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-====== Perspective ======
-In common genetic practice we cannot know of the existence of any particular gene unless we have two recognizably divergent forms (alleles). Hence an urgent need to find as many mutants as possible. Much of the logic used in past genetic terminology was derived before and during the period when radiation was thought to be a major cause of genetic changes. Many of these mutant changes were dramatic clear-cut loss of function and often clusters of both related and unrelated functions and behaved as recessive alleles which were mostly dead ends. This was true because, especially ionizing radiation was an essentially destructive process that caused breakage and rearrangement or deletion of chromosome pieces carrying the functional genes. The discovery of transposable elements that transformed the function of components of DNA in a non-destructive way and the finding of chemical reagents which induced discrete changes in the DNA molecule at high frequencies made it possible to obtain large numbers of heritable changes. Because of the nature of the DNA molecule these changes are almost limitless in variety and provide the evolutionary basis for biology.
-====== Project research history ======
-It has been the privilege and good fortune of MGN to have begun his career preparation during the pinnacle of discovery of radiation biology with its master, L J Stadler; to have followed in his footsteps and logic and eventually inherit his outstanding research project with its world class resources, traditions and goals; to have continued his own research career into the mutagenic behavior of transposable elements under the inspiration of Barbara McClintock; to have perfected a technique for chemical mutagenesis in maize which has produced the largest collection of maize mutants in existence; and to have spent the remainder of his career recording, evaluating and making good use of this marvelous resource. This and many other exciting tangential projects became available as a result of these opportunities. It was also a special advantage to have been involved in the collection and preparation of a photographic record of the phenotypes of all the early collection of naturally occurring and induced mutations in Maize with Loring Jones and Marcus Zuber and later with Ed Coe and Sue Wessler and presenting them in two editions of “The Mutants of Maize”. The opportunity to be intimately involved with cutting-edge research projects and with highly skilled colleagues working in cytogenetics, Ionizing radiation, transposon behavior and chemical manipulation of DNA all during the “Golden Era” of genetic discovery was especially rewarding. Finally, having made a good photo record of most of the mutant phenotypes over many years has provided the basis for this presentation. It all began with a Doctoral Thesis about the analysis of the progeny of a single unique colorless kernel with many purple dots on its surface which proved to be transposon activity at the A1 locus produced by the activator component Dt1. Our collection consists of the best images of a photographic record of all the mutant phenotypes that it was possible to capture in the many experiments from then until now.
-====== Earlier studies of gene mutation ======
-Earlier studies of gene mutation were based on rare wide ranging single naturally occurring events and more frequent mostly destructive radiation induced cytological events produced new phenotypes which only partially reflected the broad range, intricacy and potential value built into the DNA molecule. Having large and repetitive numbers of discrete genetic changes afforded by the discovery of the mutagenic activity of ionizing radiation (Stadler) (E G Anderson) then UV light (Stadler and Uber) then Transposable elements in the genetics system (McClintock) and finally the use of chemical reagents to directly change DNA configuration discretely in the germ line of the maize plant (Neuffer) has made it possible to have a wide array of mutant phenotypes that can be directly tied to mutational events of specific genes. It helped a great deal to have had the foresight and resources to make a good photographic record of a majority of those events.
-The treatment experiments were initially designed to test the effect on targeted well known genes that could be followed through succeeding generations so that the changed phenotypes could be objectively observed and recorded. Genes affecting anthocyanin pigments such as A1 and kernel structure such as Sh2 were selected for ease of handling. Targeting of genes in transposon studies was achieved by starting at known sites and moving to other selected loci. The frequency of events which appeared to be quite high was confused by the fact that they occurred at many stages of development of the germ lineages often having mutant sectors which produced multiple copies of a single event that could not be distinguished from single events in the test M2 progenies. The frequency as a result of transposon activity appeared to be very high when measured in the M2 but was not accurate because events happening during somatic cell divisions were duplicated many times before they were recorded. The rate per cell generation was low when multiplied by the number cell generations in the reproductive life of the plant. Also transposons rarely ever produce alleles that were dominant to wild type in the M1.
