Transgenic Alfalfa to Produce Enzymes for Environmentally Beneficial Processes

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Transgenic Alfalfa to Produce Enzymes for
Environmentally Beneficial Processes

• A Multidisciplinary Project at UW Madison

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MOLECULAR F(PH)ARMING

• The use of plants as bioreactors for the
  production of recombinant proteins
• A wide range of proteins produced in plants to
  date ranging from pharmaceuticals to commodity
  enzymes

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Second Wave of Agricultural Biotechology

• First wave considered to be agricultural traits
  e.g. herbicide and pest resistance, relatively
  simple single gene traits.
• Concept of adding value to crops by using them as
  production systems for novel proteins

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GREEN CARBOHYDRATE BASEDTECHNOLOGY

• Use renewable plant based material instead of oil
  as chemical feedstocks, concept of BIOREFINERIES
• Use transgenic plants to produce novel proteins
  such as biodegradable plastics and enzymes for
  use in biologically based processes
• Better use of waste materials and by products,
  develop technology to separate plant products
• Better land use policies, more efficient animal
  production systems and improvement of crop yields

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Overall Aims of Research

• To use plants as bioreactors(enzyme factories)
  for the production of industrial enzymes
  including animal feed enzymes and cellulases for
  use in biomass conversion
• To add value to an existing crop and recover
  other co-products by fractionation.

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Currently most commodity enzymes are produced in
capital intensive fermentation systems
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An alternative production system is to use
transgenic plants and capture solar energy
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Why Alfalfa ?

• Abundant, widespread, hardy crop capable of 3 or
  more harvests a year
• Low production costs, perennial
• Legume capable of vigorous growth without
  irrigation and with less added fertilizer
• Technology already developed to extract
  protein-rich juice from alfalfa on a large scale
• Residue useable as feed, no waste management
  problems

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Fields of Transgenic Alfalfa Will Replace
Fermentation Systems
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Production of the Animal Feed Enzyme Phytase in
Transgenic Alfalfa

• 1. Swine and poultry need phosphorus (P)
• 2. P is present in seeds such as soybean and corn
  but it is bound in the form of phytic acid
• 3. Supplemental P is added to animal diets which
  is an added cost and leads to excess P in manure
  which causes environmental pollution
• If Phytase is added to feed the animals can use
  the P in the diet, less P in the manure

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Alfalfa Transformation and Field Testing

• We made transgenic alfalfa by inserting a gene
  from a fungus that makes phytase
• We grew the alfalfa in fields, it was perfectly
  normal and the plants made high levels of the
  enzyme
• We extracted juice from the plants and made leaf
  meal which we added to animal feed

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Poultry Feeding Trials

• Feeding trials using whole alfalfa juice and
  leaf meal preparations from phytase-expressing
  alfalfa show that recombinant phytase from
  alfalfa performs as well as the microbial enzyme

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Swine Feeding Trials
Transgenic alfalfa juice and leaf meal
preparations have both been shown to effective in
swine feeding trials. Pigs actually like to eat
alfalfa!
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Phytase As a Feed Enzyme in Fish Farming

• Expanding industry
• Phosphorus supplementation required, especially
  if diet is grain based
• Phytase effective in fish

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Waste management is not a problem since the
fibrous material remaining after
juice expression is still a valuable
ruminant animal feed or can be used as a
substrate for bioethanol or fermented to lactic
acid
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Heat treatment of alfalfa juice coagulates
proteins and yields alfalfa tofu
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What This Technology Will Achieve

• Will give added value to an existing crop using
  current production capabilities
• Will allow for cheaper and more efficient use of
  animal feeds in the poultry and swine industries
• Will lead to less environmental pollution due to
  a reduction in phosphorus loading into groundwater

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Multidisciplinary Integrated Approach

• Plant molecular biology- T. Arment, T.L. German,
  D. Mathews, T. Ziegelhoffer, P. Ziegelhoffer, J.
  Raasch
• Plant tissue culture and plant physiology - S.
  Austin, J. Will
• Protein recovery and purification
• R. Burgess, M. Shahan, T. Zeigelhoffer
• Plant breeding - E. T. Bingham. Animal science -
  Mark Cook, T. Crenshaw
• Production agronomy and mechanical engineering
• R. Koegel, R. Straub
• Agricultural economics and rural sociology
• R. Klemme, R. Cropp

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SOMATIC HYBRIDIZATION IN SOLANUM

• J. P. Helgeson, Dept Plant Pathology, UW Madison
• USDA-ARS

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Overall aims of research

• To capture disease resistance from wild potato
  species
• To use segregating populations to identify plant
  disease resistance genes and transfer resistance
  into potato cultivars

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Somatic hybridization using protoplast fusion

• Isolate protoplasts (typically leaf mesophyll)
  from two parental lines
• Fuse protoplasts either chemically (PEG) or using
  electrofusion
• Cell membranes fuse forming one cell containing 2
  nuclei
• On cell division nuclear material condenses
  together and hybrids cells are formed that
  contain DNA from both parental lines.

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Potato plants growing in a test tube
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Freshly isolated potato protoplasts
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Two protoplasts ready to fuse together
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Fusion products begin to divide on
nutrient medium
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If the conditions are right, small shoots emerge
from the green calli
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Putative somatic hybrid plants
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A fertile somatic hybrid
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Phenotype of somatic hybrids clearly shows
characteristics of both parents
S. brevidens somatic hybrid
S. tuberosum
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Phenotype of intraspecific diploids of S.
tuberosum
US-W9310.3 Somatic
hybrid US-W9545.99
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Somatic hybrids have ALL of the chromosomes from
each parent plant
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Using somatic hybrids tress important genes into
potato
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Somatic Hybrids between potato and S. brevidens
are resistant to soft rot (Erwinia)
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Verticillium wilt resistance test field at
Hancock, Wisconsin
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Late blight resistance in backcross plant
derived from somatic hybrids between S.
bulbocastanum and potatoLine J101K6-A22 at
Hancock Expt. Station
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Some new disease resistances from somatic hybrids
of Solanum spp. Late blight resistance
S. bulbocastanum S. tuberosum Early blight
resistance S. bulbocastanum S.
tuberosum Soft rot resistance
S. brevidens S. tuberosum Bacterial wilt
resistance S. commersonii S.
tuberosum PVY resistance S.
etuberosum S. tuberosum PLRV resistance
S. brevidens S.
tuberosum-S. stenotomum 77-1
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S. bulbocastanum chromosome 8
TAG 101697-704 (2000) MGG 265694-704 (2001)
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Lessons learned from somatic hybrids 1.
Vigorous somatic hybrids can be obtained from
fusions of potato and wild Solanum
species. 2. The hybrids often have all or nearly
all of the chromosomes of both species. 3.
The hybrids can usually be crossed with potato
breeding lines. 4. Disease resistances and other
traits of the wild species can be
introgressed into potato breeding lines. 5.
Genes from the wild species can be mapped in
populations derived from somatic hybrid x
potato crosses. 6. Markers can be obtained from
mapping studies that will assist in breeding
for disease resistance. 7. Disease resistance
genes can be identified and cloned.
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Jim Bradeen Jiming Jiang Kristine Naess Junqi
Song Fenggao Dong Mitch McGrath Geri
Haberlach Rich Novy Susan Wielgus Chuck
Brown Greg Hunt Joe Pavek Mitch McGrath Dennis
Corsini Jeff Davis Caitylin Allen John
Pohlman Heiyoung Kim Sandra Austin Lousie
Laferriere Walt Stevenson Mark Ehlenfeldt Vaughn
James Christie Williams