Improving Plants for the 21st Century

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Improving Plants for the 21st Century

• Cell and Tissue Culture Technology and
  Applications

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Molecular Biology Applications in Plant
Breeding1/2

• PLANT CELL AND TISSUE CULTURE
• Procedures Utilizing Tissue Culture Techniques
• Tissue Culture Techniques
• Plantlet Regeneration
• CLONAL PROPAGATION VIA TISSUE CULTURE
• Commercial Applications
• PROPAGATION OF DISEASE-FREE GENETIC STOCKS
• FREEZE PRESERVATION OF GERM PLASM
• EMBRYO CULTURE, OVULE CULTURE, IN VITRO
  POLLINATION
• Embryo Culture
• Ovule Culture
• In Vitro Pollination and Fertilization

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Molecular Biology Applications in Plant
Breeding2/2

• ANTHER CULTURE AND HAPLOID PLANT PRODUCTION
• Anther Culture Procedures
• Factors Affecting Haploid Plant Production
  through Anther Culture
• Utilization of Anther Culture Derived
  Doubled-Haploids in Plant Breeding
• GENETIC VARIABILITY FROM CELL CULTURES
  SOMACLONAL VARIATION
• SOMATIC CELL HYBRIDIZATION
• PLANT GENETIC ENGINEERING
• MOLECULAR MARKERS

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Introduction to Plant Tissue Culture

• Plant cell and tissue culture includes a wide
  range of cultural techniques for regeneration of
  functional plants from embryonic tissues, tissue
  fragments, calli, isolated cells, or protoplasts
• Basic Concept is Totipotency each living sell
  of a multicellular organism would be capable of
  developing independently if provided with the
  proper external condition (white,
  1954)totipotent cell is one that is capable of
  developing by regeneration into a whole organism
  (morgan, 1901)

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Micropropagation Laboratory

• The Stages Are
• Medium Preparation and Explant Selection
• Establishment of aseptic culture
• Proliferation
• Rooting
• Acclimatization

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Basic Tissue Culture Procedures
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Procedures Utilizing Tissue Culture Techniques

• Clonal propagation the rapid multiplication of
  genetic stocks, through tissue culture, including
  procedures for isolation of pathogen-free plant
  materials and freeze-preservation of germplasm
• Embryo and ovule culture the rescue and
  propagation on a sterile nutrient medium of
  immature embryos from interspecific or
  intergeneric crosses
• Anther culture the culturing of anthers in vitro
  for the purpose of generating haploid plantlets
• Somaclonal variation genetic variation induced
  in somatic cells cultured in vitro
• Somatic cell hybridization the fusion of
  protoplasts from genetically diverse germplasms
  and
• Genetic engineering (transformation) In plants,
  the transfer of DNA from a donor species to a
  recipient species by means of a bacterial
  plasmid, virus, or other vector, or through
  microinjection or biolistic device. Plants
  receiving the new DNA are said to be transformed,
  and are regarded as transgenic plants.

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Tissue Culture Techniques Definitions and
examples

• It is the culture of isolated plant cells or
  detached fragments of plant tissue on a nutrient
  medium under aseptic conditions and their
  subsequent regeneration into functional plants
• Undifferentiated plant cells often can be made to
  develop into functional plants when appropriately
  cultured in vitro. This property is designated
  totipotency. The need to regenerate plants from
  totipotent cells makes plant cell and tissue
  culture an essential step in utilization of the
  new molecular technology.
• Model species, egs. tobacco, potato, alfalfa,
  sugarcane, rice, and various horticultural
  species, many of which are readily propagated
  vegetatively.
• Field crop species, eg. corn, sorghum, forage
  grasses, cotton, soybean, and the grain legumes,
  are difficult to regenerate.

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Tissue Culture Manipulations
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Factors Affecting Tissue Culture Efficiency

• Plant regeneration from tissue culture varies
  with the following parameters
• plant species,
• genotype within the species,
• source of the cultured tissue,
• age and health of the donor plant,
• nutrient medium,
• other factors

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In vitro Regeneration of Wheat
A Shoot emergence from callus B Profuse shoot
production C Regenerated wheat plantlet
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What are explants and how are they obtained ?