-A more comprehensive presentation of Historical Perspective of EMS mutagenesis and literature review is presented in the following publication:   Mutagenesis – The Key to Genetic Analysis; M G Neuffer, Guri Johal, and Sarah Hake.  (2009) Handbook of Maize Genetic and Genomics; Bennetzen J and Hake S (eds) Vol 2 pp 63-85; Springer-Verlag, NY.
-====== Chemical mutagenesis ======
-For protocol, see “Mutants of Maize” chapter seven, pages 395-403.
-===== Pollen treatment =====
-The frequency from chemical mutagenesis using the paraffin oil pollen method was very high and could be accurately measured in both the M1 and M2 because the pollen grain at the moment of treatment contains only 2 single germinal nuclei each of which may become an individual M1 plant and an unequivocal progeny to be counted. In addition the chemical reagents did produce significant numbers of mutants that were dominant to wild type and therefore easily seen in the M1 as whole plant cases and conveniently as half plant chimeras which permitted observation and preservation of  lethal dominant mutants. The actual frequency of mutation from treatment of maize pollen with EMS (ethyl methane sulfonate) ranged widely depending on treatment stocks, conditions, procedures, loci, etc. Our “rule of thumb” for a good average treatment was: 1 recessive mutant, per gene locus, per 1000 treated pollen grains that achieved fertilization. For dominant mutants it was, 1 per 200,000 in the M1 which is the average including a few like Oy1 (which have a uniquely high “recessive” rate of about 1/1000). The fact that all the genetic components in the rest of the genome were randomly subjected to the same powerful force for change had serious consequences. Literally hundreds of new visible mutants (and unknown numbers of invisible cases) were produced in M1 (recessives observed in M2) treatment populations of as small as 3000. This lead us to change our approach from the study of selected individuals to mutation in general.   This we did until we had examined approximately 25,000 M2’s and found  that we were getting high numbers of repeats of the high frequency recessives and  relatively fewer new ones.  We then changed our strategy to look among the M1’s for relatively rare dominant cases which were mostly unique new ones.  In doing so we found a total of 251 new dominant mutants in 100,000 treatment progenies examined. This can be compared with a total of 62 dominant mutants known to exist in maize in 1968 when the first edition of Mutants of Maize was published.  This was gratifying because most of the biologically and commercially useful mutants are dominant.
-===== Seed treatment =====
- We were able to produce good numbers of valid mutants when treating corn kernels with EMS but found that the difficulty of identifying and validating individual cases made it much less efficient so we abandoned that effort. It is apparent, however, that seed and organ treatment can be successful in other creatures whose male gametes are not as convenient as those in corn. See [[http://mutants.maizegdb.org/doku.php?id=info:topics#the_recessive_waxy_wheat_story | the “waxy wheat story” below]].
-====== Project Data ======
-MGN has had the pleasure of working with all the above forms of manipulation of the Maize genome and has collected mutant data and images from all but the “MU Transposon System”, which was treated with great respect, and deliberately kept out of our fields as though it had something that could be catching. All of this information has been brought together over the years in our own local Mutant Database (currently stored as a Microsoft Access database) which consists of a case number, symbol, name, phenotype, linkage, treatment, origin, description, seed source, photo images and other pertinent data. We periodically share our latest copies of this database with colleagues at MaizeGDB. This wiki links to data at MaizeGDB, including much of the data in Mutants of Maize. MaizeGDB, in turn, links to the Coop stock center, making it easy for interested parties to get access to seeds of the described mutants.
