1. HORT/AGRO 689: Molecular & Biological Techniques in Plant Breeding
Dr. Monica Menz
Assistant Professor
Institute for Plant Genomics & Biotechnology
Dr. Stephen R. King
Associate Professor
Department of Horticultural Sciences
HFSB 409 and Centeq 120A or
Introduction: Syllabus; slide handouts – are they preferred?; articles: “A dying breed” – news feature, will discuss at beginning of next lecture, “Transferring in vitro technology to the field” – will be discussed during next lecture – and remember: read these articles with a critical eye, don’t assume that because something is published, it must be right; Timeline handout: not interested in a lot of detail, I’ll highlight what I think is important in the lecture.
Now, I think it’s important that you learn something about me, so I wanted to tell you something about myself…

2. This Course is Not: A molecular biology course A genetics course
Students should have an understanding of basic molecular biological techniques used in plant improvement
Techniques will be covered, but focus will be on Applications of the technology, not Development
A genetics course
Students should have an understanding of the principles of genes (including structure & function) and heritability
I want to emphasize that this is not a course on molecular biology; if it were, I wouldn’t be the one teaching it. You should have a good understanding of basic molecular biological techniques used in plant improvement. In this course, we will cover the techniques from an application standpoint, and I don’t plan to go into much detail on development. For example, if we have a gene cloned into a vector, you should know that we can obtain the DNA sequence by sequencing, and use the sequence to develop allele specific markers for the gene. I don’t plan to go into detail about how you sequence the gene.
When I say that this is not a genetics course, what I mean is that you should have an understanding of basic genetic principles; this is a breeding course that applies those genetic principles.

3. This Course Is:
A review of special tools and techniques that can be applied to a plant breeding program with a focus on the role of genetics
An introduction to the applications of new technologies, including molecular biology, from a plant breeding perspective
(Hopefully) An interactive investigation of special considerations to the application of these new technologies
So, I’ve told you what this course is not, now let’s focus on what this course is, or at least what I want it to be. We will review the special tools and techniques that are available to a plant breeder, but the focus will be on the genetics of the plant and how you use these tools to manipulate the genetics of the plant, and not on the development of the technology. The focus will be on molecular biology, but there are many other tools and techniques that are considered special tools, and I’ll try to cover as many of them as I can.
And finally, for this course to work, I hope this will be an interactive (discussion) investigation of special considerations to the application of these new technologies. By consideration, I mean things like technical considerations – what germplasm are you going to use and why, economic considerations – is this the most economical approach to achieve your objective, and even public considerations – why are people so upset with GMO’s? For many of these considerations there is no right or wrong answer, so long as you have an explanation.

4. Required Reading J. Knight. 2003. A dying breed. Nature. 421:568-570
This will be your first test on being interactive. I want everyone to read this article and be ready to discuss it next Wednesday. This is a news feature and not a research article, but I think it draws some pretty profound conclusions.

5. Important Events in Plant Improvement
1865: Gregor Mendel lectures & then publishes “Experiments with Plant Hybrids” (in 1866) where he describes how traits are inherited and the Laws of Inheritance:
1) Segregation
2) Independent Assortment
1869: DNA Identified in white blood cells
1900: Rediscovery of Mendel’s work:
Tschermark: Did not understand the concepts of Dominance, Phenotypic ratios or observation & theory
deVries: Inferred Mendel’s 1st Law, but did not separate gene transmission & expression
Correns: Clearly understood Mendel’s data; Dominance = analagen; segregation is a pair of factors; understood 9:3:3:1 ratio’s; but he did confuse segregation within a trait to segregation between traits
I gave you a timeline of discoveries and advancements in plant improvement. I do not expect you to go home and memorize the people and dates, but I wanted to give you an idea of how long it can take for research to be utilized; 20 years is a very short time in research. Your handout iplies that Mendel published “Experiments with Plant Hybrids” in 1865, but he actually discussed his research findings at two meetings in 1865, but didn’t actually publish his paper until His work established the Laws of Inheritance
4 years later, DNA was first identified, although Miescher didn’t really know what he was seeing.
Then, about 1900, Mendel’s work is re-discovered independently by at least 3 researchers. It is interesting to note that these 3 didn’t fully understand all of Mendel’s findings. They all inferred Mendel’s 1st Law, that of segregation, but none of them fully grasped Independent Assortment, which was deduced by T.H. Morgan around 1910.

