GENETIC RESISTANCE AGAINST PATHOGENS IN WHEAT (Synopsis 2)

INTRODUCTION

Wheat is one of the major crop in Pakistan and occupies a position of paramount importance. It was planted over 7.2million ha annually during the period from 1981-82 to 1984-85(Shah, 1994) that yielded 11.6 million tonnes. The wheat crop in Pakistan is attacked by a large number of insects such as Aphids and Armyworm during spring after heading. the microbial pathogens that attacks wheat include Stem Rust, Leaf Rust and Stripe Rust caused by Puccinia graminis, P. recondita and P. striiformes, respectively(Akhtar, 1976). Leaf and stem rust occur all over country and become serious wherever and whenever weather conditions permit. moreover, root rot and leaf blight caused by Helminthosporium sativum occurs in parts of sind and Punjab plains. these disease epidemics cause heavy losses due to low yield and quality.
Reliance on heavy use of chemicals to control pests and microbial pathogens is no more desirable to the environment. It is becuase of their persistance and hazardous nature. Therefore, an urgent need to explore the available genetic resources for natural resistance with acceptable agronomic traits cannot be ignored. Plants are exposed to pathogens but do not have an immune system comparable to that of animals(Staskawicz, 1995). However, plants evolve defense mechanisms that involve several physical and chemical factors(Campbell, 1985). Physical defense mechanism includes tough epidermises, cuticular deposits, spines, thorns, prickles and stinging hairs whereas chemical defense mechanism includes toxins and repellents. Individual plant cells confront the disease by the combination of constitutive or induced defenses(Staskawicz, 1995). One of the earliest response is termed as Hypersensitive Response(HR), which is characterised by the oxidative burst. Release of Reactive Oxygen Species(ROS) renders toxicity to microbes and initiate cell death pathway. The second most common phenomenon is Systemic Acquired Resistance(SAR), which induces release of certain chemicals in the surrounding tissues. SAR occurs in response to necrotising infections caused by viruses, bacteria and fungi which persists for several weeks.
Disease resistance also occurs due to antimicrobial compounds such as Phytoalexins, Polypeptides and Proteins produced in response to infection. Some metabolites and plant hormones also increase in response to the fungal and viral infections in wheat(Metraux, 1990). Secondary metabolites sch as Hydroxamic acids(Niemeyer, 1988) has been reported in mediating plant defence against pathogens. Hydroxamic acid is present as an inactive Glucoside and upon mechanical injury came in contact with Glucosidase to produce 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one(DIMBOA). DIMBOA protect plants against aphids and microbial pathogen.

OBJECTIVES

1. To study the chemical defense system in Triticum aestivum L. cultivars against microbial pathgens,
2. To detect and isolate the factor responsible for resistance,
3. To study the linkage of disease resistance to the chemical defense system.
   

PLAN OF WORK

PLANT MATERIAL :
Pathogen resistance and susceptible cultivars of Triticum aestivum L.

Phase 1:-
In this phase local genetic resources will be analysed for the quantification of Hydroxamic acids and other molecules both in prescence and abscence of infection. This involves selection of suitable tissues for he extraction of Pathogenesis related(PR) compounds. This involves preparation of the protoplasts from leaf tissues. Respective metabolites may be identified using UV-TLC methods. For the quantification of Hydroxamic acids Spectrophotometric technique will be employed.

Phase 2:-
The second phase of this research involves correlation of the endogeous quantities of pathogen combating metabolites and plant resistance. For this purpose the symptomatology of the disease will be quantified in different genotypes. This may help in identifying the factors that enable a plant to resist a pathogenic attack more than a sensitive plant.