-| **Mutant images in our mutant data base**                                                       |||
-| Total image all sources                                                                           ||    8596|
-|  | “Mutants of Maize” images (includes all other sources)                   |     215|
-|  | Data base images                                                                                  |    8381|
-|  | Data base mutants                                                                                |    3483|
-| Sources; no implication of frequency just opportunity occurrences            ||
-|  | Naturally occurring of unknown causes                                              |   219 |
-|  | Radiation: Xrays, Nuclear Ionizing, Ultraviolet                                   |      13 |
-|  | Transposon: Dt, Ac, Spm, Mr, Rmb, Rst, Mu                                       |   205 |
-|  | Chemical: EMS, NG                                                                               |   3070 |
-|  | Other (ploidy, cytogenetic consequences, etc                                   |       11 |
-|  | Total, (including 35 recorded twice/ cultures with 2 active agents) |   3518 |
-===== Our Data Base =====
-All the data for our mutant collection is currently kept in a Microsoft Access Data Base in the following format and will eventually be turned over to MaizeGDB for handling:
-{{:info:database_sample_image_480px.png}}
-^ Field  ^ Definition                                                             ^
-| labno | Unique identification number for this mutant |
-| dgs    | Designated gene symbol (symbol accepted by the stock center to represent this mutant)                |
-| Chromosome arm | Chromosomal arm by linkage or cytogenetic test |
-| phenotype | Description of the phenotype for this particular mutant |
-| PT | Genetic pedigree and treatment (in this case opaque 1 ear stock crossed with opaque 2 EMS-treated pollen stock)  |
-| source | Internal identifier of our own in-house seed source. |
-| con      | Confirmed as mutant |
-| fate      | Sent to Coop (in this case envelope #2) |
-| origin | original mutant plant |
-| photos | pedigree of mutant photo(s) |
-| misc | miscellaneous notes |
-| tests | miscellaneous tests |
-| refs | references |
-| allel | allelic to |
-| linkage | linkage data |
-| filename | relative filename for image, relative to location of the database |
-| caption | The original caption for the database |
-| Short description | Description used as a caption in the online field guide |
-===== Sharing policy =====
-Soon after we recognized the great value of our material and had accumulated sizable M1 and M2 populations we knew that we could not handle even a small part of the opportunities provided:  We decided to share with others and invited them with the statement: “If you have interest in the genetic control of any biological function and can imagine what its phenotype will be, then come and sample our M2 populations of selfed M1 ears because it will most likely be there. Our calculations are that if you take a 30 seed sample from 3000 or more ears and test them you will have a 95% chance that it will be there.” Several colleagues did so and they were never disappointed.
-====== Phenocopies======
-//Not everything that looks like a mutant is a mutant//
-Visual expressions which appear to be phenotypes but are not of genetic origin (Phenocopies):   Abrupt changes in the appearance of single plants or members of a population that are unique often occur but on testing turn out to be the result of non genetic factors such as yellow leaf stripe (iron or nitrogen soil deficiency). Vivipary;  premature kernel germination (fungal infection of the kernel). Morphological changes such as twinning of growth at the nodes and abnormal structures (smut infection). Mutations causing leaf lesions, chlorophyll streaks, or strong anthocyanin pigmentation each have a counterpart phenotype produced as symptoms of a disease caused by a specific pathogenic organism, insect pest or unusual environmental event. The truth of the matter in each case requires a pedigree analysis for proof and the results often provide interesting and valuable insight into the detailed workings of Biology. Another example is the appearance of apparently dominant kernel mutants such as Sugary, Shrunken, Floury White etc whole and sectored kernels, which appear in the M1, many of which do not transmit to the succeeding plant because they resulted from the dual fertilization observed in maize where the endosperm gets a mutagenized gamete and the embryo a normal one.  The reverse is true in equal numbers but cannot be seen until expressed in the embryo progeny.
-====== Purpose of this wiki ======
-Having access to such a large collection of mutants with multiple copies of many genes and their associated phenotypes gives the observer a different perspective than that derived by observing rare single unique events. Having a single mutant event expressed as a definitive phenotype is like viewing a single frame of a motion picture film to determine the characteristics of the object framed. Each additional copy gives valuable data which leads to the complete picture. Each clue is very valuable but useful only to the extent of other knowledge available to the viewer. The purpose of this Guide is to provide as many visual clues as possible to the interested worker who may be looking at corn for whatever purpose. This is so because It also provides keyed access to the much larger collection of similar examples found with much more comprehensive data at MaizeGDB.
-When viewing new mutant phenotypes we have found that there are many aspects to consider. It is true that while things that pertain to the welfare or survival of an organism may or may not have an impact on a visual phenotypic expression there are many things that do in peculiar ways. Reporting some of these may be helpful to the user of this Guide. We add them here as text topics logically inserted with the definitions and appropriate captions accompanying the images.
