Students helping

1. Schedule CSES 5233 Spring 2011
Jan 18: Introduction/ Tissue culture
Jan 20: Agrobacterium
Jan 25: Direct DNA transformation
Jan 27: Plastid transformation
Feb 01: Floral-dip transformation (lab visits??)
Quiz
Feb 03: Genetic engineering with viruses
Feb 08:Fate of foreign DNA in plant cell
Feb 10: Exam on all topics above
Feb 15: Site-specific recombination systems
Feb 17: Transposons
Feb 22: Homologous Rec. / Zinc Finger technol.
Quiz
Feb 24: Exam on all topics since Feb 10th
Mar 01: Gene silencing I
Mar 03: Gene silencing II
Mar 08: Stabilizing gene expression
Quiz
Mar 15: Genomics I
Mar 17: Genomics II
Mar 21 - 25: Spring break
(Give the titles of the topics selected for
discussion)
Mar 29: Engineering herbicide resistance
Mar 31:Metabolic engineering
Apr 05:Other traits engineering
Quiz
Apr 07: Environmental Impact
Apr 12: Containment strategies
Apr 14: Exam on all topics since Mar 29th
Apr 19 – May 5: Student presentations
May 06: Dead day
May 7- 13: Final Exam (optional)

2. Exams
4 hour-long exams 50 each = 200
4 quizzes 25 each = 100
Student-presentations
Presentation 25
Participation (Discussion) 25
Final Exam 50 (optional)
Total 350

3. Plant Genetic Engineering
1. Plant Tissue Culture & Transformation
2. Plant Molecular Biology
3. Plant Genetics
What made biotechnology possible:
1. Ability to recover regenerated plants from tissue and organ culture. Tissue
culture provided another level of genetic variation: somaclonal variation.
2. Ability to cut and ligate DNA: gene mapping and cloning techniques.
3. Ability to introduce foreign DNA that ends up in the nucleus and ligates
with the native DNA.

4. Totipotency: ability of a cell or tissue or organ to grow and develop into a
fully differentiated organism.
Plant cells are totipotent

5. Plant Tissue Culture: historical highlights
1902: Haberlandt attempted to the culture mesophyll tissue and root hair cells.
This was the first attempt of in vitro culture.
1904: Haning attempted to culture excised embryos from mature seeds.
1922: Kotte was successful in obtaining growth from isolated root tips on
inorganic media. Robbins reported similar success from root tip and stem tip.
1920-40: First PGR, IAA, discovered by experiments on oat seedlings (Fritz Went).
1934: Used yeast extract (vit B) with inorganic salts to repeatedly culture root tips
of tomato.
1935: Importance of B vitamins and PGRs in culture of mesophyll cells.
1936: Haning experiment was repeated with IAA: works !!!
1939: Tobacco crown gall culture, callus obtained: called as Plant Cancer.
1940: WWII. Coconut milk used in plant cultures to obtain heart-shaped embyos.
1950s:Skoog used adenine sulfate to obtain buds on tobacco segments:
PGR #2 identified: kinetin
1958: Stewart and Reinert obtained somatic embryos from carrot cells using PGRs.
1950-60s: Botanists turned to plant tissue culture to study plant development.

6. 1960: Cocking isolated protoplasts from cultured cells.
1962: Murashige and Skoog developed MS media for tobacco.
1966: Guha and Maheshwari obtained first haploid plants (Delhi Univ., India)
1970: Discovery of restriction endonuclease (Daniell Nathan). Plasmids were
already known.
1972-73: First recombinant molecule created by Stanley Cohen, Stanford Univ.
1974: Discovery of Ti plasmid in Agrobacterium tumefaciens (by Zaenen in Ghent
Univ., Belgium)
1970-80s:Ti plasmid analysis (Nester, Seattle; Van Montagu, Ghent)
1983: First transgenic plant. (Monsanto, Ghent, Washington Univ).
1985: Leaf disk transformation method (Monsanto)

7. Tissue Culture:
De-differentiation to Regeneration
seeds
Wheat inflorescence
+ auxin
Callus
- auxin/ + cytokinin
embryos

8. Culture Media:
Callus (mass of parenchymatous cells)
1. Minerals
2. Carbon source
3. Plant Growth Regulators
Explant: any living tissue: leaf, root, zygotic embryos
organogenesis Somatic embryogenesis
Plant regeneration

9. Organogenesis
Unique to plants. Plant tissue in vitro may produce (de novo)
many types of primordia such as shoot and root
Explant Callus meristemoid organ primordia
Explant meristemoid organ primordia
Explant de-differentiation induction differentiation organ

10. Non-zygotic embryogenesis or somatic embryogenesis
Explant callus embryogenic callus somatic embryo plant
2,4-D BA, zeatin
Dicot: globular, heart, torpedo and cotyledonary stages
Monocot: globular, scutellar and coleoptilar stages.
PGR/ noPGR
Some direct applications of tissue culture:
1. Synthetic seed technology: encapsulation of somatic embryos
2. Seedless fruits: plants regenerated from triploid endosperm are unable to undergo meiosis

11. Protoplasts
Landmark:
1960: E. C. Cocking (Univ Nottingham) isolated
protoplasts by treating explants with concentrated
cellulase isolated from a fungus. [Commercial
cellulase and macerozyme were not available till
1968]. Tobacco protoplasts
Protoplast fusion
1. Somatic hybrids
2. Cybrids

