FACTORS CAUSING VARIETAL DIFFERENCES IN GEHMINATION OF THE COMMON BEAN (PHASEOLUS VULGARIS) By Gordon Cecil DaCosta A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture Year 19^2 ACKNOWL EDGMENT I wish to express my indebtedness and sincere appreciation to D r s . E. H. Lucas and Ft. L. Carolus for their guidance and unfailing help throughout the course of this investigation. My appreciation is also due the other members of my guidance committee as well as Drs* S. T. Dexter and D, P. Watson for thier advice and constructive cri tici sm. To the Ferry-Morse Seed Co,, Associated Seed Growers Inc,, Cornell Seed Co*, and W, Atlee Burpee Seed Co. my grateful acknowledgment for supplying seed samples used in the experiments* TABLE OP CONTENTS Pag# INTRODUCTION ............................................... 1 REVIEW OP LITERATURE . . ................................ . 3 EXPERIMENTAL P A R T ............................................. 15 Moisture Absorption Capacity of Six Varieties of Beans 15 The Effect of Temperature and Period of Soaking Been Seed as a Factor Influencing Germination • • • 21 The Influence of Variety, Temperature and Period of Cold Storage on Germination. « • • • • . • • • • 26 The Effect of the Period of Illumination on the Germination of a Black and a White Wax Bean • • • • 29 Chemical Analysis. • • • • • • • • • 32 An Anatomical Study of the Seed-coats of Beans . . . . 36 A Study of Mechanical Injury • • • • • • • • • • • • . \\Z The Effect of Varying Relative Humidity on Mechanical Injury and Germination • • • • • • • • • 1*8 The Effect of Mechanical Injury on Field Stands of Six Varieties of Beans . • • • • • • • • • • . . 51 A Study of Possible Interference with Germination by Microorganisms . . . • • • • . • • • • • • • • » Sk- Antibiotics. • • • • • • • • • • • • • • • • • • • • • 56 Germination Inhibitors • • • • • • • • • . . . • • • • 56 The Effect of Artificial Inoculation with Bacteria on the Germination of Beans • • • • • • . • • • • • 59 . . . ........... GENERAL DISCUSSION ........................................ 63 SUMMARY........................................................ 70 BIBLIOGRAPHY ............................................... 73 LIST OF TABLES Weights of Six Varieties of Beans Soaked for Six Days at 1(4° ar*d 77° F. 18 Germination of Two Varieties of Beans Subjected to Twenty-four Hours of Soaking at Three ......... ................. .. Temperatures 22 Emergence of Four Varieties of Beans Subjected to Twelve Hours of Soaking at Three Temperatures • • • • • • • • • • • • • • • • • 2i+ Germination of Beans Soaked for 3, 6, 12 and 24 Hours at Room Temperature 78° F, • • • • • « • 25 Emergence of Ten Varieties of Beans Subjected to Gold Treatment Prior to Germination • • « • 28 Percentage Germination of Two Varieties of Wax Beans under Different Periods of Illumination, 31 Pentosan Determinations In Six Varieties of Beans ............. ............... 36 Seea-ccat Measurements of Seven Varieties of Beans • • • • • • • • • • • • • • • • • • • 40 Basic Injury Counts in Six Varieties of Commercial Bean Seed • » • • • • • • • • • • • 44 percentage Germination and Abnormal Seedlings In Six Commercial Varieties of Beans • • • • • 47 Emergence of Five Varieties of Beans Subjected to Three Levels of Relative Humidity and Subsequent Mechanical Injury • • • • • • • • « 50 Influence of Selection on Emergence of Field Planted Beans........... .. 52 Percentage Germination and Contamination in Six Varieties of Beans Artificially Inoculated with Bacteria « • • • • • • • • • • 6l LIST OF FIGURLS Figure Page 1 Moisture Absorption at 41*.° F. • • • • • • • • • • 19 2 Moisture Absorption at 77° F. . . . . . . . . . . 20 Standard. Log Curve for Xylose • • • • • • • • . . 31+ 3 4 pentosan Content of Six Varieties 5 Seed-coat Structure ofBrittle Wax Bean • • • • • 38 6 Seed-coat Structure • • * 30 7 Seed-coat Structure of Rival Bean • • • • • • • • 39 9 Seed-coat Structure of Lazy Wife Bean • • • • • ♦ 39 of of Beans, Pencil Pod Wax Bean* 9 Basic Injury In Commercial Bean Seed. 10 Field Stand of Pencil Pod Wax Bean. 11 Field Stand of Brittle • • . 35 . . . . . . • • • • • • • Wax Bean . . . . , I4.5 53 , . . . 53 INTRODUCTION The problem of growing beans successfully has engaged * the attention of both growers and research workers for a considerable period of time* Germination studies In relation to different environmental conditions have been conducted but little Is known about the physiological conditions, existing In the seed, during maturation, dormancy and germination, which might Influence the sub­ sequent growth and development periods of the plant* During recent years the production areas have changed, from the humid eastern, to the seml-arid western states* This relocation was necessitated to combat the seed-borne diseases prevalent In humid areas and for greater acreage (3)* At the same time It has increased the interest of the bean growers fer the g e m i n a t i o n qualities ef the seed* It has been pointed out by Morris (i^l) that seme black seed-ceated varieties have a much higher percentage of germination In comparison to some light or white colored varieties* Because of the possible value to the bean growers and seed producers, a study ef the factors Influencing these differences in germination was undertaken* Environ­ mental factors, disease, soil organisms, mechanical injury Incurred in threshing and other possible causes, which might contribute towards this marked difference in germin­ ation, were investigated* Such a study may further indicate whether better germination ef dark seed-coated varieties Is associated with color or structure of the seed coat, or some anti­ microbial agents which might be present in such seeds* REVIEW OP LITERATURE Several prerequisites Tor the germination ef seeds ef all species ef plants are (1) water, temperature, and (3) oxygen. (2) a suitable A fourth footer, light, appears to influence the germination ef seeds ef seme species and te be essential fer the germination ef the seeds of a few species* Other factors such as the size ef seed, aeration, chemical composition, structure ef the seed-ceat and growth Inhibitors in the seeds hare been found to influence germination. The literature on these subjects has been reviewed in this paper. Absorption ef water is a primary and essential step in the germination ef a seed. The water passes through a protective seed-ceat te reach the embrye and endosperm and Initiates a series ef physloal and chemical processes which in the