-A collection of 836 of the best images of 359 phenotypes each associated with a known gene is presented at this web site as a guide to current maize mutant phenotypes. It is prepared with simple definitions and captions that will be clear and useful to the beginning student of Maize, to the worker in the corn field, to the graduate student, post doc, biologist and the seasoned  maize geneticist; to give each a clearer understanding of this especially useful plant and how it provides special insight into all biology. The photos which were taken by an amateur photographer with an ordinary camera and processed by modern electronic means, are high resolution and can be viewed in the classroom, laboratory or field using computer, tablet or even smart phone. The images can be used to get a general view or enlarged to be seen at the cellular level and can be simply printed out for paper viewing or transmitted electronically as needed.
-====== Topics ======
-===== Dominant mutants =====
-As a result of cooperatively helping colleagues treat corn pollen with EMS to find significant new mutants in their specific areas of emphasis, in their own laboratories, we have been able to view large M1 plantings and recognize new dominant mutant types, both whole plant cases and half plant chimeras. Many of these mutants were saved by selfing or outcrossing to normal and observing the progeny. From over 300 putative cases noted, 251 were validated and subjected to tests to confirm, evaluate and characterize them as mutants. Of these, 84 proved to be good, clear and viable cases which could be maintained as stocks and had relevant data and clear photographic images. This group is of special interest because dominant mutants are quite rare from EMS mutagenesis (200 times more rare than recessives) and thus are more likely to be something not previously observed. Seed samples and relevant data for each of these mutants were sent to the Maize Genetics Stock Center, and similar data along with high-resolution photo images are available at MaizeGDB. The purpose of the poster presentation was to call attention to these mutants, which are freely available to colleagues and students, and hopefully will lead to their characterization and location in the maize genome using some of the exciting new technologies now available. The details of treatment, problems, consequences of handling, and theoretical considerations are found in an earlier publication: Neuffer, etal; 2009. All our images, posted at MaizeGDB, are of high resolution and can usually be digitally enlarged to reveal often striking details about each mutant. These mutants are a unique and valuable resource, but none have been definitely placed in the maize genome. We encourage colleagues and students to join us in doing so.
-See this 2011 Maize Genetics Conference (St. Charles, Illinois) poster on Dominant Mutants from EMS Pollen Treatment (click on the image to download the PDF):
-<html><a href="/lib/exe/fetch.php?id=info%3Atopics&amp;media=posters:new_dominant_mutants_from_ems_pollen_treatment.pdf" class="media"><img src="/lib/exe/fetch.php?media=posters:new_dom_mut_from_ems_pol_treat_poster_480px.jpg" class="media" title="new_dom_mut_from_ems_pol_treat_poster_480px.jpg" alt="new_dom_mut_from_ems_pol_treat_poster_480px.jpg" /></a></html>
-===== Chimeral recovery of lethals =====
-Dominant lethal mutants that are not otherwise visible and sustainable can occur and be observed for analysis in Chimeral tissue.  The original YgD*-N2542/+ heterozygous mutant chimeric plant showed a broad pale-yellow green sector covering 1/2 of the leaf blade, sheath and tassel.  This sector appears to be physically supported and nutritionally sustained by the normal plant tissue.
-Hidden recessive lethal phenotypes can also be seen in normal heterozygotes through Chimeral loss of the normal allele by chromosome breaking Ds events caused by Ac activity.  Colorless floury defective; defective albino sectors were seen on the top leaf surface of a Ds-1S2 plant grown from the original mosaic kernel. The surface shows small long narrow sectors of indented tissue. Some larger sectors are white, indicating the absence of chlorophyll. This proved that an additional phenotype of clf1 is white albino.
-===== Pleiotropy =====
-//Pleiotropy: Single genes with multiple phenotypes.//
-Phenotype is the message we get that tells us that there has been a change in a gene controlling some biological function. What we learn from the phenotype actually depends on our ability to recognize and properly interpret what we observe. In the simplest case, it is the presence or absence of a measurable product (anthocyanin) or structure (ligule). In actual fact there are many functions involved in production of a particular product or activity and these are all a part of a complicated choreography leading to a certain display. Maize is an exceptionally well suited organism for demonstrating this point.
