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BRso DING DIswéAsu ResISTANT STRAINS OF PHASHOLUS VULGaRIS BRuGDING DIiSwasm ReSISTANT STRAINS OF PHASEOLUS VUDGARIS THESIS Respectfully submitted in partial fulfillment for a Master of Science degree at the Michigan Agricultural College Grosvenor Ward Pujnam LY¥Kae “Plate I- 108394 -TABLE OF CONTENT S- I - INTRODUCTION.- ~---- "== = er eee ew ee 1 II - PREVIOUS INVESTIGATIONS- - -----=- 2s - - - - a III - MATERIALS AND MinTHODS - - = = = ee ee ee ee 3 A-Varieties Used- - -----+--+- +e 27 2-2 = 4 l-Wells Red Kidney- ----+-+-+-+-*+-+e+e- 4 2-Robuste = === -- = gH ke eo “= = - = 4 S5-California Yellow Eye- - ----+-+«--+-+e-- 5 B-Method of Investigation- - ----+-+-+-- -~ - - 5 IV - TECHNIQUE OF HYBRIDIZATION- - - ----2----e-- 6 A-Hybridizing in the Pield- - ----+-+-e-+-#- 6 B-Hybridizing in the Greenhouse- - - - --+-=- 7 B- DATA=©§ - - 2 -e-- ee ere err rr Kr rrr te 9 A-Breeding- - -----+-"-2e# s|=f-e-- ~eer- YQ 1-Type of Cross Made and Pods Set- - - - - LO e2-Accession Numbers Assigned- - - - - - = 11 d-Progeny of FP)..1917- - ----+--- - - 13 a-Accession 166- -----*+-+e-+-e+--- 13 be-Accession 1l67- ---+-+e+e-re- 2 @& 13 4-Progeny of Po--1918- - - ------- > 13 a-Accession 166- ----+-*#-+e-ee6 13 b-Accession lL67-] - = = - +--+ - = = = & 13 Page o-Progeny of F,--1919- - - ----- = - = 14 a-Accession 166- - - - ------+e-- 14 b-Accession 167- ---*+-+e*e2- 2 = - = L5 6-Progeny of Fy--1920- - ---- = ---- 16 a-Accession 166- - - - - - - -k“- ce - 16 b-Accession 167 --- eee eg ee ee - 17 B-Disease Resistance- - -----+*--e# === - 17 l-Disease Reports- - - ---- - 19, 20, el, &2 VI - @PNCLUSIONS- - --- - -- - ee eer er ere eee ad VII - ACKNOWLEDGEMENT- - - --- 7-7-7 - - et ee te ee a4 VIII - BIBLIOGRAPHY- - - ----= ef-s e--= te ke ek eK “Oo ZL - INTRODUCTION Plant pathology or the study of plant diseases has been the subject for a great deal of original re- search work. Both the cause of disease in plants and their control or elimination have been carefully invesii- gated. The pathologists have found that certsin types of diseases do not readily subject themselves to control or eradication by the practical means available to the producer of our commercial plant products. Plant breeding, or the improvement of plants by se- lection, has also been tle subject of investigation since man first recognized that variation occurred in the plant forms. Hybridization has produced the science of genetics during the past twenty years. In the study of plant varia- tion and inheritavle characters, geneticists have recently found thst strains or varieties of the same species vary in their ability to resist and evade disease attack. In the past few years considerable attention has been given to the production of varieties of farm crops which are resistant or immune to their disease enemies. The purpose of this investigution is to develop a white pea bean that will carry the commercial importance of the white pea bean trade and the disease resistance of the Wells Red Kidney bean. Il - PRuVIOUS INVastTIGaTiIUs The most important previous work, bearing on this investigation, is that of Barrus (1915) in demonstrating the practical resistance of the Wells Red Kidney bean, and the work of Emerson (1ly0i-03-05-08-09) in his studies on bean hybrids. The work of several investigators at the Cornell laboratories on bean diseases and the securing of resistant strains by hybridization started at about the same time as did this investigation now being reported. A report of the Cornell work in which they secured resistant strains by hyb¥idization has recently been published in Phytopathology and supports many of the conclusions given in this thesis. Burkholder (1918) was able to obtain a resistant Merrowfat strain. McRostie (1¥1Y¥) working with Wells Red Kidney and Robust began his work in the fall of 1916, while the writer using the same materials started work in the summer of 1916. - 3 McRostie studied the inheritance factor of bean anthrac- nose resistance, and incidentally was looking for a commercial white pea bean resistant to the disease. In his work McRostie started in with the F, and tested each Succeeding generation for resistance. He and also Burx- holder found that resistance was dominant and that approximately seventy-five percent of his F. were resistant; twenty-five percent of which were homozygous and fifty per- cent heteroz, gous. MoRostie states in conclusion that he has been able to isolate several resistant types of white pea beans, but that considerable work is necessary before a commercially important resistant white pea bean can be properly fixed to breed true. The work of Reddick and stewart (1919) in their investi- gations on bean mosaic has established another economic phase of this particular