absence of any limiting faster results in the emergence ef the embrye from the seed, Stiles (57) compared the rate and amount ef water intake in several species ef beans and concluded that soybeans are xerlc (dry land) types and lima beans are hydrle (wet land) types. She alse compared the rate ef intake ef water into various parts ef cotton and corn seed (58), and pointed out that different varieties had dis­ tinctly different characteristics that had been breught k about by selection, either natural or deliberate* Corn with a rapid rate of Intake of water waa bettor adapted aa a dry land type than ether varieties having a less rapid rate of absorption* A wide range ef vegetable apeclea waa teated by Doneen and MaeGilllvray (19) in two types of soils; they found that the rate ef germination Increases and reaches Ita maximum at higher molature levels* Fuhr (22) working on the molature requirement of various crops reports that beans require about one and one-half percent additional molature above the permanent wilting percentage of the seedlings for development* Toole, Miles and Toele (61) used varying amounts of moisture in their germination experiments, and report that germination was significantly poorer at 60 percent moisture than at lower moisture levels* Whetzel (67) found that in saturated soils there was a greater amount of infection by damplng-off organisms than In unsaturated soils* Kidd and West (32, 33) found that soaking seeds of Phaseolus vulgaris (dwarf bean) prior to germination had a marked effect upon the subsequent course and vigor of development* The striking effects of soaking the seed are net visible In first observations, but show up later in the course of development* They found no decrease in the rate ef germination in seeds soaked for six hours, little decrease from soaking for twenty-four hours, but complete failure from treatment for seventytwo hours* Hewever, a deleterious effect of temperature 5 was visible even after shortest periods of soaking (6 hours) and the effect progressively increased with longer periods of soaking at all temperatures used, ranging from 10 to 30° C. Results obtained with Broad beans Vlcla Faba gave an absolute reversal in the effects following soaking compared to the dwarf beans* Soaking for periods up to three days gave increasingly beneficial results, not only In percentage g e m i n a t i o n , but also in the size of the plant produced* They concluded that the effect of soaking is strongly specific; quite different with similar treat* ment in closely allied species of plants* Tllford, Abel and Hibbard (60) report that soaked bean seeds that were sterilized and well aerated during soaking, germinate well under water; provided that there is sufficient oxygen for respiration, that carbon dioxide and other by-products are removed end bacteria eliminated* Bailey (5) found a decrease in germination after eight hours of soaking with aeration, and it progressively decreased as the length of soaking period increased* These results do not agree with the previous authors mentioned because they did not change the water in which the beans were soaked and also did not grow them in soil. Bailey (5) further pointed out the fact that there was an increase in the rate of production of respiratory carbon dioxide in sprouted seed that had been soaked in comparison with those sprouted without previous soaking. This is due to 6 the fact that soaked seeds contained more water than the untreated ones* This higher water content in the seed was favorable to a more rapid rate of respiration* Soaking seeds without aeration resulted In a regular decrease in catalase activity* Soaking with aeration resulted at first in a small decrease in catalase activity, but this was soon followed by a considerable increase* This modification ef the result is evidently due to the fact that the seeds when soaked with aeration pass through the early stages of germination and consequently there Is an increase in catalase activity -- characteristic of seed germination* Aeration of the soil has an Important Influence on germination and is closely related to the soil texture, moisture content of the soil, and the oxygen requirement for germination for different kinds of plants* Crocker (15) reports that seed-eoats which exclude oxygen caused a delay in germination* In nature delayed growth was brought about by the disintegration of the seed-coat structures by a longer or shorter exposure to germinative conditions of temperature and moisture* The length of delay depends on the persistence of the structure of the seed-coat* A rise in temperature lowered the minimum oxygen required for Xanthlum seeds to germinate, and Shull(53) suggested that this might be due to the increase in anaerobic respiration at higher temperatures* The temperature requirements for optimum growth maximum germination are better understood than any ether factor In the process of germination* Sub-optimum temper­ atures during the germinating period are common causes for poor stands* Planting of spinach or beet seed in soil at a high temperature will result in a low emergence due to the much more favorable conditions for the pathogens, primarily Rhlzoctonla sp*, which causes pro-emergence damping-off in vegetable seedlings (67)* Kotowskl (3*4-) has pointed out that the optimum tem­ perature for germination was in the range ef 70 to 80° P# He also pointed out the fact that the rate of production of seedlings at different levels of temperature was In accordance with van*t Hoff's law* Harrington (27) using alternating temperatures on vegetable seed found that they germinated well* The temperature changes giving best germ­ ination results* with a large number of kinds of seed* correspond rather closely with soil temperatures In the field under conditions which Induce the most prompt and vigorous production of seedlings* Field tests suggest the use ef an alternation between 18° and 32° C for optimum germination* The minimum number of days required for pro­ ducing bean seedlings in the soil was four days; and the first seedlings are produced in the smallest number ef days in moderately warm weather in the months of M a y and June* Reynolds (*4.7 ) indicated that beans require a warm sell their best development* In a cold soli* presumably* bean plants would not have as great vitality and might 8 prove particularly susceptible to species of Fuaarlum, According to Morris (41), bean varieties vary in their ability to withstand low temperature. In some varieties this was