Seed Germination Testing.pdf

Assistant Professor cum Junior Scientist
1. Apr 2023

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Seed Germination Testing.pdf

  1. Concept of Seed Germination TESTING Prof. Kumari Rajani Department of Seed Science & Technology Bihar Agricultural University Sabour, Bhagalpur-813210
  2. Structure of Monocot Seed Seed coat : Provides protection Endosperm/Cotyledon : Store food Aleurone cells : Store abundant proteins and enzymes Prof. Kumari Rajani, DSST, BAU, Sabour
  3.  The triploid endosperm is composed of two tissues:  Starchy endosperm  Aleurone layer  The nonliving starchy endosperm consists of thin walled cells filled with starch grains and it is centrally located  Living cells of the aleurone layer, which surrounds the endosperm, synthesize and release hydrolytic enzymes into the endosperm during germination  As a consequence, the stored food reserves of the endosperm are broken down, and the solubilized sugars, amino acids, and other products are transported to the growing embryo via the scutellum  The isolated aleurone layer, consisting of a homogeneous population of cells responsive to gibberellin, has been widely used to study the gibberellin signal transduction pathway in the absence of non responding cell types Prof. Kumari Rajani, DSST, BAU, Sabour
  4. Structure of Dicot Seed Radicle  Root Plumule  Shoot and leaves Prof. Kumari Rajani, DSST, BAU, Sabour
  5. Seed Germination “Germination begins with water uptake by the seed (imbibition) and ends with the start of elongation by the embryonic axis, usually the radicle” Field or Greenhouse condition “a seedling emerges from the soil” SeedTesting “development of a normal seedling” Physiologist “the emergence of radicle from a seed” Prof. Kumari Rajani, DSST, BAU, Sabour
  6. Cotyledon (provide food to the growing embryo) Epicotyl (above the cotyledon) Hypocotyl (below the cotyledon) Basis for the plant’s stem Leaves Prof. Kumari Rajani, DSST, BAU, Sabour When the radicle has grown out of the covering seed layers, the process of seed germination is completed
  7. Germination does not include seedling growth after radicle emergence, which is referred to as Seedling establishment or Field establishment Similarly, the rapid mobilization of stored food reserves that fuels the initial growth of the seedling is considered a Post germination process Prof. Kumari Rajani, DSST, BAU, Sabour
  8. Hypogeal germination of pea seed Epigeal germination of bean seed Types of Seed Germination Epigeal Germination  Cotyledons are raised out of the soil  Epigeal germination takes place by the rapid extension of hypocotyle before the growth of the epicotyle  Evolutionary more primitive than hypogeal  Ex: Bean, Castor, Mustard, Tamarind, Sunflower, Onion, Papaya, Pine etc Hypogeal Germination  Cotyledons remains underground  Hypogeal germination takes place by the rapid extension of epicotyle and the growth of hypocotyle is restricted  Ex: Paddy, Wheat, Maize, Gram, Pea, Mango, Groundnut etc Prof. Kumari Rajani, DSST, BAU, Sabour
  9. Radicle emergence: in most of species Hypocotyle emergence: Bromeliaceae, Chenopodiaceae, Onagracea, Palmae, Saxifragaceae and Typhaceae Coleoptile emergence: Maize, Oropetium tomaeum Elongation of the mesocotyle elevates the coloeoptile and its enclosed inner leaves towards the soil surface The mesocotyl is the tubular, white, stemlike tissue connecting the seed and the base of the coleoptile The mesocotyl is the first internode of the stem Prof. Kumari Rajani, DSST, BAU, Sabour
  10. The entire process of germination (water uptake by a germinating seed) may be divided into three broad phases; it shows triphasic pattern • Phase I: Imbibition phase • Phase II: Active metabolism or Plateau or Lag phase • Phase III: Cell expansion & radicle protrusion & further increase in water uptake Phases of Germination The most critical phase is phase II whereas, the physiological and biochemical processes such as hydrolysis, macromolecules biosynthesis, respiration, subcellular structures, and cell elongation are reactivated resulting in initiation of germination Prof. Kumari Rajani, DSST, BAU, Sabour