Synopsis

MOLECULAR GENETIC STUDIES OF PAKISTANI POPULATIONS

Introduction

Revolutionary discoveries in molecular biology have opened up new vistas for research. It has been known for years that genetic characters are transmitted from parents to their offspring. However, it has been few decades ago that it is possible to study this phenomen at the molecular level.
All this has become due to the abilities to cut DNA molecules into smaller fragments with Restriction Endonucleases:
to engineer them into microorganisms for producing unlimited number of a particular DNA piece,
to sequence DNA,
to identify genes and ultimately their protein products.
The genetic etiology of many diseases have been discovered due to the awesome advancement of modern Nucleic Acid technology. In order to determine the gene responsible for a particular disease two basic approaches have been employed:
If the Biochemical lesion responsible for the disease is known, a candidate gene is analysed, as in the extensively studied B-Thalassaemia(Diaz-Chico JC., 1988) and Sickle-cell Anaemia(Novitski E., 1977).
If the chemical pathway is unknown and the genetic etiology of the diseased gene is to be discovered, an indirect statistical approach called linkage mapping has been used.
The gene responsible for Cystic Fibrosis was discovered using this technique(Kerem B., et al, 1989). It depends on the availability of gene probes that provide polymorphic genetic markers which are interpersed throughout the human genome and of statistical analysis using highly sophisticated computer programmes(Ott J., 1974). Not only it is now possible to attempt to unravel causes of diseases but also to examine the extraordinary diversity between and within livings species including man. Research on all aspects of life whether it be agricultural, clinical or anthropological etc., has continuously progressed in the developed world. Unfortunately, such a tradition is in absentia in Pakistan, only few laboratories have conducted some primitive research in this field.
Pakistan has few advantages with respect to such investigations which deals with the transmission of genetic characters. For several years, cousin marriages have led to the segregation of disease gene(s) in large, multigenerational families that are still largely nuclear. in addition, the practice of marrying within ethnic groups for social, religious or logistic purposes have also created pockets of populations that inhabit various areas of Pakistan.
It was mentioned earlier that no work has been carried out on Pakistani populations whether they may be of clinical or anthropological interest. A few examples of work done on other populations of the world in lieu of conducting the similar studies to be carried out in Pakistani populations.
During the past years, the availability of genetic markers that segregate with diseases in families has allowed:
The localisation of diseased gene and
The isolation of diseased gene using positional cloning approaches(Collins, 1992).
These markers are originally detected by Southern hybridisation and were termed Restriction fragment length Polymorphism(RFLP),(Botstein et al., 1980). Frequently used technique in this domain is PCR(Polymerase Chain Reaction) which enables the rapid amplification and detection of specific genomic sequence, has rapidly replaced RFPL-based studies. For PCR-based analysis, simple sequence repeats(SSR), like di-, tri-, and tetra-nucleotide repeats are very useful markers becuase of their variable numbers throughout the genome(Weber and May, 1989; Litt and Luty, 1989; Edwards et al., 1991). They have proved to be of great value in construction of linkage maps of chromosomes(Bowcock A., 1993) and the localisation of the diseased gene e.g., Wilson disease(WD), (Stewart EA., 1993).
At the biochemical and genetic engineering division of Dr. A.K Research Laboratories, RFLP was used to search human genome for the disease susceptibilty locus(DSL) for Tubercle bacillus diseases, which has been shown to be present on chromosome 1 of the mouse(Skamene et al., 1982). These studies have shown the abscence of of such a gene in a homologus chromosome 2. Furthermore, these studies established the relative sequence of nine genes excluding the disease susceptibility locus(Lsh\Beg\Ity), on the long arm of chromosome 2(Khaliq S, 1994). On the other hand, in a large scale search using microsatellite markers, a new gene responsible for retinitis pigmentosa (arRP) was found to be proximal to the microsatellite marker D1S53 in large Pakistani family(Leutelt et al., 1998).
Migraine headaches frequently runs in families suggesting that hereditary factors are the culprits. Typical migraine is a complex neurological disorder comprises of two main subtypes: migraine with aura (MA) and without aura (MO). The Migraine is classified as a chronic, painful and debilitating disease which manifest recurrent and severe headache attacks accompany with nausea, vomiting and photophobia or sonophobia in certain cases(Rod A, 2003). The disease etiology is still unknown, but family studies provide strong evidence that defective genes play an important role. In recent years a gene for familial hemiplegic migraine(FMH), a rare autosomal dominant subtype of migraine with aura, was mapped to chromosome 19p13. The findings of mutations in the gene 19p13, coding for the pore-forming subunit (alpha1A) of neuronal voltage-dependent P/Q-type calcium channels (FHM1), and in the ATP1A2 gene (1q21-23), encoding the alpha2-subunit of the Na+, K+ ATPase ionic pump (FHM2). A dysfunction of these channels modifies neuronal function and resulting in migraine with aura(Fumal and Schoenen, 2004).