partly inherent* while in all varieties tested it was related to a reduction in their ability to withstand the attack of soil pathogens. Gilman (23) observed that at higher temperatures the plants may possess a lower degree of vitality and hence should be more susceptible to facul­ tative parasites. Axentjev (if) found that the light inhibiting effect on germination of a large variety of soeds was entirely dependent on the presence and integrity of the seed-coats. The coats evidently act by restricting the oxygon avallablo to the embryos. In some cases* light apparently Interferes with oxidation processes within the seed* while in others it favors these processes. Rudolfs (49) studied the rate of growth of bean seeds from large and small seeds in the dark* and concluded that large seeds gave by far the better growth. Seed—coats differ in their structure from seed to seed, and may also differ in the same variety of seed (45) J and some of these differences result in "hard" seeds. Barton (6) describes these modifications in the legumes as a cutlnlzatlon of the ends of the palisade cells or the formation ef a water impermeable cuticle. Gloyer (24) and Lebedeff (37) point out that the tendency to develop "hard" seeds in field beans is a genetic character* but 9 the degree to which this Is developed Is a function ef envlrenment* Swanson and Hunter (59) correlated field stands of Sorghum with the thickness ef the starchy mesocarp layer, and pointed out that soft grain types with a thick meso­ carp averaged thirty-nine percent field stands, whereas Sorghum types with a thin mesocarp averaged fifty-seven percent* They state that water intake was one-third greater in the soft grain types but molds and rots accounted for the decreased stands* Watson*s (66) werk on the structure of the testa in relation to germination in several species of Paplllonaoiae tribes Indicates that there is much variatlen in the structure and chemical nature ef the seedcoats of different species* The amount ef suberlzed and outicularized sub-outlcular and Malpighian thickening found in individual species of the group seems to justify a high general rate ef impermeability and difference in germination* Ott and Ball(43) working with Red Kidney beans suggest that there is an indication that polyuronides and "true pentosans" are Involved in water retention in the dried seed-ooats* Schulze and Godot (50) point out that seed-oeats contain a large percentage ef hemicellulose, also some brown unrecognizable substances which they termed "Holzgumral"* Mechanical injury in seed beans has attracted considerable attention of research workers in recent years* 10 Asgrew* (3) research workers began an Intensive research program In 1940 to determine the causes and means of prevention of mechanical damage to bean seed* Injury is brought about by mechanical harvesting, threshing, pro­ cessing and undue handling or improper storage* The Aagrow workers have divided seed Injury inte two categories: (1) visible or external Injury, and (2) internal injury to the embryo, detectable enly by a viability test* The first type of injury ranges from slightly cracked seedcoats, hardly discernible to the naked eye, te severely cracked er chipped seed* Chipping and cracking can eocur anywhere on the seed-coat, but are more commonly found on the ends* A sample of seed which at first appears to be of normal viability reveals upon closer examination these external injuries which are indicative ef broken embryos and consequent lowering of germination* Internal injuries give rise to abnormal seedlings which are consequently weak and, if lucky te survive, produce a poor plant of little er no value te the grower (3)* Goss (25) indicates that the poor germination of Fordhook lima beams la due to cracking Injury of the seed-coat* Fungus pathogens are more apt to cause infection of the seeds and cause a lower percentage of germination* According to Morris (41), under optimum conditions for germination and with a "»•* of mechanical Injury, there Is no significant varietal oAsgrow - abbreviation for Associated Seed Growers Inc* This abbreviation will be used throughout the paper* ■?-u 11 difference In germination* Low germination ef some lima varieties he attributes to mechanically injured seed obtained from some sources* Virgin (62) has reported that baoterla may be found underneath the seed-coats ef large wrinkled type peas* finding entrance during threshing through cracks in the seed—ceats* Morris (41) further points out that seed-ceat color in certain varieties is associated with the ability te withstand sell infection* This resistance is considerably greater in dark seeds than those with white or light colored seeds* Soil pathogens take a big toll of seeds and young seedlings* Among the most important soil pathogens causing pre-emergence damping-off are Rhlzoctonia solanl and Pythlum debaryanum. Excessive soil moisture* poor aeration and favorable temperatures are ideal conditions for development of soil pathogens* especially fungi which cause damping-off• Burkholder (10) found that the bean plant appears te be little affected by the pH of the soil and thrives well I** aoid or alkaline soil* Severe infection by Pusarl«"» martli may be produced in a soil of fairly low pH, 5*0, and in an alkaline soil of pH 0*0 as well* Generally speaking, fungi thrive in a fairly aoid soil, while bacterial path­ ogens favor a neutral or slightly alkaline medium. There ar® a large number of microorganisms present in the soil, and most ef them are saprophytes er faoultatlve parasites 12 causing secondary infection# Fawcett (21) points eut that mixtures of pathogenic organisms, i#e#, the presence ©f two or more of them, cause the greatest amount of damage in seeds and seedlings* Likewise, according to Christensen (II4-) association of microorganisms are the cause of seedling Injury especially when the seeds are infected* When the seeds are planted in the soil, the seed-borne pathogens first cause infection and later on secondary organisms come in and oause death ef the seedlings* Walker (6 3 ) states that no effective seed treatment has been perfected for seed-borne diseases on beans, especially bacterial pathogens# Arason and Spergon have proved beneficial on some varieties of beans and other vegetable seeds* McGuffy (39) reports that lima beans treated with a fungicide failed to germinate# This he attributed te injury reoeived during the treatment* Hay (28) found that Infection by the common soil fungi and bacteria lead to decreased germination and pointed out the fact that chemical seed treatment with the fungicide Uspulun tends te reduce the amount of injury both under laboratory and field conditions* Morris (IjJ.) ooncluded from his experiments that light colored or white bean seed planted in soils are significantly benefited by treatment with Spergon* Evanarl (20), in his review on "germination inhibitors", 13 states that many plants produce substances which Inhibit er delay the germination of seeds of the same or different species* Xt is doubtful whether germination Itself or merely the first phase of subsequent growth is inhibited* Therefore* he defines them as substances which* from observation* have been found te Inhibit germination* Germination and growth inhibition are nearly always associ­ ated with one another (20)* When seeds in different stages ef germination were transferred from water to tomato juice* their growth was inhibited* Many authors (20) have found auxins in seed and some have shown that the auxin content of seed decreases germin­ ation* If the auxin in seeds were to function as a "germination hormone"* the action ef inhibitors could be explained by auxin destruction or inactivation* Evsnarl (20) doubts whether auxins can be considered germination hormones* Siegel (54)* using Red Kidney beans* obtained extracts which showed an inhibitory effect on the germination of oat seed. Barton and Solt (7) found inhibitors of both ger­ mination and enzyme activity in a number of seeds* Using water extracts ef the seed—ooats* cotyledons and embryos of Phaseolus vulgaris* they found that it inhibited root growth ef wheat seedlings* The presence ef antibiotics in seeds has been re­ ported* It is presumed that these substances provide them with a measure of protection against plant pathogens* li* In an Intensive study of resistant and susceptible strains of* black mustard and turnip, Stahmann et al (56) refute the olaims that allyl isothiocyanate (mustard oil) in black mustard seed was responsible for resistance against the clubroet organism. Quantitative estimation of the amount ef mustard oil in the roots ef these plants failed to show a correlation of 1 sothiocyanate content to resistance or susceptibility to clubroot* Lucas et al (33) in their search for antibiotic substances have found that extracts from seme varieties of Brasslca showed a marked degree of inhibitory action against many bacteria and some fungi* Berrlss (9) ooncludes in a discussion on germination by saying "that the germination promoting effect ef the soil is due not to the presence ef any stimulating factor, but rather te the removal of an inhibiting substance from the seeds by the absorptive power of the soil complex"• EXPERIMENTAL PART Commercial stock of beans of several varieties were obtained from seed companies, which, will be referred to as A, B, C, and D, whenever the source of the seed is mentioned# Incidentally, some of the varieties were the same as those used by Morris (1±1) In his Investigation on the factors influencing the germination of snap, and lima beans in 19^9* Of the ten varieties selected six, namely Lazy Wife, Dixie White, Brittle Wax, Black Valen­ tine, Plentiful and Pencil Pod Wax, were further selected for more detailed study# The seed when received from the seed companies had been treated with a fungicide for protection against soil pathogens. Since untreated seeds were to be used in the investigation, the treated seeds were washed with a detergent solution, rinsed with distilled water, and quickly dried on cheesecloth. This washed seed was used throughout the Investigation unless specified. Experiments were carried out In the laboratory, greenhouse, and in the field* Moisture Absorption Capacity of Six Varieties of Beans Absorption of water Is a primary and Initial step in the germination process of a seed# In order for the 16 moisture to reach the dormant embryo* It has to pass through the protective layers of the seed-coat* Barton (6), Gloyer (2ii)* Lebedeff (37)# and Watson (66) who have made a study of the seed-ooats of leguminous seeds point out that there Is a difference in the structure of the seedcoats of different varieties* which accounts for their differences In moisture absorption and subsequent germin­ ation* Stiles (57# 58) working with corn and cotton* studied the rate ef water Intake into various parts of the seed* and pointed out that various varieties had distinctly different oharaeteristies in relation to affinity for moisture absorption* She also compared the rate of amount of water Intake in several varieties of beans* and classified soybeans as xerlc types* and lima beans as hydric types* This experiment attempts to make an examination ef the moisture absorption capacity of six varieties of garden beans in order to determine whether there is any difference between dark and light varieties* Procedure, Six varieties ef beans* three black- seeded and three white— seeded* were selected for the experiment* Duplicate samples of each variety were tested for their moisture content* Average readings wore taken of the moisture content of tho sample* by means of a Steinlite Moisture Tester* 17 Two sets of approximately equal numbers of uniform seeds of each, variety were selected and weighed in a * beaker. Equal quantities of water were added to each beaker; one group was held at room temperature 77° F» the other in the refrigerator l^0 P. After a period of twenty-four hours had elapsed, the seeds were quickly taken out , dried on cheesecloth to remove excess moisture, and weighed. Increase over the original weight was assumed to express the amount of water absorbed in a twenty-four hour period. This procedure was repeated every twenty-four hours for a period of six days, and the total Increase over the original weight was calculated. Results of water absorption are given in Table 1 and Figures I and XI. The initial level of moisture oontent was higher in all black varieties than in all the white varieties. Results and discussion. Results indicate that there is a significant difference In the amount ef water absorbed by different varieties. Black Valentine absorbed the largest amount of water in the first 2i± hours, both at 77° and i|4° F* followed by Dixie White, Pencil Pod Wax, Brittle Wax, Plentiful and Lazy Wife. The variety Lazy Wife is inconsistent because of the large percentage ef "hard" seeds. Black Valentine and Dixie White reached their peak ef absorption on the fourth day. At a temper­ ature of 77° F# the black varieties absorbed a larger