  11. Phase I : Imbibition phase  The initial rapid uptake of water by the dry seed during Phase I is referred to as Imbibition  It is the first key event that moves the seed from a dry, dormant organism to the resumption of embryo growth  The extent to which water imbibition occurs is dependent on three factors: composition of the seed seed coat permeability water potential  Species produce seeds with impermeable testa called hard seeds (hardseededness) Ex: Leguminosae, Cannaceae, Chenopodiaceae, Convolvulaceae and Malvaceae Prof. Kumari Rajani, DSST, BAU, Sabour
  12.  Imbibition Phase is relatively shorten and characterized by rapid water uptake Chief changes during imbibition phase: • Absorption of water • Absorption of other substances • Release of gases • Increase in volume of seeds due to swelling • Leakage of solutes  The initial period of imbibition induces an immediate and rapid leakage of solutes such as sugars, organic acids, amino acids, proteins, phenolics, phosphate and ions, from the seed tissues but it rapidly decreases and becomes negligible within about 30 min to 1 hr  The leakage results in loss of enzymes like glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, cytochrome oxidase and fumarase Prof. Kumari Rajani, DSST, BAU, Sabour
  13. “The release of non-respiratory gases as a result of very rapid seed imbibition” • It is immediate and last only a few minutes • It occurs by the release of adsorbed atmospheric gases (Oxygen, Nitrogen, Carbon dioxide) retained in the dry porous structures of the seed coats Wetting Burst Prof. Kumari Rajani, DSST, BAU, Sabour
  14. Imbibitional chilling injury is defined as sensitivity to a combination of low seed-water content and imbibition at cold temperature The severity of injury depends upon several factors such as (i) The species or the cultivars involved (ii) The initial water content of the seed (iii) The temperature to which seed is exposed (iv) The duration of chilling exposure (v) The period during the course of germination when the chilling exposure takes place Ex: Cotton, Soybean, Limabean, Maize Imbibitional Injury Prof. Kumari Rajani, DSST, BAU, Sabour
  15.  Water uptake by imbibition declines and metabolic processes, including transcription and translation, are reinitiated  The seed volume may increase as a result embryo expands and the radicle emerges from the seed coat  The emergence of the radicle through the seed coat in Phase II marks the end of the process of germination  Radicle emergence can be either a one-step process in which the radicle emerges immediately after the seed coat (testa) is ruptured, or it may involve two steps in which the endosperm must first undergo weakening before the radicle can emerge Phase II : Plateau or Lag phase Prof. Kumari Rajani, DSST, BAU, Sabour
  16.  Dry seeds contain several enzymes, which are desiccation tolerant and can become active only after sufficient hydration of seeds  Major metabolic pathways affected respiration, protein synthesis, DNA replication, RNA synthesis Prof. Kumari Rajani, DSST, BAU, Sabour
  17. Mobilization of Stored Reserves  The major food reserves of angiosperm seeds are typically stored in the cotyledons or in the endosperm  The massive mobilization of reserves that occurs after germination provides nutrients to the growing seedling until it becomes autotrophic  At the subcellular level, starch is stored in amyloplasts in the endosperm of cereals  Two enzymes responsible for initiating starch degradation are α- and β- amylase  α-Amylase hydrolyzes starch chains internally to produce oligosaccharides consisting of α (1,4)-linked glucose residues  β-Amylase degrades these oligosaccharides from the ends to produce maltose, a disaccharide. Maltase then converts maltose to glucose Prof. Kumari Rajani, DSST, BAU, Sabour
  18.  Protein storage vacuoles are the primary source of amino acids for new protein synthesis in the seedling  In addition, protein storage vacuoles contain phytin, the K+, Mg2+, and Ca2+ salt of phytic acid a (myo-inositol hexaphosphate), a major storage form of phosphate in seeds  During food mobilization, the enzyme phytase hydrolyzes phytin to release phosphate and the other ions for use by the growing seedling Prof. Kumari Rajani, DSST, BAU, Sabour
  19.  During Phase III the rate of water uptake increases rapidly due to the onset of cell wall loosening and cell expansion  Protrusion of radical during germination is caused by cell expansion or elongation before cell division Ex: Maize, Barley, Broad beans, Pea etc  Pinus lambertiana: cell division and cell elongation occur simultaneuosly  Prunus cerasus: cell division precedes cell elongation Phase III : Cell expansion & Radicle protrusion Prof. Kumari Rajani, DSST, BAU, Sabour
  20. Phases of Germination Prof. Kumari Rajani, DSST, BAU, Sabour
  21. Phases of Germination Prof. Kumari Rajani, DSST, BAU, Sabour
  22. STAGE EVENTS PREGERMINATION (a) Rehydration – imbibition of water. (b) RNA & protein synthesis stimulated. (c) Increased metabolism – increased respiration. (d) Hydrolysis (digestion) of food reserves by enzymes. (e) Changes in cell ultrastructure. (f) Induction of cell division & cell growth. GERMINATION (a) Rupture of seed coat. (b) Emergence of seedling, usually radicle first. POST GERMINATION (a) Controlled growth of root and shoot axis. (b) Controlled transport of materials from food stores to growing axis. (c) Senescence (aging) of food storage tissues. © 2008 Paul Billiet ODWS Major Events occurring during Germination Prof. Kumari Rajani, DSST, BAU, Sabour
  23. Process: Seed Germination 1. Imbibition - water uptake, softens inner tissues - causes swelling and seed coat rupture - more water uptake 2. Gibberelic Acid - plant hormone (similar to steroids) - dissolved & distributed by water Prof. Kumari Rajani, DSST, BAU, Sabour
  24. 2. Gibberelic Acid - arrives at aleurone cells - activates certain genes 3. Transcription Transportation Translation  amylase 4. Amylase accelerates hydrolysis of starch Process: Seed Germination Prof. Kumari Rajani, DSST, BAU, Sabour
  25. Hydrated starch moves to the cotyledon and radicle to initiate growth Process: Seed Germination Prof. Kumari Rajani, DSST, BAU, Sabour
  26. Factors affecting Germination Internal Factors External Factors  Seed Vitality  Seed Age or Maturity  Seed Dormancy  Mechanical Damage (The effect of mechanical injury is greater when it affect the embryo)  Water  Air (Oxygen & Carbon dioxide)  Temperature  Light Prof. Kumari Rajani, DSST, BAU, Sabour
  27. Water  Water is clearly the most important factor in germination  An adequate continuous supply of water is necessary for assumption of the physiology, metabolism and molecular processes that drive germination  Water functions as triggering enzyme for starch conversion into sugar, turgor pressure for moving the radicle root down and the cotyledons up, and for transporting nutrients and enzymes within the seed  Recalcitrant seeds usually do not require external water for germination since their natural water content is sufficient for them to complete germination  Germination on parent tree before shedding: Spp. of Mangrove swamps such as Rhizophoraceae, Rhizophora spp, Bruguiera gymmorrhiza, Cerops tagal, Avicennia marina  In fleshy fruits within which they are enclosed: Mango Prof. Kumari Rajani, DSST, BAU, Sabour
  28.  Excess of water is harmful and seeds don not germinate when immersed in water  Sugarbeet seeds: A thin layer of water around the seed inhibits germination  Barley: Germination is affected by excess water, which is called as water sensitivity (Excess of water intervenes indirectly by depriving the embryo of oxygen)  Typha latifolia (Aquatic plant) and Paddy: Germinate well when covered with water (under reduced oxygen levels)  Cynodon dactylon: Germinate in low oxygen levels  Oldenlandia corymbosa (tropical weed): Germinate only when completely immersed Prof. Kumari Rajani, DSST, BAU, Sabour