investigation, in that they have determined that the Robust bean is resistant to the mosaic disease, so that strainag from the Wells Red Kidney by Robust cross have the possibility of being resistant to both anthrac- nose and mosaic. III - MATERIALS AND METHODS In undertaking this problem the writer has followed the suggestions of Professor FP. A. Spragg in selecting varieties -4- and in method of procedure in the investigation. In determining to what extent the work has been successful, the writer wishes to acknowledge the cooperation of Dre G He Coons, Pathologist, who Kindly consented to give the desired strains a test as to their resistant qualities, and whose report is incorporated ss a part of this thesis. The varieties used in the first attempt at this problem consisted of some twenty-eight varieties and strains then on test oy Professor Spragg; reciprocal crosses were made and in each case Wells xed Kidney was one of the reciprocals. A - Varieties Used The varieties used in the tinal attempt were Wells Red Kidney, Robust and California Yellow Eye. The following is @ complete description of these three varieties:- I - Wells Red Kidney Tis is a typical bush type, green podded, self colored, red kidney bean, which Barrus (1915) demonstrated to be resistant to the A strain of Colletotrichum lLindemuthianun, and practically resistant to the F strain. MThese two strains, according to Dr. Barrus, constitute the occurring disease under any trials that he thas made covering a wide range of distribution. «-Robust This is a typical navy pea bean of the intermediate indeterminant sort, having originated as a single plant -5- selection by Professor Spragg. McRostie (1914) states that Dr. Barrus reports it as being resistant to the F strain of Colletotrichum lindemuthianum, but as being very susceptible to the A strain. Reddick and Stewart (191%) report it as being resistant to bean mosaic. 4-California Yellow Eye This is a self color yellow pea bean of the intermediate indeterminate sort. The name seems to be a misnomer, as the bean has no distinct eye color, but is a sulphur color. fo the best of our knowledge it is a pure line, but has not been tested for its resistant qualities. It was used in this investigation because of its reputed excellence ss a coocing bean. B-Method of Investigation The method pursued in this investigation of obtaining resistant strains of beans has been somewhat different from Otuer investigators worxing with similar ma terial, in that they have subjected their early generations to epidemics of the disease, and then proceeding to isolate desirable com- Mercial strains from these resistant types. The writer hus attacxed the problem in reverse order to this, that is, by first selecting out of the segregating progenies desirable commercial types, and then applying the disease test. In this way as soon as a resistant strain has been found among -6- the desirable commercial types it is a gimple problem of increasing for distribution. IV - TxCHNIGUE OF HYBRIDIZATION The method of hybridizing is so well understooé that Lt hardly seems necessary to go into detail, and yet every plant and every new set of field and climatic conditions gives to each problem and to each investigator an individual Case. Beans are naturally self-fertilized, though the writer observed a condition reported by Techermak and others, which accounts for some cross-fertiilization in beans. In a few instances, cases were observed where the pistile hed pene- trated the Keel of the flower and thus become separated from any chance of receiving its own pollen, and liable to receive the wind blown pollen of mature flowers, or contamination from the pollen carried by visiting insects. A - Hybridizing in the Pield The problem of securing "sets" ig a good deal more difficult umder field conditions than under greenhouse conditions. Under field conditions so much depends on the weather. When the writer arrived at kast Lansing in July of 1916 to begin this hybridizing, the weather was decided- ly unfavorable,as a great many of the normal flowers were 7 - turning yellow and dropping because of excessive heat. The emasculated flowers were even more susceptible to the heat and considerable difficulty was encountered be- fore sny pods would set. During the first three weexs crossing was attempted on a great many flowers without obtaining a single pod. In desperation an attempt was made to use a weak sugar solution (one-half of one percent) to aid in securing fertilization. xtreme care was used to emasculate just before the anthers would normally discharge their pollen, in ordur to faciilate fertilization. Fresh polien was collected at frequent intervals but with no avail. In early august there came a week of cool, moist weather and the difficulty was solved as the pods