TABLE 1 WEIGHTS OP SIX VARIETIES OP BEANS SOAKED FOR SIX DAYS AT Air-dry weight (grams) moiB&urv content (percent) (a) (b) 19.0 19.0 7.75 Black Valentine (a) (b) 15.5 15.0 Pencil Pod Wax (a) (b) 16,0 17.0 (a) (b) Dixie White Lazy Wife Variety Black Plentiful White brittle Wax P (a ) AND 77* F (b) Weight In grams after soaking for 1 day 2 days 3 days 1+ days 5 days 6 da; 22.5 24.5 28.0 32.5 25.5 35.5 28.5 38.5 29.5 39.5 30.5 1+0.5 25.9 29.9 30.5 32.0 31.5 32.5 32.0 33.0 32.0 33.0 31.5 32.5 7.75 19.3 21.5 28.5 32.5 26.5 35.5 29.5 37.0 30.1 38.0 31.3 38.5 16,8 16.5 1+.75 20.5 23.5 27.0 31.5 25.2 33.5 27.0 31+.5 28.5 35.0 29.5 35.5 (a) (b) 9.9 10,0 5.35 14.5 16.0 18.5 19.0 16.1 19.0 164 20.0 16.5 20.0 16.5 20.0 (a) (b) 22.0 22.0 5.35 23.8 27.0 32.1 34.1 27.5 35.1 28.0 36.5 28.5 37.0 28.7 37.5 10.1 1BLACK VALENTINE 2 PENCiL P O O W A X PERCENT INCREASE OVER DRY W- 3 PLENT/FVL \ br/ ttle M X 5 DIXIE WHITE 6 LAZY W/PE % $ o / Figure I* 3 S DAYS MOISTURE ABSORPTION AT 4-4- C / P E N C I L P O O WAX 2 BLACK VALENTINE 3 PLENTIFUL 4 B R I T T L E WAX 5 DIXIE WHITE - PERCENT 3 5 \ INCREASE OVER DRY WT 6 L A Z Y WIFE s / Figure II. D A YS M O /S T O R E A B S O R P T /O N A T 7T °C 21 amount of water in comparison with the white varieties at 1^° P. The amount of water absorbed at 77° F is significantly greater than at i+ij.® P in all the varieties* Nevertheless* the results were considered inconclusive* Fermentation set in sooner in the white than in the blaek varieties* If such were true in the soil it might explain deterioration and consequently poor germination ef the white varieties. Similar and confirming results were obtained in experiments conducted to show the effect of soaking on germination and presented subsequently* .e trrect or Temperature ana Period or soak ing Bean Seed as a Factor Influencing termination In the case of phaseolus vulgarla * the common bean* soaking seeds prior to germination has a marked effect upon the subsequent course and vigor of development. Kidd and West (32* 33) observed that soaking of seeds for only eight hours reduced the total growth considerably as compared to that of seeds sewn dry,on damp sand. This was true in spite of the fact that those whloh had been soaked had a considerable start in germination and sprouted earlier* They further pointed out that the period of soaking bears no relation to the percentage germinated* Bailey (5 )* on the other hand* found a decrease in germin­ ation after eight hours of soaking* and it progressively decreased as the length of the soaking period Increased* The object of the present experiments was to examine the effect ef temperature and pe rlod of soaking on the 22 germination of some varieties of beans* Effect of Soaking for 12 and 24 Hours at 3 Different Temperatures on Germination Procedure * Pour varieties of beans. Pencil Pod Wax* Black Valentine, Brittle Wax, and Dixie White were selected for this experiment* A preliminary experiment with one black variety, Pencil Pod Wax, and one white variety, Brittle Wax, was set up in the laboratory. Fifty seeds of each variety were soaked in distilled water at three different temperatures - 44° » 02°, and 96° F; dry seed was used as a control at 02° F* After a period of twenty- four hours the seeds were removed from the water and placed on filter paper, In Petri dishes* Germination counts were made at Intervals of three and six days* Results ef germination are given in Table 2* TABLE 2 GERMINATION OF TWO VARIETIES OF BEANS SUBJECTED TO TWENTY-FOUR HOURS OF SOAKING AT THREE TEMPERATURES Fifty seeds of each group were treated Variety Black Pencil Ped Wax White Brittle Wax Period of observation Control 02° F Number germinated after soaking 214. hours at W* F 02° F 96° F 3 days 48 15 48 20 6 days 50 43 50 41 3 days 40 0* 9 10* 6 days 48 13* 9* 12* ^Contaminated by growth of microorganisms Results. Germination results Indicate a clear-cut difference between tbe black and white varieties. A del­ eterious effect of soaking was very noticeable in the white variety, whereas the black one was little affected. Brittle Wax was Injured at all temperatures. Germination was considerably slewed down in both varieties at very low and high temperatures. These results are in agreement with those ef Bailey (£) who worked with Early Valentine beans. Effect of Soaking for 12 Hours at 3 Temperatures on the Emergence of Beans Planted in Soil Procedure. Four sets of 100 seeds of each of the four varieties of beans were soaked in distilled water at three temperatures - l|.0o , 5f>°, and 73° F , with one set of dry seed as a control. Temperatures at which the beans were soaked had been selected on the basis of soil tem­ perature levels possible under field conditions during the growing period. After a period of twelve hours, the seeds were taken out of the controlled temperature cham­ bers and planted in flats in the 60° F greenhouse. Emergence counts were made after a period ef one week. Results and discussion. Results (Table 3) indicate that soaking for twelve hours had an injurious effect en all the varieties tested; however, two black varieties were much less injured than the two white varieties. effect of temperature on soaking did not indicate any The TABLE 3 EMERGENCE OP FOTJR VARIETIES OP BEANS SUBJECTED TO TWELVE HOURS OP SOAKING AT THREE TEMPERATURES* Percent emergence after soaking 12 hours at 55° P 73° P Control Black 61a!ok Valentine 100 78 76 72 92 79 60 79 White ""Elxie White 88 3 % 1 Brittle Wax 96 20 10 27 significant differences among the varieties. It would Pencil Pod Wax 0 o -d Variety eSeeds planted in soil appear that the period of soaking is more important than the temperature at which the seeds were soaked. The white varieties again responded to the treatment by a far greater decrease in germination than the black ones* Effect of Soaking at Room Temperature for 3* 6 , 12, and 21± Hours Respectively on the G e m i n a t i o n ef Bean Seeds Procedure* Pour sets of 100 seeds each, of twelve varieties, were soaked in distilled water at room temper­ ature <70® P) for 3» 6 , 12, and 2I4. hours respectively. The seeds were removed after the above mentioned periods of soaking and placed in moist chambers. control. Dry seed was used as The amount of germination was noted; the data are expressed as percentages. Results of germination are given in Table 4* 25 TABLE 4 GERMINATION OF BEANS SOAKED FOR 3* 6 . 