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' "4,? 44.4,..14. 4M “' 4" 4:» cm ‘p'fil .1. 34".. .""‘.:.:,.’" _ {'4' “4,41 4.1r.fs.'LI-.._... ..',1' Mr I:4 '., a» v -. .n. 4’. r_ v . ‘3' ”P‘“ 7' 4.44.4»;V nor-:4: v t ,4" i,”(':'.' L'." WW" 4, .."L¢,;u. (I, “.412'“, ”4.. I. ’34" 4-4" ”.3444... art—"44 r .4 :1: .. .ru-I“. h a 2.4“... . 544-11.:uiyvy . .142 “'1 ;0(0335@ LIBRARY Michigan State University This is to certify that the thesis entitled .Evaluation of Doubled Haploids of Wheat (Tritlcum aestivum). for Stability and Gametaclonal Variation Utilizing Disease and Insect Resistance presented by Dwight E. Bostwick has been accepted towards fulfillment of the requirements for __M.S_.__degree in CSS g'e 7 ~€JWN\ Major professor I‘Date 8/9/58 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution .«—q_._—.. wfl- .. .—.—-v-—r7 -__._.._ .47, MSU LIBRARIES .—c—. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. EVALUATION OF DOUBLED HAPLOIDS OF WHEAT (TRITICUH AESTIVUH), FOR STABILITY AND GAHETACLONAL VARIATION UTILIZING DISEASE AND INSECT RESISTANCE BY Dwight E. Bostuick A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTERS OF SCIENCE Department of Crop and Soil Sciences 1988 ABSTRACT EVALUATION OF DOUBLED HAPLOIDS OF WHEAT (TRITICUH AESTIVUN), FOR STABILITY AND GAHETACLONAL VARIATION UTILIZING DISEASE AND INSECT RESISTANCE BY Dwight E. Bostwick This study was undertaken to evaluate wheat doubled haploids for gametaclonal variation and stability. The haploid plantlets, were developed by anther culture, subdivided into individual isolates (plants) and were doubled to the diploid level (doubled haploid) using colchicine. The isolates were tested in the seedling stage for leaf rust, powdery mildew, Hessian fly resistance and scored for the awned/awnless morphological marker. A total of 1,131 doubled haploid isolates, subdivided from 362 anther-derived plantlets, were recovered. A small percentage of these lines exhibited variation for disease resistance, with a higher amount of variability observed in the Hessian fly tests. Environmental, experimental, and biological influences, along with possible mutations and/or chromosomal alterations induced by culturing were discussed as possible sources of variability, emphasizing the need for replicated testing in subsequent generations. Lines could not be tested for field adaptation due to insufficient seed. ACKNOWLEDGMENTS I would like to express my appreciation to Dr. Everett H. Everson, my major professor, for the opportunity to conduct this research under his guidance. I would also like to thank Dr. Joseph Saunders and Dr. Ken Sink for their critical evaluation and contributions to this thesis. This research could not have been conducted without the work of Dr. Joe Petilino, Dr. Jim Nelson and Alice Jones, of United Agriseeds, in making the crosses and developing the haploid plantlets. I would also like to thank Joe Clayton for his assistance with the leaf rust and powdery mildew tests. Also, the assistance of the USDA Entomology lab at Purdue University, headed by Dr. John Foster, with the Hessian fly tests is greatly appreciated. I would like to thank the members of the Wheat breeding Project, Dr. Nasarat "assimi, David Glenn, Dr. David Livingston and Ann Harantette-Sears for their technical help and friendship. And lastly, I would like to thank my parents for their continual support and encouragement. ii TABLE OF CONTENTS List of Tables . . . . List of Figures. . . . Introduction . . . . Review of Literature Introduction . . . Genotype . Physiological state of donor plant. Developmental stage of pollen Pretreatment of anthers Media . . Physical factors involved in plantlet Production of dihaploids Evaluation of dihaploids Gametaclonal variation and chromosome Conclusion. . . . Materials and Methods Anther culture. . . Transfer to soil . . Vernalization . . . Colchicine Treatment . Production of seed. . Seed increase . . . Powdery mildew testing. Leaf rust testing . . Hessian fly testing . regeneration. stability . Test for stability between generations. . . . Test for gametaclonal variation Test for 4-hour vs. 5-hour colchicine treatment . Results and Discussion . . Summary and Conclusions. . Literature Cited . . . iii Page iv 17 23 23 23 24 25 25 27 29 30 31 31 32 63 68 LIST OF TABLES Page 1. Parents used in developing F1 hybrids for anther culture and their leaf rust and powdery mildew ratings . . . . . . . . . . 18 2. Pedigrees of the 3-way and 2-way crosses and their anther-derived plantlet identification numbers . . . . . . . . . . . 19 3. Composition and types of media used for anther culture . . . . . . . . . . 22 4. The survival and colchicine treatment effects on the anther-derived plantlets and subdivided isolates . . . . . . . . . . . 33 5. Summary of 3-way cross experiment . . . . . 36 6. Comparison of 4 and 5 hour colchicine treatments . 38 7. Isolates tested for stability between DHl and DH2 . 40 8. Isolates tested for stability for the H7H8 gene. . 43 9. Isolates tested for gametaclonal variation derived from same anther-derived plantlet . . . 48 10 Plantlets and isolates tested for the H3 gene . . 50 11. Plantlets and isolates tested for the H6 gene . . 51 12. Plantlets and isolates tested for the H7H8 gene. . 52 13. Doubled haploids having superior resistance to leaf rust than best parent . . . . . . . 6O 14. Doubled haploids having superior resistance to powdery mildew than best parent. . . . . . 60 ’iv LIST OF FIGURES Page 1. Photograph of a haploid cell from one of the regenerated plantlets (N=x=21) . . . . . . 33 INTRODUCTION Anther culture is a technique for developing haploid plants from pollen grains. These haploid plants can be doubled to the diploid level (doubled haploid) using colchicine. A series of haploid plants, derived from several 2-way and 3-way Fl’s, were developed by United Agriseeds of Champaign, Illinois (UAS) and transferred to MSU for chromosome doubling and line evaluation studies. These studies were designed to examine the doubled haploid plants for stability between generations and for gametaclonal variation, among isolates originating from the same plantlet, for disease and insect resistances. The doubled haploids were scored for resistance in the seedling . stage to leaf rust, powdery mildew and three biotypes of Hessian fly with comparisons made on the awned/awnless morphological marker. REVIEW OF LITERATURE Introduction Since the initial induction of haploid plants from anthers in Datura innaxia (Guha and Haheshwari, 1964), this technology has been utilized in many different crop species. By 1982, Maheshwari et al. (1982) reported that 171 species had been anther cultured of which 121 had advanced to the embryo or plantlet regeneration stage. Included in the later list are rice (Niizeki and Gone, 1968), barley (Clapham, 1973), triticale (Sun et al., 1980) and wheat (Ouyang et al., 1973; Chu et al., 1973; Picard and DeBuyser, 1973; Craig, 1974). The advantage of this technique is the rapid development of homozygous breeding lines. For example, several wheat varieties have been developed in China that are higher yielding and of better quality than current cultivars. These new cultivars include "Jinghua No. 1" (Hu, 1986), ”Huapei No. 1" and "Lunghus No. 1" (Hu et al., 1978). Although many reviews have been written on androgenesis (Maheshwari et al., 1980; Maheshwari et al., 1982; Collins and Genovesi, 1982; Baenziger and Schaeffer, 1983), only a few discuss androgenesis in wheat (Wenzel and Foroughi-Wehr, 1984; Ouyang, 1986). Ouyang (1986) discusses the 3 methodologies of inducing wheat haploids by anther culture. The objective here is to review the methodologies used to produce and evaluate the doubled haploid products of wheat anther culture. Genotype A key factor when inducing haploids by anther culture is the selection of parental material. The genotype of the parental material is directly related to the androgenic capacity. Many studies have shown that cultivars differ in androgenesis and that androgenic capacity is genetically inherited (Picard and DeBuyser, 1973; Research Group 301, 1977a; Schaeffer et al., 1979; Henzel and Uhrig, 1981; Foroughi-Wehr et al., 1982; Bullock et al., 1982; Liang et al., 1982). Recently, Bullock et al. (1982) studied the inheritance of androgenesis by crossing poor and good androgenic wheat lines; the resulting Fl’s were intermediate. Studies in corn (Hiao et al., 1978), rye (Wenzel et al., 1977) and rice (Niizeki and Oono, 1968) substantiate the findings of Bullock et al. (1982). Several hypotheses have been proposed to explain why cultivars differ in degree of anther culture response. Dunwell (1978) and Sangwan (1978) state that cultivars differ in endogenous amino acid profiles that enhance or suppress induction of embryogenic tissue. A second concept is that maternal cytoplasm plays a direct role in androgenic capacity. However, other researchers believe androgenesis is controlled exclusively by nuclear genes (Picard et al., 4 1978; Foroughi-wehr et al., 1982; Bullock et al., 1982; Lazar et al., 1984; Charmet and Bernard, 1984). Androgenic capacity has also been linked to callus stabilization (Lazar et al., 1984 a and b). Other possible factors include genotypic responses to different culture media, hormone levels and growth conditions that may inhibit or enhance anther culture. Physiological state of donor plant Success of haploid induction also depends on other factors aside from parental genotype. Researchers agree that the physiological state of the donor plant, prior to anther culture, is important for successful induction of androgenic calli. However, they do not agree on what conditions produce the best donor plants. Schaeffer et al. (1979) found that donor cultivars responded better when grown in the greenhouse rather than in the field. Others have found the exact opposite to be true (Wang and Chen, 1980; Ouyang et al., 1983). Wang and Chen (1980) obtained their highest induction frequencies when they vernalized the donor plants in a growth chamber and transplanted them to the field in the spring. Ouyang et al. (1983) also found that winter wheat planted in the field produced better donor plants than greenhouse grown plants. Thus, conflicting data exists on what physical conditions produce the best donor plants. 5 A possible explanation for the conflicting data is believed to be associated with anther thickness. Zhong and Liang (1981) and Ouyang (1986) showed a direct correlation between greater anther thickness and higher induction frequencies. Anther thickness may play a role in addition to environmental conditions affecting plant growth. They concluded by stating the conditions generating the thickest anthers should be utilized in developing anther cultures. Another important physiological factor includes the age of the donor plant. As a plant matures, many investigators found that the production of androgenic calli was reduced (Sunderland, 1971; Picard and DeBuyser, 1973; Foroughi-Wehr et al., 1976; Dunwell, 1976). Photoperiod also plays a role as anthers tend to respond better when the plants are grown in a shorter photoperiod (Dunwell, 1976). Dunwell (1976) compared donor plants grown in 8 hours of daylight versus 16 hours. He discovered that 8 and 16 hour days generated a 56.68 and 34.9: induction frequency, respectively. The effect of temperature on donor plants has also been investigated. Picard and DeBuyser (1975) and Sunderland and Dunwell (1977) reported that moderate temperatures, 20-30 0, produced the best donor plants. Applications of hormones also have numerous effects on callus induction (Picard and DeBuyser, 1973; Wang et al., 1974; Wilson, 1977). 