6. Important Events in Plant Improvement
1904: Gene Linkage demonstrated
1905 – 1908: Modifier genes described
1909: Relationship between genes & proteins
1913: First genetic map constructed
1920’s: Hybrid cultivars adopted
1926: Pioneer Hi-Bred formed
1928: Transformation observed in bacteria
1935: Pure DNA isolated
1941: One gene – One enzyme hypothesis
1953: Molecular structure of DNA discovered
As I point out these advancements, again, I won’t ask you what year gene linkage was demonstrated, but I might give you a list, like Gene linkage demonstrated, first genetic map constructed, and one gene - one enzyme hypothesis formed, and ask you to put them in order. You should be able to do that in most instances just by understanding the basis for these principles: you can’t construct a map without knowing gene linkage. I think I’ve listed most of what I feel are the important discoveries on the slides.

7. Important Events in Plant Improvement
1953: Plasmids observed to transfer genetic markers between bacteria
1959: Gene regulation established in the DNA sequence
1966: Genetic code deciphered
1969: First gene isolated
1972: First recombinant DNA created
1972: First successful DNA cloning performed
1973: First recombinant DNA organism created
1978: RFLPs are discovered
1980: PCR technique invented
1984: DNA fingerprinting developed
Go over these;
I also would like you to look over the other events in the handout, in particular the commercial products on pages 6 and 7. I might ask you on the test what was the first commercially grown, genetically engineered plant? If you look on page 6, you will see that China grew commercial acres of genetically engineered tobacco in 1988, 6 years before Calgene grew commercial acres of Flavr-Savr.

8. Discoveries Usually Take Time to Reach Potential
1838: Theory of totipotency developed
1939: Carrot callus cultures cultivated
1959: Plants regenerated from carrot cultures
1946: Source of dwarfing gene sent to US
1962: Dwarfing gene used to start the Green Revolution
1943: Mexican Agricultural Program initiated
1957: Mexico became self-sufficient in wheat production
1951: Barbara McClintock reported her work on transposable elements in maize
1983: Barbara McClintock received Nobel Prize for work on transposable elements
As I mentioned before, the most important thing I want you to get from this timeline is that discoveries often take a very long time to be recognized or applied if they are recognized. An extreme example is with tissue culture development. In 1838, it was theorized that all plant cells have the ability to develop entire plants – or are totipotent. It was 101 years before carrot callus cultures were cultivated, and another 20 years before entire plants were regenerated from these cultures.
A breeding example is with the discovery of the dwarfing gene used to develop green revolution wheat varieties; the source was discovered in 1946, but it was 16 years later before the genes were used on a commercial scale, actually a fairly quick application of a new gene.
An even quicker application of technology happened in Mexico, when the Rockefeller Foundation helped the Mexican government form the Mexican Agricultural Program in years later Mexico became self-sufficient in wheat production.
And a perhaps extreme example is with Barbara McClintock, who first reported on transposable elements, but it was 30 years before the significance of her work was recognized.

9. History of Modern Plant Breeding
Mendelian Genetics – early 1900s
Resulted in Hybrid Cultivars
Chemical Agriculture – 1940s
Allowed more freedom for breeders to select high yielding, high quality genotypes
Green Revolution – 1960s
Combined Modern Varieties with Chemical Fertilizers
Now what I would like to do is go into some detail on what I consider to be the most significant advances of Modern Agriculture over the past 100 years. I consider these advances to be the re-discovery and application of Mendelian genetics, which occurred in the early 1900’s, the advent of chemical agriculture which began in the 1940’s using technology that was probably developed during WWII, and the Green Revolution, which in a way combined Mendelian genetics and Chemical agriculture.

10. World’s Food Supply vs. Increasing Population
Green
Revolution
Chemical
Agriculture
I’ve been working on this theoretical graphical representation of those advances in modern agriculture. It has been proposed that food production gains are limited to arithmetic (or linear) increases. It has also been proposed that the world’s population will increase at a geometric rate. This is the basis for the Malthusian theory, which some people don’t agree with because they say it’s been refuted. I tend to agree with the principles behind the Malthusian theory, and I would like to explain why with this graph. I’ve plotted a possible arethmetic increase with the dotted line here, and a theoretical population growth curve which shows a tendency to be geometric. It’s not purely geometric because we’ve had some pretty serious wars and disasters (predicted by the malthusian theory), as well as some political policies that have been directed at reducing population growth. Still, we do know that the world’s population is getting larger each day, and that the rate is faster than a linear increase. With improved health care, we may be in for a period of geometric population growth. So, why has the malthusian theory, which predicted wide-spread disaster, not been realized? I would argue that one possibility has to do with unforseen advances in modern agriculture. Applying the principles developed by Mendel resulted in huge yield gains, application of chemical agriculture also created a steep rise in the world’s food production, and not so much in developed nations as in the developing world, the green revolution had a dramatic impact on the world’s food production. You will also note that this model shows that yield gains tend to stabilize after the introduction of these new technologies.
Mendel

