some FACTORS INHUENCING me FORMATION OF I, saw BETWEEN VAmmes OF OAT: Mskforfiubcgman.& MiCHQGA-N STATE COLLEGI Samuel Linea: Hagar 1948 THEsls This is to certify that the thesis entitled "some Factors Influencing the Formation of F1 Seed Between Varieties of Cats." presented by Samuel L. Eager has been aecepted towards fulfillment of the requirements for u -I as degree finialth—I‘st Major professor Date Avgust 28, 1948 M-795 A -r.._".-‘.. .v —r '- WM-“ SOME FACTORS INFLUENCING THE FORMATION OF F1 SEED BE'I‘WEDI VARIETIES 0F OATS By Samuel Lineaa Hagar M A THESIS Suhnitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requiranente for the degree of MASTER OF SCIENCE Department of Farm Crops 1948 THEg [S ACKNOWLEDGMENT The writer is grateful to Dr. E. E. Down for guidance and aid in conducting this experiment and for the review of this thesis. The helpful suggestions given by Dr. S. '1'. Dexter are appreciated. 206047 TABLE OF CONTENTS I . INTRODUCTION A. Purpose of experiment B. Review of literature II. PRELJJHNARY GREENHOUSE EXPERIMENT 1947 A. Method B. Data 0. Discussion III. FIELD EXPERDIENT 191.7 A. Plan B. Materials C. Method D.Data E. Discussion IV. FIELD EXPERIMEJT 1948 A. Plan B. Materials Co MethOd D. Data E. Discussion V. COMPARISON OF RESULTS 191.7 AND 1948 VI. CONCLUSIONS VII. REVIEW OF LITERATURE DON dumb 10 E 18 S 83888 R a t INTROIUCTION In plant breeding, it is recognized generally that the more material there is with which to work, especially during the earlier phases of the project, the greater are the chances that the desired ends will be accompolished. When oats, a plant normally self-pollinated, are cross-pollinated artificially, a very small percentage of the florets produce grain. The difficulty with which seeds are secured following cross-pollination of cats is undesirable not only because it results in Just a few seed with which to start the 1'1 generation but also because it makes it impractical, if not impossible, to carry on a program of back-crossing. Back-crossing is a very useful method that plant breeders may employ to concentrate and to render in pure lines, with a substantial saving of time, desired characteristics of the varieties involved in the cross. Without back-crossing, several extra generations would be required before chance segregation and combination would unite the sought after characteristics. For a back- cross program to be very successful, new pollinationa would have to be made followed by a reasonably high per- ' contage of set. Why oats fail to produce seeds after the flowers -2. have been cross-pollinated artificially is not thoroughly understood. Oat florets, anaeculated and pollinated, are subjected to a process that visibly injures than. A partial drying of the outer floral parts is generally evident. The glunes can not be replaced about the pistil to give it the protection that they afforded before the flower was manipulated. Of the many things that could influence the setting of seeds after cross-pollination, air moisture was considered to be one of then. 0n the foregoing postulation, this experiment was based. The purpose of this experiment was to determine whether it would be possible to increase the set of seed following cross-pollination of cats by enclosing the heads with a suitable container along with a chenical compound designed to have some influence on the moisture content of the encased air. It is the property of any salt in a saturated solution to maintain an equilibrimn vapor pressure (1. e. a constant relative humidity) with a confined atansphere at a constant temperature ( 7 ). The vapor pressure maintained will depend upon the compound in solution and the temperature. This eccperiment is to follow closely the theory and the procedures used by Dexter ( 2, 3 ) in the curing and processing of farm products to the desired moisture content by the use of suitable salts impregnated into wooden blocks. REVIEW'OF.LITERATURE Much data are available dealing with the correlation of various phases of plant growth to airnmoisture. Some of the data concerns plant responses under conditions of controlled air moisture; other data show the relation of the natural atomspheric humidity to various plant responses. lbrk that has dealt with controlled humidity in relation to seed set, or with the correlation of seed set with humidity after cross-pollination will be cited. Hollowell (6), working with red clover, came to the conclusion that atomspheric humidity did not affect the setting of seed following cross-pollination under greenhouse or field conditions. Grandfield (4), working with alfalfa, found that the number of flowers setting pods increased as the relative humidity decreased from 90 to 10 percent. Dexter (2) and Dexter and Creighton (3) describe methods to bring certain farm products to a desired moisture content by the use of wooden blocks impregnated.with suitable salts. The blocks function to decrease, or increase, the relative humidity in the surrounding atomsphere. mamas! O‘HOUSE EXPERIMENT To obtain preliminary information on the problem as stated, work was started in the greenhouse during the winter and spring of 191.7 to determine 1. what type of bag should be used to cover the pollinated heads and 2. what method should be used to apply the salts. An attanpt was made to keep the greenhouse temperatures around 60 degrees F. during the night and 75 1'. during the day. Occasionally during the winter and early spring because of steam pressure failure, the temperature fell to 48-55 F. at night; and on the coldest days, the tuperature would not go above 60 1“. During the late spring, the temperature on the warm, sunmr days would range frequently from 80 to 90 degrees F. Several strains of oats were used to make the crosses. No attanpt was made to use the same variety for the fanale throughout the experimmt. When pollen was needed ,it was secured from m variety that was shedding pollen at the time as long as this variety was not the same as the one one being pollinated. Pollination was made immediately after emasculation, but care was takai not to pollinate am floret an anther of which had showed signs of shedding pollen even after the anther had been tapped against the thumbnail. Briefly the treatments in the greenhouse were as follows: No. 1. Heads were covered with a 2% by 6 inch glasine enclosing a vial filled with a saturated salt solution with a 1% inch cotton wick extending from the vial. No. 2. Heads were covered with a cellophane bag, 2% by 3 by 8 inch with pleated sides, and the salts were applied by the vial and wick method as outlined above. No. 3. Heads were covered with a cellophane bag of the type mentioned above, and the salts were applied impregnated into wooden blocks 3/4 by 3/4 by 3% inches. No. 4. Heads were covered with a pliofilm turkey bag, 10 by 12 by 24 inch pleated side, and the salts were applied using the wooden blocks. No. 5. Heads were covered with the pliofilm turkey bags, and the salts applied by the vial and wick method. No. 6. Heads were covered with the glasine bag,5 by 3}; by 11 inches pleated side , and the salts applied using the wooden block method. A few heads were left in the open after they had been pollinated. In some instances water was used in the vials without any salt. DATA FOR GREENHOU SE The results of the treatments in the greenhouse for 191.7 are as follows. (Table on following page) .6- Table No. 1. Results of greenhouse experiment. Florets No. Salt Humidity: ngLinated set Z set Method 1. Glasine bag 2% by 6 inches with wick. Water 100 27 O 0 0.3804 98 71 7 9.9 Na2303 95 36 0 O NaZSOL 93 68 12 17.7 211304 90 82 6 7.3 KBr 84 10 1 10.1 NHACl 79 17 0 0 Left in open - 1.6 l 2.2 Total - 357 21 5.9 Method 2. Cellophane bag with wick. Water 100 36 2 5.2 113(02 H302)2 65 1.4 l 2.3 K02H302 20 52 2 3.6 Left in open - 42 0 0 Total - 171. 5 2.8 Method 3. Cellophane bag with blocks. Mg(023302)2 65 14 0 o CaClg 32 20 O 0 Total - 34 0 0 (7% The humidity as given shows the equilibrium relative tunnidity that will be maintained in a closed atomsphere over a saturated solution of the given salt at a tenperature of 20 degrees C. No claim is made that these humidities were maintained within the bags covering the heads. Table No. 1. Continued firsts Salt Humidity pgllinated N9. set zest Method 1.. Turkey bags with blocks. NsZHP04 95 24 4 16-7 Na02H302 76 21 0 O M23302»; 65 24 3 12.5 NaBr 58 20 l 5.0 CaCl2 32 21 9 43.0 Total - 110 17 15.4 Method 5. Turkey bags with wicks. Nazi-POL 95 27 4 14-2 ZnSOL 9O 17 8 47.1 Ms(02H302) 65 9 1 11.1 KCzH3OZ 20 83 15 18.1 Total - 136 28 20.6 Method 6. Glasine bag 5 by at by 11 inches with blocks. Na02H302 76 10 O O Mgfczfigxoz) 65 27 7 25.9 NaBr 58 31 8 25.8 08.012 32 10 .6 60.0 Bag alone - 15 7 46.6 Total - 93 28 30.2 Several different kinds of salts were used in the preliminary experiment. The same ones were not used necessarily with all of the different methods of applying the salts. -8- The different methods are listed in the chronological order that they were applied. Method 1 was used in the colder part of the year. The last one, number 6, was used during the warmer part of the spring in May. Temperature differences in the greenhouse may have had some effect on the number of seed set following the cross-pollinations. Moisture would collect on the inside of the pliofilm turkey bags and on the inside of the cellophane bags soon after the bags had been used to cover the pollinated heads. This water apparently had been transpired by the head. The adsorbing area seeded to be too small to take up the water as fast as it was given off by the plant. The observation, that water collected within the bags that were impervious to moisture, led to the conclusion that the plant was capable of giving off sufficient moisture through the enclosed head to bring the air within the bag to the saturation point. It seemed , therefore; that the problem would be to get rid of the excess moisture rather than to provide other means to raise the moisture content within the bags. Grandfield and Zink (5) found it necessary to introduce dry air into humidity control cabinets during the daylight hours to counterbalance the effect of the moisture transpired by the plant. In the greenhouse experiment, the 5 by 3% by 11 inch glasine gave the best set after cross-pollination. .9- FIELD MWT 191.7 After profiting from the preliminary experiment in the greenhouse, a new study was made in the field during the smmner of 191.7. The varieties selected for use in this experiment were as follows: females , two white seeded oats, Eaton and 3909 (a variety being tested at the Michigan State College Meriment Station); males, two yellow seeded oats, Benton and Clinton, and two reddish seeded oats, Bonds and Bonham. These varieties were chosen because the yellow color of the seed of Clinton and of Dalton and the reddish color of Bonds and of Bonham could be used as an index of crossing when either of these varieties were crossed on to one of the white seeded varieties. On May 6th, seed of the foregoing varieties were space planted in separate rows three feet apart. The seeds of Eaton and 3909 were planted at approximately 8 inches apart in the rows. The seeds of the varieties used to supply the pollen were sown about 1. inches apart. The planting was made in such a manner in order to faci- litate working around the plants later in the season. Also, it was designed this way so the plants would tiller . much more than normal to give more heads over a longer period of time than would have been 'usual following a drilling of the seeds in rows. ~10- As stated earlier, the object of this experiment was to get an increased set of cross—peninated oat flowers. An attenpt was to be made to influence favorably the moisture content of the atomsphere surrounding the cross-pollinated heads by the employment of suitable salts and by covering the heads with a suitable bag. The salts selected for use during the summer of 1947 were £32503, ZnSOL, KBr, NHACl, Na'Br, llg(02 H3 02), CaClz, and NazflPOA. These salts were impregnated into western yellow pine blocks which were 3% by 3/1. by 3/4 inches in size. The blocks were soaked in a saturated, boiling solution of a particular salt for twenty-four hours during the first impregnation. When the blocks were made reach for re-use, they were soaked for only three or four hours. After soaking, the blocks were dried overnight in an oven at a temperature near 150 degrees C. The blocks of the different salts, after drying, were wrapped in separate cellophane bags to protect thm from adsorbdng moisture from the atomsphere. The blocks renained wrapped until they were used in the field. The type of covering used to put over the pollinated heads were glasine bags: Size No. 1 white, pleated side, ' 5 by 3% by 11 inches, weight 25 lbs. This bag is semi- transparent. It is neither water proof nor air proof. It is somewhat water and air resistant. This type of bag was used in order that some of the water transpired by the plant could escape to the free atomsphere. On July 7th, the oats had started to head. The variety, 3909, was a few days earlier than the Eaton; therefore the 3909 was used as the female for the first two replications of the experiment . At the beginning of the experiment, those heads that were the fartherest out of the boot were masculated first. The florets selected for emasculation were located near the top of the head. As the season advanced, the florets anaeculated were located farther and farther down the head so that near the end of the flowering period the florets wasculated were those which were located at the basal portions of the head. As the season advanced,there was a general reduction in the number and size of the florets available for was culation and pollination. A pair of curved nosed forcepts that had been filed down to make a flat point were used to emasculate the oat flowers. A description of the actual mode of masculation followed during the smaller will be given. Florets that had shed pollen, or that were near to shedding pollen, were cut from the plant with a pair of scissors. Then the florets on the plant that appeared to be in such a stage of development that the anthers in them would have shed pollen in the next day or two were emasculated. The remainder of .12.. the florets that had small immature stigmas and anthers were cut from the plant. This procedure left on the average 15 to 22 florets per head. Sometimes there were more ; sometimes, less. ‘ To masculate, the innermost of the outer glumes was folded back with the aid of the forcepts. The axillary oat was thus exposed. Next the axillary floret was moved. Then the palea of the main floret was pulled back exposing the anthers . The three anthers were removed. Finally, the palea and the outer glume were replaced to the normal position that they had occupied. Imediately after the emasculation of all of the florets left on the head, the head was covered with a small glasine bag of the type commonly used to cover corn ear buds. The bag was folded at the bottom and fastened with a paper clip. The heads were kept covered so that the chances of stray pollen fertilizing the florets would be reduced. The masculated heads remained covered until the time of pollination. Part of the enasculation was done in the forenoon and part, in the afternoon whichever fitted into the schedule of work for the day the better. All through the process of masculation, care was taken to prevent the florets being emasculated from becoming accidentally pollinated. The time allowed to elapse between enasculation and -13.. pollination varied from one to three days. No attempt was made to systuatize the length of time between enasculation and pollination with the subsequent treatments. A block of heads of the same variety were anasculated in the morning before the work of pollination for that day had begun, or in the afternoon after the pollination had been completed. These heads were pollinated during the next three days until all had been used. To get pollen, a mall handfull of florets were collected from the male; and were used to supply pollen until their glumes began to wilt. The flowers were opened, and enough ripe anthers were collected to pollinate the florets on one head. These anthere were put in a small cardboard box while being collected, and then used imediately to pollinate only one head. In this manner, an attempt was made to insure fresh anthers for use on each head. Pollen from Bonds was used to cross with the 3909; and pollen from Bonham, to pollinate Eaton. In case there was no pollen being shed by the variety required, pollen was gathered from any one of the varieties designated as male. In this experiment , it was considered that the source of pollm made no difference. It may be well to note that late in the afternoon, it became difficult to find sufficient pollen for crossing the heads. Generally by 3 to 5 o'clock in the afternoon, all of the oat flowers that were going to shed pollen that afternoon had dehisced; and the florets had opened up scattering their pollen in the wind. Near the end of the crossing season, some difficulty was experienced collecting pollen. Then only a few flowers were shedding pollen at a given time. The anthers , too , were smaller and not as well matured as the ones used in the earlier part of the season. In the act of pollinization, the florets to be crossed were opened individually using the process described previously for enasculation. Then a ripe anther was taken from the box, dusted over the stigma, and left in the floret. The floret was then carefully closed by putting the glmnes back in place before proceeding to the next floret. As soon as a head had been pollinated completely, the head was subjected imediately to an assigned treatment. Before proceeding, an explanation of the treatments applied to the pollinated heads will be given. There were eight salts used in the eaqaerlment. The heads were kept covered for three, four, and five days. Heads were covered for a similar period with the glasine bag alone. This made a combination of twenty-seven treatments . Three heads were left in the open. All of these gave a total of thirty individual treatments. Hereafter in this experiment, a combination of these thirty treatments will be spoken of as a replication. Imediately after a head had been pollinated, a .15. western yellow pine stake, three-quarters inch square, was driven into the ground to an agreeable height beside the head. From a cross am at the top of this stake, were suspended two salt impregnated blocks that had been tied together with a string. This gave an effective . moisture adsorbing area of. approximately 23 square inches. One of the glasine bags was fitted down over the stake so that it enclosed the blocks and the cross-pollinated head of oats. Care was taken to insure that the head was not touched by the blocks. Host of the salts would have been tofic to the plants if the salts had touched than. A‘ wad of cotton was wrapped around the stun of the oat. Then the lower part of the bag was tied with a string in such a manner so as to close the bag as tight as possible around the stake and the eta without injuring the stan. In the process, the stem was drawn up close to the stake, and the passage of air into and out of the bag through the lips was cut down to a minimum. For the heads that were covered with the glasine bag alone, a similar process was followed except that the salt impregnated blocks were omitted. Both blocks used with a head were impregnated with the same salt. Within the individual replication, the order of assigning the treatments to the thirty heads was accompolished by drawing lots as soon as the head had been pollinated. The first one of the thirty treatments that was drawn was applied to the first head pollinated. The remainder of the treatments in the replication followed in chronological order the sequence in which they were drawn. In this manner, a replication was completed over two or more days. The various treatments were applied in the morning, in the afternoon, and at the time of day determined by the time the ones drawn before then were completed. Each head except those left in the open was covered with a bag with the appropriate treatment within two to five minutes after pollination was completed. The bags renained on the heads for three, four, or five days. Then they were removed. The removing of the bags took place in the afternoon from five o' clock to dark depending on the time the rest of the work for the day had been completed. The heads renained uncovered from the time the bags were removed until harvest several weeks later. Only one of the two female varieties was used throughout a single replication of the thirty treatments in the enmeriment. Replication 1 was completed first; replication 2, next; and the others, in numerical order. There were four different replications completed during the summer. Part of a fifth was done. The results of each are tabulated in charts appearing later in this thesis. The seeds that were harvested from the eicperiment -17. were planted in the greenhouse in the fall of 191.7. The plants that resulted from these seeds could not be identified as hybrids because the grain color, the index or crossing relied upon for this experiment, could not be detennined. Red seeded and yellow seeded oats growing in the greenhouse at the same time appeared to be white seeded oats. The seeds from this F1 generation were planted in the field during the first of June 191.8. A preliminary check on the resulting material shows that segregation for date of maturity is taking place in each of the progeny rows except for about five that have one to three plants each. These results indicate that practically all of the seeds were actually a product of cross-pollination, and they will be treated as such in the folling discussion. The heads that had been cross-pollinated in the field in 191.7 were tagged for identification so that the following information could be checked against the records: 1. the day of emasculation 2- the (193' 01’- pollination , and the number of florets 3. the time of anasculation and pollination, whether morning or afternoon 1.. the treatment used on the head including the salt and ' the length of time that the head was covered. Explanation of table 2: Table 2 shows the percentage set of the cross—pollinated heads for the sumer of 191.7. 0.3 ass a H309 ad has Hesse 0s«« seesaaoa .8 dance H \m.MH _ Nammr, 1w.