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Plant Biotechnology
Nono Carsono, PhD
Dr. rer. nat. Suseno Amien
Anas, PhD
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Understand application of biotechnology forplant science, including:
- DNA isolation and cloning,
- Making DNA and cDNA library,- Gene construct design
- Molecular marker technique (overview)
-
Transfer gene techniques (transgenic plants)- Analysis of transformants
- Current application: examples of transgenicplants with improved traits
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Overview Biotech product- animal, plant, etc
Tissue culture for crop improvement/production
(overview)
Marker assisted selection
Genetic transformation
Transgenic plant: an example
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Variety improvement
Conventional breeding
SelectionHybridization
Mutation
New developed tools(complement)
In vitrocultureMolecular marker (marker
assisted selection)Transfer gene
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Source of Genetic Variation The Ultimate Driving Force Behind All New
Technologies
To Speed Variety Development Faster Source for Genetic Variation
Faster, more Efficient Assimilation of Traits
High Through-put Screening
To Improve Quality Purity/Hybridity Testing
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Identity-preservedor specific-attributecrops (functional
nutrient: vaccines,higher oil or starchcontent, additionalamino acids)
(Molecular marker,Transfer gene)
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Animal growthhormones, e.g., bST(bovine GrowthHormone- to enhance
milk production)
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Herbicide tolerant crops, e.g., RoundupReady soybeans and corn and LibertyLink corn
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Insect resistant crops commercially available,
e.g., Bt corn, cotton, and potatoes Corn rootworm resistance in 2001?
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Insect resistantcrops commercially
available, e.g., Btcorn, cotton, andpotatoes
Corn rootworm
resistance in 2001?
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Herbicide tolerantcrops, e.g.,Roundup Readysoybeans and corn
and Liberty Linkcorn
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Engineered to produce more vitamin A precursor, beta-carotene
In Southeast Asia, 70% of children under the age of five suffer
from vitamin A deficiency
Golden RiceWild type
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1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000
PredictedProduction
Actual
Production
Population
GrowthMendel
ChemicalAgriculture
GreenRevolution
?
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30,000
metabolite
30,000
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What does the term cloning mean?
What is gene cloning? How does it differ
from cloning an entire organism? Why is gene cloning done?
How is gene cloning accomplished ?
What is a DNA Library? A cDNA Library? What are some of the ethical considerations
regarding gene cloning?
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From the Greek - klon, a twig
An aggregate of the asexually producedprogeny of an individual;a group of replicas of
all or part of a macromolecule (such as DNA oran antibody)
An individual grown from a single somatic cellof its parent & genetically identical to it www.m-
w/cgi-bin/dictionary
http://www.m-w/cgi-bin/dictionaryhttp://www.m-w/cgi-bin/dictionaryhttp://www.m-w/cgi-bin/dictionaryhttp://www.m-w/cgi-bin/dictionaryhttp://www.m-w/cgi-bin/dictionaryhttp://www.m-w/cgi-bin/dictionaryhttp://www.m-w/cgi-bin/dictionary7/31/2019 Plant Biotech New2
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When DNA isextracted from anorganism, all its
genes are obtained In gene (DNA)
cloning a particulargene is copied
(cloned)
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A particular gene can be isolated and itsnucleotide sequence determined
Control sequences of DNA can be identified& analyzed
Protein/enzyme/RNA function can beinvestigated
Mutations can be identified, e.g. genedefects related to specific diseases
Organisms can be engineered for specificpurposes, e.g. insulin production, insectresistance, etc.
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DNA isolation
Cutting DNA with Endonucleases (cutting)
Join DNA with Ligases (ligation)
DNA entry into cell (competence cells;transformation)
Identifying recombinants from non-transformants (Screening)
Identifying the correct recombinant colony(confirmation)
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DNA is extracted
Restrictionenzymes, e.g.