Plan of Work:

It is the objective of this work to conduct similar studies among Pakistani population, using modern and complementry gene-mapping technologies.

Materials:

Blood samples from the screened patients. Lymphocytes will be separated by overlaying blood samples on Ficoll hypaque, centrifuging and collection of cells from the interface(Boyum A., 1968). Immortalised B-Lymphoblastoid cell lines will be generated by transformation with EBV as a substantial amount of DNA will be required to analyse the samples. the cell lines will be cryopreserved in a freezing mixture containing 10% DMSO, 45% heat inactivated foetal calf serum and 45% RPMI-1640(Walls EV., et al., 1987).

DNA Preparation:

DNA will be prepared from periphral blood cells and EBV-transformed cells by the method described in Maiatis et al., 1982. The techniques described below will be used to search disease gene.

RFLP Analysis

PCR

SNP and dinucleotide Repeat Marker

Agarose gel Electrophoresis.

Scientifc Paper

Writing for Publication:

I'm particularly mentioning a 'research paper'. According to standard defination research paper is "a piece of academic writing that requires an abstract, crtical and thoughtful level of inquiry". Such a Writing involves comparison of research work conducted and published in past with your own.

There are mainly two types of research papers:

1. Analytical and
2. Argumantative

The main difference between the two types of writing is that the Analytical research paper is an attempt to use your research to give an objective picture of what is known about the work. While the argumentative research paper is an attempt to convince your readers of the validity of a certain view you hold of a research.

Writing a Thesis:

A thesis statement points toward the course of an argument.
General guidelines:

• A thesis statement is always placed in the Introduction,
• It should be arguable,
• A thesis should argue on a specific point.
  

In brief, a thesis statement should draw attention of your readers towards an unknown side of the question.

For Scientific Publication:

Every science journal have their own style but generally they all carry following parts(they are also the 'part and parcel' of thesis but in detail):

Introduction:

It introduces your piece of work, provide a brief background, mention aims and objectives and the result of the investigation carried out.

Materials and Methods:

Mention specific information about your materials and methods in chronological order but related methodologies can be grouped as a section.

Results:

Mention the big picture of experiment and direct your readers towards the unknown solution of the problem. Data can be presented in the form of tables, charts or graphs but must follow sequential order, otherwise it will be incomprehensible for the readers.

Discussion:

• Do NOT rephrase the Results but Compare your results and conclusions with published scientific papers.
• Clearly contrast and compare your inferences with previous work and findings.
• Discuss the practical aspect of your work and its application that you foresee.
• Mention a summary of evidence for each major finding. Make concise concluding statements at the end of the discussion.
  

Summary:

• The Summary is a brief and a complete report of a scientific work that "stands
  on its own" without any explanation.
• Lengthy discussions and references to the literature must be omitted.
  

References:

References can be written in different formats depending on the requirement of the journal,

for a detail understanding pls. visit: http://www.liunet.edu/cwis/cwp/library/workshop/citation.htm

Hypothesis and its testing

Hypothesis(from Greek):
It consists either of a suggested explanation for a fact or of a reasoned proposal suggesting a possible correlation between multiple facts. Scientists generally base such assumption or hypotheses on prior readings or on outcome of scientific theories. Even though the words "hypothesis" and "theory" are often used synonymously, a scientific hypothesis is not the same as a scientific theory. A hypothesis requires either a confirmation or disproval by researchers. In due course, a confirmed hypothesis may become part of a theory or can grow to become a theory itself. Now-a-days, scientific hypotheses have the form of a mathematical model.