-Ideally one might expect a fairly consistent relationship between phenotypic expression and each causal genetic event but reviewing large numbers of mutations (including both numbers of loci and repeats of single loci) has shown that such is not the case. Duplicate factor genes which generally appear alike usually produce mutants that are demonstratively different from each other. Likewise repeated mutants of the same locus arise in different allelic forms either because they are different or because they have different immediately adjacent neighboring genes which modify their activity.  Some loci have mutants that regularly express variations of only one phenotype (i.e. white seedling) but some mutant genes have multiple phenotypes. For example repeated mutants at the clf1 locus on the short arm of chromosome #1 have four distinct phenotypes: a colorless (no anthocyanin because the aleurone layer where the pigment should reside is completely missing) kernel, a white floury endosperm, a small round defective embryo and lethal albino (can only be seen as chimeral loss in DsClf/clf leaf tissue) plant.
-Examples of pleiotropy:
-(1) clf1(dek1); EMS induced recessive mutant; Ac Ds-1S2,4 Clf1 transposon analysis.
-(2) PgD; EMS induced dominant chimera case.
-There are many important genetically controlled activities that regulate expression of a phenotype without being in the biochemical pathway that leads to the observed phenotype.
-See also this poster on Pleiotropy from the 2012 Maize Genetics Conference in Portland, Orgeon (click on the image to download the PDF) :
-<html><a href="http://mutants.maizegdb.org/lib/exe/fetch.php?media=posters:pleiotropy_2012_03_12.pdf" class="media"><img src="/lib/exe/fetch.php?media=posters:pleiotropy_2012_03_12_480px.jpg" class="media" title="pleiotropy_2012_03_12_480px.jpg" alt="pleiotropy_2012_03_12_480px.jpg" /></a></html>
-===== Number of genes per phenotype =====
-We were surprised at the large number of lesion mimic mutants obtained (82). Our rough estimate was that there were more than 200 such loci regulating this sort of disease symptoms.  Also there were a few loci in which the dominant rate was in the same range as the common recessive rate (1/1000; Oy1 for example which also has only one site in the corn genome. Another surprise was the fact that there were 19 different hcf (Miles) loci with mutants affecting a single operation: the transport of a single electron across the chloroplast membrane. There were many other interesting things that have been or still are available for inquiry.
-===== Lesion Mimics =====
-The disease lesion (Les) mimics are the most frequently occurring dominant mutants from EMS mutagenesis in maize. From the screening of over 50,000 M1 plants for all variations of the lesion phenotype, we have identified 51 separate dominant cases. A much smaller, though probably comparable number of recessive les mutants, have been seen but not considered in this report.
-The mutants range widely in expression, but have the common phenotype of leaf lesions that are strikingly similar to those caused by various leaf blight diseases. In all cases tested, the phenotypes have occurred in the absence of a pathogen. They are initiated by sunlight and certain chemicals, and can be chlorotic, necrotic, or sequentially both. The lesions of different mutants vary in size, shape, color, frequency, distribution, time of onset, position, rate of expansion, sharpness of boundaries, etc. In some mutants, the lesions expand to cover the leaf, resulting in senescence. It appears that particular cells on the leaf surface are, at specific developmental stages and within certain specifically variable temperature ranges, highly susceptible to damage by sunlight.
-We hypothesize that two signals are involved. The first arises from dissolution of the cell membrane, which releases highly active cell contents that cause lethal damage to neighboring cells. This damage spreads continuously outward, forming a necrotic lesion that stops growing when conditions change. The second signal is revealed by the “Target Spot” oscillatory phenotype; a central spot of dead tissue surrounded by alternating rings of healthy and dead tissue. This phenotype suggests signaling between dead and living cells, across living tissue, that causes lesion formation. This signal may proceed more rapidly than the first, through several ranks of normal cells without damaging them during a diurnal cycle of conditions that do not favor lesion formation.
-See also this <html><strong><a href="/lib/exe/fetch.php?media=posters:lestargetspot_2014_03_06.pdf" class="media mediafile mf_pdf" title="posters:lestargetspot_2014_03_06.pdf">poster on Les Target Spot (in English and Chinese) from the 2014 Maize Genetics Conference in Beijing, China</a></strong></html>.