6 Developmental stage of pollen Another critical factor for successful anther culture is the selection of the pollen at the appropriate developmental stage. Wheatley et al. (1986) discovered that the correct pollen stage can be selected phenotypically in wheat. This can be accomplished by establishing what plant growth stage contains pollen at the proper developmental stage. In wheat, pollen is in the appropriate stage when the spike is still located within the leaf sheath, at the leaf below the flag leaf. Many researchers have examined a wide range of pollen stages. The general consensus is that for wheat anther culture, the mid-to-late uninucleate stage induces the highest frequency of calli (Ouyang, 1973 et al.; Wang et al., 1973; Cytological Laboratory of Lanchow University, 1975; Pan and Kao, 1978; He and Ouyang, 1984). He and Ouyang (1984) examined the frequency of androgenic callus formation from the meiotic stage to the binucleate stage. They found all stages induced calli, but the mid-to-late uninucleate stage yielded the highest frequency. An exception to this observation is in some tobacco cultivars the binucleate stage produced the highest induction frequency (Herberle-Bors and Reinert, 1979; Sunderland and Roberts, 1977 and 1979). Other cereal crops have been studied and the best stage for induction was also the mid-to-late uninucleate stage (Collins and Genovesi, 1982). Pretreatment of anthers Certain physical treatments applied to the anther, spike or donor plant can be highly conducive for the induction of androgenic plants. One of the most common treatments is cold treatment of the spike and anthers. In most cereal crops a standard treatment has been developed that produces optimal results. However, in wheat, this is not the case. Many researchers have worked out their own treatment procedures that they believe provides the best results. Pan et al. (1975) pretreated spikes at 3-4 C for 48 hours, while Picard and DeBuyser (1977) pretreated for 2- 8 days at 3 C. Other methods include 2-3 days at 4-5 G (Schaeffer et al., 1979) and 1-4 C for 48-96 hours (Amssa et al., 1980). These pretreatments were also conducted in the dark, possibly indicating that pretreatment may be enhanced by the absence of light. However, other researchers (Liang et al., 1982; Ouyang, 1986) could ascertain no beneficial effect of a cold pretreatment. Sunderland and Roberts (1979) expressed several theories on the significance of pretreatment. One stated that the cold delays the development of mature microspores while allowing many immature microspores to mature. This would lead to a greater percentage of microspores in the proper developmental stage. A similar theory stated that the pretreatment arrests the microspores at the first meiotic stage allowing more gametophytic microspores to develop into sporophytic microspores. Another concept is CU that the pretreatment alters the amino acids in the anther wall, favoring embryogenic formation. In general, the pretreatment induces some unspecified "shock" that enhances calli formation. PM. Probably the most important component in developing anther cultured haploids is the culture medium. The first two commonly used media for anther culture were MS medium (Murashige and Skoog, 1962) and Miller's medium (Miller, 1963). Many modifications have been made on these initial media since their introduction. Ouyang (1986) lists the three main components of anther culture medium as the basal salts, sucrose and hormones. Ouyang (1986) described a basic medium as composed of inorganic salts, organic growth substances (mainly B vitamins) and agar or other solidifying compound(s). The second component is the sucrose concentration. MS and Miller’s medium contain 38 sucrose, but many researchers today use a range from 3* - 12% sucrose. Many different hormones have been examined, but only auxins and cytokinins consistently gave significantly higher induction frequencies compared to media without these two hormones (Ouyang, 1973; Research Group 301, 1977a). Many types of media have been used for wheat anther culture. Some of the more common media include Miller and MS (Picard and DeBuyser, 1977), N6 (Chu et al., 1975 and 1978; Rives and Picard, 1977), and potato medium (Chuang et c; al., 1978; DeBuyser and Henry, 1979 and 1980). N6 medium is a synthetic medium originally reported by Chu et al. (1975 and 1978). N6 differs from MS and Miller’s media by lowering the inorganic salt and nitrogen contents. Chuang et a1. (1978) introduced potato medium that substitutes potato extract for many of the commonly used chemical additives. Ouyang (1986) also specifies that within these general media, modifications are made for the different stages of anther culture. These stages include embryoid or initial callus induction from the anther, calli differentiation and plantlet regeneration. The response media had its sucrose concentration at 10x as a high sucrose concentration was found to produce a higher frequency of embryoids or initial calli formed from within the anther (Ouyang, 1973; Cytological Laboratory of Lanchow University et al., 1975; Pan et al. 1975; Research Group 301, 1977a). The sucrose concentration is usually lowered for callus formation as Zhuang and Jia (1983) found that a high sucrose concentration inhibited callus differentiation. Ouyang (1986) reported that media for calli and embryoid induction often contains auxin, in the form of 2,4- D and a cytokinin, in the form of kinetin. These hormones are lowered considerably in concentration for plantlet regeneration compared to embryoid and callus formation. Many times, 2,4-D is substituted by IAA or NAA, weaker auxins, for plantlet regeneration. 10 Physical factors involved in plantlet regeneration The two main physical components for calli induction and plantlet regeneration are light and temperature. Ouyang (1986), was not able to determine the role light plays in the induction procedure._ However, he stated that light is required for post-induction development of embryoids and for plantlet regeneration. Culture temperature, on the other hand, is known to have a significant influence on callus induction. Early workers in anther culture used a range from 20-27 C for callus induction (Ouyang et al., 1973; Research Group 301, 1977a). In these cases, it was shown that higher temperatures increased the number of calli formed, but ultimately reduced the number of green plantlets regenerated. Hu et al. (1978) showed that in the temperature range 28-32 C, the highest frequency of green plants was obtained. Ouyang et al. (1983) reached several conclusions on the importance of culture temperature. First, a difference of 2-3 0 can significantly influence callus formation. Second, the temperature requirements differ depending on genotype, with the common ranges from 26-32 C. Sensitivity to culture temperature is a heritable trait. Finally, as culture temperature increases, the time required for embryoid or initial callus formation is reduced. 11 Temperature is not as critical for plantlet regeneration as it is for callus induction (Ouyang 1986). Researchers have shown that regenerating plants are less sensitive to temperature than the steps included in the induction of calli (Li, 1978; Ouyang, 1986). Ouyang (1986) showed that from 17-30 C, the regeneration percentage increased slowly but significantly. Picard and DeBuyser (1975) demonstrated that 25-27 0 gave slightly better Fesults than 21-22 C for regenerating plantlets. Proggction of dihaploigg Jensen (1974) and Hinkle and Kimber (1976) have developed two similar methods for doubling haploid plants. The doubling procedure can be accomplished by using colchicine in combination with dimethyl sulfoxide (DMSO) and Tween 20. Colchicine inhibits spindle fiber formation during mitosis, allowing the genome to double (Jensen, 1974). DMSO acts as a carrier for colchicine to enhance cell membrane transport (Jensen, 1974). Cell wall membrane contact is enhanced by the addition of Tween 20 (Schaeffer et al., 1979). Colchicine is the active agent while the other compounds are present to improve efficiency. Evaluationggfggihaploidg The last step in anther culture is the evaluation of the dihaploids. There is a need for critical evaluation of the dihaploid method versus conventional plant breeding methods such as pedigree and single-seed descent. The 12 application and significance of anther culture among investigations is diverse. Several investigators have found that anther culture dihaploids of tobacco and wheat derived from pure lines were inferior in many characteristics, including yield (Burk and Matzinger, 1976; Arcia et al., 1978; Baenziger et al., 1983; Friedt and Foroughi-Hehr, 1983). Recently, Baenziger et al. (1983) showed that some individual dihaploid lines derived from the self-pollinated wheat cultivar “Kitt” were equal to the pure line cultivar in yield. However, the mean yield of the dihaploids was significantly lower than the pure line cultivar. Many investigators believe this reduction in vigor is associated with the loss of residual heterozygosity maintained in lines developed by conventional breeding methods (Burk and Matzinger, 1976; Baenziger et al., 1983). Another possible explaination for the vigor loss is the deleterious effects that media and colchicine may induce (Burk and Matzinger, 1976; Arcia et al., 1978). These investigators also found that variation among dihaploids is high when compared to the pure lines, indicating that calli were affected differently by the anther culture procedure. However, when comparing dihaploids obtained from F1 hybrids, the data does not always favor conventional breeding methods. Several investigations have found that F1 derived dihaploids are as good as lines developed by pedigree or single-seed descent methods (Park et al., 1976; Choc et al., 1982; DeBuyser et al., 1985). Park et al. 13 (1976) stated that pedigree lines always seemed to be slightly higher in yield, although seldom significantly. Some of the studies compared dihaploid lines to released varieties and found several superior dihaploid lines (DeBuyser et al., 1985). Schnell et al. (1980) stated in their studies, dihaploid lines were inferior to lines developed by pedigree and single-seed descent methods. Gametaclonal Varigtion and Chromosome Stability A final area of review is the effects that the anther culture procedure can have on the plants and their progeny. In tissue or cell culture, genetic variation has been observed which can be directly associated to the procedure itself. The term somaclonal variation was implemented by Larkin and Scowcroft (1981) to define genetic variation in plants regenerated from tissue or cell cultures. In the review by Larkin and Scowcroft (1981), they discuss and reference several examples of this phenomenon occurring inoa wide range of crops. Anther culture is similar to tissue and cell culture in several phases of the procedure. In a paper by Powell et al. (1984), they termed the variation that arose due to anther culture, gametaclonal variation. This term was used because the product of anther culture is derived from the male gametophyte. The variation that arises due to anther culture has been studied by many investigators. 