12. Inter-specific fusions
Datura innoxia X D. stramonium = D. straubii (O. Schieder)
Tomate X Kartoffel = Tomoffel (G. Melchers)
Cybrid Technology
Mixing two cytoplasm without hybrid formation.
2n x 2n
4n Synkaryon
2n Heterokaryon
Fusion of haploid protoplasts (derived from anther cultures)
n + n= 2n
Somatic hybrids

13. Haploid Culture
Haploid plant (n) = recessive mutations displayed
n+n= double haploid
Landmark
1964 Guha and Maheshwari cultured Datura innoxia anthers and
found that large portion of culture contains haploid cells.
Later: Microspore cultures.
Occur spontaneously in inter-specific cross or induced by
irradiating pollen prior to pollination. Extremely poor efficiency.

14. Protoplast fusion: gametic hybridization
Haploid cells Protoplast (n)
n X n 2n (synkaryon)
Anther culture techniques/ fusion has been
extensively used in rice breeding program

15. Applications of tissue culture to plant breeding
1. Haploid production (rice, wheat and barley)
2. Triploid production (fruits and poplar)
3. Embryo Rescue/ Wide hybridization (numerous examples)
4. Somatic hybridization (scientific examples, few commercial products)
5. Somaclonal Variations (Tomato with altered color, taste and texture by
Fresh World Farms; Imidazolinone resistant maize, American Cyanimid;
Bermuda grass (Brazos R-3) with increased resistance to fall armyworm
etc.)
6. Production of disease free plants.
7. Clonal propagation
8. Secondary metabolite production (eg. Taxol production from cell cultures
derived from the bark cuttings of pacific yew tree)
9. Germplasm conservation (cryopreservation)

16. Food Prospects
Malthus’s 1798 book: Essay on population: population growth will soon outpace
food production.
Marx Das Kapital: Agric will follow the experience of manufacturing, becoming an
increasingly concentrated sector with many workers per farm with each worker
specializing in small fraction of the tasks involved in farm operation. The USSR and
China tried to implement this vision.
Ecologist Paul Ehrlich’s 1968 book: The Population Bomb predicted that the world
will undergo famines in 1970s, hundreds of millions of people will starve to death in
spite of any crash programs embarked upon now. It is too late!!!
William and Paul Paddock’s 1967 book Famine 1975! America’s Decision: Who will
survive? advocated a triage approach to foreign aid. The “can’t be saved group” that
included India and Philippines should not receive any aid.
Biologist Garrent Hardin became famous for coining the term “the tragedy of the
commons” to describe the problems that can arise from conflicts of interest when
there is open access to exploitation to natural resources. In 1977 he published The
Limits of Altruism in support of “tough-minded” approach recognizing that countries
such as India had exceeded their “carrying” capacity.

17. Yet over the past century growth in productivity of both land and labor has
enabled world food supplies to outpace the unprecedented increase in food
demand caused by jumps in the growth rate of world income and by
doubling and redoubling of population.
All these theorists were wrong!!

18. What contributed to this phenomenal increase in ag productivity in last 50
years?
1. Selection of plant varieties: sophisticated genetics based breeding
technique.
2. Crop management.
3. Improvement of animal breeds.
4. New methods of controlling pests and diseases.

19. Despite doubling and redoubling of crop yields seen in some developing countries,
any absolute yield ceiling seems far off.
Scientists have estimated yields that can be generated if a plant is given all the
inputs it needs. For most cereals, potential yields are several multiples of the
present average US yield.
How far from a yield ceiling?
Year
1866 1936 1956 1996
1
2
3
4
5
Tons/hectare
Yield of a crop is a function of biomass x harvest index (HI). Hence yield can be improved by
increasing biomass or HI or both. Since HI of many crops is approaching a ceiling value, so to
increase yield potential we have to increase crop biomass, i.e. there will have to be more
photosynthesis. The theoretical limits of solar energy utilization efficiency in photosynthesis
and the efficiency attained by crop plants provide possibilities and scope for improvement of
photosynthetic productivity.

20. What role might biotechnology play in sustainable agriculture?
"Sustainable agriculture" is both a term and a concept whose definition has varied a great
deal. As articulated in the 1990 "Farm Bill" Food, Agriculture, Conservation, and Trade Act of
1990, P.L. 101-624, Title XVI, Subtitle A, Section 1603) sustainable agriculture means "an
integrated system of plant and animal production practices having a site-specific application
that will, over the long term: (A) satisfy human food and fiber needs; (B) enhance
environmental quality and the natural resource base upon which the agricultural economy
depends; (C) make the most efficient use of nonrenewable resources and on-farm resources
and integrate, where appropriate, natural biological cycles and controls; (D) sustain the
economic viability of farm operations; and (E) enhance the quality of life for farmers and
society as a whole."
Biotechnology has the potential to assist farmers in reducing on-farm chemical inputs and
produce value-added commodities. Conversely, there are concerns about the use of
biotechnology in agricultural systems including the possibility that it may lead to greater
farmer dependence on the providers of the new technology. Where these two new
developments will lead agriculture is open for debate.