Schedule CSES 5233 Spring 09 Jan 13: Introduction/ Tissue culture Jan 15: Agrobacterium Jan 20: Direct DNA transformation Jan 22: Plastid transformation Jan 27: Floral-dip transformation (lab visits) Quiz Jan 29: Genetic engineering with viruses Feb 3: Fate of foreign DNA in plant cell Feb 5: Exam on all topics above Feb 10: Site-specific recombination systems Feb 12: Transposons Feb 17: Homologous Rec. / Zinc Finger technol. Feb 19: Gene silencing I Feb 24: Gene silencing II Feb 26: Stabilizing gene expression Mar 03: Genomics I Mar 05: Genomics II Mar 10: Exam on all topics since Feb 5th exam Mar 12: Engineering herbicide resistance Mar 16 - 20: Spring break (Give the titles of the topics selected for discussion) Mar 24: Metabolic engineering Mar 26: Other traits engineering Mar 31: Quiz Apr 07: Environmental Impact Apr 09: Containment strategies Apr 14: Exam on all topics since Mar 10 Apr 16 - 30: Student presentations May 1: Dead day May -- : Final Exam

Exams 3 hour-long exams 50 each = 150 3 quizzes 25 each = 75 Student-presentations Presentation 25 Participation (Discussion) 25 Final Exam 25 Total 300

Plant Genetic Engineering Plant Tissue Culture Plant Molecular Biology Plant Genetics 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.

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

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.

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)

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

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

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

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 Somatic hybrids Cybrids

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

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

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

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 etc.) Production of disease free plants. Clonal propagation Secondary metabolite production (eg. Taxol production from cell cultures derived from the bark cuttings of pacific yew tree) Germplasm conservation (cryopreservation)

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.

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 years? Selection of plant varieties: sophisticated genetics based breeding technique. Crop management. Improvement of animal breeds. New methods of controlling pests and diseases.

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 photosynthetic productivity. 1866 1936 1956 1996 1 2 3 4 5 Tons/hectare Year 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.

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.