14 Two noticeable types of genetic gametaclonal variation that have been studied are albinism and aneuploidy (Burk and Matzinger, 1976; Picard et al., 1976; De Paepe et al., 1977; De Paepe and Pernes, 1978; Arcia et al., 1978; Collins and Legg, 1980). Albinism often occurs in anther culture studies at varying frequencies, up to 908, in terms of the percentage of total plantlets regenerated (Henzel and Foroughi-Hehr, 1984). Albinism is thought to be due to the emergence of recessive genes caused by the haploid method of developing plants (Wang et al., 1973; Clapham, 1977). Other possible causes could be the lack of functional proteins, the lack of ribosomal RNA in the plastids (Sun et al., 1979) or that transcription is blocked in the chloroplast. These would interfere with the conversion of DNA into RNA which in turn could prevent proper protein formation (Hang et al., 1978). Another characteristic of anther culture-derived plants is aneuploidy. Since haploid and doubled haploid plants of common wheat should contain 21 and 42 chromosomes respectively, investigators found that aneuploidy occurred around these two focal points (Hu, 1986; DeBuyser and Henry, 1986). Shimada (1983) also found that the longer a line is held in culture by subculturing, the greater the occurrance of aneuploidy. The expression of quantitative traits in anther-derived plants has also been investigated. In a study by Picard et al. (1986), doubled haploids developed from homozygous 15 cultivars were investigated. They compared yield, height, heading date, quality factors, disease resistance and ctDNA and mtDNA between the parental cultivar and the doubled haploids. They found significant differences, in yield, height and disease responses. These results were similar to the findings of Powell et al. (1984) in barley. However, they did not find any differences in the ctDNA and mtDNA in the restriction digest patterns between the original cultivar and the doubled haploid derivatives. Several investigators have studied genome or DNA stability within the doubled haploids. Rode et al. (1985) examined organelle DNA stability and found no differences in ctDNA or mtDNA between the parent and doubled haploids derived from that cultivar. In a follow up paper (Rode et al., 1987), they examined ribosomal DNA and found variation in the nuclear rRNA between the parent and doubled haploids. They found that once the variation was present it was stable within the population. In a paper by De Paepe et al. (1982), a series of recurrent androgenesis was done by continually regenerating haploid individuals from previously anther-derived plants. They found that with several cycles of anther culture the average amount of DNA in the cell increased. They also found that the proportion of tandemly repeated DNA sequences increased. Thus, variation can be introduced from anther 16 culture at the phenotypic, nuclear, plastid level and possibly within ctDNA and mtDNA, although the later has not been proven. Conclusion However one views the data, several dihaploid varieties have been developed and released in several countries. A high yielding rice cultivar called “Tenfeng No. 1“ was released in China, which is 21: higher yielding than control varieties (Yin et al., 1976; Heilungkaing Academy of Agricultural Sciences, 1978). Three tobacco cultivars, “Tanyu No. 1,“ No. 2“ and "No. 3“ are 10-308 higher yielding and of 10-15% higher quality (Research Laboratory of Breeding, 1978). Also, three wheat varieties have been released in China, “Huapei No.1” and “Lunghus No. 1," (Hu et al., 1978) and "Jinghua No. 1” (Hu, 1986) which are better yielding than control cultivars. Though investigators differ on the uses and potential of anther cultured dihaploids, several very good varieties have been developed. Thus, anther culture is a multi-procedural process which takes careful planning and execution within each~phase to complete successfully. MATERIALS AND METHODS Anther Culture Anther culture was performed by United Agriseeds of Champaign, Illinois. The parents used were chosen for several criteria including yield, quality, disease and insect resistance and most importantly androgenic capacity. Table 1 lists the parents and their ratings for leaf rust and powdery mildew resistance. From these parents, several two-way and three-way F1 crosses were made in 1985 (Table 2). The spikes of the F1 plants, containing the anthers, were selected while still within the leaf sheath, at the leaf below the flag leaf. The stem containing the spike was cut and the spike excised. The spike was then given a cold and dark pretreatment by wrapping the spike in aluminum foil and placing it in a cold chamber without lighting for one week at 5 0, similar to Shimada’s procedure (1981). The spike was removed from the cold chamber, unwrapped and placed in a sterile hood at 25 C. The remaining procedures were carried out under sterile conditions (within a laminar flow hood). The spike was sterilized in 108 Clorox for 15-20 minutes, then rinsed in sterile water for 15 minutes. 17 18 Table 1. Parents used in developing F1 hybrids for anther culture and their leaf rust and powdery mildew ratingsl. Varietal Michigan Leaf Powdery name[number Accession number Rust Mildew 1N6919509 13000 2 3 MOW10501 ‘13005 4 5 w1x1372-6 13007 3 5 1L79-1457 13009 3 4 OH256 13418 2 4 ‘1L81-3737 13419 3 5 1L81-3869 13420 5 3 YAYLA 305 13548 4 O ELGIN/P1178383 13549 4 5 LEHJAIN 13551 4 5 P1466705 13553 3 5 84REA-PSB75 13556 4 5 UAS 8 13814 3 5 MD55-220-76 13956 NASH76180 13957 3 5 SALUDA 14128 2 4 MD72001 14129 1 0 H1x1349-6 14131 3 5 MOW9965 14134 4 80770096 14137 3 5 FL74265-5-10-A2-Bl-D1 14142 1 O FL791-G161 14143 1 3 FL7223-3-3-A2 14144 5 5 NASH78-171 14148 2 5 FL301 14154 3 1 IHHPN14 14234 1 5 INHPN15 14235 3 3 121-49-1 14604 2 3 NFCl7 14605 2 4 NFC27 14606 2 0 NF029 14607 2 5 NFC33 14608 3 1 NFC35 14609 2 1 UASl 14610 3 4 UAS6 14611 3 5 UAS7 14612 3 5 ROLAND 14613 3 4 OASIS M0274 2 4 AUBURN M0289 2 3 FRANKENMUTH M0290 3 3 CALDWELL M0291 3 3 SCOTTY M0293 3 3 HILLSDALE M0295 4 0 HILLSDALE (WHITE) M0299 AUGUSTA M0300 3 3 PIONEER 2550 M0305 3 4 MSU LINE 87321 3 5 1 Disease ratings described on pages 25-28. Table 2. 19 Pedigrees of the 3-way and 2-way crosses and their anther-derived plantlet identification numbers Cross Number 865007 865037 865053 865063 865088 865123 865127 865152 865160 865175 865194 865200 865211 865232 865251 865257 865267 865279 865317 865334 865368 865389 865394 865417 865443 865459 868012 868041 868054 868066 868077 868087 868143 868145 868156 868158 868164 868173 868174 868192 868265 868304 868311 868323 868326 868461 868473 Anther-derived plantlgt 12 numbers Pedigree 1-46,296-298,307-319,324-328 M0293/I4144/IM0305 46-98 99-129,302,329,330 130-142 143-148 149-187,331-335 188 189-198 199-223 224-257 258-261 262,357,358,461,462 364,426-430 263-276 277-279 280 352,457 281-284 285-286 287-295,321,431 411 362 339-344,386-398 367-368,453-456 365 369-372,422-425 412-414,459 359-361,399-405 349-351,460 366,415-421 373-374,432-452 353-356,409-410 345-346 375-376,406-408 363 348 338,458 377 347,378-382 336-337,383-385 I4148/I4613/IM0305 I4613/I4143/lM0305 I4137/I4131/IM0305 I3005/I4144/IM0305 I3956/M0291l/M0305 I4144/I4612/IM0305 I4128/I4148//M0305 I4128/I4129/IM0305 I4610/M0300/IM0305 I4611/M0300/IM0305 I4611/I4142/IM0305 I4611/M0295//M0305 I3814/I4142/lM0305 M0299/I4613/IM0305 M0299/I3005/lM0305 M0299/I4154/IM0305 B7321/I4154I/M0305 I4143/I3548//M0305 I4613/I3548//M0305 M0295/I3549I/M0305 I3009/I3556/IM0305 M0299/I3549/IM0305 I3007/I3548/IM0305 M0290/13551I/M0305 M0290/I3553/IM0305 I3005/M0305 I4134/M0305 M0293/M0305 I3420/M0305 M0295/M0305 M0289/M0305 I3957/M0305 I3419/M0305 14612/M0305 I3814/M0305 I3000/M0305 M0274/M0305 M0305/14137 I4604/MO305 14605/M0289 14606/M0289 I4607/I3418 I4608/M0291 14609/M0293 14234/M0291 I4235/M0305 20 The anthers were removed from the spike under a dissecting scope. Each floret was dissected individually removing three sets of three anthers. The best set of anthers, from each floret, was selected. This was done for 12 florets per spike, plating 36 anthers per petri dish. The anthers selected had a light green color, with a glossy or shiny appearance (not translucent or dark green). Anthers at the proper stage adhered readily to the dissecting tools, while anthers that were too mature usually fell off. The anthers were plated in 60 x 15 mm petri dishes on approximately 10-12 ml of response medium, AC-2 (Table 3), placed approximately 4 mm apart in 3 rows of 12 anthers. The petri dishes were wrapped with Parafilm and placed in the dark at 26 C for one week. Following this treatment, the dishes containing the anthers were placed in a 16-hour photoperiod under fluorescent light at approximately 1,500 lux at 26 C. In three to four weeks "responses” from the pollen grains became visible, varying in organization from embryoid to a semi-Organized callus. These "responses“ are herein termed embryo-like structures (ELS) to encompass the mixture derived from the microspores within the anther. The ELSs were transferred to callus forming medium, C1 (Table 3), at the same temperature and photoperiod. In 4-8 weeks two types of calli formed: 1) Non-embryogenic: 21 white, crystalline, friable and with very little structure and 2) Embryogenic: not translucent, yellow (not brown), not friable and with organization. The non-embryogenic calli was discarded and the embryogenic calli were transferred to shoot regeneration medium, W4 (Table 3). Through embryogenesis, plantlets containing roots and shoots began to develop from the transferred embryogenic calli within 4-8 weeks. Often roots developed poorly so the regenerating plantlets were transferred to a liquid regeneration medium, W6 (Table 3), that enhanced both root and shoot growth. The anther-derived plantlets were placed in 250 ml Erlenmeyer flasks containing W6 medium and placed on a shaker at 120 rpm supplemented with a 16 hour photoperiod under fluorescent light at approximately 1,500 lux at 26 C. In 4-8 weeks the roots developed sufficiently to enable transfer to soil. At this stage the material was sent to Michigan State University in nine shipments over a 5 month period. The plantlets were transferred in stoppered plastic vials containing some residual W6 root medium to avoid desiccation. After four weeks of culturing, the anthers, calli or regenerating plants were transferred to fresh medium to insure proper chemical concentrations. Also, the growth conditions for all stages of anther culture were similar at 26 C for a 16-hour photoperiod under fluorescent light at approximately 1,500 lux. 22 Table 3. Composition and types of media used for anther culture (mg/l) Compound AC-2lCl1 51? W9? KNO; 2,830 1,900 1,900 NH4N03 --- 1,650 1,650 (NH4)2SO4 463 --- --- KH2P04 ‘ 400 170 170 CaClz*2H20 166 440 440 Mgsoq*7H20 185 370 370 Fe304*7H20 27.8 27.8 27.8 NazEDTA 37.3 37.3 37.3 Mn804*H20 4.4 22.3 22.3 Zn804*7H20 1.5 8.6 8.6 CuSO.*5H20 --- 0.025 0.025 Coolz*6H20 --- 0.025 0.025 H3803 1.6 6.2 6.2 K1 0.8 0.8 0.8 Na2M004*2H20 --- 0.25 0.2 Glycine 2.0 0.2 0.2 Pyridoxine*HCL 0.5 0.05 0.0 ThiaminetHCL 1.0 0.01 0.0 Nicotinic acid 0.5 0.05 0.0 Glutamine --- 146 146 Myo-inositol --- 100 -100 2,4-D 2.0 --- --- 1AA --- 1.0 5.0 Kinetin 1.0 1.0 0.1 Sucrose 100,000 30,000 30,000 Agar 8,000 8,000 --- pH 5.8 5.8 5.8 l AC-2. Initial response media (Chu, 1978), 01. Calli induction (Chen and Wang, 1979) same as A0- 2 except with 38 sucrose 2 Shoot regeneration (Schaeffer et al., 1979) 3 Rooting and propagation (Schaeffer et al., 1979) 23 Transfer to Soil Each anther-derived plantlet could usually be subdivided into individual plants, herein termed isolates, averaging 4-5 isolates per plantlet. The isolates were planted in a sterile planting medium containing a 1:1:1 ratio of soil, sand, and peat in 6 oz. styrofoam cups with a hole in the bottom for drainage. Root tip chromosome counts of the haploid plants, using the aceto-carmine squash method (Tsuchiya, 1971), were also made on approximately ten different anther-derived plantlets. The isolates were placed under fluorescent lights with a 12-hour photoperiod at approximately 1,500 lux at 24 C. After a week they were given a dilute fertilization with Rapid Gro (23-19-17 plus micronutrient fertilizer). After 2-3 weeks the plants were vigorous enough to be moved to the vernalization chamber. Vernalization Since all of the parents used in this study had a winter growth habit, the plants were vernalized for 10 weeks at 4-5 C. The vernalization chamber contained two high intensity sodium lamps providing the plants with a 9 hour photoperiod. Colchicine Treatment The plants were removed from the vernalization chamber and placed in a 12-hour photoperiod at 24 0. They were in this environment for 8-10 days to initiate regrowth before 24 being treated with colchicine. The aqueous solution contained 2* (v/v) dimethyl sulfoxide (DMSO), 0.18 (w/v) of colchicine, a few drops of Tween 20 and .035mg/l gibberellic acid(GA3). The colchicine solution was usually made in 1 liter batches just before use; this was sufficient to treat approximately 100 plants. The plants were removed from the soil mixture and the roots were washed thoroughly with water. The roots were trimmed to approximately 1-2 inches in length, blotted dry, and each plant was placed in a 30 ml test tube containing approximately 10 ml of colchicine solution, with the crown and roots completely submerged. Most of the plants were left in the solution for 4 hours but two sets were treated for 5 hours. The plants were removed from the colchicine solution, rinsed 2-3 times with distilled water and the leaves were cut to approximately one-half their length. The plants were then repotted using the same type of sterile planting medium, as previously described (in 4-5 inch clay pots). Prgggction of Sggg, 1 After repotting, the plants were grown in the lab under fluorescent light with a 12-hour photoperiod at 24 C for 2-3 weeks, then moved to the greenhouse for seed production. The seed was harvested at maturity and notes were taken on the number of spikes per plant, fertile spikes and the total 25 amount of seed produced. Since the period of maturity varied due to the time spread of the material, all of the plants were not harvested at the same time. Seed increase After the initial seed yield had been collected from the doubled haploids, a seed increase was necessary. This was done by planting up to seven seeds of each isolate. Seeds were planted in the same type of planting medium in 2 foot by 1 foot cedar flats. The seeds were allowed to imbibe water for 36-48 hours and then placed in the vernalization chamber at 4-5 0 for 48-72 hours. This was required because most of the lines were soft red wheat which have a dormancy period of several weeks. The flats containing the seeds were removed and returned to the greenhouse for germination. 1n the first leaf stage, approximately 4 inches tall, the plants were vernalized for 10 weeks at 4-5 0. After 10 weeks, the plants were transplanted to sterile soil in 4-5 inch clay pots and grown under standard greenhouse conditions of fertility, disease control and 18 hour photoperiod. A rough estimate was made on the amount of seed produced by each line and the plant was scored for being awned or awnless. Powdery Milgsw Tssting Seeds of the isolates to be tested were planted in cedar flats. Approximately 10-12 seeds of each isolate line 26 were planted. Seeds were given a cold treatment to break dormancy, as described earlier, and then placed in the greenhouse for germination. The plants were grown in a natural lighted greenhouse supplemented with fluorescent light at 22 C. When the first leaf of the plants had developed, the plants were inoculated by gently dusting the lines with powdery mildew spores, using a pot of plants containing the inoculum. The method of innoculation and rating of disease severity was similar to the method described by Ellingboe (1968). The plants were immediately placed in a environment controlled chamber at 22 C, utilizing only incandescent light for the first six hours at an intensity of approximately 2,000 lux, after which they were placed in the dark for eight hours. During and after inoculation, the relative humidity was maintained at 858 or higher to enhance spore germination and infection. Following inoculation they were grown in a greenhouse with 14 hours of daylight, supplemented with incandescent and fluorescent lights at approximately 2,000 lux with a DT/NT of 22/17 C. The above conditions were maintained for seven days after which time the plants were ready to read for infection. Powdery mildew resistance was rated on a 0-5 scale: 0 - Plant remains free of any disease; completely immune or resistant 1 - Flecked; small yellow-tan necrotic spots on the leaf 27 2 - Hypersensitive reaction; the resistance mechanism within the plant over-reacts to the fungus, causing a necrotic breakdown on the plant. Large pustules may be present 3 - Small pustules present on leaves; called slow mildewing'. The resistant genes allows the fungus to develop very slowly. Three to five days slower than a susceptible variety. 4 - Large pustules on the leaves; leaves are not completely covered. 5 - Large pustules on the leaves; leaves completely covered. Plant is very susceptible. , Four checks or control lines with known resistance and/or susceptibility to powdery mildew were planted within each flat. They were Little Club (completely susceptible), Augusta and Frankenmuth (intermediate or “slow mildewing”) and Hillsdale (resistant). LséLLRtJfit Tsfii he The seeds of the isolate lines to be tested were planted, given a cold treatment to break dormancy, as described earlier, and grown in the same manner as the material grown for powdery mildew testing. Four checks with known resistances to leaf rust were planted within each flat. The checks were the varieties Little Club (completely susceptible), Augusta and Frankenmuth (intermediate) and Hillsdale (susceptible). Spores, in the uredospore stage, were collected from infected plants of the cultivar Little Club and suspended in 28 Sol 170 oil from the Phillips Petroleum Company. At the first leaf stage, plants to be tested were sprayed with the inoculum mixture from a glass atomizer. After being sprayed with the inoculum, the plants were placed in a mist chamber for a minimum of eight hours to enhance spore germination. The plants were allowed to slow dry for two hours following the mist treatment. The plants were subsequently placed under continuous high output fluorescent light at approximately 5,000 lux until the plants began to fleck from the inoculum. At this time the plants were removed from the high output light and placed in natural greenhouse lighting. The plants were maintained at a temperature at or above above 25 C for disease development. Ten days after inoculation the plants were read for infection. The inoculation procedure was similar to the method used by Long et al., (1988), which was modified from the method used by Dyck and Samborski, (1968). A second reading was also taken at 14 days after inoculation to determine the presence of “slow rusting" genes and to reduce the possibility of reading errors. Leaf rust severity was rated on a scale from 0-5, similar to the rating of Stakman et al., (1962): 0 - Plants remain free of disease, immune or completely resistant. 1 - Flecked; small yellow-tan necrotic spots on the leaves. 29 2 - Hypersensitive reaction; the resistant mechanism with the plant over-reacts to the fungus, causing a necrotic ring around the pustule, trapping the fungus and robbing it of food, thus preventing the pustules from producing uredospores. 3 - Small and large pustules on the leaves. A hypersensitive reaction may occur on the leaves. Also called “slow rusting.” The resistant genes cause the fungus to develop very slowly. Pustules do produce uredospores but at a slower rate than a susceptible plant. 4 - Small pustules with no hypersensitive reaction showing. The pustules are two to three days slower developing that of a very susceptible plant. 5 - Very large pustules covering the leaves. No resistance, plants are completely susceptible. Hessian Fly Testing The Hessian fly testing was done at Purdue University by the USDA Entomology lab or at Michigan State University. Ten to twelve seeds from each isolate along with the check varieties were planted, given a cold treatment to break dormancy and allowed to germinate in the greenhouse. The ‘plants were infested with Hessian fly at the first leaf stage. The flies were maintained in wheat stubble contained in a covered cedar flat. The flies were uncovered and released in an enclosed netted area (9’ x 3’ x 2’) containing the lines to be tested. The flies remained in the netted area for 36-48 hours for emergence and infestation. Following infestation, the plants were kept well watered and maintained at approximately 24 c in high humidity for 3 weeks. After this time the plants were rated for resistant or susceptible reactions. 30 Susceptible plants were stunted, had very short internodes, usually darker green than resistant plants and pupae could be seen within the leaf sheath. Resistant plants had normal internode lengths, were lighter green and no pupae could be seen within the leaf sheath. A different fly biotype and set of checks were used for each of the three genes tested. For biotype 0, controlled by the H3 gene, the check varieties were Monon, Abe and Caldwell. For biotype D, controlled by the H6 gene, the check varieties were Frankenmuth, Caldwell and Abe. Biotype E, controlled by the H7H8 gene, using the check varieties of Monon, Abe and Seneca. Test for stability_sstwesg_generations Of the 1,131 isolate lines that were recovered, 72 lines from 46 different anther-derived plantlets produced enough seed to be tested for disease and insect resistance with enough seed remaining for seed increase. These 72 isolate lines were tested for leaf rust, powdery mildew and Hessian fly in both the first generation (DH1) after anther culture and the second generation (DH2) after the seed increase. These lines were planted in sterile soil in cedar flats. The same doubled haploid isolate from different generations were planted adjacent to minimize receiving unequal amounts of inoculum or visitation from the fly. The testing was carried out as described earlier in each of the tests. 31 Test for gametaclonslvvariation As indicated, many of the anther-derived plantlets could be subdivided into individual plants (isolates). Plantlets that yielded at least four fertile isolates were tested. These isolates were tested for powdery mildew, leaf rust and some for Hessian fly resistance. The seeds of the isolates were planted adjacent to each other to minimize possible inconsistency in inoculum and/or fly infection. Only those isolates having a parent with some resistance to a particular race of Hessian fly were tested. All isolates were also examined for the awned or awnless character. Isss_for 4-hogr vsgsgs 5-hogr colchicins treatment Most of the colchicine treatments were 4 hour treatments. Two sets of haploid isolates were treated for 5 hours to see if any difference could be detected in the percentage of fertile plants produced. Only those plants having isolates from the same anther-derived regenerant represented in both 4 and 5 hour treatments were compared. RESULTS AND DISCUSSION Root tip chromosome counts, completed on approximately 15 different plantlets, verified their haploid status, containing 21 chromosomes (Figure 1). Since some smears were not completely conclusive, aneuploidy could exist in some plants as it has with anther-derived plantlets in other investigations (Hu, 1986 and DeBuyser and Henry, 1986), but was not investigated. The number of haploid plantlets and their isolates surviving the various stages of production in this research are shown in Table 4. Of the 2,201 isolates obtained from the original 460 anther-derived plantlets, over 150 died following the colchicine treatment and almost half (1,070) were found to be sterile. Some of the plants treated with colchicine were weak prior to treatment, possibly contributing to a lower survival rate. The main cause of sterility is probably the result of unsuccessful doubling, as haploid plants will not produce seed. In a study by Kudirka et al. (1986), they found that most of the sterile plants examined after treatment with colchicine were haploid. They also found a small percentage of plants that were doubled but were sterile. This finding leads to the conclusion that other forces are involved in 32 33 Figure 1. Photograph of a haploid cell from one of the regenerated plantlets (N=x=21). Table 4. The survival and colchicine treatment effects on the anther-derived plantlets and subdivided isolates. Plantlets obtained by anther culture . . Plantlets whose isolates were treated with colchicine . . . Plantlets whose isolates survived colchicine treatment . . . . . . . Plantlets whose isolates produced seed . . Isolates obtained by subdividing plantlets . Isolates treated with colchicine . . Isolates that survived colchicine treatment . Haploid isolates converted to doubled haploids Number 486 460 448(978) 362(79‘) 2775 2201 2048(93‘) 1131(528) 34 conferring sterility. Karyotypic changes such as anueploidy and/or polyploidy could induce sterility (Kudirka et al., 1986). Cryptic changes such as chromosomal breakage, additions, deletions, rearrangements, etc., could also be involved in conferring sterility (Hu et al., 1978; Mix et al., 1978). These forces along with others, will be discussed in detail in a later section dealing with the possible sources of variability. 1t suffices to say that the major cause of sterility is probably the result of unsuccessful doubling with a small percentage being related to karyotypic or cryptic changes in the number and/or structure of the chromosomes. The percentage of fertile anther-derived plantlets and fertile isolates after doubling is also indicated in Table 4. Each plantlet was usually subdivided into an average of 4 to 5 isolates. The proportion of plantlets that produced seed bearing isolates was 798 while only 528 of the isolates were fertile. The difference of 278 can be attributed to each plantlet having several isolates treated with colchicine. Thus, through subdividing, the efficiency in obtaining fertile (doubled haploid) anther-derived plantlets was increased. As indicated in Table 2, there were several 2-way and 3-way F1 crosses used for anther culture. The results were recorded for all phases of anther culture from the procedure at UAS to the production of doubled haploids at MSU (Table 35 5). The totals indicate that from 12,096 plated anthers, only 2.08, or 236 anther-derived plantlets, produced fertile isolates after doubling. This low production of doubled haploids is probably the main disadvantage of anther culture. From a breeding standpoint, the cost of developing breeding lines in the United States through anther culture is much more expensive than conventional methods (S.R.M. Pinson, per. comm.). In a study with rice, Pinson found that anther culture was 3 times more expensive for line development than conventional breeding methods conducted in the field. Thus, increased production of doubled haploids is needed to make anther culture more cost-effective compared to conventional breeding. The data in Table 5 also indicates that individual crosses respond differently to anther culture. Many researchers have conducted experiments comparing cultivars and found that they often differed significantly in androgenic capacity (Schaeffer et al., 1979; Bullock et al., 1982). The results in this study would substantiate these findings as some crosses are more responsive to anther culture than others, such as cross 865007 versus 865211. It is difficult to ascertain if these differences are significant as these 3-way crosses were not organized as a controlled experiment. 36 Table 5. Summary of 3-way cross experiment Anthers1 Plantlets2 Producing Total1 Treated Plantlets2 Cross Anthers ELS’s ELS’s With Producing Number Plated (51 (31 Colchicins, Seed 865007 1080 88(8.1) 158(14.6) 70(6.5) 51(4.7) 865037 1008 93(9.2) 208(20.6) 52(5.2) 40(4.0) 865053 900 52(5.8) 84 (9.3) 35(3.9) 26(2.9) 865063 396 13(3.4) 18 (4.5) 10(2.5) 7(1.8) 865088 360 16(4.4) 22 (6.1) 5(1.4) 5(1.4) 865123 288 7(2.4) 7 (2.4) 0(0.0) 0(0.0) 865127 828 53(6.4) 83(10.0) 45(5.4) 31(3.7) 865152 36 0(0.0) 0 (0.0) 0(0.0) 0(0.0) 865160 216 16(7.4) 29(13.4) 1(0.5) 1(0.5) 865175 108 6(5.6) 9 (8.3) 0(0.0) 0(0.0) 865194 900 34(3.8) 45 (5.0) 10(1.1) 6(0.7) 865200 1296 81(6.3) 128 (9.9) 25(1.9) 18(1.4) 865211 720 17(2.4) 17 (2.4) 0(0.0) 0(0.0) 865232 828 55(6.6) 91(10.1) 29(3.5) 22(2.7) 865251 180 8(4.4) 11 (6.1) 9(5.0) 2(1.1) 865257 576 16(2.8) 21 (3.6) 1(0.2) 1(0.2) 865267 36 0(0.0) 0 (0.0) 0(0.0) 0(0.0) 865279 612 20(3.3) 29 (4.7) 8(1.3) 6(1.0) 865317 576 28(4.9) 48 (8.3) 14(2.4) 6(1.0) 865334 72 2(2.8) 3 (4.2) 3(4.2) 3(4.2) 865368 288 15(5.2) 31(10.8) 1(0.3) 1(0.3) 865394 288 18(6.3) 30(10.4) 4(1.4) 3(1.0) 865417 36 2(5.6) 3 (8.3) 1(2.8) 1(2.8) 865443 216 4(1.9) 5 (2.3) 0(0.0) 0(0.0) 8__65459 2_5_2_ _L_)_9 3 - 6 big-12.). ALA). m Totals 12096 652(5.4) 1092(9.0) 329(2.7) 236(2.0) l The ELS stands for "embryo-like structure“ which is used to define the mixture of structures that arise from within the anther, encompassing the range from an embryoid to semi- organized callus in appearance. 2 The plantlets were not directly treated, but the plants (isolates) that were subdivided from them. 37 The length of time that plants were treated with colchicine was also investigated. The isolates which received a five hour treatment produced a higher percentage of fertile doubled haploids (70.9) than those receiving a 4 hour treatment (56.3) (Table 6). The difference between treatments (14.68) is significant at the 0.01 level using the Chi-square contingency test. The higher fertility level of the 5 hour treatment is probably the result of the meristematic region remaining in the colchicine solution for a longer period of time. Since the 5 hour treatment was superior to the 4 hour treatment, longer colchicine treatments should be investigated to see if the conversion rate of haploids to doubled haploids can be increased further. The main emphasis in this study was to examine and test the doubled haploid isolates for leaf rust, powdery mildew and Hessian fly resistance. Since several of the parents were awned, the anther-derived progeny were also examined for the awned or awnless morphological marker. The first part of the study was designed to compare the doubled haploid isolates for stability between generations. Due to time involved in advancing the material to this stage, there was not enough remaining for field evaluations. The tests used were chosen because they could be performed in a greenhouse under a controlled environment with the limited amount of seed. 38 Table 6. Comparison of 4 and 5 hour colchicine treatments 4 Hour 5 Hour chi-square Treated plants 798 526 Fertile plants 449 373 Percent fertile 56.3 70.9** 8.86 **significant at the 0.01 level As mentioned in the materials and methods section, there were 72 isolates, from 46 plantlets, having enough seed in both the DHl and DH2 generations to conduct the three disease and insect tests. There were also 11 other isolates producing enough seed to test for leaf rust and powdery mildew in both generations. Doubled haploids should be homozygous, and as wheat is self-pollinating, homozygosity and stability for all traits should be maintained. The tests were implemented to determine if these isolates were indeed stable between generations. Table 7 lists the 83 isolates with their leaf rust and powdery mildew ratings. As the data indicates, most isolates had identical ratings in the first (DH1) and the second (DH2) generation. The first set of readings in the leaf rust column were taken 10 days after inoculation with the second reading taken at 14 days. The 10 day reading is probably more accurate because the leaves of some plants in the later reading were beginning to dry up. If drying occurred between the 10 and 14 day reading, the later leaf rust score could be read lower, which was apparent in several isolates. Of the 78 isolates tested for leaf rust in both generations, 74 had the same score on both dates. Three isolates (0306, 0481 and 0488) were off 1 point in the reading score, with only one isolate (1059) showing mixed resistance to leaf rust. For powdery mildew, 58 isolates were tested of which 52 had identical readings. Four isolates (0006, 0188, 0815 and 0961) showed a deviation of 1 point in the reading score between generations. Only one (0439) showed a difference of 2 points with the highest reading in the first (DH1) generation. One isolate (0115) exhibited a variable reading in the DHl generation but not in the DH2 generation. Only, 136 isolates had enough seed to be tested for leaf rust and powdery mildew in both generations. Of these 136 comparisons, 126 had identical readings, with 7 deviating by only 1 reading score. One isolate (0439) differed by 2 points in the powdery mildew reading with two (0115 and 1059) exhibiting variable resistances for powdery mildew and leaf rust, respectively. Comparing isolates tested for leaf rust and powdery mildew, only 3 (0006, 0815 and 0961) exhibited variation between generations in both tests. These isolates will need to be examined in future generations to see if they are the 4O Table 7. Isolates tested for stability between DH1 and DH2 Leaf2 Leaf2 Rust Powdery Rust Powdery Line1 19 ;g_ Mildew Linel ;9_ L1 Mildew 2-0001-1 4 3 4 230-0464-1 2 2 4 2-0001-2 5 3 4 230-0464-2 2 2 4 3-0006-1 3 3 4 230-0467-1 2 2 5 3-0006-2 3 2 5 230-0467-2 2 2 5 5-0014-1 2 2 5‘ 230-0468-1 2 2 5 5-0014-2 3 2 5 230-0468-2 2 2 5 5-0015-1 2 2 5 235-0481-1 3 2 5 5-0015-2 2 2 5 235-0481-2 4 3 5 5-0017-1 3 2 4 238-0483-1 3 2 4 5-0017-2 3 2 4 238-0483-2 3 2 4 5-0018-1 3 2 4 238-0486-1 3 2 4 5-0018-2 3 2 4 238-0486-2 3 2 4 56-0115-1 1 1 0-5 239-0488-1 4 3 5 56-0115-2 1 1 3 239-0488-2 3 2 5 66-0140-1 1 1 4 241-0495-1 4 3 5 66-0140-2 1 1 4 241-0495-2 3 3 5 90-0182-1 2 2 0 242-0497-1 3 3 5 90-0182-2 2 2 0 242-0497-2 3 2 5 90-0183-1 2 2 0 271-0527-1 2 2 0 90-0183-2 2 2 0 271-0527-2 2 2 0 92-0188-1 2 2 1 333-0628-1 5 3 0 92-0188-2 2 2 0 333-0628-2 5 3 0 97-0197-1 3 