12, AND 21* HOURS AT ROOM TEMPERATURE 78° F Variety Percent germination after soaking for Control 3 hrs • 6 hrs • 12 hrs* 96 98 99 96 85 100 100 100 100 99 86 86 80 82 71 96 81* 68 1*8 18 Rival 96 61* 72 60 12 Bountiful 9k 61* 76 60 1*8 Landreths* Stringless 96 92 86 88 90 Full Measure 98 96 96 76 70 90 96 82 66 1*9 Lazy Wife* 1*8 16 28 28 32 Dixie White 89 1*8 32 21* 17 Mlchellte 96 96 68 52 6 Blaok pencil Ped Wax Black Valentine Plentiful Brown fop Crop White Brittle W ax 21* hrs* *Re suits ef germination In this variety aro inoonalatant because or the large pereentage of "hard." seeds* All results are the average of 3 experiments performed consecutively* He standard deviation was neoessary as the results being close, the simple arithmetic mean served as an average* Results and disoussion* The deleterious effect ef soaking is clearly evident In this experiment* Soaking periods affected germination ef different varieties to 26 varying degrees. As previously noted the black varieties germinated better than the white varieties. varieties were intermediate. The brown Besides the difference in germination another fact became evident in this experiment that is, the black beans were not contaminated by micro­ organisms whereas the v&ite beans had a high percentage of affected seeds even after only 6 hours ef soaking* This effect of soaking may be compared to that of a saturating rainfall immediately after planting. The saturated condition of the soil may have the same effect as the immersion in water* The Influence of Variety. Temperature and POI*l°d of Cold Storage on Germination The investigation was undertaken on the basis of results of Morris (ip.)» *s reported in the review of liter­ ature* Seeds of ten varieties of beans were selected at random and planted in nine-inch pots in August, 1950* The experiment was arranged as a two-way classification design with three periods - three, six and nine days - at ifO° F in cold storage, and a control. After planting, the pots were watered and covered with wax paper to prevent evap­ oration and drying out of the soil* in the cold storage at l^O0 P* outdoors. The pots were placed Ten control pets were placed Ten pots,of each ef the ten varieties were removed from the cold storage at Intervals ef three, six and nine days respectively and placed outdoors* Prom then 27 on normal watering was done and emergence counts made at regular intervals. Outdoor night temperature during the month of August averaged 67° P» Results of germination and emergence are given in Table 5* Results and discussion. The results show a signif­ icant difference in the emergence of the ten varieties tested and the effect of the length of the period of cold treatment on germination and emergence. These results are in agreement with those reported by Morris (i+1) that "Varieties vary in their ability to withstand low temperatures, due to a reduction in their ability to combat infection by soil pathogens," Seeds that failed to germinate were found on examination to be infected by microorganisms. The organisms were isolated in pure culture and identified, Seed-coat color seemed to be associated with resistance to infection at low temperatures. There is little varietal difference in emergence in the untreated controls, except in the variety Brittle W a x which had many "hard" seeds* Reynolds (Aj-7) pointed out the fact that beans require a warm soil for their best development. In a cold soil presumably bean plants would not have as great a vitality and might prove susceptible to Fusarlum sp. Two different species of Fusarlum were Isolated from the beans which failed to germinate. Aspects of fungus infection are 28 TABLE 5 EMERGENCE OP. TEN VARIETIES OP BEANS SUBJECTED TO COLD TREATMENT PRIOR TO GERMINATION* Variety Number emerged after days In cold storag e 6 Average 9 3 20 18 20 5 15.75 Pencil Pod Wax 20 20 11 13 16.00 Black Valentine 19 16 15 1 12.75 19.6 18.0 15.3 6.3 11*.80 19 15 18 16 O . tH Black Plentiful Control Top Crop 19 16 1*4- 1 12.5 Lows* Champion 16 16 10 1 10.75 Red Valentine 18 12 11 2 10.75 17.5 1^.7 13.2 5 12.75 Average Brown tandreths' Stringless Average White Lazy Wife 18 6 k k 8.0 Dixie White 20 3 15 3 10.25 Brittle Wax 6 3 0 0 .2.25 6.3 2.3 11.8 ^•6 Average 11+.6 k General average 17.5 12*5 6.80 #Twenty seeds planted per pot. Least significant difference fer varieties - 5% » 5.95 1% * 8.03 Least significant difference for treatments- 5% = 3.7$ 1% = 5.07 29 discussed in a later section of this paper. The period of cold treatment had a decided effect on the germination of all varieties. As the period of cold treatment increased, the germinating capacity of all the varieties progressively decreased* The longer the beans were in the cold soil, the greater were the chances of infection (ifl)* Leach (35>) compared the growth rates of host and pathogen for several crops and found that the relative rates of growth deter­ mine, to a considerable degree, the severity of pre-emer­ gence infection at different temperatures. Seed treatment with fungicides provides a measure of protection against soil pathogens. The degree of protection afforded by fungicides is very important during the critical period of germination; it was absent in this experiment since all the seeds had been washed* The Effect of the Period of Illumination on the termination of a Black And a White Wax Bean Axentjev (If) found that the light inhibiting effect on germination was entirely dependent on the presence and integrity of the seed-coats in Amaranthus retroflexus and other species of plants, but not entirely due to the coats in Cucumls melo and related species. Apparently, in some cases, light Interferes with the oxidation processes within the seed, while in others it favors these processes* Lettuce seeds are frequently light sensitive and the germination of these has attracted much attention. Accord­ ing to Shuck ($1) American lettuce seed is in a physio- 30 logically unstable condition that makes it particularly sensitive during germination to the effects of light, moisture and temperature* On the basis of these reports it was thought to be of interest to test the influence of illumination on tho germination of black and white bean varieties* Procedure* One black variety, Pencil Pod Wax, and one white variety, Brittle Wax, were selected on the basis of results ef the previous experiment* The former had proved to be the best black variety and the latter the poorest white variety in regard to emergence* Three hundred uniform seeds ef each variety, free from external Injury, were selected and placed in square glass dishes, each containing one hundred seeds* Another set of dishes was prepared