began to set. aA total of fifteen pods wag Obtained during the summer from rive hundred and six attempted crosses. Because of the lateness of the cool weather and of the sets obtained, fourteen of the fifteen were caught by frost before reaching maturity; and the one that matured proved to be a self, so that the summer had given some valuable experience in technique but no beans. B ~ Hybridizing in the Greenhouse Realizing that it was necessary to get some crosses made during the winter if the problem was to get undcrway, ete arrangements were made with the Lansing Floral Company of West Karnes Avenue, Lansing, wichigan, for space to make some winter plantings, no space being avaiiable in the college greenhouse. On October 17, 1¥16, sixty-five pots of beans were planted in three inch pots, fillea with ordinary green- house potting soil, and these pots set in flats and filled in around with dirt to better hold the moisture. The sixty- six pots were planted as follows: - 25 pots of 630302. (Robust) 13 pots of 60901. (Wells Red cidney from Cornell) le pots of Crimson Rummer. 4 pots of 61v0801. (California Yellow Eye) 4 pots of 61090%. (California Yellow Eye) 4 pots of 61410¢. (California Yellow Kye) 4 oots of 614103. (California Yellow iye) On January 20, 1¥17, seventy-three additional pots were planted as follows:- 20 pots of 60501 - 3 - 4 = 6. (Wells Red Kidney) 20 pots of 613700. (California Yellow Eye) 28 pots of 630501. (Robust) 6 pots of Crimson Rummer. With the controlled conditions of the Leat ana moisture in thw greenhouse it is a much easier inatter to -J- get "sets" than in the field, except as the weather happens to be favorable out-of-doors. Some difficulty was encountered in the greenhouse because the house was used largely for the propogation of flowers and was msually too cool for ripening beans. ‘The technique was the same ag used in the field in the summer, except no Sugar gsOlution was used. Crossing was faciliated by placing the plants side by side, thus saving the carrying of the pollen of the male plant around. aA special pair of forceps, obtained from a dentist's set, was usea, which enabled the remov- ing of anthers from the mother flower with a minimum of mutilation. A camels-hair brush was found convenient for dusting on the pollen, care being used to avoid any con- tamination by sterilizing when changing pollen parents. V- DAA In working up the data of this: thesis it has been found rather difficult to offer it in table form, so that most of it is given by steps in the order in which the results were obtained. A - Breeding Te method of hybridizing having been discussed, we are now concerned with the results obtaineu from the -l0O- crosses made in the greenhouse during the winter of 1916-17. I - Type of Cross Maée and Pods set Mother Parent 613904 610906 60901 605038 60503 640501 60504 650501 630501 60 506 613801 60 504 60506 Male Parent 630501 Crimson Runner 600501 600501 Crimson Runner 605 (Tag blurred) 650601 Tag blurred 60506 63050 1 630501 Crimson Runner 615801 Number of Pods Set me PF RP BH NO KF KF # ~ PF NM F&F RK In the spring of 1917 those plants in the green- house that had crosses setting on them were taxen up and transplatted in the alfalfa nursery plat east of the M. A. Ce Dairy Building. The writer found it necessary to give up his investigation for a time and, on leaving, -|ll- arrangements were made with Professor Spragg to have the beang harvested when mature and planted. The writer was unable to return to this investiga- tion until the winter of 1918-19. In the mean- time Professor Spragg had Kept the material going, having raised the FP, seed in 1917 and the Ps seed in 1918. The writer was stationed at the Upper Penin- Sula Experiment Station during the summer months of 1919, 1920 and l¥cl, but was at the college during the winter months, at which time the bean material was studied and the next year's seed made ready. By this arrangement much more complete data has been ob- tained than is usually possible in an investigation of this kind. e-Accession Numbers assigned Early in June, 1lvl7, the F) beans were harvested and given accession numbers. They were then ready for planting. A register was made up and the following ~lz- notes taxen:- Register Cross Acces- Type Color Date Date Number. Sion No. of of plan- har- bean. bean. tea.vested. 