3 4 334-0629-1 5 3 0 97-0197-2 3 3 4 334-0629-2 4 3 0 100-0210-1 3 2 4 344-0669-1 3 2 0 100-0210-2 3 2 4 344-0669-2 3 2 0 108-0233-1 5 4 3 348-0686-1 5 5 4 108-0233-2 5 4 3 348-0688-1 5 5 4 109-0237-1 2 2 0 364-0752-1 2 2 5 109-0237-2 2 2 0 364-0752-2 2 2 S 109-0239-1 2 2 0 367-0765-1 3 3 0 109-0239-2 2 2 0 367-0765-2 3 3 0 149-0305-1 3 3 0 367-0768-1 3 2 0 150-0306-1 5 5 0 367-0768-2 4 2 0 150-0306-2 4 3 0 367-0775-1 3 2 0 150-0307-1 4 3 0 367-0775-2 3 2 0 150-0307-2 4 3 0 376-0811-1 2 2 4 161-0335-1 3 2 5 376-0811-2 2 2 4 161-0335-2 2 2 5 378-0815-1 5 4 4 170-0363-1 3 2 0 378-0815-2 5 3 5 170-0363-2 3 2 0 378-0816-1 4 4 4 189-0381-1 3 2 4 378-0816-2 5 4 4 189-0381-2 3 2 4 379-0817-1 1 1 0 190-0382-1 3 3 4 379-0817-2 1 1 0 190-0382-2 3 2 4 379-0818-1 1 1 0 217-0439-1 2 3 5 379-0818-2 2 1 0 217-0439-2 2 3 3 387-0840-1 5 3 3 41 Table 7. (can’t) Leaf Leaf Rust Powdery Rust Powdery Line Ag 11. Mildew Line ;9 11 Mildew 387-0840-2 5 4 3 433-1039-1 1 1 406-0930-1 3 3 0 437-1047-1 1 1 406-0930-2 3 2 0 437-1047-2 1 1 406-0931-1 3 2 0 441-1059-1 1 1 406-0931-2 3 3 0 441-1059-2 1,5 1,5 406-0933-1 4 3 0 443-1063-1 2 2 406-0933-2 3 3 0 443-1063-2 2 2 406-0936-1 3 3 0 457-1120-1 5 5 406-0936-2 3 3 0 277-0535-1 5 5 406-0937-1 3 2 0 277-0535-2 5 4 406-0937-2 3 2 0 280-0539-1 5 4 407-0938-1 1 3 0 280-0539-2 4 4 407-0938-2 1 3 0 347-0681-1 2 2 414-0961-1 4 2 4 347-0681-2 2 2 414-0961-2 4 3 3 347-0682-1 3 2 415-0963-1 2 2 5 347-0682-2 3 2 415-0963-2 2 2 5 352-0698-1 5 5 415-0964-1 2 2 5 352-0698-2 5 5 415-0964-2 2 2 5 352-0706-1 5 5 415-0965-1 2 2 5 352-0706-2 5 5 415-0965-2 2 2 5 432-1038-1 1 1 415-0966-1 2 2 4 432-1038-2 1 1 415-0966-2 2 2 4 435-1043-1 1 1 416-0968-1 2 2 4 435-1043-2 1 1 416-0968-2 2 2 4 442-1060-1 2 2 416-0970-1 2 2 442-1060-2 2 2 416-0970-2 2 2 442-1062-1 2 2 416-0972-1 2 2 442-1062-2 2 2 416-0972-2 2 2 453-1097-1 3 3 416-0974-1 2 2 453-1097-2 416-0974-2 2 2 416-0975-1 2 2 Checks 416-0975-2 2 2 419-0982-1 5 4 Frankenmuth 4 4 3 419-0982-2 5 4 Hillsdale 5 5 0 419-0984-1 5 4 Augusta 3 3 3 419-0984-2 5 4 Little Club 5 5 5 l The first number is the plantlet identification number. The second number is the isolate number. Lines ending with -1 represent seed obtained from the first generation (DH1), while lines ending with -2 represent seed from the second generation (DH2) after the seed increase. 2 The first column are isolates read 10 days after inoculation with the second reading taken after 14 days. 42 result of possible outcrossing in the greenhouse or are indeed genetic variation possibly induced by anther culture. Also, two isolates (0115 and 1059) had variable readings within the line. In these cases, they did not vary in both generations and could be explained by variation in test procedures and will also need to be re-tested. Isolates tested for stability between generations for the H7H8 Hessian fly gene are listed in Table 8. The data expresses much more variability between generations than the leaf rust and powdery mildew tests. From the 63 isolates tested for the H7H8 Hessian fly gene, 26 exhibited substantial variation between generations when compared to the checks. The checks in Table 8 should be completely susceptible or resistant, but were not. The susceptible line Monon has 28 variation; whereas, the resistant lines Abe and Seneca vary by 5 and 13 percent in fly reaction, respectively. Thus, over 408 of the lines were more variable between the DH1 and DH2 generations for the H7H8 Hessian fly data than the highest deviating check reading. The environmental and biological influences that can cause variablity will be discussed in detail later. The second area of study was to investigate whether or not isolates derived from the same plantlet would exhibit variability. Isolates originating from the same plantlet should be identical to one another, unless some chromosomal 43 Table 8. Isolates tested for stability for the H7H8 gene. Linel gssding.Scors Linel gssding Score 2-0001-1 1-9 217-0439-1 1-7 2-0001-2 2-7 217-0439-2 1-8 3-0006-1 0-8 230-0464-1 3-5 3-0006-2 3-4 230-0464-2 6-4 5-0014-1 0-8 230-0467-1 4-6 5-0014-2 1-7 230-0467-2 7-3 5-0015-1 0-9 230-0468-1 2-6 5-0015-2 2-5 230-0468-2 8-1 5-0017-1 1-4 235-0481-1 2-8 5-0017-2 2-7 235-0481-2 9-1 5-0018-1 0-10 238-0483-1 5-3 5-0018-2 1-11 238-0483-2 6-4 56-0115-1 2-7 238-0486-1 2-6 56-0115-2 7-2 238-0486-2 6-3 66-0140-1 7-3 239-0488-1 3-7 66-0140-2 8-2 239-0488-2 9-1 90-0182-1 0-9 241-0495-1 7-3 90-0182-2 3-6 241-0495-2 7-3 90-0183-1 2-7 242-0497-1 7-3 90-0183-2 3-6 242-0497-2 6-3 92-0188-1 1-7 271-0527-1 0-9 92-0188-2 7-3 271-0527-2 4-3 97-0197-1 4-5 333-0628-1 1-8 97-0197-2 3-1 333-0628-2 5-6 100-0210-1 0-9 334-0629-1 3-4 100-0210-2 5-5 334-0629-2 2-7 108-0233-1 2-8 344-0669-1 0-9 108-0233-2 0-10 344-0669-2 6-3 109-0237-1 0-9 364-0752-1 4-5 109-0237-2 0-9 364-0752-2 7-3 109-0239-1 0-9 367-0765-1 6-3 109-0239-2 0-9 ' 367-0765-2 4-4 150-0306-1 0-10 367-0768-1 5-3 150-0306-2 0-9 367-0768-2 4-5 150-0307-1 0-10 367-0775-1 1-7 150-0307-2 1-8 367-0775-2 4-4 161-0335-1 2-7 376-0811-1 4-4 161-0335-2 8-1 376-0811-2 1-8 170-0363-1 1-7 387-0840-1 1-7 170-0363-2 0-9 387-0840-2 2-7 189-0381-1 3-6 406-0930-1 10-1 189-0381-2 7-3 406-0930-2 6-3 190-0382-1 1-6 406-0931-1 6-3 190-0382-2 4-5 406-0931-2 8-3 Table 8. (con’t) Line Reading Score Line Reading Score 406-0933-1 5-5 419-0984-2 0-10 406-0933-2 5-3 437-1047-1 9-1 406-0936-1 5-5 437-1047-2 11-0 406-0936-2 6-2 441-1059-1 8-1 406-0937-1 6-3 441-1059-2 2-7 406-0937-2 6-4 443-1063-1 4-1 407-0938-1 2-8 443-1063-2 7-1 407-0938-2 0-7 414-0961-1 5-3 Checks 414-0961-2 6-2 415-0963-1 0-10 Monon 0-11 415-0963-2 0-9 0-9 415-0964-1 0-10 1-11 415-0964-2 0-9 0-7 415-0965-1 0-8 0-11 415-0965-2 0-10 415-0966-1 0-11 Abe 10-0 415-0966-2 0-9 6-1 416-0968-1 0-11 10-0 416-0968-2 0-9 7-0 416-0970-1 0-9 10-0 416-0970-2 0-9 7-1 416-0972-1 0-9 12-1 416-0972-2 0-9 416-0974-1 2-8 Seneca 8-1 416-0974-2 0-10 7-1 416-0975-1 1-6 10-2 416-0975-2 0-9 11-1 419-0982-1 0-9 8-1 419-0982-2 0-9 8-2 419-0984-1 0-6 11-1 1 The first number represents the number of resistant plants and the second number represents the number of susceptible plants. 45 change has occurred, as they originated from a single haploid pollen grain. After doubling, the isolates should be completely homozygous. Only plantlets that yielded at least four fertile isolates were tested for leaf rust and powdery mildew (Table 9), and Hessian fly with comparisons on the awned or awnless trait. The material was tested for the presence of the H3, H6 and H7H8 Hessian fly gene depending on the parental background of the F1. There were a total of 122 doubled haploid isolates tested from 21 plantlets for leaf rust (Table 9). The data indicates that isolates from 2 different plantlets (352 and 400) displayed a deviation of more than one point in the reading score. The comparisons were based on the first reading, as some leaves began to dry out before the 14 day reading. In the powdery mildew data (Table 9), isolates from 2 plantlets (400 and 404) exhibited a difference of more than one point in the reading score. Comparing the data of the two tests, only one plantlet (400) was variable by 2 points in both the leaf rust and powdery mildew test. In all cases, the four check varieties exhibited consistant readings. Uneven inoculation, environmental variation or human error in scoring could contribute, in part, to the observed variation. Although plants should respond similarly to leaf rust or powdery mildew regardless of inoculum quantity, slight variation in the seedling growth stage can affect 46 infection levels and pustule distribution on the leaves. Inoculations are made when the plants are in the single leaf stage. If only the leaf tip of a plant has emerged from the coleoptile before inoculation, spore distribution will occur on only the tips of the blade which can confuse ratings (Law and Johnson, 1967). A replication of these tests would have been helpful in determining if variation was caused by inoculum distribution or if it is genetic variation, however, there was not enough seed in most cases to run a second test. Another possible source of variation could also be related to the existence of multiple races of leaf rust. As of 1980, 35 different leaf rust races and resistance genes have been identified (Browder, 1980). In Michigan, many races of leaf rust can be found in close proximity to one another (Schafer and Long, 1988). The inoculum used for these leaf rust tests are developed from spores collected from several sites in Michigan. This is done to try and adequately represent the leaf rust races occurring in Michigan. These multiple races could induce variation if the doubled haploid lines received inoculum containing races in different proportions. Also, comparing tests completed at different times could induce variation, as leaf rust races can shift in terms of frequency to one another over a period of time (Long et al., 1988). This source of 47 variation is less likely to occur in the powdery mildew tests as a specific race is maintained and used for all tests. Plantlets having Hessian fly resistant parents were tested using the fly biotypes described in the materials and methods section (Table 10, 11 and 12). Isolates from 26 different anther-derived plantlets were tested. Only one plantlet (5) was tested for two genes, the H3 and H7H8. None of the three tests for Hessian fly resistance gave consistant results. In each test there was at least one plantlet having isolates with both resistant and susceptible reactions. In total, 6 of the 27 plantlet readings yielded opposing ratings. The data are perplexing because some isolates from the same plantlet gave intermediate resistant- susceptible readings. Examples of this response are in plantlet 386 whose isolates read 6-4, 5-4 and 5-4, or plantlet 406 whose isolates read 10-1, 6-3, 5-5, 5-5 and 6-3 for tests to the H3 and H7H8 gene respectively. The fly readings are difficult to interpret as many factors can contibute to the observed variability. The readings of the check varieties (Tables 8, 10, 11 and 12) illustrate the problems; they should have uniform reactions to the various Hessian fly biotypes. The variations in fly readings may be due to escapes often due to uneven plant emergence, slight mixture of biotypes used in testing, or temperature sensitivity of certain biotypes inducing a genetic x environment interaction. Table 9. 48 Isolates tested for gametaclonal variation derived from same anther-derived plantlet. Line1 5-0014 5-0015 5-0017 5-0018 277-0532 277-0533 277-0534 g77-0535 280-0538 280-oss9 347-0681 347-0682 347-0683 347-0684 352-0697 352-0698 352-0699 352-0700 352-0702 352-0703 352-0704 352-0705 352-0706 352-0707 352-0708 352-0709 364-0747 364-0748 364-0749 364-0750 364-0751 364-0752 364-0753 364-0754 370-0793 370-0794 370-0795 370-0796 370-0797 370-0798 s7o-0799 382-0824 382-0825 382-0826 382-os27 388-0847 Leaf Rust .1219. 4 2,4 2,3 4 #15 MN JNNNNNNNNMNNUG' - NNOIOIUHNNNNUIUIMUIUUI NNNNNUNNNN 2 4 3 NNNNNO‘NMNN NMNNNNNNO‘AUAMNAA“NAUNNMNUIOIAUIAA Powdery Mildew OOPOAAU'UI “HOOOUMUNUUNIUIU'MUIUIUIUIUIbtfibbAAAAAACOOOO 388-0848 388-0850 388-0851 388-0852 see-0853 400-0887 400-0888 400-0889 400-0890 400-0891 400-0892 400-0893 400-0894 400-0895 400-0896 402-0900 402-0901 402-0902 402-0903 402-0904 402-0906 404-0907 404-0908 404-0909 404-0910 404-0912 404-0913 404-0914 404-0915 404-0917 404-0918 405-0919 405-0920 405-0921 405-0922 405-0923 405-0924 405-0925 405-0926 405-0928 415-0963 415-0964 415-0965 415-0966 423-0994 423-0995 mwnnnnaemaeaaueeaaeuuuuuu UUQAAAWUUhU UbthUIUl UIUI amnnnnaaaaewuuuaaeaaeunnn narrowest-await» #0101010! 