EcoRI, HindIII, etc.,cut the DNA intosmall pieces
Plant DNA
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Restriction enzyme action
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Bacterial plasmids(small circular DNAadditional to a bacteriasregular DNA) are cut
with the same restrictionenzyme
A chunk of DNA canthus be inserted into theplasmid DNA to form a
recombinant
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The recombinantplasmids are then
mixed with bacteria
which have beentreated to makethem competent,
or capable of taking
in the plasmids This insertion is
calledtransformation
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The plasmids havenaturally occurringgenes for antibioticresistance
Bacteria containingplasmids with thesegenes will grow on amedium containing theantibiotic- the others
die, so only transformedbacteria survive
Bacteria containing an Amp resistant plasmid
Bacteria with no Amp resistant plasmid
Culture medium containing Ampicillin
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The transformedbacterial cells formcolonies on themedium
Each cell in a givencolony has the sameplasmid (& the sameDNA)
Cells in differentcolonies have differentplasmids (& differentDNA fragments)
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A gene libraryis defined as a collection ofliving bacterial colonies that have beentransformed with different pieces of DNA from
the organism that is the source of the gene ofinterest
The gene library then must be screened to findthe colony with the gene in which the
researchers are interested
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Screening can involve:
1. Phenotypic screening-the protein encodedby the gene changesthe colour of the
colony2. Using antibodies that
recognize the proteinproduced by a
particular gene
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3. Detecting the DNA sequence of a cloned
gene with a probe (DNA hybridization)
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Once colonies areidentified, they arecultured in broth toincrease numbersand therefore the
amount of DNA Samples are also
prepared forstorage at -80degrees. They canbe kept for manyyears this way.
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Eukaryotic DNA differs from bacterial(prokaryotic) DNA in that it has introns(intervening sequences) and exons(expressed or translated sequences).
In order for a eukaryotic gene to beexpressed, the introns are edited out of
mRNA after transcription
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A simplified diagram of transcription in eukaryoteshnRNA = heterogenous nuclearRNA in the process of being cut and spliced into
messenger RNA
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Bacteria cant deal with introns, so in caseswhere a product (e.g. insulin) is to beexpressed by the bacteria, an uninterruptedcoding sequence is needed.
Also, since introns can account for up to90% of an eukaryotic gene, and cloninglong fragments is difficult, it is sometimesdesirable to work only with the expressed
sequences (exons)
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To deal with this, special DNA is synthesizedusing mRNA as a template. This process alsorequires a primer and an enzyme, reverse
transcriptase (a DNA polymerase thatsynthesizes a DNA strand from the mRNA)
This complementary DNA is called cDNA
cDNA may be attached to a vector such as aplasmid and then introduced into bacterialcells.
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Cc the cat, slide 5 courtesy of Texas A & M University,College of Veterinary MedicinePhotos of sheep & tissue culture, slide 5, L. Macdonald 2003Graphics on slides 8, & 12 , V. Ward, University of Auckland
DNA Hybridization, slide 15, courtesy of Texas Tech UniversityDNA cloning & screening, slides 10 & 14, courtesy of The University of Arizona
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Micropropagation
Germplasm preservation
Somaclonal variation & mutation selection
Embryo Culture Haploid & Dihaploid Production
In vitro hybridization Protoplast Fusion
Industrial Products from Cell Cultures
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PERBANYAKAN MIKRO
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PERBANYAKAN MIKRO
Tahap III
Tahap I Tahap II
Tahap IV
PERBANYAKAN MIKRO
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PERBANYAKAN MIKRO
Tahap III
Tahap I Tahap II
Protocorm Like Bodies
Anggrek
Tahap IV
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What are molecular markers?
Genetic information resides in the genome
A molecular markeris based on DNA sequence Polymorphisms arise by mutation
ATP5L2 ATP synthase
C22orf11 chromosome 22 ORF
PANX2 pannexin 2
gtataagtgaaccactcagggtcctggccgggcacagtggctcacgcctgt
aatcccagcctttgggaggccgaggtgggtggatcatgaggtcaggagttcaa
gaccagcctggccaaatggcgaaacattgtctctactaaaagtacaaaaattag
ccagacgtggtggtgctcactgtaatcccagctactcgggaggctgaggcagg
aaaatcacttgaacccgggaggcggaggtcacagttagccaagatcacaccac
tgtactccag
DNA Fingerprinting Basics
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=retrieve&dopt=default&list_uids=267020http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=retrieve&dopt=default&list_uids=26150http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=retrieve&dopt=default&list_uids=56666http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=retrieve&dopt=default&list_uids=56666http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=retrieve&dopt=default&list_uids=26150http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=retrieve&dopt=default&list_uids=2670207/31/2019 Plant Biotech New2
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g p g
Different individuals carry different alleles.
Most alleles useful for DNA fingerprinting differ on thebasis of the number of repetitive DNA sequences theycontain.
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o Co-dominant (distinguish between homozygotesand heterozygotes)
o Nondestructive assay
o Complete penetrance
o High polymorphism
o Random distribution throughout the genome
o Assay can be automated
Ph i k
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non-waxy waxy
Phenotypic markers
hullednaked
2-rowed 6-rowed Black white
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Restriction digest
Polymerase chain reaction (PCR)
Sequence analysis
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A Site With Three Alleles Useful for DNA Fingerprinting
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A Site With Three Alleles Useful for DNA Fingerprinting
DNA fragments of different size will beproduced by a restriction enzyme that cuts at thepoints shown by the arrows.
