influenced by a complex interaction of factors, including plant species, osmoticum, water potential of the priming agent, duration of priming, temperature, seed vigor, and dehydration and storage conditions following priming or invigoration (Parera and Cantliffe, 1994) ; more study is needed, especially regarding the optimum duration of presoaking and the interaction of 'presoaking duration at different temperatures. In addition, to achieve consistent and beneficial results from priming or seed invigoration, it is advisable that only seed of high quality be used. To further refine invigoration methods and obtain greater and more consistent benefits from the process requires an understanding of the physiological and molecular effects of priming, which remains a largely' unelucidated ‘topic, regardless of the 62 quality and quantity of studies that have been conducted (Parera and Cantliffe, 1994). LITERATURE CITED Abbott, J.A. and T.A. Campbell. 1982. Sensory evaluation of vegetable amaranth (Amaranthus spp.). HortScience 17:409-410. Bewley, J.D. and M. Black. 1994. Seeds: Physiology of development and germination. 2nd ed. Plenum Press, New York. 445 pp. Bittenbender, H.C. 1983. Handbook of tropical vegetables. Department of Horticulture, East Lansing, Mich. 118 pp. Bradford, K.J. 1986. Manipulation of seed water relations via osmotic priming to improve germination under stress conditions. HortScience 21:1105-1112. Campbell, T.A. and J.A. Abbott. 1982. Field evaluation of vegetable amaranth (Amaranthus spp.). HortScience 17:407-409. 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