0101 Powdery Bilge! AfithbhbbUMAAWAUUUMbbbbb U' 0"“ AAbfiu & U|U|U|U| 49 Table 9. (con’t) Leaf Leaf Rust Powdery Rust Powdery Line Ag, ;1_ Mildew Line 19. L1 Mildew 423-0996 5 5 3 455-1105 3 3 4 423-0997 5 3 3 455-1106 3 3 4 423-0998 5 3 3 455-1107 3 3 4 figs-0999 ésngi 454-1108 3 3 5 425-1013 4 3 454-1109 3 §, 4 425-1015 4 4 456-1110 4 4 5 figs-1016 5 4 456-1111 4 5 5 446-1068 4 456-1113 3 4 5 446-1069 2 2 4 456-1114 3 3 5 446-1070 2 2 4 456-1115 4 5 5 519-1071 2 2 . 4 456-1116 4 5 5 447-1072 4 447-1078 2 2 Checks 447-1079 454-1099 5 5 0 Augusta 3 3 3 454-1100 5 5 0 Hillsdale 5 5 0 454-1101 5 5 0 Frankenmuth 4 4 3 454-1102 5 3 0 Little Club 5 5 5 1 The underlining within the table defines the division between plantlets and their isolate lines. 2 The first column are readings taken 10 days after inoculation with the second reading taken after 14 days. 50 Table 10. Plantlets and isolates tested for the H3 gene Linel Reading Score2 Linel Reading Score2 5-0014 6-4 428-1022 0-10 5-0015 9-2 428-1023 3-7 5-0017 10-1 528-1024 5-4 5-0018 7-3 429-1025 5-5 277-0532 1-9 429-1026 0-8 277-0533 0-13 429-1027 0-9 277-0534 3-8 431-1029 7-2 277-0535 0-9 431-1030 9-1 277-0537 5:; 431-1031 9-0 364-0747 10-0 364-0748 9-2 Checks 364-0749 8-2 364-0750 10-1 Monon 10-1 364-0751 7-4 7-2 364-0753 8-3 14-0 M-0754 §:§ 386-0836 6-4 Abe 9-0 386-0838 5-4 7-0 386-08§9 s;g_ 11-1 387-0841 8-2 387-0843 8-2 Caldwell 3-7 387-0844 7-3 1-5 396-0876 6-3 0-6 396-0877 5-3 1-7 396-0879 5-3 5-3 '427-1018 10-1 1-5 427-1019 6-3 1 The underlining within the table defines the division between plantlets and their isolates. . 2 The first number represents the number of resistant plants and the second number represents the number of susceptible plants. 51 Table 11. Plantlets and isolates tested for the H6 gene. Linel Reading Score2 Line1 Reading Score2 345-0672 10-0 379-0817 2-7 345-0673 9-0 379-0818 3-7 345-0675 11-0 s79-0819 1-10 346-0676 9-2 382-0823 6-0 346-0677 3-8 382-0824 11-1 346-0678 4-5 382-0825 10-1 346-0679 0-9 382-0826 8-2 346-0680 4-s 382-0827 9-1 347-0681 1-11 347-0682 0-11' Checks 347-0683 1-11 s47-0684 0-11 ‘ Frankenmuth 0-11 378-0814 10-0 Caldwell 12-0 378-0815 11-0 Abe 8-4 378-0816 7-§ l The underlining within the table defines the division between plantlets and their isolates. 2 The first number represents the number of resistant plants and the second number represents the number of susceptible plants. 52 Table 12. Plantlets and isolates tested for the H7H8 Gene Linel ggsdingsgcorsz Linel Reading Score2 5-0014 0-8 415-0965 0-8 5-0015 0-9 415-0966 0-11 5-0017 1-4 416-0968 0-11 5-0018 0-10 416-0970 0-9 90-0182 0-9 416-0972 0-9 90-0183 2-7 416-0974 2-8 109-0237 0-9 416-0975 1-6 109-0239 0-9 419-0982 0-9 150-0306 0-10 419-0984 0-6 150-0307 0-10 230-0464 3-5 Checks 230-0467 4-6 g30-0468 2-s Monon 0-11 238-0483 5-3 0-9 238-04§§ 2-6 367-0765 6-3 Abe 10-0 367-0768 5-3 6-1 §§7‘0775 1-7 10-0 406-0930 10-1 7-0 406-0931 6-3 406-0933 5-5 Seneca 8-1 406-0936 5-5 7-1 406-0937 ézé. 10-2 415-0963 0-10 11-1 415-0964 0-10 1 The underlining within the table defines the division between plantlets and their isolates. 2 The first number represents the number of resistant plants and the second number represents the number of susceptible plants. 53 Differential plant growth during the infestation period can influence the readings. The Hessian fly deposits the eggs on the upper leaf surface of the young plant, but cannot if the leaf has not yet emerged from the coleoptile (Foster et al., 1986). In this case, lines that exhibit mostly susceptible plants, with a few plants exhibiting what appears to be resistance, may actually be escapes caused by uneven germination and leaf blade emergence. Biotype admixture of the fly may also contribute to the observed variability as it is very difficult to maintain a completely pure fly strain (J. Foster, per. comm.). The biotype mixture may result from a mechanical mixture or mutation occurring within the biotype. Admixture could prevent lines from exhibiting complete resistance or susceptibility. Some wheat cultivars have a temperature sensitive reaction when tested for Hessian fly in the greenhouse (Sosa, 1979). At one temperature a plant may exhibit resistance, but will be susceptible when grown in a slightly different temperature. This type of sensitivity in known to occur in the check variety Abe (Maas et al., 1987), used in the H6 gene test, yielding both resistant and susceptiple plants (8-4 reading). This is one of the possible causes of variation found among isolates from the same plantlet in Table 11 since the pollen which gave rise to these plantlets originated from crosses which had one or more parents containing the H6 gene. 1n the study of stability of the H7H8 Hessian fly gene reactions between the DH1 and DH2 generations isolates from the same generation were tested together. Although the tests were run simultaneously, the isolates were in separate flats possibly introducing environmental differences. Allowing for this error, the differences reported in Table 8 between generations is still too great in several instances to be explained by the design flaw as the checks varied much less between flats. The variation in the fly studies reported in Tables 8, 10, 11 and 12 are often seen when screening early generations of plant breeding materials. In conventional breeding lines the variation has often been envisioned as being caused by heterozygosity at individual loci and possibly by minor modifying genes. It was believed that doubled haploids with the accompanying homozygosity would lead to more consistant resistant or susceptible readings. It did not. This opens a very fertile area for future research for both the doubled haploids variability and the accuracy of these Hessian fly tests. Thus, many environmental and biological factors can contribute to the apparent variability in the Hessian fly test making the data difficult to interpret. Since seed quantity was limiting, replications 0f the Hessian fly tests could not be completed. Further testing is necessary to 55 determine the non-genetic as well as the genetic forces involved in the observed variability in the Hessian fly data. Many of the environmental, experimental and biological causes of variability have been discussed for the insect and disease tests. The other source of observed variability is probably the consequence of the anther culture procedure. The different types of forces that can cause change within these doubled haploids is numerous. One possible explaination could be the the ELS (embryo-like structure) formed from multiple pollen grains which grew together to form one. This was stated as a possibility in a study by Kudirka et al. (1986). If plants arose from the same calli but from a different set of cells, natural genetic variability would exist. This is possible since more than one ELS can develop from an anther. The plantlet could also have been formed from somatic tissue, such as the anther filament (Kudirka et al., 1986). However, plantlets derived from somatic tissue of an F1 hybrid would vary for a number of characters in the F2 generation. This possibility is unlikely since no lines exhibited extreme variation in morphological characters or disease resistance. Spontaneous genetic variation could have arisen in the anther culture procedure or during the doubling procedure in several ways. One possible explaination for the observed variation could be the result of a mutation(s). A mutation during early callus development, referred to as sectoring or 56 chimera formation, has caused variation in tissue culture investigations (McCoy and Phillips, 1982; Buiatti et al., 1985; Barwale and Widholm, 1987). In the quoted studies some plantlets or isolates derived from the same calli were believed to have originated from different cell origins. These cell origins were believed to differ due to mutations occurring within the callus during development. The possibility of a chimeral effect, occurring in the embryogenic callus formation stage, could be partially responsible for the gametaclonal variation observed in this study. Mutations could also be induced by the chemicals in the medium and doubling procedure which are known mutagens, such as 2,4-D (Mohandas and Grant, 1972) and colchicine (Jensen, 1974). Several other factors could also be responsible for the observed variability. In tissue culture studies (Larkin and Scowcroft, 1981; Larkin et al.,1984) and pollen culture studies (Hu et al., 1978; Mix et al., 1978) karyotypic and cryptic changes have been observed. These types of changes include anueploidy, polyploidy, chromosomal breakage, deletions and replacement and rearrangements which can lead to variable expression of genes. A study by Ashmore and Gould (1981) utilizing Giemsa C-banding also found that chromosomal abnormalities occur both in number and structural changes. These alterations in chromosome structure could be involved in expressing variability directly or indirectly. One indirect pathway that has been discussed elsewhere is a chromosomal change such as a deletion, which could cause genes to be expressed that otherwise showed no penetrance. This phenomenon was termed hemizygosity by Siminovitch (1976) and also transposition by Larkin and Scowcroft (1981). They state that genes could be turned "on“ or "off" by the deletion or addition of DNA segments, causing genes that were once silent to become expressed. Thus, many factors could be involved in causing the observed variation within these doubled haploid isolates. Also, variability within these doubled haploids probably exists in other forms that could be detected using more specific tests involving cytogenetic and restriction enzyme studies. With all the possible sources of variability, it emphasizes the need for more testing to detect the source and extent of variability within these doubled haploids. Only comparisons for the awned/awnless morphological marker was conducted on the entire set of plants. Of the 1,131 DH1 isolates from 362 plantlets examined after heading, none exhibited variation for the awned/awnless character. However, in the DH2 generation, two awnless isolates from 2 different plantlets, gave rise to single plants with awns. Since the awnless gene is dominant, and most mutations occur from the dominant to the recessive, it is very likely that a mutation from the awnless to the awned trait occurred in both cases. 58 The mutations could have occurred in two possible stages of development. The mutations could have occurred between the DH1 and DH2 generation in the form of a double mutation from homozygous dominant to homozygous recessive. However, a more likely possibility is that the mutations occurred during the anther culture or doubling procedure. The awned characteristic could exist in the heterozygous state and would not appear in the DH1 generation as the recessive gene would be masked by the dominant gene. This is possible as Oono (1981) observed mutations occurring in rice pollen culture in the heterozygous state. Also, work done by Gu (1986) found that the phenotypic variability observed in his study was probably the result of recessive mutations. However, it is still posSible that other forces described earlier could also be responsible for this apparent mutation. 