The DNA Fragments Are Separated on the Basis of Si e
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The DNA Fragments Are Separated on the Basis of Size
The technique is gel electrophoresis.
The pattern of DNA bands is compared between eachsample loaded on the gel.
Possible Patterns for a Single Gene With Three Alleles
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Possible Patterns for a Single Gene With Three Alleles
In a standardDNAfingerprint,about a dozensites are
analyzed, witheach sitehaving manypossiblealleles.
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1. What for Plant Breeder use biotechnology
2. What are the new products of agricultural
biotechnology use Plant Biotechnology ?
3. What does the term cloning mean?
4. What is gene cloning? How does it differ from cloning an
entire organism?5. Why is gene cloning done?
6. How is gene cloning accomplished ?
7. What is a DNA Library? A cDNA Library?
8. What are some of the ethical considerations regardinggene cloning?
R f
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References
Kreuzer, H., Massey, A.,2001, Recombinant DNA & Biotechnology,ASM Press, Washington
Turner, P.C., et al, 1997, Instant Notes n MolecularBiology, Bios, Oxford
www.agbiosafety.unl.edu/education/clone.htm
http://avery.rutgers.edu/WSSP/StudentScholars/Session12/Session12html
http://www.pssc.ttu.edu/pss3421/gene%20cloning%20Strategies.htm
http://www.uic.edu/classes/phar/phar331/lecture7
http://www.biology.arizona.edu
http://www.agbiosafety.unl.edu/education/clone.htmhttp://avery.rutgers.edu/WSSP/Studenthttp://www.pssc.ttu.edu/pss3421/gene%20cloning%20Strategies.htmhttp://www.uic.edu/classes/phar/phar331/lecture7http://www.uic.edu/classes/phar/phar331/lecture7http://www.pssc.ttu.edu/pss3421/gene%20cloning%20Strategies.htmhttp://avery.rutgers.edu/WSSP/Studenthttp://www.agbiosafety.unl.edu/education/clone.htm7/31/2019 Plant Biotech New2
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Accelerate the breeding process
Introduce/enhance desired trait in an established geneticbackground
Extend the gene pool
Select genes from any Kingdom (with care, especially ifpotential for entry into the food chain)
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Research Largest number of transgenic plants are
currently created for research purposes Knock-outs, over-expression, modified proteins
K. Yamaguchi-Shinozaki, JIRCAS, Japan
stress-induciblepromoter drivingdrought- andcold-responsivetranscription factor
wild type
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Bioreactors / Molecularfarming
Therapeutic proteins
Human lactoferrin to treat
iron deficiencies Antibodies
Vaccine production
Antigen expression
HepC, HIV
Dow AgroSciences Achieves Worlds First Registration for Plant-Made Vaccines
Indianapolis, IN - January 31, 2006
Dow AgroSciences LLC, a wholly owned subsidiary of The Dow Chemical Company, (NYSE: DOW), announced today that it
has received the world's first regulatory approval for a plant-made vaccine from the United States Department of Agriculture
(USDA) Center for Veterinary Biologics. This approval represents an innovative milestone for the company and the industry...
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How do we transfer a gene (genes) to the plant?
What is the requirement for this purpose?
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Nature Biotechnology 25: 271 (2007)
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Nature Biotechnology 25: 271 (2007)
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Metode konvensional (persilangan+seleksi)menghadapi kendala teknis;
Tidak dijumpai sumber gen pengendali karakter ygdituju pada kerabatnya, spt gen ketahanan thd hama &
penyakit, tahan herbisida, penghasil vit. tertentu; Manipulasi karakter baru/unik, yg tidak dapat
dilakukan dgn metode konvensional, misalnya tnmpenghasil antibodi, dll
Metode konvensional relative membutuhkan waktu yg
lebih lama (time consuming); Gen pengendali karakter yg dipindahkan tidak
melanggar etika, agama ataupun sistem nilai yg sudahada.
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Parameter PT konvens Rekayasa gen
Tingkat Tnm utuh Sel/molekul
Ketepatan Sekumpulan gen Satu gen
Batasan taksonomi Satu species/genus Tdk ada batasan
Kepastian Perubahan genetika
sulit diestimasi
Perubahan
genetika dpt
diestimasi
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