OEIECTWE MEASUREMENT OF HARDINESS 3N AZALEA Theda far the Dogma of Ph. D. MECHlfiAN STATE UNIVERSITY Rob’ar’f L. Gondeman T962 T724399; LIBRARY Michigan State University LECTRSCT OBJECTIVE EfiAfiUhEEEET O? fiAhDIfiESS IN AZALEA by Robert L. Genderman Azaleae are among our best landscape materials, but are reetricted to limited ecological sites. In breeding Azalea: to adapt them for diverse new habitats, the deeired characters must be found among the parents and recombined in their probeny. These must then be tested to determine their qualities eo that selection of superior clones may be made. Hardiness ie one trait of major importance to be added to epeciee with otherwise desirable landscape charac- terietice for satisfactory performance in colder areas. Hardiness, on internal, physiologically developed, sequentiel prooeae ie not an easily diacernahle overt character. There- fore, come means to determine its presence and degree or intensity is needed. Thie work is an investigation into n method to devise a usable technique for plant breedere. To furnieh e base, plants of known hardineee estab- lished in campue plantings were meeeured every two week: through the major part or the year, go that quantitative standards might be established. Azaleee chosen as base eclece tione were specimens of celenQfilgccvn, godfflorc, panhanenee and cultivere of 'Kaxwelli alba','Jemes Gable,‘ 'Alacke,‘ Robert L. Gondermnn 'Pclnr Beer'. ‘Gloekey Pink', and 'Corecgc'. Honouremcnte were node by employing e week flow or direct electricity through.e twig portion one centimeter long. leeeurenente were recorded in.kilo—ohne of reel-tonne, ueins c smell betterrponred, porteble multimeter. Contacts of three types were employed. The envil coneieted of two:netcl blades epcced one centimeter cpcrt, and firmly supported by*e cork bore. Twigs were cut to length, and pieced in contact with the electrodes. The eecond typo coneieted of two electrolytic rubber pod: under teneion or d epring clip. Here the rubber pod: served on contacting electrodes. The third device consisted of two needle electrodes one centimeter epcrt firmly secured through c hend grip. Thin woe merely plugged into the twig. Heceurenentc could be and. or rapidly on they could be recorded. Differenticl reading: otter freezing ere lcrgely due to excemoeie which follow: treat injury to cello. The bone plant. were tented for degree or resistance to electriccl conduction every two week: throughout tell, winter, end spring. This revecled differencee in their hardineee pat- tern: over thin period. Thee. measuremente constituted d bee. or qucntitctivc vclnce cgninet which measurement: or other plants were campered, to predetermine their degree of hordineee. The technique was then applied to hybrid plant: or Robert L. Gandermnn unknown parentage. Recovery end survival observetions teken in spring corresponded closely to the predictions of herdiness derived from an neesurements. This method for neesurements of plant hardiness is rcletively repid, inexpensive, convenient, quantitative, end rcpseteble. OBJECTIVE ERASURWEW 0F Elf'mlflmfi IN AZAIEA 5! Robert L. Gondermen A THESIS Sunnitted to the School of Graduete Studies of lichigen atete University in pertiel fulfillment of the requiremente for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1962 97 hula» /V :3 r? J ’ll'); TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . 11 TABLE OF C ONTEKTS C O O O O O O O O D O O O O O O O O O 1 1 1 LIST OF TABLES e e e e e e e e e e e e e e e e e e e e V IETRODUGTIOH e e e e e e e e e e e e e e e e e e e e e 1 OBJECTIVE e e e e e e e e e e e e e e e e e e e e e e e 4 HEVIEK OF LITERATUHE e e e e e e e e e e e e e e e e e 5 Hardening P’OOOBI e e e e e e e e e e e e e e e e e 5 FTCGZlng PTOOOII e e e e e e e e e e e e e e e e e 17 Th. 0011 W‘ll e e e e e e e e e e e e e e e e e s e 21 Placma K8flbr3n. e e e e e e e e e e e e e e e e e e 22 PTO. SplOI e e e e e e e e e e e e e e e e e e e e 2‘ 001101dl e e e e e e e e e e e e e e e e e e e e e 26 Permeability e e e e e e e e e e e e e e e e e e e 28 H‘TdOfliflg Conditions e e e e e e e e e e e e e e e 31 Breeding e e e e e e e e e e e e e e e e e e e e e 32 MATERIALS AND KETHODS e e e e e e e e e e e e e e e e e 58 Technique of Heasurement . . . . . . . . . . . . . 36 EXprcacions of Hctcurement e e e e e e e e e e e e 57 Plant MaterlIl e e e e e e e e e e e e e e e e e e 39 Standflrdizntlon e e e e e e e e e e e e e e e e e e ‘1 TEMPORAL HAIIDIK"°S . . . . . . . . . . . . . . . . . . 44 EFFECTS OF NUTRIENT LEVELS . . . . . . . . . . . . . . 48 EARDIHESS PHEDICTIONS . . . . . . . . . . . . . . . . . 51 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . 59 Results of Temporal Hardiness . . . . . . . . 60 Reeults of Growth.et Different nutrient Levels . . 62 V‘ri‘ticn e e e e e e e e s e e e e e e e e e e e e 55 iii Page DIEE'CUE-SIICI'I OF RFLULTS O O O O O 0 O O O O O I O O 0 O O 66 D1.0Ul§103 Of Temporal Hardiness e e e e e e e e e 68 Discussion 0! Effects of fiutrient Levels . . . . . 69 Discussion of Hardiness Prediction . . . . . . . e 70 Discussion of Hardiness Prediction aemplesz November 8 end November 12, 1961 . . e . . . . . 71 Discussion of Hardiness Prediction Samples: November 26, 1961 e e e e e e e e e e e e e e e e 72 Discussion of Herdinese Prediction Samples: December 28, 1951 e e e e e e e e e e e e e e e e 73 RELATIONSHIP BETfiEER SURVEY OF LITERATUHE T0 mEf‘iIfiLNTSeeeeeeeeeeeeeeeeeeeee 75 0013C USIONS O O O O O O O O O O O O O O O I O O O I O O 80 BIBLIOGRAPHY e e e e e e e e e e e e e e e e e e e e e 82 iv Table 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. LIST OF TEB'73 Ratio Sequence for Base Plants . . . . . Nutrient Stock Solutione . . . . . . . . Hicronutrient Stock fioiutiona . . . . . October 26 Hardiness Prediction Sample . November 8 Herdineee Prediction fiemple . November 12 Hardiness Prediction Sample November 25 Hardiness Prediction Sample December 28 Hardiness Prediction Sample Temperatures, Fall, 1961, East Lansing, mien. Ratio of Summetione of Resistance: of Twigs From Plants Grown at Different Nutrient LOVOereeeeeeeeeeeeeeeee Pogo 45 48 49 55 54 55 56 57 5B 62 AC XI; 02. 1.333854“: JETS The writer eiehee to expreee his deep gratitude to thoee who here been or euch.perceptire helpfulneee during hie eduoetion, reeeerch end theeie properetion. To Dr. Weteon, genuine gretitude ie expreeeed for hie aeneroue help over the long end devioue pethxey to enlightenment ever end ebove the cell or ecedemio nettere alone. To Dr. fiche: eincere eppreoietion ie expreeeed for hie valueble eeeietenoe with.verioue probleme of technical neture end timely encouragement. The writer eleo eithee to extend his eppreoietion to the membere or the grecuete committee: to Profeeeor F.L.3. O'Rourke for hie well-rounded epproech.to problems or horti- cultural complexity; end to Dr. 8.0. Eeeekoe for hie concern Ind guidence through pethsaye reaching into the unknown. The valueble euggeetione given.by Proreeeor Olien ie elee gratefully ecknoeledged. Acknowledgement ie eleo due to Proteeeor 0.3. Leeie for‘hie philosophy end friendship throughout the tic yeere of our Pleeeent eeeeeietion. 