-<html><a href=" http://mutants.maizegdb.org/lib/exe/fetch.php?media=posters:lestargetspot_2014_03_06.pdf" class="media"><img src="/lib/exe/fetch.php?media=posters:lestargetspot_2014_03_06_480px.jpg" class="media" title=" lestargetspot_2014_03_06_480px.jpg " alt=" lestargetspot_2014_03_06_480px.jpg " /></a></html>
-==== Porphyria a Dominant Mutant Phenotype in Maize and in Humans ====
-Hu, Yalpani, Briggs, and Johal, reported that the maize lesion mimic gene Les22 which is defined as dominant mutation characterized by the production of minute necrotic spots on leaves in a developmentally specified and light-dependent manner.   Phenotypicaly, Les22 lesions resemble those that are triggered during a hypersensitive disease resistance response of plants to pathogens. They cloned Les22 using a Mutator-tagging technique.  It encodes uroporphyrinogen decarboxylase (UROD), a key enzyme in the biosynthetic pathway of chlorophyll and heme in plants. Urod mutations in humans are also dominant and cause the metabolic disorder porphyria, which manifests itself as light-induced skin morbidity resulting from an excessive accumulation of photoexcitable uroporphyrin. The phenotypic and genetic similarities between porphyria and  Les22 and their observation that Les22 is also associated with an accumulation of uroporphyrin presents what appears to be a case of  porphyria in plants.
-Gongshe Hu,a Nasser Yalpani,b Steven P. Briggs,b and Gurmukh S. Johala,1 aDepartment of Agronomy, University of Missouri, Columbia, Missouri 65211  bPioneer Hi-Bred International Inc., 7300 N.W. 62nd Avenue, Johnston, Iowa 50131 The Plant Cell, Vol. 10, 1095–1105, July 1998, www.plantcell.org © 1998 American Society of Plant Physiologists
-===== Chromosome breaking Ds phenotypes =====
-The relative position of Ds and associated genetic markers produce unique genotypes and phenotypes that can be determined by observing AcDs induced loss and revertant chimeras.
-(see [[http://maizegenereview.maizegdb.org/doku.php?id=chromosome_breaking_ds | Chromosome Breaking Ds Part I]] and [[http://maizegenereview.maizegdb.org/doku.php?id=chromosome_breaking_ds_sites | Chromosome Breaking Ds Part II]] at [[http://maizegenereview.maizegdb.org/ | Maize Gene Review]]).
-Ac Clf1 Ds-1S4 kernel chimeras with proximal location, show a lack of cell autonomy, have lacy colored-colorless areas and leaf chimeras and otherwise hidden morphologically changed albino tissue.  Ac Ds-1L6 Bz2 shows breaking Ds at the Bz1 locus producing both losses and intermediate revertant alleles.  Ac/-, b1-m: Ds2S3 (at B1) shows non breaking function with suppression and reversion and also red streaks of P-vv (Ac reversion); colorless (no B function) kernels with many tiny purple (B) aleurone dots, at 3 levels of Ac dosage and recovery sectors of red maternal pericarp due to 1 dose Ac in the female plant..  Ac/-, Ds-3L2 A1 Sh2 shows behavior of closely linked genes governing anthocyanin and sugar accumulation.  Ac/-, Ds-4L6 C2 shows frequent loss of C2 color in pale Ccc and dilute CCc aleurone heterozygous kernels display twin color/colorless spots; distal location.  Ac/-, A2 Ds-5S1 Bt1 Pr1 show special phenotypes when both the Ds breaker and the centromere are between the gene A2 on the short arm and Bt1 Pr1 on the long arm.  Our challenge is to interpret them properly.  Ac/-, O5 Ds-7L1 has frequent collapsed opaque sectors and pits on mutant kernels and slender white streaks on seedling which are variations of the known phenotypes of different mutant o5 alleles.