724000 60506 16D Navy White 6/e2 10/5 GO0DUL 7a81LU0 600501 l66 «idney Red 6/a2 10/1é BO50S— 748000 615801 167 Kidney Red 6/22 10/15 7283800 660501 l6s Navy White 7/lo Frosted Y Tag biurred 728400 60501 169 Kidney Co lox - 7/lo Frosied BS0501 & 728500 60506 170 Kidney Color- 7/lo Frosted e 748600 ? Tag blurred 171 Navy White 7/15 FProsted * fag bdlurred 728700 60500 i7a Kidney Color- 7/ls Frosted 60050 L ed It is oosgerved that five of the plots were caugkt by frost and only three were harvested. Of these tlree, accession number 165 proved to be a self, as it bred true to the Robust type in lyls. There were left two crosses with which this paper is concerned. They are accession number 166, the Robust pollen on a Wells Red Kidney flower and accession number 167, California Yellow kye pollen on a Wells Red Kidney flower. Mese two crosses will be referred to as -13- accession 166 and 16”, thrcugh Succeeding generations of the resulting progeny. S-Progeny of Fi--1917 a. Accession lsoo--Ten red Kidney beans produced ten red Kidney viney plants. The F., seeds had the appearance of red kidney beans because the F, plants that produced them were of the Red Kidney type and, Since the bean seed coat is mother tissue, they appesr to be red kidney. b. Accession 167--Three red kidney beans produeed three vine plants that developed 1<0 FP, seeds. 4-Progeny of F,,~- L916 a. Accession 166--Kight of the 10 FL plants were selected in 1918. The seed of each selected plant was planted in @ separate piot. In all there were 550 PF, Seeds planted. Some of these produced bush type and cthers indeterminate plants. There were in all 4,605 F., seeds saved from these F. plants. The writer, returning to the investigation during the winter of 1918-ly had an opportunity of studying the Fz seed in detail. One of the interesting characters -13 ae- -Plate II- Pana | rie. mA des a The 1919 Bean Nursery. The Fz plants of accession 166 and 167 are located at the back of the nursery. brought out in this generation is a black color factor. tmerson explains this condition by assuming that the white oean carries either the pigment factor or the color factor; in the absence of one the pigment fails to deveiope. The red Kidney carries another color factor and the pigment factor. In the recombination of characters any bean that carries the black pigment factor and also the color factor would develop into 4 blac« bean. In working up these F, seeds for 1yl9 planting, something of the complexity of the problem could be ascertained by the great diversity of types into which this generation could be grouped. b. Accession 167--The seed of the three F, plants were 1 pianted in separate plots in 1918. The one hundred and twenty FP. seeds produced vine type plants that developed only 370 Fa seeds. Many of these F. plants were very weak and failed to mature seed which accounts for the small increase in this particular cross. o-Progeny of FPu--ivly a. Accession i66--Of the Lodl Fy plants saved in 1918 for iy1y9 planting, eighteen proauced white beans, sixty- five produced red Kidney and sixty-eight produced some -~15- Other color of bean. Since the problem was to select out desirable comnercial types, ail of the variable characters had to be considered. Many of those that Lad apparently oecome homozygous for color were still heterozy- gous for size and shape, also heterozygous for bush and vine character. In arranging the Register for the i9ly planting, the F., plants of 1¥18 with their progeny of P., beans were grouped together for the purpose of field study into homozygous whites, homozygous reds and heterozy- gous other colors. Since part of the population had been dropped it was not necessary to classify them into any Mendelian ratio. Something over 4,000 Fa plants were planted in 19149. MThe plants were carefully studied during the summer of 1y1¥. It was observed that many of the strains had apparently taken on all of the weak characters of both parents. These strains were dropped because they gave very little if any promise of having commercial value. Pifty- three of the 151 plats planted in this year were discarded in the field. Ninety-three strains, representing 1460 P, plants were harvested for study during the winter. b. Accession 167--The 370 B,; Seeds of 1918 developed sonething over SOO Fs plants jin 1419. Many of these plants were very weak und failed to produce seed. Only fifty-eight -15 Ger ~Plate III- The 1920 Bean Nursery. Many of the apparent- ly desirable strains of 166 and 167 were retsined for increase. plants were harvested for study during the winter. 