  29. Oxygen  Oxygen in presence of enough moisture causes respiration to start metabolism and it creates energy for the germination process  Respiration rates for germinating seeds are very high; adequate oxygen is necessary to complete respiration  Oxygen concentration higher than air: promotes germination  Carbon dioxide concentration higher than air: retards germination Lettuce and Timothy grass (Phleum pratense)  If oxygen supply is limited during germination, emergence may not occur due to inhibited growth Prof. Kumari Rajani, DSST, BAU, Sabour
  30.  The germination percent of most seeds will be retarded if the oxygen percent goes below 20 percent (Normal air is 20 percent oxygen)  Typha latifolia (Aquatic plant) and Paddy: Germinate well under reduced oxygen levels  Cynodon dactylon: Germinate in low oxygen levels  Oxygen removes metabolic waste from the cell; without oxygen, waste is not removed and the cellular metabolism is slowed Prof. Kumari Rajani, DSST, BAU, Sabour
  31. • Light is another key germination factor; it can either stimulate or inhibit seed germination • Both light quality (light intensity) and quantity (duration of exposure) influence seed germination • Promotion of germination is generally through breaking the seed dormancy • Some crops have a requirement for light to assist seed germination (e.g.Tobacco, Lettuce, Petunia, Begonias, Impatiens) Light Prof. Kumari Rajani, DSST, BAU, Sabour
  32. Photoblastic: Seeds respond to light for germination Three categories of photoblastic seeds: (a) Positive photoblastic: Seeds that are stimulated to germinate by light Ex: Lettuce, Tobacco, Poa pratensis, Poa nemoralis, mistletoe, Petroselinum crispum (Parsley) etc. (a) Negative photoblastic: Seeds whose germination is inhibited by light Ex: Onion, Lily,Amaranthus, Nigella, etc. (a) Non-photoblastic: Seed which germinates in light as well as dark Prof. Kumari Rajani, DSST, BAU, Sabour
  33. Visible light radiation is required by seed for germination Maximum promotion of germination occurs at 660 to 670 nm with a peak at 670 nm (red area) since phytochrome has an absorption maximum at this wavelength Wavelengths >700 nm and <290 nm: inhibit germination Prof. Kumari Rajani, DSST, BAU, Sabour
  34.  Phytochrome is a plant pigment found in cytoplasm that senses the presence of red light  Phytochrome absorbs light in two inter-convertible forms 1. Phytochrome-red (Pr) is metabolically inactive & absorbs red light (660 nm) 2. Phytochrome-far red (Pfr) is metabolically active and gets transformed from Pr  The Pfr promotes germination and other phytochrome- controlled processes in plants  Pfr reverts back to Pr after absorbing far-red (730 nm) Photoreversible Germination Prof. Kumari Rajani, DSST, BAU, Sabour
  35. This reversible effect of red to far- red light was first reported in 1952 in lettuce and also shown by other plant spp. like tobacco, pepper grass, elm, birch etc Inactive form Active form The light intensity should be approx 750 to 1250 lux in seed germinator for light requiring seeds and 250 lux is sufficient for non-dormant seeds Prof. Kumari Rajani, DSST, BAU, Sabour
  36. This is determined by how the seed would naturally be sown  Small seeds must sprout on the surface of soil because they lack a suitable endosperm to supply the needed nutrients; these are typically aided by light exposure  Large seeds contain enough nutrition to grow underground when photosynthesis is not possible. These seeds are more likely to germinate in dark conditions Prof. Kumari Rajani, DSST, BAU, Sabour
  37. Temperature  A favorable temperature is necessary to allow for plant growth  Temperature not only affects the germination percentage but also the rate of germination  For every species of seed, there is an optimal temperature for germination; at that temperature, the maximum number of seeds will germinate and in less time than at any other temperature  The optimum temperature for most seeds is between 15°C and 30°C  Kharif crops: 25°C and Rabi Crops: 20°C  The maximum temperature for most species is between 35°C and 40°C Prof. Kumari Rajani, DSST, BAU, Sabour
  38.  At some point, the seed becomes sensitive to the presence of “trigger” agents  A “trigger” agent can be defined as a factor that elicits germination but whose continued presence is not required throughout germination  A “trigger” agent such as light or temperature alterations shift the balance of inhibitors to favor promoters such as gibberellins  In contrast, a “germination” agent is a factor that must be present throughout the germination process; an example is Gibberellic Acid Trigger and Germination Agents Prof. Kumari Rajani, DSST, BAU, Sabour
  39. Seed Germination Testing
  40. SeedTesting “Seed Germination is the emergence and development of the seedling to a stage where the aspect of its essential structures indicates whether or not it is able to develop further into a satisfactory plant under favorable conditions in the field” (ISTA, 2015) Seed Germination Physiologist “the emergence of radicle and plumule” Prof. Kumari Rajani, DSST, BAU, Sabour
  41. Germination paper/sand Wax or butter paper Petri plate Seed germination chamber Plastic boxes or tray or pots Rubber band Seed counting board Marking pencil/pen Materials Required for Germination Testing Prof. Kumari Rajani, DSST, BAU, Sabour
  42. Composition: the growing medium can be paper, pure sand or mixtures of organic compounds with added mineral particles Characteristics of germination paper  It should be porous in nature  It should have maximum water holding capacity to ensure continuous supply of water during the test period  Free from bacteria, dirt, fungi and toxic substances  Made out of 100% cellulose  pH should be 6-7.5  Paper should posses sufficient strength to the prevent penetration of root in to the paper  Paper size is 46 X 29 cm  It should have reasonable cost  Should not serve as suitable media for saprophytic Fungi Growing Media Prof. Kumari Rajani, DSST, BAU, Sabour
  43. a) Paper substrata  The paper substrata are used in the form of top of paper (TP) or between paper (BP) tests  In most of the laboratories, paper-towel method (Roll towel test) is most commonly used for medium sized and bold seeds  The paper substrata are not reusable b) Sand substrata  The sand substrata have advantage of being relatively less expensive and reusable  The results in sand media are more accurate and reproducible in comparison with 'roll towel‘ tests especially in case of seed lots that are aged or heavily treated with chemicals Prof. Kumari Rajani, DSST, BAU, Sabour
  44. Methods of seed germination using paper A. Top of paper (TP): the seeds are germinated on top of one or more layers of paper which are placed B. Between paper (BP): the seeds are germinated between two layers of paper C. Pleated paper (PP): the seeds are placed in a pleated paper strip with 50 pleats, usually two to a pleat Prof. Kumari Rajani, DSST, BAU, Sabour
  45. i)Top of sand (TS), Top of organic growing medium (TO): the seeds are pressed into the surface of the sand or the organic growing medium. ii) Sand (S), Organic growing medium (O): the seeds are planted on a level layer of moist sand or the organic growing medium and covered with 10–20 mm of uncompressed substrate, depending on the size of the seed Methods using sand or organic growing media Prof. Kumari Rajani, DSST, BAU, Sabour
  46. i) Top of paper covered with sand (TPS): the seeds are germinated on top of a moistened sheet of cellulose paper which is covered with a 2 cm layer of dry sand ii) Soil: Soil is generally not recommended as a primary growing medium However, it may be used as an alternative to organic growing media when seedlings show phytotoxic symptoms or if evaluation of seedlings is in doubt on paper or sand Methods using a combination of paper and sand Prof. Kumari Rajani, DSST, BAU, Sabour
  47. The accuracy and reproducibility of the germinator result are very much dependent on the quality of the substrata (paper and sand) used for germination testing The germination substrata must meet the following basic requirements:  It should be non-toxic to the germinating seedlings  It should be free from mould sand other microorganisms  It should provide adequate aeration and moisture to the germinating seeds  It should be easy to handle and use  It should make good contrast for judging the seedlings  It should be less expensive Prof. Kumari Rajani, DSST, BAU, Sabour