11 INTIKODUCTION Kan has long been intrigued by the problems of lack of hardiness in plants. Since our prhnordial ancestors moved from.their trapical epicenter and began t0»e1change their nomadic says for the more stable pastoral existence, freez- ing and death of desirable plants has plagued them. Present archeological findings (Anderson, 1946; Vavilov, 1925; Bone- ecn. 1956) reveal that man practiced plant selection in pre- historic these to obtain craps with desirable adaptations. As civilisation.noved northeard, hardier plants became in- creasingly important for survival during adverse seasons. Selections over long periods of time have changed several genetically plastic seeds into desirable plants adapted to cold climates (chshansky, 1955 and Harlan, 1958). Although all plants respond in various ways to enrironmental stimuli, hardy plants differ from tender plants .in their physiological adaptation to toleration or freezing (Duhemel and Burton, 1757; Sachs, 830: Levitt, 1951; Gorke, 1924). Breeding programs are in the process of increasing hardiness of various food, fiber, feed, drug and landscape plants today. The introduction of hardiness into woody orna- mentals has lagged, eith many or them being discovered by chance, rather than being the result of art and science of the geneticist. Versatility of plants for the landscape has created a horticultural commodity that demands increasingly more attention than it fonmerly did. The psychological effects or the various facets of the art of landscaping are well- knovn as yet only to a comparatively to. people. This is changing. Our technology gives man.more leisure time to de- vote to interests outside of his primary field. The endeavor to assure survival through food, clothing and shelter is not the totality of man's needs. in our society, recreation is important. Public parks with an organized program save money for the community in terms of alternative costs for crime prevention and Juvenile delinquency. Hardiness in plants is a complex trait, consisting of a balance between external conditions and internal physi- ological processes that result in a cumulative response (Luyet and Gehenio, 1940). Comprehensive accounts of frost history such as those of Scarth (1944), Levitt (1940) and Vasilyev (1955) shoe that hardiness is the result of a number of factors such.as rapidity of ice ronmation, culture, light, nutrition, temperature and genetic factors influencing physi- ological processes. Considering the overall complexity of the conditions and processes which induce hardiness, it is necessary to fol- low the organismal approach. The chief methods for determining hardiness in the past have been: 1. 2. 3. 4. Degree of injury as determined visually (Sachs, 1860). Leaching or loss of pigments or electrolytes (Dexter, 1930). Changes in electrical resistance of exosmosed electrolytes (Dexter, Trottinghsm and Graher, 19323 and Fillinger and Cardwell, 1941). Percent of regeneration after treatment (Greenham and Daday, 1957’s Generalizations about the amount of natural hardiness to be expected {ran a plant can only be valid within the limitations imposed by the vagaries of the environment. Largely, the range of wild and cultivated plants are deter- mined by their resistance to frost damage toward the extremes in the distance from the equator, and the degree and duration of rest period toward the areas approaching the equator (Searth, 1944) many of these phases still defy scientific investi- gation--theories changes new facts are discovered; and new postulations promulgated. OBJECTIVE Because of a need for increased hardiness in plants, attempts have been.made tovard discovering a.method for determining hardiness. In recent years, several methods have been develOped, but present methods inculcate various working deficiencies which cause them to be of relatively small use to the average plant breeder. Time, laboratory equipment, training and expense discourage use of many methods. Investigations were initiated in an attempt to discover a rapid, inexpensive, convenient, reproducible method of determining hardiness, and one which could be stated in quantitative terms. hr'NIht’i' 0F LITEM'MHE Hardening Process Resistance to frost depends largely on the character of the organized protoplasm, as revealed by the study of living cells in the intact organism (Stuart, 1940; Dexter, 1933; Haximov. 1929 and Kessler, 1935). Early reports of cell size, ploidy, plasmolysis, rapid thawing, conductance of electricity by electrophoresis have not proven to be reliable guides. deveral other corre- lations have been investigated with.varying degrees of suc- cess. In such.attempts, utilisation of the organism as an organised biological entity, embodying the syndrome of inter- related processes is essential. The parts, as cytOplasm, cells, vascular system, hormonal system, enzyme system, transpiration, and activities of the roots are important as factors. To explain the accumulated effect of hardiness, the entire plant must be projected as a unit against the background of its microuclimate, and related to its physi- ological responses gg_tg£g. Metabolism coupled with its physiological reactions is affected by the environment impressed on the genetic con- stitution of the plant (Dexter 1933a). The standard hardening conditions induce physiological changes which resist frost injury (Maxhnov, 1929). Since energy from food reserves are need in this process, it follovs that healthy, vigorous plants are more apt to become successfully hardened, to main- tain life over winter, and to burgeon into new growth in spring (Dexter, 1933 and tilhelm, 1935). The key factors appear to be protoplasmic or at least cellular, but include integrally the associated factors which reinforce cytOplasmic responses (Luyet and Gehenio, 1940). Among these is the enzyme system as suggested by Downs and Butler (1960). The role of colloids in the protOplasm may be more important than has been generally reported. Dexter and his associates have investigated hardiness rather extensively, and have concluded that a major precondi- tion for frost resistance is an increase in dry weight and a decrease in free water, associated with increased protOplasmio permeability to salts. The course of the process is related to the retention of efficient leaf area into the cold inducing period. Cul- tural factors, as low nitrogen level, restricted soil moisture, adequate summer growth, early fall cover crOps, and mulch after soil temperatures have decreased to forty-five degrees or after freezing, do.modify the microolimate, with attendant reactions by the plant toward stimulation of processes lead- ing to the hardened condition (Daddy and Greenham, 1960; and Tysdal, 1933). Several morphological regions of hardiness have been delineated, such.as bud, hark, cambium, root and shoot. In this paper, the entire organisn.eill be the unit under study. Due to the reactions of several physiological pro- cesses, the overwintering plant becomes dormant at the same time that hardiness develops as is inferred from the reports of Chandler (1941a), Harvey (1918) and Rosa (1921). Studied further, one is led to the conclusion that this condition is a prerequisite leading to the resting condition, which is resistant to environmental fluctuation. Environmental conditions are complexes of triggering mechanisms for the stepwise sequence of hardening. It appears clear that no one factor is all important, but that the organ- ized physiological response is due to the combined forces involved during the onset of the standard hardening conditions found at the approach of normal freezing temperatures. Environmental Effects:--Plant reactions leading to winter hardening are induced by the interplay of external environ- mental forces and physiological conditions (Flatt, 1937). The physiological conditions are in turn governed by the genetic constitution and its influence exerted throubh control of the enzymatic system, according to Harris (1934), soolley and ailsie (1961) and Samish (1954). A great diversity of environmental stimuli, as edaphic, biotic, nutritional, and insolational, effect the hardening process. Seymour (1944) observed that the steady frosts of midvinter do less damage than late spring or early fall frosts, which find plants unprepared. Most hardy soody plants respond to hardening condi- tions of the environment in several ways: new growth hardens; loses its excess moisture; winter buds form with protective coatings; and leaves drop. One change induces subsidiary changes, and all combine into the reaction chain of the organ- ism. Tumanov (1951) reported that decreased photOperiodic duration as well as decreased temperatures were necessary for the hardening process to begin. Long and Melcher in 1943 found that leaves exposed to shortening days deve10ped a growth.inhibitor, Seymour (1944) believes that much alleged tenderness of Azaloas is due to the deleterious effects of late summer drought, rather than prhmarily winter cold. The colder, shorter days of fall reduce the availability of ni- trogen and soil moisture. The cooler nights decrease trans- portation of carbohydrates out of the cell, but bright sunny days produce a good supply, so that an accumulation and con- versions result in the cell. Tysdal in 1933 reported that short days increased frost resistance and that this was more important than intensity of insolation. Harvey (1930) suggested that hardening reaponees to temperature began at five degrees Centigrade. He also found that more rapid changes followed a twelve hour alternation between ten and zero degrees