6-Progeny of Fya--196e0 @. Accession 166--With the evidence of some strains having segregated into a homozygous condition for the more important characters involved, it seemed desirable to study the material during the winter of 1919-20 with the view of making the final test to determine whether or not resistance had been fixed in any of the types that gave evidence of having commercial value. The 1,260 Fz plants of 1919 were classified according to the way in which the characters had segregated into a fairly well fixed type. It was found that 188 were intermediate white navy type, 175 were bush type red xidney, 897 were still breaking up, some were white navies on bush plants, some red kidneys on vine plants, and in fact all gradations that we would expect coming from a population of this sort. Seventy progenies of Fz Plants, that were apparently breed- ing true, were turned over to Dr. G He. Coons, Pathologist, to be tested for their resistance to disease. These P, seed were tested during the summer of 1920 by Dr. Coons and under date of September 15, 1920, he submitted a report as to their performance. -17~- b. Accession 167--The fifty-eight Fg plants and their progeny of P, seed of this cross were carefully studied during the winter of 191ly¥-cO to determine whether or not this population should be discarded or preserved for further study. Nothing of importance had come out of this cross in the previous generation, but in studying these Fa plants it was observed that some of them were producing white Fy seed. Since this is a cross between two pigmented varieties and since the California Yellow Kye is reputed to have exceptional cooking qualities, it was thought that a white strain might have some commercial importance, so that this population along with the remnant of the 166 was turned over to Professor Spragg for further consideration #s a station problen. B-Disease Resistance It is only in the past few years that investigators have been working on this problem of disease resistance. kKven yet it is not very clearly understood as to what con- stitutes resistance. For tunately there has been consider- able work on the bean diseases in the past few years. McRostie (1919) states that there is a single factor dif- ference between resistance and susceptibility to bean -~18- anthracnose and that resistance acts as a dominant. If this is the case, then we would expect it to act as any other single factor difference and under autogamous no selection, segregate itself into homozygous pure lines at about the fourteenth generation. By applying the formula 2 n-1, where n refers to Fr, we find that in the en fourth generation we have a ratio of seven homozygous resistant to two heterozygous resistant to seven homozygous susceptibles. By analyzing Dr. Coons' report,we find 55 showing resistance and 15 susceptible to bean anthracnose. If these 70 strains have been uninfluenced,so far as resistance susceptibility is concerned, by the selections previously made and if they are in the fourth filial generation, so far as this disease character is concerned, the genetic expecta- tion would be that approximately 39 would show resistance and Si: susceptibility. If this is the case we would expect that 16 of the 55 on further test would prove to be susceptible. Considering the data from another angle,it is probable that in discarding certain strains from time to time,whole series of homozygotes have been eliminated and probably some of the seventy strains tested are actually in the Fo generation so far as the segregating of this disease character is concerned. We find in the F, generation, the genetic expectation would be 52.5 showing resistance and 17.5 showing susceptibility. -l8 Qae> -Plate-IV- The 1920 Bean Nursery at harvest time. The vacant spots in the rows show where the desirable strains have been harvested. ‘The bulk of the mater- ial was found unsuited for further increase. ~19= It is probable that all of these influences are at work and the population does not coincide with any one genetic expectation. REPORT ON PUTINAM'S BeaN CROSSHS-SiPT. 15, 1920 Made by Dr. G. He Coons Regis- Anthrac- Blight Yield Matur- MType Remarks ter No. nose ity 90203 0 t Good Good Navy Save 90206 0 t Heavy Fair Navy Save 90213 O t Fair Poor Navy 90219 t tT Poor. Navy Two plants (Broad) only. t ( Pods) 904026 0 Poor No pods set. One plant. 90515 O t kxcel-Gvod Navy Broad Pods lent 90508) ) One poor plant only on each. 90 501) 90417 | Poor Navy Stand Poor | tr 9041lz ? Poor Late Leaves liv- ed on vines. 90407 0 t Heavy Late R. Ke 90524 O Slight Fair Late R. K. 90702 O severe Poor Late R. K. 90811 ? tv eoor Fair R. K. 90708 O t Heavy Fair Navy 90713 t One weak plant. 90716 t Few weak plants. @iO- .Regis- Anthrac- Blight Yield Matur- Type Remarks ter No. nose ity 90801 on Slight Good Good Strong Plants 3 pods by leaves (out) 90806 0O + Low Fair Re ie 90814 O t Poor Late Low growth 91104 0 O Low Fair Re Ke 91107 + 0 Fair Late Upright. 91111 O t Good Late Navy 91115 + t Fair Fair R. K. 91402 0 t Fair Good R. K. 91406 0 t Fair Good Rk. K. 91409 Oo t Fair Good R. K. 91414 0 ¥ Good Late Navy 0. xk. 91602 0O ? Low Karly Navy 91801 0O t Fair karly Navy 91805 0 ? Fair Good Rk. K. 91811 O t Low Karly Navy 91821 O Slight Low harly Navy 92704 tr Fair Fair RR. K. 92713 O t Good Good R&R. K. 93408 f¢ t Poor R. K. 93211 + t Poor R. X. cut. 93213 + t Fair Late Three strong plants. 95302 O t Good Good Rk. K. 95309 0 t Good Good R. K. egl- Regis-