  48. Important facts to be remembered… pH: the growing medium must have a pH value within the range 6.0–7.5 when checked in the substrate Conductivity: the salinity must be as low as possible and no more than 40 millisiemens per metre Measurements of conductivity can be replaced by biological tests Cleanness and freedom from toxicity: the growing medium must be free from seeds, fungi, bacteria or toxic substances, which may interfere with the germination of seeds or the growth or evaluation of seedlings Seed sample: 400 seeds are used for germination testing Seedlings evaluation: is done on two days: (Different for various crops) 1. First Count 2. Second or Final Count Prof. Kumari Rajani, DSST, BAU, Sabour
  49. Re-use of substrates: it is strongly recommended that the growing medium is only used once Counting boards: Counting boards are often used for large seeds such as Zea, Phaseolus and Pisum Vacuum counters: Vacuum counters can in principle be used for all species, but are mostly used for species with regularly shaped and relatively smooth seeds such as cereals or species of Brassica or Trifolium Prof. Kumari Rajani, DSST, BAU, Sabour
  50. Procedures of Routine method (Between Paper)
  51. Place 100 seeds on soaked paper at equal distance in 8 rows (12 seeds: 1, 3, 5, 7 13 seeds: 2, 4, 6, 8) Place another soaked paper Roll in wax paper Prof. Kumari Rajani, DSST, BAU, Sabour
  52.  Normal Seedlings: Seedlings that possess essential structures that is indicative of their ability to produce useful mature plants under favorable field conditions  Abnormal Seedlings: Seedlings that exhibit some form of growth but have insufficient plant structures to maintain a healthy plant, such as missing roots or shoots  Fresh Seeds: Seeds that have failed to germinate but have imbibed water. They appear firm, fresh and capable of germination, but remain dormant  Dormant Seeds: Viable seeds (other than hard seeds) that fail to germinate when given the prescribed or recommended germination conditions  Hard Seeds: Seeds that remains hard at the end of the prescribed test period, because their seed coats are impermeable to water  Dead Seeds: Seeds that cannot produce any part of a seedling Evaluation of germination test Prof. Kumari Rajani, DSST, BAU, Sabour
  53. Evaluation of seedlings Normal Seedlings Seedlings with all essentials structures, well developed, proportionate root and shoot, healthy AS Prof. Kumari Rajani, DSST, BAU, Sabour
  54. NS Abnormal Seedlings Damaged Seedlings: missing essential structures Deformed Seedlings: unbalanced development Decayed Seedlings: diseased Prof. Kumari Rajani, DSST, BAU, Sabour
  55. Dead Seeds Hard Seed Prof. Kumari Rajani, DSST, BAU, Sabour
  56. Replication (100 seeds) No. of Normal Seedlings No. of Abnormal Seedlings Ungerminated Seed Germination (%) No. of Hard Seeds No. of Fresh Seeds No. of Dead Seeds R1 89 4 3 0 4 92 (89+3+0) R2 92 2 1 2 3 95 (92+1+2) R3 90 3 2 2 3 94 (90+2+2) R4 87 3 5 0 5 92 (87+5+0) Average % 89.5 3 2.75 1 3.75 93.25 Calculations and Reporting of Results The results of the germination test are reported as percentage of normal seedlings, abnormal seedling, hard seeds, fresh seeds and dead seeds The sum of the normal, abnormal and ungerminated seeds must be 100 (90+3+3+1+3) Germination percentage is calculated based on number of normal seedling The percentage are rounded to the nearest whole number Normal seedlings + Hard seeds + Fresh seeds Germination (%) = X 100 Normal seedlings + Abnormal Seedlings + Hard + Fresh + Dead Seeds Prof. Kumari Rajani, DSST, BAU, Sabour
  57. Minimum Seed Certification Standard for Seed Germination Percentage Recommended in Field Crops Field Crops Foundation and Certified Maize Hybrid (Sweet Corn Hybrid, Synthetic, Composite, OPV) 90 Barley, Wheat, Triticale, Bengal gram, Rapeseed, Mustard 85 Paddy, Maize (Inbred lines, Single cross FS), Horse gram 80 Sorghum, Pearl millet, Minor millets, Black gram, Cowpea, Green gram, Indian bean, Lathyrus, Lentil, Moth Bean, Pea, Rajmash 75 Castor, Groundnut, 70 Prof. Kumari Rajani, DSST, BAU, Sabour