eentigrade. Thus hardy plants are induced to begin the hardening process by effects of the environment. m figle of Estabolim in the Hardening Process s-v-Hetabolic activities vhich prasote and conserve accumulation of soluble earbchydrates serve as one or the initial processes leading to changes vhich induce hardiness. This in turn increases osmostic pressure and hydrophily (S-carth, 1944 and Levitt, 1951). hiring the advent of tall veather, sugars are stored in the cells at the same time that they are decreasingly eon- med from the same cells by respiration and translccation. The balance gradually changes to favor sugar accumulation. Chandler (1941a) and Chandler and Chandler (1943) found that sugar content increased as starch content decreased. Harvey (1950) and Tyedal (1933) found the best temperature to he a serc to ten degree Centigrade fluctuation. This gives the best level for metabolic activities on sugar acctmulation and its conversion to other plastic substances. (Manon 19:51 and Hedlund, 1917). Sisakjoh and Rubin (1939) and 'ryedal (19:53) concurred. They found the greatest activity of invertase to be at sere Centigrade. Thus metabolism is necessary not only to supply plastic substances but also to furnish energy for conversion of soluble proteins, and oils which also play a role in cyto- plasmie resistance to freezing temperatures. 99;; Permeability in the Hardening ProcessxooPemeability or the cell to water is important in that eater from the vacuole passes through the cytoplasm, along with eater from the 10 cytoplasm itself, to tone ice crystals interoellularly. This appears to be a protective mechanism against lethal intra- cellular crystallisation. Western investigators have long agreed that permeability increased with hardiness (Scarth, 1944 and Levitt, 1955), and nov belief appears to be gener- ally accepted (Vasilyev, 1956). hearth.(1944) reported that the hardier the cell, the greater the permeability. Dexter's asperhsents have led hin.to the conclusion that an increase in dry substance and an increase in protoplasmal permeability to salts is essential to the process or hardening. Hon~Solvent Spaces-The fraction of the cell not in solution extends to hair the volume. In.seeds and spores, vhich are ordinarily very hardy, this increases to a very large frac- ticn of the volume, and little space for freeaable eater remains. Sulukadse (1965) reports that seventy to eighty percent or the water contained in the cell at freesing tem- peratures becomes frozen. Lidforss (1896) followed by John (1951) and Harvey (1933) state that cold temperature intensifies hydrOphilio processes. Several insoluble compounds become colloidal in nature to constitute nonvsolvent space, leaving very little water to treese. Thus, this is a contributing factor in hardiness. Protoplasma;_Viscositlz--Scarth in 1944 reported on the dif- ficulty or measuring the viscosity of the prctOplasm, as sell 11 as the fact that it varies with.the physiological condition of the plant (Wilhelm, 1936). Kittsley and Noble (1955) and Kessler (1935) also report variation with differences in specific gravity, solute concentration, osmotic pressure, and pH. Ross (1921) observed that hardiness was determined mainly by hydrophilic colloids. His work was amplified by newton (1924) who concluded that hardiness was concomitant eith.the water holding capacity of the cell colloids. Harvey (1933), Kartin (1934), Scarth.and Levitt (1957) and Kesslsr and Ruhland (1938) found increased viscosity of the proto- plaam upon hardening. Simincvitch and Levitt (1941) believed that the protoplasmal permeability and consistency change with in- creasing hardiness, and that increasing hydrOphily precedes than. Increasing viscosity decreases vital activity, which induces relative dormancy under which cells are more resistant to unfavorable external conditions. The Role of Osmotic Pressurewin the Hardening Process:-- Scarth.(1944) reports that increasing osmotic pressure is due largely to conversion of starch to soluble sugars, with the greatest change being in.hardiest cells. Ackerman (1927) felt the osmotic pressure was a function of the concentration of hydrophilic sugars. The work of Dexter over a period of years showed that hardening is related to the amount of plastic substances present. hetabolic activities, which promote the increase of sugars, promote hardiness (Ross, 1921). This increasing concentration of plastic substances attracts and holds water through an increase in the osmotic pressure (flew- ton, 1924a) Kessler (1935) found that osmotic pressure in the cells rises with increasing frost resistance in fall. Increase of osmotic pressure without other related factors in the hardening process appears to have little affect on hardiness (Searth, 1944 and Levitt, 1951). Role_of Physico-chemioal Factors in the Hardening_Process¢-- Biological physico-chemical changes accompany the changes of organised cellular resistances leading to hardiness. Cell sap characters are related to frost resistance as moisture content, sap concentration and sugar content, which forms an inter-related group (Levitt, 1951). However, correlations with survival in nature indicate that more fundamental factors must be operative. As early as 1896, Lidfcrss found that fats and oils in the cell resisted freezing. These lipids fluctuated, appearing to be independent of temperature. hearth in 1944 reported that lipids regulate cell wall permeability. Siminovitch (1949) found twice as many soluble pro- teins at pH 5 than at pH 7 in hardy trees. He also noticed that increasing hydrophily increases protein and lipid content. 13 Proteins undergo changes during the hardening process, split- ting into simpler, more soluble, less coagulable forms. Only the water soluble proteins increase . Rich storage of re- serves occur when.growth stops, but the manufacture of carbo- hydrate nutrients ccntinues in the processes of natural re- sistance to cold according to Hedlund (1917). Various inves- tigators have reported on soil solution uptake, as a.morc or less passive process combined with semipermeability of root tissue membranes (Epstein, 1955; Eylmo, 1953; hitchell at al., 1960 and Hoagland and hroyer, 1956). Harris (1954) was convinced that properties of tissue fluids were due to soil solution properties as well as geneti- cally controlled synthesis. Later, Hurd-Karrer in 1959, reported that liming the soil had no effect on the pH of plant juices, and that little difference was found in plants under good growing conditions. Location effects related to soil nutrient concentrations were also reported. tilheln.(1935) found increased potassium, when com- bined with deficiencies of nitrogen and phosphorus, contrib- uted to prolonged duration of sugar concentration in proto- plamm. Gladwin (1917) was unable to influence the degree of winter-killing of grapes by nitrogen, potassium or phos- phorus fertilisation, but Yssuda (1927) did so by using high potassium combined with low temperatures. Dickey and Poole in 1961 found a direct relationship between soil nutrients 14 and their concentration in leaf tissues. Inverse relation- ships existed between concentration of the nitrogen-calcium- magnesium group and the potassium-phosphorus group. In 1956, Levitt reported that while potassium and phosphorus increased hardiness, an excess produced adverse effects on physiological processes. This was corroborated to a degree by the work of Arland in 1952. Goodall and Gregory (1947) reported on the complicae tions entailed in determining soil solution concentrations by foliar testing, but other factors remain to be discovered as yet. Vasilyew, writing in 1961, concluded that pH is a concomitant factor, and changes to a relatively small degree with season or hardiness. Hewitt (1952) found low tolerance to salt accumulation, and that high osmostic pressure depres- sed growth. (Haywood and Long, 1943 and Osuch and nndleigh, 1945). The information obtained from the chemical approach is of interest as it applies to the problem. figle of Boundgfiater in the Hardening Procese:~-Chandler (1941b) postulated that a11.nolecules of“bound water in a nieelle are strictly orientated and polarised. Heston (1924b) reports that tender and hardy plants have approximately the same amount of bound water in summer. Searth (1944) reports a consistent increase in waterbinding hydrophilic colloids as a feature of hardening. 