with unselected seed* The filter paper in the dishes was moistened with distilled water, and each set of dishes was exposed to illumination as described below* Seeds of both varieties were grown in the dark as controls* Either variety was placed on a greenhouse bench under conditions of normal daylight* The second set was placed on another bench with overhead fluorescent lights, which were kept on continually thus extending the period ef illumination to 2l± hours. The third set was placed in a box completely sealed to prevent the entry of light* Germination counts wore made after an interval of four days, 31 the normal period in which beans should germinate at the temperature of the experiment* did not Tall below 60° F* Greenhouse temperatures Results of germination are given in Table 6 * TABLE 6 PERCENTAGE GERMINATION OP TWO VARIETIES OP WAX BEANS UNDER DIFFERENT PERIODS OF ILLUMINATION Variety Normal daylight Black Pencil Pod W a x (Selected seed) (Unseleoted seed) White Brittle Wax (Selected seed) (Unselected seed) Light for 2 ^ hours Darkness 14 52 92 28 52 96 70 88 92 54 92 96 Results and discussion* The germination of the black beans under normal daylight and under continuous light was significantly poorer than that of the white variety* The unselected seed of the white variety did better than that of the black under light* in darkness there was ne sig­ nificant difference, both varieties having a high percent­ age of germination* Seedlings of both varieties, in total darkness, had larger roots than those germinated under light* The hypo- cotyls of the white variety were well developed and had 32 emerged from the seed-coats* wh.ile very few of the black variety had appeared* The strong inhibition exhibited at exposure to normal daylight followed by darkness* and the less pronounced inhibition caused by continuous llluxnlnation* seem to indicate that not light alone but also photoperiod may be involved* The white variety was less subject to this influence than the black variety* Chemical Analysis Schulze and Godet (50) found hemlcelluloses to be the largest constituent of the bean coats; the content of the hemicellulose reaching a maximum in the ripe bean* Besides hemicellulose* the coats consist of nitrogen-free substances insoluble in water* significant quantities of llgnin* and a brown unrecognizable gummy substance which Schulze and Godet called "Holzgununl" • The cell walls consist of pectlc substances which are extremely hydrophilic compounds. These pectic substances are composed* for the most part* of uronlc acids* Ott and Ball (U-3) suggest that a knowledge of the polyuronide and pentosan content of the coats points towards an ex­ planation of varietal, difference in water absorption and retention* One of the most important components of the hemlcelluloses is pentosan* which is closely related to the polyuronides* This investigation sought to determine whether there was any difference in the pentosan content in the seed-coats of different varieties and whether this 33 difference is related to the water absorbing capacity of these varieties, Procedure# and has any Influence on germination. The method used in this experiment is that of McRary and Slattery (i|_0)«, Briefly, it involves removing the soluble sugars from the dry material by alcohol ex­ traction, hydrolyzing the residue to make the pentosans soluble, and assaying the resulting solution by a simple colorimetric test# The colorimetric determination is based upon one of the color reactions of the five-carbon sugars, namely, the interaction of pentose with orcinol in the presence of ferric chloride and concentrated hydrochloric acid (Bials reaction) producing a green color# Concentration of xylose in the unknowns was read off the xylose standard curve (Figure III) previously worked out from the readings of the standard xylose solution. Concentration of pentosans in the unknown samples was calculated and the values expressed as percentage xylose, presented in Table 7* A graphic presentation of the results is given in Figure IV# Results and dlaoussion. The percentage of pentosans In the white varieties is slightly higher than in the black vari e tie s • The results obtained are in disagreement with the expectations, just as Ott and Ball found and reported in their paper (1+3) • The'black varieties with a lower pentosan content would normally be expected to have a % T R A N S M IS S IO N so sa -jo so so *00 JPO CONCENTRATION OF XYLOSE < to o ------- ---------------------- ------- -------- V A R IE T Y F i gure IV. PENTOSAN CO NTENT 36 TABLE 7 PENTOSAN DETERMINATIONS IN SIX VARIETIES OP BEANS Sample number Variety Percent transmi s si on Percent concentration of xylose Remarks White 1 Laz y Wife 78 16.19 2 Dixie White 87 12.88 3 Brittle W a x 91 12.11 k Pencil Pod Wax 92 11.92 5 Plentiful 914- 10.89 6 Black Valentine 95 10.70 lower retentivity for water. Black This is not the case according to the results obtained In the experiment dealing with water absorption* An Anatomical Study of the Seed-coats of Beans Considerable work has been done in the past on the anatomical structures of the seeds of the Legumlnosae (21*., 37, I4I4., 6 6 ). Most of the work, however, has been concerned with the permeability of the seed-coat and its influence on germination. Watson (66 ) has correlated impermeability with poor germination in his investigation on the papilionaceous tribes Trifoleae and Loteae. This investigation is primarily concerned with exam­ ining the structure of the seed-coats of some bean var­ ieties In an attempt to correlate thickness with their 37 ability to withstand mechanical injury, and consequently explain varietal differences In germination* Procedure* Ten seeds of approximately the same size, color and maturity representative of the variety in question were soaked in water for twelve hours* The seed-coats were then easily removed by hand and small squares were cut out from the same plaoe of each seed-ooat to insure un i ­ formity in the sections* The material was placed in a formalin-glacial acetic acid-5>0# alcohol killing solution (5 *5 j90 parts) for eighteen hours and embedded in paraffin for sectioning* Sections were cut twelve microns in thickness on a rotary microtome, stained with Conants* quadruple stain, and mounted permanently in Canada Balsam* Measurements and camera lucida drawings (Figures V, VI, VTI, VIII) were made using a magnification of ij.30X* The drawings were enlarged to twice the original size and photographed* Seed-oeat measurements are given in Table 8 * General Description ef the Seed-coats The outermost layer of the coat consists of a row of palisade cells, called Malpighian cells, ranging from forty to sixty microns in length* Their outer ends are pointed and the exposed surfaces are covered by a thin cuticle which forms a continuous layer of unequal thickness and extends between the projections of the Malpighian cells* 38 SEED-HOAT STRUCTURE 0^ POUR REAN VARIETIES CUTICLE MALPIGHIAN •A SUBEPfDERMAL Figure V Brittle V.’ax CUT/CLE MALPIGHIAN SUB-EPIDERMAL PARENCHYMA Figure VI. Pencil pod Wax 39 - CUTICLE MALPIGHIAN 0 SUB-EPIDERMAL PARENCHYMA Figure V I I • Rival CUTICLE MALPIGHIAN SUB-EPIDERMAL PARENCHYMA Figure VIII. Lazy Wife ko TABLE 8 SEED-COAT MEASUREMENTS OP SEVEN VARIETIES OP BEANS Measurement in microns Variety Malpighian layer Black Pencil Pod Wax Sub-epi dermal layer 60.97 14.74 Plentiful £6.28 17.42 Black Valentine 51.59 11.39 48.24 14.74 ^ 6.90 12.06 White brittle Wax 46.90 20.10 Lazy Wife 44.22 24.79 Brown •flop Crop Rival Coefficient of correlation between layers within varieties significant at level -0 *5 4 . The lumen of the Malpighian cells is large at the base and tapers toward the top, being very narrow in the upper part of the cell owing to pronounced thickening of the cell walls. Beneath the palisade cells Is a sub-epidermal layer of closely packed cells, with no intercellular spaces, and greatly thickened walls with little or no lumen. Under­ neath the sub-epidermal layer Is a parenchymatous zone which consists of several layers of much compressed thinwalled cells. The inner layers are spongy In their arrange­ ment, while the outermost layers are flattened and without Intercellular spaces. Drawings were made of four varieties kl of beans — Pencil Pod Wax (black) Figure VI, Rival (brown) Figure VII, Brittle Wax (white) Figure V, and Lazy Wife (white) Figure VTII* Results and discussion* The over-all picture does not indicate any differences in the basic structure of the seed-coats. Individual parts of the different varieties examined showed decided variations In thickness. The cuticular layer is thin, uneven and coninuous. The Malpighian layer varies in depth In the different var­ ieties examined. Pencil Pod Wax has the thickest Malpighian layer measuring 60.97 microns, Figure VT ; Plentiful and Black Valentine 56.28 and 51*59 microns respectively. The two brown varieties, Top Crop and Rival (Figure VII), and the white variety Brittle Wax (Figure V), have Malpighian layers Intermediate In thickness between the black and white varieties. Lazy Wife (Figure VIII), a white variety, has the thinnest Malpighian layer measuring L1I4..22 microns with an exceptionally thick sub-epidermal layer 2i|..79 microns in* depth. The sub-epidermal layer In all the var­ ieties examined contain crystals o f v a r i o u s shapes and sizes. The thickness of the Malpighian layer explains to some extent the amount of mechanical Injury incurred in thresh­ ing and handling. Generally speaking, the black varieties which have a much thicker Malpighian layer appear to be more resistant to mechanical damage. Plentiful, however, The black variety although having a thick palisade layer, had a high percentage of basic injury. This anomaly may 1*2 ba explained by the fact that the samples examined could have come from lots which were harvested and threshed when too dry, thus making the seeds more brittle* Hand-shelled seeds of this variety showed no signs of mechanical injury and germinated just as well as the two other black varieties* A study of the variety Lazy Wife was prompted by the fact that the commercial sample contained a large per­ centage of "hard" or impermeable seeds* The sub-epidermal layer is extremely thick, preventing the absorption of water and accounting for the large number of impermeable seeds* Within varieties there is a significant negative correlation between the thickness of the Malpighian and sub-epidermal layers* A Study of Mechanical Injury The subject of mechanical injury in bean seeds has been investigated in the past decade (3 # 1 7 ) but specific information on seed-coat color in relation to injury is lacking* The present studies were undertaken with a view to discover the relative resistance of black and white seeded varieties to mechanical injury* Basic Injury In Commercial Bean Seed Procedure * Samples of seed were taken at random from the bags In which they had been shipped, and examined for U3 basic injury — external injury incurred in threshing and handling operations prior to sale to the growers* Broken or chipped seeds and those with large cracks were first picked out* The remaining seeds In the sample were further examined for invisible cracks by placing them in a flat dish containing water* Seeds with fine cracks had a wrinkled appearance after 10 minutes in the water* The results of such studies in six varieties of commercial bean seed are given in Table 9 and graphically represented in Figure IX* Results and discussion* Basic injury varies between varieties, different lots of the same variety, and different sources* The white varieties Brittle Wax and Dixie White had a higher percentage of injury than the two black varieties Pencil Pod Wax and Black Valentine* The anatomical study of the seed-coats of these varieties showed that white ones have a thinner seed-coat than the black* Although there are indications that mechanical injury may be Inversely proportional to the thickness of the seed-coat, the exceptions of the black variety Plentiful, which shows a high percentage of injury, and of the white variety Lazy Wife having the least amount ef Injury, make It impossible to draw definite conclusions* 2*4 TABLE 9 BASIC INJURY COUNTS IN SIX VARIETIES OP COMMERCIAL BEAN SEED Source of seed Variety Black Black Valentine Pencil Pod W ax Plentiful White Brittle W a x Dixie White Lazy Wife Number of seeds in sample Percent basic Injury (cracked, broken and shrivelled seed) A 529 A*-.3 B 2+26 3.2 A 525 4*9 B 617 3.0 C 705 2.5 A 772 12.9 B 663 5.2 A 529 8.5 B 62+7 6.4 C 820 5.3 A 557 1 0 .2+ B 799 6.5 A 667 4.9 C 52+5 1.0 3.5* 9.0* 6.7* 0.4* 2 .5* • ■p 0 o «Average basic injury 3.7* Mechanical Damage Experimentally Induced Types of abnormality resulting from mechanical injury have been described by Asgrow (3)* The object of* this experiment was an attempt to examine the varietal resistance exhibited by black and white beans to internal injury resulting from undue handling 45 1 plentiful 2 3 4 5 6 Dixie White Brittle Wax Black Valentine Pencil Pod Wax Lazy Wife