• Plant tissue cultures are generally initiated
  from multicellular tissue fragments, called
  explants, obtained from living plants.
• Explants may originate from a wide range of plant
  tissues, such as
• leaf,
• stem,
• root,
• petiole,
• hypocotyl,
• cotyledon,
• embryo, or
• meristem

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From explants to callus on a solid medium

• The explant is commonly cultured on a nutrient
  medium solidified in agar. Explants from most
  species of plants may be induced to divide in an
  unorganized manner on specifically formulated
  nutrient media
• An undifferentiated mass of cells, known as
  callus (plural, calli), is formed within 4 to 8
  weeks.
• The callus may be divided, with clusters of cells
  transferred to fresh agar media to form
  subcultures. Repeated subculturing of the callus
  permits rapid multiplication of the cultured
  material.
• Plant regenerability may decline, and genetic
  stability of the plant material may be altered,
  with successive subculturing.
• Callus cultures are incubated under aseptic
  conditions, normally in dim light, with
  temperatures around 25C.

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Nutrient medium and the role of growth hormones?

• The nutrient medium commonly contains
• inorganic salts, sugar as a source of carbon, and
  vitamins to maintain high growth rates
• Phytohormones such as auxins and cytokinins may
  be added to control cell growth and division
• The ratio of auxin to cytokinin has an important
  role in the initiation of shoot and root
  primordia.
• a low auxin cytokinin ratio stimulates
  initiation of shoot buds and suppresses root
  initiation
• a high auxin cytokinin ratio leads to
  dedifferentiation and favors root initiation
• an intermediate ratio favors continued division
  of cells as undifferentiated callus
• The optimum culture medium may vary with the
  species, the genotype within the species, and the
  origin and age of the cultured tissue.
• The preferred physical state of the culture
  medium, whether a liquid medium or a solid agar
  gel, may vary with the species and the culture
  environment.

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PhytohormoneStructures
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Plantlet Regeneration

• Plantlets can be initiated
• Indirectly from callus via
• adventitious shoots
• somatic embryos OR
• Directly from explants such as
• Axillary buds
• the culture is transferred to a rooting medium to
  induce root initiation and subsequently plantlets
  
• Somatic embryos have both root and shoot apices
  present and can develop directly into plantlets.
• Adventitious shoot initiation (organogenesis)
  occurs with a wider range of plant species than
  initiation of somatic embryos few major field
  crop species can be routinely induced to form
  somatic embryos.

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Establishment of regenerated plantlets

• Establishment It refers to the successful growth
  and development of plantlets regenerated through
  tissue culture techniques in soil.
• The establishment of a healthy plantlet in soil
  with minimum mortality is as essential for
  success in tissue culture propagation as
  obtaining a high frequency of plantlet
  regeneration.
• Difficulty in establishment Species differ in
  their capability of adjusting to the new
  environment. During this period the plantlet must
  change from the heterotrophic state to the
  autotrophic state, where it synthesizes its own
  organic food requirements.
• Water loss from the regenerated plantlet is high,
  due to inadequacy of the root system formed in
  culture to maintain the plant in soil, and a
  reduced presence of epicuticular wax on leaves
  and stems of regenerated plantlets. The
  regenerated plantlet must be protected from
  desiccation and hardened to attain some tolerance
  to moisture stress.
• the new plantlets, which have been developed
  under aseptic conditions, should be protected
  from soil pathogens so that they can grow and
  develop into healthy plants.

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CLONAL PROPAGATION VIA TISSUE CULTURE

• clonal propagation, cloning, or micropropagation
  It is the use of tissue culture technology for
  rapid regeneration of particular plant genotypes.
  
• Some potential uses of clonal propagation in
  agronomic crops are
• large-scale increase of a heterozygous genotype,
• increase of a self-incompatible genotype,
• increase of a male-sterile parent in a
  hybrid-breeding program,
• propagation of disease-free genetic stocks, and
• preservation and international exchange of
  germplasm.

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Advantages of clonal propagation

• Shoot tips cloned from axillary buds or
  meristem tissue produce fewer genetic variants
  than cultures from more mature tissues.
• If, in addition to the meristematic region, one
  or two leaf primordia are included in the
  shoot-tip explant, the explants will be larger,
  require less time for excision, and have a higher
  survival rate than the smaller explants cloned
  without the leaf primordia.
• Axillary shoots produced on the shoot-tip
  explants can be subcultured until the required
  number of potential plantlets are obtained. The
  plantlets are transferred to a rooting medium and
  later transplanted into soil.

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Commercial Applications of Clonal Propagation

• Clonal propagation has the potential for
  propagation of thousands of plantlets from a
  single genetic stock.
• Examples
• orchids,
• pyrethrum,
• potato,
• asparagus,
• strawberry, and
• various flowers or herbaceous ornamentals that
  set seed poorly.
• This may not be suitable for seeding field crops
• In vitro propagation may have application for
  early generation increase of breeding materials
  in crop species with sparse seed-setting,
  provided that efficient tissue culture procedures
  that can be routinely employed have been
  developed for those species and that genetic
  identity can be maintained in the plants
  propagated.