15 The fall weather with.bright sun and cool nights increase the concentration of sugars which in turn binds water to its molecular framework. This bound water is less subject to freesing. haximov (1929) and Chandler (1941a and 1941b) found that the soluble carbohydrates, especially the pentosans, were instrumental in binding water against the pressures of crystallization. John (1951) and Siminovitch and Levitt (1941) agree with this conclusion. According to _Frey-Wyssling (1950) and Phillis and mason (1951), water bound more closely to the micelles by proximity attraction is less likely to freeze than the more distal, loosely-bound molecules. This factor is of importance as a matter of degree within its major role in protection. Holecular Lewg;;--Since physiolOgical processes can often be readily approached and comprehended at the molecular level, some considerations are pertinent4here. Decrease in temperature decreases the kinetic energy of the molecule, and activities are reduced accordingly. The less soluble a substance, the slower is its diffusion, transportation rate, and reaction capacity (Newton, 1924b). Solutions and colloids thus become more viscid as the Brown- ian movement slackens its activity, as colloids gel, and all associated activities decrease their activity (Chandler, 1941a gnu dorthen, 1942). The higher energy water molecules are lost to intercellular space, and only the more sluggish remain 15 to bombard and cembine with protein molecules, which leads to'a greater measure or dessication. increased propinquity causes greater attraction.and cohesion between chemical bonds in the cyt0p1asm, which shrinks, thickens and becomes more gelatinous (Chandler, 1941b and Derjaguin, 1960). The ions of salts and acids‘remaining are more concentrated and bind polar substances more firmly (John, 1931). This in turn raises the osmotic pressure, which resists greater dessica- tion until an equilibrium point is attained. Lipids appear and coat the surface or the plasmolysed cytOplaem, further encysting it against adverse effects (Samish, 1954). Since all cells must respire sufficiently to provide energy for life processes, there appears to be a threshold beyond which.m1nlma1 vital processes of the cell cannot Operate. For hardy plants this may be the balance between degree of hardiness necessary to resist freezing conditions, yet maintain life. All the factors involved in induction of hardiness must be considered in a research program.conccrncd with the “physiology of hardiness. Environment, metabolism, permeability, viscosity, physico-chamical reactions, bound enter, and molecular behav- ior are all factors affecting the process. Times the harden- ing effect is achieved throuah their combined activities, an understanding is essential for planning of experimental work and interpretation of results. Eeoults of work by previous 17 investigators in relation to the overall problem or hardiness can be used as a'basis of consideration‘when.attempting a new attack on the problem. Without the knowledge obtained through the survey of literature, this investigation into better methods for detection of hardiness would not have been possi- ble. Through knowledge obtained in this manner, the basic natural reading was established as a part of the measurement of hardiness. Freezing Process The conditions which the hardening process impresses upon the normally hardy cell are present in nature at the onset or usual freezing temperature. It is upon this ayn~ drone of cellular conditions that freezing influences are exerted, the shrunken protoylaem, gelled colloids, lipidi~ nous coating, high soluble carbohydrate content, concentra- tion of soluble proteins, and reduced metabolism (Samish, 1954). All cells must reepire foods to iain energy for metabolism to sustain life processes. Eeaeurable respira- tion has been demonstrated at minus twenty degrees Centigrade. This has been generally believed to be commensurate with the minimal state of activity during the hardened and resting condition (Hewton.and Anderson, 1951 and Dexter, 1933). During the usual course or natural freezing, ice crystals form first in the intercelluler spaces. This 18 crystallisation provides a concentration gradient which draws water from the cells. The lower the temperature, the greater is the proportion of water which freezes, according to Siminovitch and Levitt (1941). Chandler and Chandler (1943) agree with fiaximov (1929) that plants Which do not store cellular carbohydrates are much.more susceptible to treat injury. Both concur that the amount of water that does freeze under high soluble sugar content, does so only after considerable undercooling. In a Eolcr solution of sucrose, approximately half of the water freezes at minus 4.3360 0., but at two holar strength, only traces of ice occur. Akerman (1927) computed changes in sugar solution concentration when water was frozen out of it. From in initial 1/4 H, the concentration doubles st minus o.ss° c., quadruples st minus 1.850 0., increases twelvefold at minus 7.44 and sixteenfold at minus l4.88° 0. He believed the process theoretically would be accelerated by metabolizing living cells. There is an increase due to hydrolytic pro- cesses, which cause dissolution of previously insoluble com- pounds, and an increase in plastic, water-binding. substances. Various other physiological processes affect the freezing process inter-relatedly. These processes {all into the organismsl concept here also. In freezing, water from the vacuole is drawn through the cytoplasm and to the ice crystals of the intercellular 19 space. Increasing cell permeability protects against intra- cellular fressing, as the cutflcs of water serves to con- centrate cell solutions till their freezing points drop to the actual temperature as observed by Searth (1944). WP-Frcst injury to plants endemic to the tem- perate sons depends on ice formation. The usual locus of ice is outside of the cell in intercellular spaces. The also of crystals decreases and their dispersion increases with increased rate of freesing. Upon slow fressing, large extracellular ice masses cantmechanically disrupt transport and collapse dehydrated cells sith.high.osnotic pressure. Intracellular freesing is usually fatal to the cell. Deathris probably due to crystals'shich.ranify through.the cytOplasn, disorganiss essential structure, and possibly cause precipitation of proteins, as was postulated by Scarth in 1944. Losino-Losinskiy (1948) micrcmanipulatsd cytoplasm, shoving that it was somewhat resistant to.nechanical damage. Frost injury produces several visible changes. The calls may be completely separated from each other. Proto- plasts undergo plasmolysis, coagulation and contraction. The dead protOplasm may shot fluidity. The «11 loses 1:. “er- psrneahls properties and solutes are subject to equilibrium with.the external media (Layet and Gehenio, 1940). Frequently a frothy structure may be seen (Searth, 1944). Injury to Cellular Componentss--In testing, it appears that 20 cubic: tiuuee and ray parenchyma are especially waceptible. Allen and Mai (1943). upon nicroecOpioal examination found. the pericycle and phloem rays to be injured, in addition to cambium. Injured celle econ become discolored. Protoglaemal Damage zo-lnability of roots in winter to abecrb sufficient coil noieture often reaulte in deuication, plu- nolyeie and impairment of vital functions leading to death. Thin type or inability to resist winter freezing damage any be attributed to drought or frozen coil moieture. Death or teminal parts are also hastened by sun and wind: which aid in deeeication or cell contente. when half the water freezes out or the cell, the resulting deeaication, plamolyeie, and ionic concentration: may cause nevere (image or death. Damage may occur in epring due to late i'roete after dormancy has been broken by the burgeoning proceee an a con- eequenoe or early spring warn periode.B-;1rgeoning may induce colloid hydration in the cytoplasm (Keceler, 1935 and Keuler and Ruhland, 1958). Vanilyev (1956) reporte the general acceptance now in Rueeia of tho underlying idea or chemical destruction or protoplaem through concentration by hydrolysis, deecication and plunolyeie. Vasily" reports that the commonest tom of winter-killing in cell dehydration caused by freezing of the ntcr necessary for the prot0plamal activity necessary to mulntain intracellular life processes. 21 The factors involved in the freezing process are complex, and must be studied intensively to understand the physiological processes entailed. Pressing injury or cellular components and death of the cell are matters affecting the experimental readings. Limitations could be placed upon readings‘eith.decth or cells by realising the affects upon extracellular moisture. It is treesing damage to the plasma membrane which allows exosmosis of intracellular solutions that is measured in terms or lowered resistance to conductivity of electricity. The Cell hall Several investigators have studied the cell wall intensively, and ascribed to it no major factor in the physi- ology or the cell nor contribution to hardiness. Chambers and Chambers (1961) report the primary cell wall to be very thin network of cellulose fibrils embedded in.a matrix or enorphoue pectic material. it the end of the elongation phase, internal apposi- tion or fibrils in parallel orientation were found. These A are layered in.a series of three rotations. alignment shifts true one rotation to the next inra regular pattern, but all converge at the poles. This forms the secondary rigid cell wall. The calcium pectate matrix, as a colloidal gel, becomes rigid as it is affected‘by different chemicals at this stage (Proline and Preston, 1961) 22 The stretching of the primary wall is due to the prop- erties of the long chain macromolecules, formed of helical, longitudinal repeating units, according to Swanson (1960). The cell call has come to occupy less importance in physi- ology as its main contribution is mechanical. Plasma hembrane The general features of the molecular structure of the plasma.nenbrane and the fundamental pattern of organi- sation have been revealed by Robertson (1962), Swanson (1960), Chambers and Chambers (1961), Walker (1958), Bedford, Meyer, and Preston (1958) and others. Studies of ionic exchange between the enveloping solution of the free space and the interior of the cell in- dicate that molecules of various kinds enter or exit against the concentration gradient through the expenditure of energy in active transport (Epstein, 1955 and Harris, 1955). The plasma.membrane forms the outer boundary of the cytoplasne This membrane is common to all cells of biological organisms, but plants, in addition, have a cell wall for sup- port. The plasma membrane may be elastic and pliable, or rigid and unyielding, depending upon the function of the cell (Brachet, 1961 and Robertson, 1962). Models generally indicate the presence of a double lay- er of lipid.nolecules sandwiched in right-angled orientation to, and between, two layers of long-chain protein.molecules. 23 The cell membrane has been reported to be strongly acidic and well buffered (Halter, 1961; Harris, 1954 and Doty, 1957). Considerable work has been done on this subject, and cosposites of fora and function are gradually forming. 'the structure of the plasma membrane is characterised by an internal double layer of lipids with the non-polar end groups of the molecule adjacent to each other. The polar end groups meet with the polar groups of the amphoteric protein molecules at the outer faces. “the proteins on the surfaces are orientated in a reticular network of chains which allows elasticity, mechanical strength, filtration, and enzyme localization, in the opinion of Halter (1961). The latter may be important in conversion of insoluble to soluble deriva- tives to pass the cell nenbrane (Levitt, 1951 and Levitt and Sininovitch, 1940). The thickness of the aeusbrane has been reported to be fru seventy-five to 100 angstrcn units. Diameter of the pores also have been estinated variously, with Solonan (1960) euputing a disaster of seven Angstroms in erythrocytes. (Robertson, 1962) Taperary imbalances of the equilibrium may tend to cause variability in the degree of permeation. Thus the “brace is semi-permeable, pemitting entry of some ions but excluding others (Rothstein, 1966a). Through such selective Permeability, the ionic content of the cell is maintained at ‘ relatively constant level. Dynamic equilibrims achieves a 24 balance under‘which.metabolisn can produce carbohydrates for energy, growth, and processes leading to hardiness. Since the semieperneable plasma.membrane largely controls the amount and kind of ions which accumulate in the file of moisture on the cell wall, the effects upon conduct- anee under an electro-motive force would varyzw with condi- tions prevailing at the time of testing. The post freesing readings would be affected if the plasma membrane was damaged and its selective permeability destroyed, as this*wculd allow equalization of intra and extra cellular solutions. Free Space The apparent free space has been definedbas that fraction of tissue into and out of which ions can.move freely by diffusion without any permeable membrane between this vol— uae and the external medimn. While there have been controversies over the subject and its potential anatomical ramifications, they will not be covered in the scope of this paper. The freely diffusable ions in the solution which adheres as a thin filn.over the outer layer of the cell wall and which is essentially the end point of the circulatory transport system of the organism has been.useful in explaining biological phenomena of physiological nature (Jpnes, 1961). This ,upface film within the organism gn.eitu under ordinary growing conditions varies little. The ion accumu- lation is narrowly variable under nonmal growing conditions. Under the stress of dessication, the film thins and resist- ance rises. The accumulation of excess salts may also have a degree of influence upon the conductance of electrons, but this eould normally be.a constant from plants in the same ‘ uniform area. The transport of ions from root to shoot has been generally considered passive in nature (Epstein, 1955; Hylmo, 1953 and Mitchell, 1960). It is generally accepted that ion absorption and accumulation are independent (Kramer, 1957). The ions move passively over the moist film.interface of the cell wall. This extra-cellular solution is considered to be part of the continuous liquid system in plant tissues (Clien, 1961). It is this space which.allows conduction of electrons by ions. Levitt (1956) postulated surface film of 0.02 millimeter from tests of solutions absorbed on experimental tissues. Robertson (1959) and Cole and Curtis (1938) reported that cells were separated by a space of 110 to 150 Angstrom units which contains a material of low electron density. This space varies with.metabolic changes. Since the cellular membrane resists passage of direct currents of electricity, the electrical resistances used in this study do not include varilblcc Of 03t09188m or cellular inclusions. Thus the flow of electrons 26 from the electrical forces applied are limited to the free space (Kramer, 1957). Colloids The majority of biological colloids are 1y0philio (Semish, 1954; Wilhelm, 1935 and Beaten, Broth and Anderson, 1951). The viscosity of colloids derives from amphoteric molecules with.hydr0philic forces which.align at interfaces and produce surface activity (Livingston, 1938 and Fisher, 1924). Gelation results from numerous strong linkages be- tween protein.molecu1es and their attracted spheres of water (Frey-Wissling, 1948: Derjaguin, 1960 and hedelsky, 1945), eithin a reticulated.nesheork of intertwining fibrils (Robertson, 1962; Francis and horse, 1956 and Swanson, 1960). Several workers have investigated gelation and vie~ cosity. Eewton (1924b) found no'difference between.colloids of tender and hardy plants during the growing season. He noted an increase in viscosity of colloids associated'eith lowered temperatures, as did La Verne,(19d9), John (1951), Buhlert (1906) and hudra (1932). The rapid increase in viscosity of hydrOphilic sole with increasing concentration of solute is ascribed to the hydration sphere of the micelles in a review by Meyer and Anderson (1952). Scarth (1944) states that a high colloidal content of cyt0plasm is necessary for resistance by hardened cells, as does Dexter (1955) and Levitt (19:55). 27 Daniels and Alberty (1955) reported that colloids under dehydration shrink to a certain extent, than tens- eionely maintain the remaining liquids over a long period or time. Lehedincev (1930) found the eater-binding power of colloids to be directly related to both frost and drought resistance. Hisenjkov (1939), however, round the inverse relationship'vith.drought resistance. Gels appear to have an increasing effect on diffusion, conductivity, and velocity or reactions as rigidity increases (Langley, 1960; danish, 1954 and firminovitch and Levitt, 1941). Increases in viscosity lead to gelation. Since pro- tein sols are amphoteric, the activity or their gel-sol re- lationships depends on the pH of the media (Anderson, 1939; Chandler, 1941a and Mndra, 1932). Generally in hydroPhilic colloids, stability is determined by the electrical charge on the molecule and the hydration sphere (Anderson, 1939; Chandler, 1941b and Derjaguin, 1960). Too great a degree of stability leads to an undesir- able rigidity. In the investigations or Simindvitch and Levitt (1941) it was found that protOplasm of hardy cells does not become rigid as does that of tender plants at freezing temperatures. The most generally agreed characteristic change accompanying hardiness was a sharp increase in.vater-ho1ding capacity or cellular colloids (Heston, 1924; nartin, 1954; Chandler, 1941a and Heyer, 1952). It1night be postulated fron.reports in the body of the literature that more viscid colloids permit a slow and steady rate of respiration whichhnaintains life in proto- plasm.during adverse periods. It is apparent that colloids play a basic role in the resistance of protoplaen.tc frost injury, to rest period, and to the gradual return to the burgeoning of’grovth in spring. They also play a part in dessication of extracellular solutes upon hardening, as well as gradual release during the depths of winter's cold. These affect readings of conduct- anoe of electricity as used in.neasurenents of hardiness. Permeability Continued cell life depends upon a dynamic equil- ibriun of water, salts and organic matter in the cytoplasm. Control is largely maintained by a.membrane only one ten~ nillionth of a.nillineter thick. Ions and molecules of various kinds pass in.and out under a controlled system. Pressure from the vacuole pushes the cytoplasm outward for intimate contact with the cell wall, shich.faoilitates ex- change through.the membrane by the aqueous phase containing food, tastes, andxnetabolic products. More rapid exchange occurs in small cells due to the higher surface to volume ratio. The plasma.nembrane is capable of controlling the 29 processes of energy conversion and expenditure in accordance with the complex pattern of cellular nutrient and energy exchange (Halter, 1961). Molecules in the solution in the plant normally dis- sociate into ions which carry an electrical charge. This forms a force in.permeability equal to the differential electrical potential. Passive transport is derived from cellular environ- ment. If energy from the cell is employed in passing an ion across the membrane, this is active transport. Ions can be attracted, held or released against the concentration gradient. Soluble substances can dissolve in the lipid layer of the~nembrane for penetration. Others enter through the pores, so that the size of the ion may be a regulatory factor. In ad- dition, the pores may contain ionic charges. Holter (1961) reported that the factors altering permeability were mainly ionic. In.his Opinion, the movement of solutes through the cell membrane is affected by molecular size, partition coef- ficient, concentration gradient, electrical chsrge, and active transport. Other workers generally agree with this vies. Harris (1954) adds the diameter of the hydrated ion and os- motic pressure. Tumanov (1951) inferred that food exhaus- tion and accumulation of’metabolic wastes could lead to an acid condition which could become toxic and thus affect permeability of the cell. Kctchalsky and fiiller (1951) reported that an increase in.acidity of the medium.increased 30 ionisation or electrical charges. Permeation is proportional to the percent of undis- sociated molecules, and this is influenced by pH changes to and from the isoelectric point. Easier entry of monovalent ions as compared to divalent ions has lead Glass (1952) to postulate larger hydration spheres due to the attraction and orientation of eater molecules. Rothstein (1955b) postulated an enzyme type carrier with localised areas in the cell wall. hepe and Robertson (1953) postulated that increasing frost injury to cell mem- branes might lead to leaching and dcssication resulting in death to the cell. Permeability could also be affected by the increasing concentration of acids and salts during plas- molysis of cold hardiness conditions. Volume computed as 4/3 Pi r cubed, leads to possible increases in concentration by eight to sixty-four times. This magnitude could dissolve the pectocellulose matrix in the cell walls or the lipids of the membrane and allow rapid passage of solutions such as is seen after freezing. Siminovitch and Levitt (1941) stated that permea- bility and consistency change with alteration in frost re~ sistance, and that hydrOphily appears to control these changes. lewton'e experiments (1922) showed that hardened plants re- leased very little Juice under pressure. Later, Newton (1924) found that frozen hardened plants also retained their Juice equally as well, while tender frozen plants released their 31 Juices freely. Using this infonmation, Dexter, Trottingham, and Graber (1930) expressed the belief that this could be used as a means of measuring the injury to the semipermeable cell membrane. Dead plants, whether tender or hardy, re~ leased approximately the same quantity of Juice. These fac- tors are basic foundations for modern investigations of plant hardiness. Hardening Conditions Adequate conditions for hardiness of some plants can be induced by gradually lowering temperatures and decreasing daylight hours (Angelo, 1958; Harvey, 1950; Scarth, 1944 and Stuart, 1940). Duration of treatment and degree of intensity of application are the function of the growing stage of the plant and its innate capacity to respond to hardening environments. Adequate hardening conditions appear to‘be tempera~ tures fluctuating ton to twenty degrees diurnally, just above the freezing point, combined with light duration reduction to approximately eight hours. Standard Hardening Conditions:--The term standard hardening conditions will be used as denoted above. Valid measurements of hardiness can only be made under the influence of these environmental effects. 32 Breeding In selecting species and varieties of plants to grow in.the north or south, factors of winter hardiness or rest period must be given attention. Breeding methods can recom- bine desirable features into a composite plant to meet the demands of increasing urbanisation and the need for better landscape plants. Asaleas at present are enjoyed for their pyscholog- ical impressions in landscaping almost exclusively in limited areas of natural adaptation. Azalea species are known that are indigenous to set or cold or neutral soils or exposure to sun, and have various degrees of environmental adaptation. Thus gene pools exist for these characteristics. In the control of a competent breeder, recombinations of gene patterns can genetically adapt azaleas for greater service to man. Phenotypic effects of gene frequencies for size, color, foliage appearance and form are easily selected. Physiological hardiness and rest periods are not so easily detected. Breed- ers may have to carry thousands of seedlings till a ”test winter“ to determine hardiness before introduction or use in breeding. Weather records indicate that winter conditions are variable. The search for'methods to determine the hardiness of plants has been the subject of several investigations in recent years. If possible, the desirable plants should be 33 selected and tender plants discarded before large amounts of time, labor, and land use has been expended. This would also speed the cycle for a more successful breeding prOgram. Programs have been recently developed, but have not proven as applicable as might be desired due to the need for laboratory equipment, training, time and expense involved. A rapid, inexpensive, non-complicated system is needed to adequately measure the hardiness of segregating generations of progeny, new introductions, and seedlings of superior merit phenotypically. Such has been the object of the basic research of this paper. DistributionzooThe northern limits of distribution of Rhododendrons appears to be the cold of Lapland, Kamchatka and Siberia according to Watson (1911). The southern range extends to the humid tropics. Of the reported species of the genus, fev are hardy in Zone V and VI which comprise a large portion of the densely populated northern United States (Skinner, 1962; Lee, 1958 and Lewis, 1961). Breeding lines descended from hardy varieties are new being deve10ped. Background for fireeding:--The three species from the arctic and the three from Siberia should contain adequate germ plasm for cold resistence. Vavilov (1926) postulates paral~ lel development such as has attended introduction in Alfalfa and Eye. Axelrod (1959) purports that evolution of‘modern plants has often been toward hardiness. atebbine (1950) and 34 Darlington (1939) also theorize that the variation and evaluation of plants such as the Azalea could lead to re- combinations including hardiness, as most of the genes are in a plastic stage of diversionary evolution. Jain and Allard (1960) reported on the genetic background of polymorphism, homeostasis, and coadaptation which.may be encountered in various species. Daday and Greenhan (1960) have shown the distribution of hardiness in interbreeding gene pools, and that polygcnetic inheritance 1. indicated. Paris (1960), in tracing the parentage of hybrid azaleas revealed the breed- era choice of hardy parents to produce progeny selected for introduction to the trade. Geneticistc' results point to several independent Mendelian factors which effect this stepwise process accord- ing to Levitt (1941). It is of interest to notice the over- dcninance which.alloss selection of progeny either'more tender or more hardy than either parent. Since the basis is for several genes which regulate this quantetive physiolog- ical process, and these are variably espressed in the phone- type as inter-related to vagaries of climatic and edaphic factors, the problems of determination of most suitable paren- tal material.are considerable. (Hagberg, 1952 and 1955; Rollins and Hewlett, 1955 and Chambers, 1961). Application in Breedingz-obhile overt physical traits may be relatively easily observed, noted, and duly utilised in a pedigree program, the internal innate physiological mechanisms 55 are a vastly different type to distinguish. If these traits can be recognised and especially if they can.be determined to a degree of intensity, the breeder is in a.much.bettcr position to manipulate 11s parental selections for better results. Such.is the case with plant hardiness. With the results of the experiments described in this paper, the breeder has a readily available tool for determining the hardiness of his stock. Thus, fortified vith.knowledge of inherent hardiness, the work of the breeder will be simpli- fied and reduced. Hardiness canflbe incorporated as an inte- gral part of the program to introduce this characteristic into plants with many large flowers, evergreen foliage, desirable form, texture, foliage gloss, fall color and sea- son of bloom. It is confidentally expected that improved Azaleas will be produced by breeders through application of the measurement of hardiness. MATERIALS. AND EETHODS Technique of fleasurement Twigs were selected from healthy plants which appeared to be in normal condition for the season. Twigs to be taken were out above a node, labeled with masking tape, placed in a polyethylene bag which was humidified with damp paper towels, and sealed for transportation to the laboratory. Twigs were allowed one-half hour at room temperature to thaw, if necessary. Following this,twigs were removed individually and measured by the Ohmzneter, after which they were placed in a second polyethylene bag with the same humid- ifying materials and sealed. The lot was then placed in a tuperature of minus eighteen degrees Fahrenheit, on a mesh bench with adequate air circulation at all times. Materials were frozen for approximately eighteen hours. a batteryupcwered, portable Otmmeter manufactured by the Supreme Instrument Cmpany, Model 345, at Greenville, Mississippi was used to measure resistances. The low voltage, 1 1/2, was selected to avoid intra-cellular complications and avoid injury to membranes of the living cells. Contacts of three types were used, anvil, spring-clip and matrix. The anvil was comprised of two metal blades set in 56 37 a basal block. Lead wires from the Ohmmeter were soldered to these conducting surfaces. Twigs were out to 1 cm. length and placed finely between flhese contacts for reading. The second device consisted of electrolytic rubber cushions fused into a spring-clamp arrangement. The electro- lytic rubber was supplied by the hhell Chemical Company. The rubber was reduced to a solution with organic solvents and allowed to harden in cushions in the jaws of the spring-clamp arrangement. Twigs were cut as above, placed between the jaws, tension allowed to close the circuit, and readings made. The matrix consisted of a non-electrolytic hand grip pierced by two needle electrodes placed 1 cm. apart, and at- tached to the lead wires or the Ohmmeter. These were merely plunged into the twig. leasurmments could be made as rapidly as they could be recorded. Honsally four.neasurements were taken.fram each.twig, and two to three twigs used per plant during seasons of greater variation. As techniques and equipment improved this was reduced to three measurements on two twigs, even though at times it was no longer necessary. EXpreesions of Measurements Resistance to conductance of electrons through the intercellular solution is the basic factor determining read- ings on the Ohmmeter. Two readings were necessary to establish the measure- ment or a sample twig. One reading (a) was taken from the 58 natural state twig. At times it was taken from the living plant gg_g;533 The second reading (b) was taken after the freezing treatment period. The measurement was at first expressed.as an a/b relationship. Later this was reduced to a ratio by the division process. To be valid, it was found that the division process entailed a conversion of diameters to a constant unit size. The 2.0 mm modal class was chosen. Later it was found that expressions of readings by ratio did not reflect the magnitude of readings in some cases, and alternate methods were investigated. ‘ The formula yen m, a + b, where m is a base constant reading: a, an after-freezing reading: and b, the reading prior to freezing minus 3a, was suggested. A second formula, b =- bl "’ t’2 ‘1 " ‘2 c + n, where o is a class level, and n is a base constant, was also considered and tested. Both formulas have certain.merits and certain un- desirable features. Upon trial and examination, it was found that a more convenient formula based on the above could be employed which Oerved the purpose more closely. This was e l 2 run our where n1 is a constant relating to a, and n2 is a constant relating to b. Since this measurement derived from the two readings on a basis of equality, the resulting figure is of magnitudinal character. A further step in the process of trial and examination was to use base constants of frost killed twigs. Then it was revealed that any pertinent a number could be employed. Thus, for ease in computation the second step of this formula was selected as a + b 1000 100 with n1 and n2 falling roughly into classes accompanying hardiness in climatic sons V. In the final stage of evolution, it was seen that the two readings could be reduced to percentages and added, as the most rapid and convenient method. This was adopted as the percentage summation.method of expressing hardiness. Plant Material Plant material of a wide scope of hardiness was chosen as base plants to establish a range of basic values of measurements. Rhododendrons, Series Azalea, used in the program or 40 possibly used as parents of hybrids fall into climatic zones of hardiness as follows: Zone III. R. ggdiflore, roseum. Zone IV. E. vesezi, Ghent Hybrids. Zone V. R. calendulaceum. Zone VI. h. poukanesgq(yedoense poukanense), Iaponicum. Zone VII. R. Kaempferi Hybrids, Gable Hybrids, mgllg. Zone VIII. K. obtusum, macrantha, Kurume hybrids. More specifically, the hare Plant List published annually by the Hichigan State University Grounds Department, under the direction and cooperation of Hilton Baron, is a guide for this area. Dr. Baron reports the following hardi- ness evaluations: 'James Gable' - hardy here several years. 'Gloskey Pink' - has done well here. Few years of establishment. 'Polar hear' - believed hardy. 'Corsage' - probably tender here. 'Kaxwelli alba' hardy here for many years. 'Alaska' - tender. poukanense - hardy over many years, several locations. calendulaceum - dependably hardy. nudiflora - very hardy. 41 Standardization Rigid standardization of techniques, timing, and growth stages was found to be a necessary factor in estab- lishing a measure of reliability to readings. Severed plant parts continually change and soon can no longer adequately reflect living conditions. Techniques:--Apical twigs or stems to be measured were re- moved frdm intact plant and immediately placed in a poly- ethylene bag containing a damp paper towel to maintain humid- ity. These were carried to the laboratory, and, if frozen, allowed a.minimum.of thirty minutes to thaw at room tempera- ture. Twigs were removed from this microenvironment one at a time as they were measured, after*which they were again placed in the same type of container and frozen at minus 18° F. for approximately 18 hours and then read again. Timing:--When measuring the degree of hardiness, the plant material must conform to the stages of standard hardening conditions as earlier outlined. Otherwise the interplay of the developmental phases of hardiness are prone to be mis- leading. After material has been severed, it should be used for'measurements within a half hour after removal or thawing. Growth Stagee:~-For all purposes, it is best to select com- parable material from the same growth stage. Obviously green and brown, water sprouts and spurs, seedlings and 42 mature plants are not easily equated. It is also best to select twigs of even size if possible. However, the differences in diameter can be trans- fenced to a given dimension--2.0 mm--by arithmetical computa- tions. Standardisation as to loci of measuring was important during physiological changes of fall and spring. During this period, marked changes in hardiness between the tips and lower portion of a twig were apparent, as the twig changed from one condition to another. During periods of stable growing or hardiness conditions, this factor was negligible. Exposure to air was also controlled, as trials indi- cated that a moisture loss of approximately one-half milli- gram per 100 milligrams occurred each fifteen seconds from twigs with leaves. This loss was considerably reduced in plants without leaves, and further reduced with deciduous, winter hardened tissues. Plants growing under a moisture stress were quick to increase resistance to unfrozen measurements. Thus adequate moisture control is a prerequisite for measurement, unless base correlated plants are included as a standard. Temperature controls are also important, for, while hardiness is gained slowly, it is lost rapidly under elevated temperatures in the early stages-~often in a matter of a few hours. Twigs selected must also be in a healthy condition. 43 fleasurement of tender twigs with previously frozen tops revealed that injury had occurred internally from one to three centimeters basipetally, and that this was soon fol- lowed by dessication. Salt in concentrations leading to death of the plant from soil applications, had only a small effect on readings of resistance to conductance of electricity. While large differences in soil fertility and alkalinity can affect readings, the usual variations found in nature and gardens were negligible. Thus, the more precisely variables are controlled, the more precise are the measurements, and the more reliable the predictions will be. TEMPORAL m EDINESS In order to establish a foundation of known values for comparative evaluations through the year, Azalea plants of known hardiness were selected as 'base' plants. Selected plants were of a broad range of hardiness, established in the trade, and known to the majority of horticulturists in their respective areas. Azalea 'Polar Bear' was growing in a flower bed with living ground cover, without overhead shade, but with.some protection from a north tree belt. Variety 'Gloskey Pink' was grown in a raised peat bed in the open. These were approximately three to four year plants with one year of growth in the bed. 'Alaska' ‘was under heavy shade, and mulohed heavily. 'Corsage' was in a cold frame with.lath.ehading. The others were dis- persed at borders of wooded slopes with leaf mulch over the soil. The soil acidity, measured at two, three and five inches ranged from pH 6.0 to 6.5. While this is comparatively high, all except nudiflorg appeared to be healthy and vigor. ous. Leaves of nudiflora exhibited chlorosis during drier parts of summer, but otherwise plants were healthy and grew well. Natural state and post~freezin3 readings were made 44 45 following techniques previously described under 'Haterials and Hethods.’ Measurements were taken each two weeks during fall, winter and spring. During periods in which standard- ised hardening conditions were absent, readings were largely suspended, as of little value in this study. a few aberrant ratios were found, in which it appeared that either the pre- or post-freezing physiological processes were advanced temporally over the other in a particular twig. RATIO SEQUENCE FOR BASE PLANTS ‘6 EABLE l Day 13 27 11 86“ 24 5 20 4 is 261.: Bear 2.2 3.5 3.5 3.6 5.7 4.2 6.4 4.6 4.0 414.25? 11.7 4.7 3.0 3.6 -~- --- -.- -- -- 016424: Pink 3.7 5.0 4.5 6.3 6.6 4.7 6.5 7.3 7.6 6561:1624 -- 2.5 2.5 3.6 7.6 7.0 6.6 5.0 4.0 peak-lease .-- -- -~- 3.2 7.6 6.2 6.0 6.5 6.7 ealendulaeeu- --- —- --- ~- 5.6 3.2 3.5 3.9 2.9 Janos Gable 2.2 3.4 3.3 -- 6.4 4.2 5.6 3.6 5.1 laawelli Alba. 2.3 3.4 3.3 3.2 7.6 4.0 5.6 6.3 7.4 mic-lib 7;. Dee. Jun. Feb. war. was, 3161153118269239h3w 2514: Bear 3.4 4.0 5.6 2.5 2.5 1.3 1.3 1.0 -- 2.5!2.2 4114.24 --- -- -- --. -- --»-~ --- -- -.- -~ 016424: Pink 2.1 6.1 6.0 4.3 2.3 2.2 2.6 2.6 --- ---~3.6 Indiflora 3.2 4.2 2.6 1.0 1.0 0.3 1.0 1.0 0.5 1.0 2.7 9662464644 4.1 4.0 2.3 1.5 2.0 1.0 1.7 2.4 2.3 2.3 3.6 salendulaeeul. 3.4 4.5 1.2 1.5 1.2 1.0 1.2 11.0 1.0 1.0 2.9 as... 0451. 4.4 5.0 3.3 4.4 3.9 3.1 3.1 2.3 2.4 2.6 3.6 Iaawelli 4154 7.5 5.6 3.6 4.0 3.3 3.6 3.7 2.6 2.7 3.7 6.0 “Twigs injured by freesing were discarded. 'Polar Bear'eAsalea on February 23 were under three feet of packed damage had killed it. 80 readings taken. 'Corsage' is not shown, as frost .oQHH snow phonwuu.exmofia. “chad Ufiaomnu.ednoc meson. MonHH seen wconncoAOHmHusc .m answn< Haamd nonsounom no 7 4. £0962 nopouoo unneeboz nonaeooz haescoh nonaboon mdmnoz haeonsn homeroom nore viscous with the dehydration of hardening, it was postulated that a gradation of resist- ances would be found commensurate with differences in hardi- ness. Combining the ideas implied in the above reports, the two readings were postulated to express measurements. CONCLUSIONS The nethod of comparison of resistance to electrical conduction may be used to compare the degree of hardiness of plants with an unknown hardiness to measurements of plants of known hardiness for determination of relative hardiness. With this information, the reaction of a plant in regard to winter cold resistance may be pro-determined on a relative basis. Plants may be tested for any degree of cold tolerance desired. To be reliable, testing,must be done after harden- ing conditions have induced the physiological reactions re- sulting in tolerance to a given degree of cold. Cold hardiness is gained slowly and is easily dis- sipated by elevated temperatures during early phases of the hardening process. The degree of hardiness increases over a long period until limited by the physiological capability of the plant to resist freezing dmmage. Conclusions for Nutrient Levels-«The differences in concsn~ tration of soil nutrients appears to affect readings of electrical conductance to some extent. Under the concentra- tions found under average growing conditions in gardens, this effect probably 1. negligible, but could be important. This indicates that measurements of plants growing in areas of high. 80 81 or low soil nutrient concentrations should be measured with a ”base selection" plant from the same area so that adequate comparisons of hardiness may be made. Measurements of Asaleas tested in the fall corre- sponded closely with results of recovery and survival in spring. 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