-===== Practical Applications =====
-==== Pollen Mutagesis –The Dominant Id Maize Story ====
-Using pollen mutagenesis, in the summer of 1987, Dr M T Chang, shown in photo with windmill type vial mixer for multiple treatment EMS oil pollen mixture, found a dominant IT gene mutation. .  IT stands for Imazathaphy Tolerant.  A total of 3,000 ears were pollinated by EMS mutagenized pollen, and about 250,000 M1 seeds obtained.  An estimated progeny of 100,000M1 seedlings were grown in sand benches and sprayed with Pursuit (Imidazolinone) herbicide producing an extreme bleached lethal seedling phenotype among which were found more than 10 tolerant normal mutant seedlings.  These were saved and proved to be dominant resistant to the herbicide. The mutation rate is 1 per 10,000.
-{{:info:vial_mixer_480px.jpg}}
-{{:info:id_screening_480px.jpg}}
-{{:info:id_screening_success_480px.jpg}}
-==== The Recessive waxy Wheat Story ====
- In 1995, Dr. Chang found that IKE wheat had 2 waxy genomes and 1 non-waxy genome. He reasoned that one could mutagenize the single non-waxy gene and thereby produce a waxy wheat variety.  An estimated 80,000 IKE wheat seeds were soaked 8 hours overnight in an EMS solution with air bubbled through the solution, then washed 2 hours with water, then mixed with Polite to dry, and then planted. Seeds were harvested on a single plant basis and were shelled and put on a light box and examined for segregation of non-transmitting dark seeds.  The light non-transmitting seeds were stained with iodine solution and observed under microscope to see whether the starch stained red (waxy) or blue.  From less than 10,000 plant heads examined, more than 10 cases were found segregating for waxy.  The mutation rate was about 1 per 1,000.
-===== Diurnal cross banding =====
-Many genes controlling seedling and plant phenotypes have mutant alleles that are sensitive to daily extremes of temperature and/or sunlight.  These express zebra like cross bands of albino, Homozygous cb*-N1620A pale green mutant seedlings, showing white and green in crossbands and green leaf tips.  These are indicators of sensitivity of this mutant to environmental conditions at time of emergence and were seen for chlorotic, and necrotic mutants as well.  In the poster two nec3-N409, M2 tan necrotic mutant plants and 1 normal seedling with; 1st 2 leaves (left) manually unrolled mutant leaves and intact tightly rolled tan necrotic seedling (middle) showing alternate diurnal bands of tan necrotic and narrow dark brown crossbands which contain a dark fluid released in the dying cells.  This fluid is often extruded as dark brown drops on leaf margins and tip or anthocyanin tissue that can be related to the timing of the conditions causing them.
-===== Filial Phenotype expressed by maternal tissue on kernel =====
-A clear example of this is seen in the duplicate factors producing orange pericarp:  A selfed M1 ear was found to be segregating 15 normal to 1 for orange kernels.  A genetic analysis established that two mutant genes on separate chromosomes (4S and and 10L) were responsible.  It was further determined that 4S gene (orp1) was present in the untreated female parent (Parker Flint) and the other orp2 arose in the EMS treated male parent and could not be recognized until they were brought together in the treatment M1. The unique thing about this pair of duplicate factors is that they are expressed in the female pericarp tissue which shows orange only on those filial kernels which are recessive for both genes.  Which were brought together by the high mutation rate of the treatment.  For another example  See orp*-N888D.
-===== Genetic modifiers =====
-The phenotype of induced dominant  mutant alleles of most genes are highly responsive to many different genetic modifiers found in diverse breeding stocks such as Mo17, Mo20w, B73, and W23.  Outcross will produce enhancement, suppression, or even elimination of the phenotype depending on the mutant gene and stock.  For example Les1 produces very few lesions when crossed on Mo20w but an almost lethal profusion of lesions on W23.  Almost the reverse is true with when Les10 is crossed on the same stocks.  Similarly when heterozygote’s for certain recessive lethal defective kernel mutants are selfed there are recessive segregants in some of the F2s that are viable and produce viable though modified mutant plants. This is probably true because, especially, the elite inbreds all carry an assortment of dominant alleles of genes that promote vigor, yield etc. or cover weak alleles of other genes when making hybrids.
-===== Genetically  Modified Organisms (GMO’s) =====
-The thousands of mutant genes produced on this project certainly are each could be considered an example of a “genetically modified organism”.  We, the Authors and Associates and our many competent colleagues have handled all of this material daily in many ways; pollen in our faces and on our hands, plant dust and residue in our lungs and on our clothing; and we have eaten our crop when appropriate.  No mutant corn plant has ever bitten, stung, poisoned or injured its handler in any logical way that we know of.  Stadler and some of his peers died as a result of the biological effect of radiation which he pioneered with and some of the handlers of chemical reagents may have been injured by accidental mishandling of dangerous fluids but not by prudent use thereof. Some of the chemicals are dangerous carcinogens which react rapidly when applied to cells of living tissue to cause the mutations but which are promptly de-activated by powerful protective enzymes in all living cells (or pollen grains).  We went to great lengths to prove that there was no residual activity in growing tissue and that dead tissue and equipment was properly de-activated or disposed of. Thousands of humans have worked intimately with corn plants in the fields producing hybrid seed and growing the crop for commerce with no discernible damage except for work stress and pollen allergies (more common in women than men,  we suspect hormonal interaction).  In our operations we literally copied what nature is doing all the time but we just increased the frequency of one major aspect a **thousand** fold.  We as intelligent creatures are personably responsible to handle wisely those things which our world provides and learn to accommodate those extremely rare, possibly dangerous events just as nature has done in the past. Such is a fundamental tenant of Natural Selection.
-===== Greening of plant tissue =====
-//Virescent, delayed greening of emerging leaf tissue//
-The most common (**258 cases**) mutant phenotype induced is that of variations in the timing and characteristics of development of chlorophyll and related pigments following emergence of the seedling and growth of the plant.  Typically seedlings emerge devoid of such pigmentation but it develops quickly after exposure to light.  Mutants occur which delay that response giving white seedlings with gradually greening of older leaf tips.  This phenotype is more pronounce under cool weather conditions or reduced sunlight.  Other phenotypic variables are different distribution patterns of pigment development such as between veins only or not, in irregular patches or streaks and in different colors (i.e. green or yellow green).  Unique sensitivity to various environmental conditions is common.
-===== Navajo Spot Silk Attachment =====
-In this phenotype mutant kernel tissue immediately around the base of the silk attachment sometimes show a different, “Navajo type” phenotype from the rest of the kernel.  Typically a sun red or purple aleurone colored spot appears on an otherwise colorless R1-nj kernel.  Similarly, spots of normal tissue appear in the same place on collapsed dek17 and dek33 mutant kernels. floury dent navajo spot defective kernel:  See the selfed ear of a + + fldntnj*-(dek33)-N1299, + /a2 bt1 + pr heterozygous plant segregating for large floury dent dek33 kernels and also for linked (in repulsion) a2 (colorless aleurone), bt1 (brittle endosperm), and pr1 (red aleurone) kernels showing intricate phenotypic interaction between the 4 genes involved. Note colored plump "navajo" spot of apparently normal tissue around the site of the silk attachment on top of several of the dek33 kernels indicating the conditional nature of dek33's blockage of anthocyanin production in this case.  These are highly suggestive of tissue response to sunlight which could only arrive in such a covered dark place if conducted there by the possible fiber optic properties of freshly exposed silks.
-===== Warm plant =====
-Leaves of maturing plants that are warm to touch:  Plants in shoulder high M2, segregated for individuals that were warm when brushed against worker’s bare arm.  Closer examination reveals a slight almost imperceptible, yellow cast of mutants but no reduction of vigor.
-===== Leopard Spot pattern =====
-Pale green background with dark green spots arranged in a systematic pattern.  Leaves of original Spt*-N2597/+ heterozygous mutant plant showed a light pale green background with dark normal green spots arranged in a systematic pattern.   Good pollen but no ear was produced.  No progeny was obtained because no pollination was made!  This case is reported here as possibly valid because another similar plant was seen some years later but too late to produce progeny.
-====== Disclaimer: Scientific Validation ======
-MGN’s comments about the many interesting things seen in this huge collection of images and the experiences of gathering and reporting the data that go along with them reflect the fact that not all observations could possibly be validated by carefully prepared scientific proof instead they often point to the many aspects of biology that are uncovered there from for further more careful investigation.  We do present good strong data but also provide some information on a show and tell basis in hopes of exciting the reader to carry on where we left off.