Plant Biotech New2

7/31/2019 Plant Biotech New2

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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/dictionary


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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=267020


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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.htm


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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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