Types:

1. If and then in case of dependent variables.
2. Statistical
   Null Hypothesis- H0: coin-tossing operates "fairly" (equally likely to fall "Heads" or "Tails")
   Alternate Hypothesis- H1: coin-tossing operates in a biased manner to give a 90% probability of falling "Heads"

Testing:
The purpose of hypothesis testing is to test the viability of the null hypothesis in the light of experimental data. Depending on the data, the null hypothesis either will or will not be rejected as a viable possibility. The null hypothesis is often the reverse of what the experimenter actually believes; it is put forward to allow the data to contradict it.

However, various statistical approaches (such as Bayesian statistics and classical statistics (i.e. t-tests)) can quantify the strong intuition that H1 appears much less likely than H0 let us suppose that if, in 1,000 tosses, 495 came out "Heads" — and much more likely if 895 came out "Heads". Researchers generally evaluate experiments statistically.

1. After specifying the H0, the next step is to select a significance level(it is the criterion used for rejecting the null hypothesis). The level of significance is chosen as 0.05 level(also referred as 5%) or 0.01 level(also referred as 1%).


2. Then the difference between the results of the experiment and the null hypothesis is determined.


3. Next, assuming the null hypothesis is true, the probability of a difference that large or larger is computed .


4. Finally, this probability is compared to the significance level. If the probability is less than or equal to the significance level, then the null hypothesis is rejected and the outcome is said to be statistically significant.

For further understanding pls. pay a visit to http://statpages.org/

From Researching to Publication

How to perform scientific experiment

INITIAL OBSERVATION
You notice something, and wonder why it happens. You see something and ponder what causes it. You want to know how or why something works. You ask questions about what you have seen.The first step is to write down what you have noticed.

INFORMATION GATHERING
Find out about what you want to investigate. Read books, magazines, search internet or ask professionals who might know in order to learn about the effect or area of study. Keep track of where you got your information.

TITLE THE WORK
Choose a title that describes the effect or thing you are investigating.

AIMS & OBJECTIVES OF YOUR EXPERIMENT
What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.

MAKE HYPOTHESIS
Make a list of answers to the questions you have.Hypothesis must be stated in a way that can be tested by an experiment.

DESIGN AN EXPERIMENTAL PROCEDURE TO TEST YOUR HYPOTHESIS
Design an experiment to test each hypothesis.

OBTAIN MATERIALS AND EQUIPMENT
Make a list of the things you need to do the experiments, and prepare them.

PERFORM THE EXPERIMENT AND RECORD DATA
Do the experiment and record all numerical measurements made. Data can be amounts of chemicals used, how long something is, the time something took, etc. If you are not making any measurements, you probably are not doing an experimental science project.

RECORD YOUR OBSERVATIONS
Observations can be written descriptions of what you noticed during an experiment, or problems encountered. Keep careful notes of everything you do, and everything that happens. Observations are valuable when drawing conclusions, and useful for locating experimental errors .

CALCULATIONS
Perform any math needed to turn raw data recorded during experiments into numbers you will need to make tables, graphs or draw conclusions.

SUMMARIZE RESULTS
Summarize what happened. This could be in the form of a table of numerical data or graphs. It could also be a written statement of what occurred during the experiments.

DRAW CONCLUSIONS
Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.
Other Things You Can Mention in the Conclusion are:
If your hypothesis is not correct, what could be the answer to your question?
Summarize any difficulties or problems you had doing the experiment.
Do you need to change the procedure and repeat your experiment?
What would you do different next time?
List other things you learned.

COURTESY: http://www.isd77.k12.mn.us/resources/cf/SciProjIntro.html

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