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??)
Feb 03: Genetic engineering with viruses
Feb 08:Fate of foreign DNA in plant cell
Exam on all topics above
Feb 15: Site
specific recombination systems
Feb 17: Transposons
Feb 22: Homologous Rec. / Zinc Finger technol.
Exam on all topics since Feb 10
Mar 01: Gene silencing I
Mar 03: Gene silencing II
Mar 08: Stabilizing gene expression
Mar 15: Genomics I
Mar 17: Genomics II
25: Spring break
(Give the titles of the topics selected for
Mar 29: Engineering herbicide resistance
Mar 31:Metabolic engineering
Apr 05:Other traits engineering
Apr 07: Environmental Impact
Apr 12: Containment strategies
Exam on all topics since Mar 29
May 06: Dead day
13: Final Exam (optional)
50 each =
25 each =
Plant Genetic Engineering
Plant Tissue Culture & Transformation
Plant Molecular Biology
What made biotechnology possible:
Ability to recover regenerated plants from tissue and organ culture. Tissue
culture provided another level of genetic variation: somaclonal variation.
Ability to cut and ligate DNA: gene mapping and cloning techniques.
Ability to introduce foreign DNA that ends up in the nucleus and ligates
with the native DNA.
: ability of a cell or tissue or organ to grow and develop into a
fully differentiated organism.
Plant cells are totipotent
Plant Tissue Culture: historical highlights
: Haberlandt attempted to the culture mesophyll tissue and root hair cells.
This was the first attempt of
: Haning attempted to culture excised embryos from mature seeds.
: Kotte was successful in obtaining growth from isolated root tips on
inorganic media. Robbins reported similar success from root tip and stem tip.
: First PGR, IAA, discovered by experiments on oat seedlings (Fritz Went).
: Used yeast extract (vit B) with inorganic salts to repeatedly culture root tips
: Importance of B vitamins and PGRs in culture of mesophyll cells.
: Haning experiment was repeated with IAA: works !!!
: Tobacco crown gall culture, callus obtained: called as Plant Cancer.
: WWII. Coconut milk used in plant cultures to obtain heart
:Skoog used adenine sulfate to obtain buds on tobacco segments:
PGR #2 identified: kinetin
: Stewart and Reinert obtained somatic embryos from carrot cells using PGRs.
: Botanists turned to plant tissue culture to study plant development.
: Cocking isolated protoplasts from cultured cells.
: Murashige and Skoog developed MS media for tobacco.
: Guha and Maheshwari obtained first haploid plants (Delhi Univ., India)
: Discovery of restriction endonuclease (Daniell Nathan). Plasmids were
: First recombinant molecule created by Stanley Cohen, Stanford Univ.
: Discovery of Ti plasmid in
(by Zaenen in Ghent
:Ti plasmid analysis (Nester, Seattle; Van Montagu, Ghent)
: First transgenic plant. (Monsanto, Ghent, Washington Univ).
: Leaf disk transformation method (Monsanto)
differentiation to Regeneration
(mass of parenchymatous cells)
Plant Growth Regulators
: any living tissue: leaf, root, zygotic embryos
Unique to plants. Plant tissue
may produce (
many types of primordia such as shoot and root
Explant Callus meristemoid organ primordia
Explant meristemoid organ primordia
differentiation induction differentiation organ
zygotic embryogenesis or somatic embryogenesis
Explant callus embryogenic callus somatic embryo plant
: globular, heart, torpedo and cotyledonary stages
: globular, scutellar and coleoptilar stages.
Some direct applications of tissue culture:
Synthetic seed technology:
encapsulation of somatic embryos
plants regenerated from triploid endosperm are unable to undergo meiosis
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
Tomate X Kartoffel = Tomoffel (G. Melchers)
Mixing two cytoplasm without hybrid formation.
2n x 2n
Fusion of haploid protoplasts (derived from anther cultures)
n + n= 2n
Haploid plant (n) = recessive mutations displayed
n+n= double haploid
1964 Guha and Maheshwari cultured
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.
Protoplast fusion: gametic hybridization
n X n
Anther culture techniques/ fusion has been
extensively used in rice breeding program
Applications of tissue culture to plant breeding
Haploid production (rice, wheat and barley)
Triploid production (fruits and poplar)
Embryo Rescue/ Wide hybridization (numerous examples)
Somatic hybridization (scientific examples, few commercial products)
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
Production of disease free plants.
Secondary metabolite production (eg. Taxol production from cell cultures
derived from the bark cuttings of pacific yew tree)
Germplasm conservation (cryopreservation)
Malthus’s 1798 book:
Essay on population
: population growth will soon outpace
: 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
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
Limits of Altruism
in support of “tough
minded” approach recognizing that countries
such as India had exceeded their “carrying” capacity.
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!!
What contributed to this phenomenal increase in ag productivity in last 50
Selection of plant varieties: sophisticated genetics based breeding
Improvement of animal breeds.
New methods of controlling pests and diseases.
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?
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
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
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
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
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.