1f the plants in the DH1 generation were indeed heterozygous for this trait, the awnless to awned ratio should segregate into a 3:1 ratio in the DH2 generation. In both cases only six seeds were planted yielding two observed ratios of 5:1. Due to the low number of plants involved, this ratio does not differ significantly from a 3:1 ratio using the Chi-square contingency test. The appearance of an awned plant in two separate instances is difficult to explain by outcrossing as plants would exhibit variation for a number of characters. In both cases the 59 plants with the mutant character were similar in growth habit and other head and grain characteristics to their sister plants. The possibility of a single mutation could be tested by examining the awnless progeny of these two isolates. 1f the awnless lines give rise to plants expressing the awned characteristic in the next generation, it is very likely that the mutation occurred during the anther culture procedure and was maintained in the heterozygous state. If no plants exhibit the awned characteristic in future generations, the mutation may have been a double mutation or could be a result of the forces discussed earlier. There are many genes involved in conferring leaf rust, powdery mildew and Hessian fly resistance and in most cases the genes conferring the resistance are dominant (Flor, 1971; Everson and Gallun, 1980). Dominant resistant genes are utilized almost exclusively by plant breeders as they are easy to recognize and transfer when developing resistant varieties, especially in self-pollinating crops such as wheat. In this study a number of doubled haploid isolates exhibited very good resistance to leaf rust and powdery mildew. In several instances, the doubled haploid isolates had disease resistance superior to their parents. From 174 isolates tested for leaf rust 24 exhibited a reading score of 1 point better than the best parent (Table 13). This could be explained by inoculum or environmental variation as the parents and doubled haploid lines were not 60 Table 13. Doubled haploids having superior resistance to leaf rust than best parent. Best Best Parental Line Parental Line Line Ratigg Rating Line Ratigg Rating 5-0015 3 2 415-0964 3 2 56-0115 2 1 415-0965 3 2 66-0140 2 1 415-0966 . 3 2 364-0747 3 2 416-0968 3 2 364-0748 3 2 416-0970 3 2 364-0749 3 2 416-0972 3 2 364-0750 3 2 416-0974 3 2 364-0751 3 2 416-0975 3 2 364-0752 3 2 433-1038 2 1 364-0753 3 2 435-1043 2 1 364-0754 3 2 437-1047 2 1 415-0963 3 2 441-1059 2 1 Table 14. Doubled haploids having superior resistance to powdery mildew than best parent. Best Best Parental Line Parental Line Line Rating Ratigg Line Rating Rating 90-0182 4 0 370-0796 4 3 90-0183 4 0 370-0798 4 3 92-0188 4 0 370-0799 4 3 109-0237 3 0 379-0817 3 0 109-0239 3 0 379-0818 3 0 150-0306 4 0 382-0824 3 0 150-0307 4 0 382-0825 3 -0 170-0363 4 0 382-0826 3 0 333-0628 4 0 382-0827 3 0 334-0629 4 0 406-0930 3 0 344-0669 3 0 406-0931 3 0 347-0681 3 0 406-0933 3 0 347-0682 3 0 406-0936 3 0 347-0683 3 0 406-0937 3 0 347-0684 3 0 407-0938 3 0 367-0765 3 0 423-0996 4 0 367-0768 3 0 423-0997 4 0 367-0775 3 0 423-0998 4 0 370-0793 4 3 454-1099 3 0 370-0794 4 3 454-1100 3 0 370-0795 4 3 454-1101 3 0 3 0 454-1102 61 tested at the same time. However, of the 146 isolates tested for powdery mildew, 34 were completely resistant of which 8 were better than the best parent by 4 rating points with 26 better by 3 rating points (Table 14). In the powdery mildew case and possibily for leaf rust, it is possible that a recessive gene(s) may be responsible for this improved resistance. One possible way to test for the existance of a recessive gene(s) is to set up controlled matings. This could be done by crossing the doubled haploids having superior powdery mildew resistance compared to their parents, with a doubled haploid isolate exhibiting complete susceptibility. After hybridization, no plants should exhibit resistance or even partial resistance in the F1 generation. However, in the F2 generation, if only one gene is involved, the plants should segregate into a 3:1 ratio of susceptible to resistant or 15:1 if two genes are involved. Although no field evaluations were completed, some positive points can be derived from this study. Several isolates have been identified that have combined leaf rust and powdery mildew resistance. It appears that recessive genes for leaf rust and powdery mildew may be operating. Some isolates have a combination of Hessian fly genes along with disease resistance genes. Through anther culture, homozygous lines could be rapidly developed that have 62 resistant genes for different diseases and insects existing in combination with one another. These lines could be used for varietal development or for parental line improvement. Another similar type of utilization would be to combine several resistant genes to the same disease or insect. This type of gene combining has been discussed as a breeding strategy by Everson and Gallun (1980). This could be very important in combining a number of different genes for Hessian fly resistance. Currently, Hessian fly resistant genes are usually overcome relatively quickly through the devolpment of new biotypes (J. Foster, per. comm.). 1f the fly had to overcome several resistant genes simultaneously to infest a plant, it would be much more difficult. This would slow the emergence of new biotypes as well as add to the life of a variety containing the complex of genes. Anther culture is a way to fix combinations of genes in the homozygous condition. Probably the weakness of the anther culture technique, alluded to earlier, is the low number of doubled haploids produced with much effort and high cost. Tests for agronomic characters, adaptation and yield require large population for selection. Unfortunately, anther culture cannot provide homozygous lines in large numbers on a cost- effective basis compared to conventional breeding. ' I 1 '. ‘-ha "gi'QL- cat! " i mat-IL!— SUMMARY AND CONCLUSIONS A total of 486 haploid plantlets were developed by anther culture, of which 362 were doubled to the diploid level (doubled haploid) using colchicine. Each plantlet was subdivided into several individual isolates (plants). A total of 2,201 haploid isolates were treated with colchicine of which 528 were successfully doubled. However, plantlet doubling was 798 attributed to plantlets having several representatives in the colchicine treatment, thus increasing the efficiency of plantlet doubling through subdividing. Two different colchicine treatments were compared. The 5 hour treatment was more effective in doubling the haploid plants (70.98) than the 4 hour treatment (56.38); significant at the 0.01 level using the Chi-square contingency test. Since the 5 hour treatment was more effective than the 4 hour treatment, longer colchicine treatments should be explored to increase doubling efficiency. The production of fertile doubled haploid plantlets in the 3-way cross experiment was 2.08 from 12,096 anthers plated initially. The low production of doubled haploids is probably the main disadvantage of anther culture when comparing the time and effort expended. Efforts to increase 63 64 anther response, embryogenic callus formation and better doubling procedures are necessary to increase the utilization and potential of anther culture by making it more cost-effective. The main emphasis of this study was to evaluate these doubled haploid isolates utilizing disease and insect tests. The first phase was designed to examine whether doubled haploid isolates would be stable between generations using these tests. Doubled haploids should be homozygous theoretically, and therefore traits should be stable between generations. However, in all of the tests between the DH1 and DH2 generations variation occurred. For the leaf rust and powdery mildew test the variation observed was very low, at most 10 comparisons varied from a total of 136. Seven of these comparisons deviated by only one reading score and are probably the result of environmental influences leaving only three variable comparisons. However, in the H7H8 Hessian fly gene test between generations, much more variability was observed (408 higher than the highest deviating check reading). An experimental design flaw, uneven germination, biotype admixture and a temperature-sensitive interaction were all discussed as possible forces contributing to the observed variation. The high variation in these doubled haploids opens an area of further study on these doubled haploids for insect resistance and for testing the accuracy and effectiveness of the current Hessian fly testing procedures. 65 The second area of study was designed to see if isolates derived from the same plantlet would exhibit identical resistance and/or suceptibiltiy to these tests. Isolates from the same plantlet should be identical, unless some chromosomal change has occurred, as each plantlet was developed from a single haploid pollen grain. 1n the leaf rust and powdery mildew tests only two plantlets in each test exhibited variation greater than one in reading score with only one line deviating in both tests. In the three Hessian fly tests, at least one plantlet in each test exhibited a wide range of variability. The possibilities of environmental and biological influences and human reading errors were discussed as contributing to the observed variability. Variation was also attributed to the anther culture and doubling procedures. Many different sources may be involved. The embryogenic calli may have been formed from multiple microspores or possibly from somatic tissue generating natural genetic variability. Variation could also be induced by mutations caused by 2,4-D or colchicine. Karyotypic, cryptic or other chromosomal alterations, which have been observed in numerous studies, could also occur within these doubled haploids. Thus, with all of the possible causes of variability, the need for further testing is necessary to establish the extent and sources of variability. (26, The only comparison made on all of the isolates was for the awned/awnless morphological marker. From 1,131 doubled haploid isolates, two isolates, from different plantlets, varied within the line for this character, almost certainly caused by a mutation. The mutation probably occurred during or shortly after the anther culture procedure and was masked in the heterozygous condition and did not appear until the DH2 generation. The mutation should segregate into a 3:1 ratio of the dominant awnless to the recessive awned. However, a 5:1 ratio was observed in both cases, but because of the small number of plants involved, these ratios do not differ significantly from a 3:1 using the Chi-square contingency test. The cause can be established by examining these lines in future generations to see if variation still exists or not. One of the last points of the study was that several of the doubled haploid isolates tested exhibited superior leaf rust and especially powdery mildew resistance when compared to their parents. The presence of a recessive gene(s) could be responsible for this improved resistance. The anther culture procedure followed by doubling creates instantly homozygous lines which would fix the recessive gene(s). Further testing of these doubled haploid lines is needed to substantiate this possibility utilizing controlled matings between the resistant doubled haploids and homozygous 67 susceptible lines. 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