11. Modern Agriculture has not been readily accepted
LUTHER BURBANK
"We have recently advanced our knowledge of genetics to the point where we can manipulate life in a way never intended by nature."
"We must proceed with the utmost caution in the application of this new found knowledge.“
1906
Luther Burbank was a well-known plant breeder that developed over 800 varieties of fruits and vegetables, including the burbank potato.

12. Resistance to the Green Revolution
India resisted the importing of “exotic” wheat in 1965:
These varieties would “destroy Indian agriculture” warned scientists.
The Minister of Agriculture allowed for the use of the new varieties because of the crisis facing Indian agriculture:
Predictions gave the country two years before wide-spread famine engulfed the country.
Within two years, a bumper crop helped feed the nation (
There was also resistance to the green revolution.

13. Resistance to Chemical Agriculture
No References to resistance prior to wide-spread use (acceptance)
Indiscriminate use of Chemical Agriculture probably poses the greatest risk to public health of all modern farming practices
Interesting, I could find no references to the resistance of chemical agriculture prior to wide-spread use.

14. World’s Food Supply vs. Increasing Population?
Green
Revolution
Chemical
Agriculture
So back to the graph, if we look at the major advances I’ve outlined, you’ll see that these advances have occurred about every 20 years or so, and yet it’s been almost 40 years since the last major advancement. Now, I can’t predict when the actual production line will intersect with population growth, but I can predict that while production levels remain on a linear growth line, and population growth remains on a geometric path, they are coming closer together. So the question remains, where will the next major agricultural advance come from?
Mendel

15. Where will the next major advance in Agricultural Production come from?
Plant Breeders will likely play a major role:
2 of the 3 major advances in the 20th Century were directly attributable to plant breeding
Modern Biotechnology is poised to provide a major advance:
But only if this basic science is understood and used by the applied sciences
Plant Breeders are the logical avenue for the application of biotechnology
I can’t answer that question, but I can say that plant breeders will likely play a major role:
I can also say that modern biotechnology is poised to provide a major advance, but there are conditions:

16. Uses of Cell & Molecular Biology in a Breeding Program
Source of Genetic Variation
The Ultimate Driving Force Behind All New Technologies
To Speed Variety Development
Faster Source for Genetic Variation
Faster, more Efficient Assimilation of Traits
High Through-put Screening
To Improve Quality
Purity/Hybridity Testing
What can cell and molecular biology do for a breeding program?
The ultimate driving force behind all new technologies is genetic variation.
It has been said that you can find any genetic variation you want if you look hard and long enough, but we may not have enough time to find that corn plant that yields 1000 bu/a with no chemical inputs, so molecular biology can help us go down that road faster than we would be able to using conventional techniques. It may also allow us to assemble these new traits with the other 20,000 or 30,000 traits more efficiently, and it may allow us to look at thousands of more genetic combinations than are currently possible.

17. Modern Plant Breeding Tools
Tissue Culture Applications
Micropropagation
Germplasm preservation
Somaclonal variation & mutation selection
Embryo Culture
Haploid & Dihaploid Production
In vitro hybridization – Protoplast Fusion
Industrial Products from Cell Cultures
The next few slides outline what I hope to cover in this course. I plan to spend more time on the technical aspects of tissue culture applications than on molecular biology because I believe you are more likely to have support for molecular biology, but when it comes to some of these tissue culture applications, you may be on your own to apply them.

18. Reading Assignment:
D.C.W. Brown, T.A. Thorpe Crop improvement through tissue culture. World Journal of Microbiology and Biotechnology. 11(4):
D.R. Miller, R.M. Waskom, M.A. Brick & P.L. Chapman Transferring in vitro technology to the field. Bio/Technology. 9: