Pengantar Bioteknologi Pertanian

Rani A. Wulandari
Fskultas Pertanian UGM
 Gene – transfer methods

Conventional
breeding

Tissue culture Plants

Genetic engineering

karthikumarbt@kcetvnr.org 2
 Bioteknologi Tanaman
1) Budidaya Jaringan Tanaman (Cloning)
2) Rekayasa genetika (Genetic Engineering)
Perkembangan tanaman
transgenik

Periode Tujuan Tahap

I ketahanan tanaman Komersialisasi

terhadap cekaman biotik
dan abiotik
II perbaikan kandungan Pengembangan
nutrisi dari produk
pertanian
III pendukung industri Pengembangan
 Transgenic Plants In Use or
About to be on a Large Scale

 Herbicide-resistant plants
 Pest-resistant plants
 Vaccine plants (just starting to be used)
 Kedelai Roundup Ready™

Permasalahan dalam budidaya tanaman adalah gangguan gulma. Di negara maju

karena tenaga kerjanya yang sedikit untuk mengendalian gulma digunakan
herbisida. Herbicida seperti RoundupTM dan Liberty LinkTM dapat membunuh
beragam gulma dan mudah terurai. Perakitan varietas tanaman tahan herbisida
akan mengatasi permasalahan gulma di lahan pertanian tanpa menghambat
tanaman yang dibudidayakan.
 Peningkatan daya hasil
Tanaman dapat dioptimalkan
pertumbuhaannya dengan
memperbaiki asimilasi
nitrogennya, penyerapan
oksigen, efisiensi alur
fotosinthesis dan biosintesis
patinya..
Tanaman transgenik Tetua

Tahan hama
Varietas tahan hama dapat
dirakit dengan menyisipkan
gen Cry yang berasal dari
bakteria Bacillus thuringiensis
(Bt). Gen ini menyandi suatu
protein yang dapat membunuh
beberapa serangga.
Tetua
Jagung transgenik dengan
gen bt
 Beras “Emas”

Padi transgenik yang dapat mengandung beta-

carotene dalam berasnya. Apabila dikonsumsi,
beta-carotene akan berubah menjadi vitamin A.

Beras biasa Beras “Emas”

Tanaman Tahan Penyakit
 Produksi bahan obat
Tanaman transgenik daapt digunakan sebagai “bioreactors”
untuk menghasilkan protein yang dapat digunakan sebagai alat
terapi dan diagnostik di bidang kedokteran (Vaksin mulut).

Vaksin mulut adalah vaksin yang dihasilkan dalam jaringan

tanaman yang dapat dikonsumsi langsung. Mengkonsumsi tanaman
transgenik penghasil vaksin dapat menyebabkan tahan penyakit.
Recombinant miraculin - tomatos

leaves (102.5) &

Fruits(90.7) μg/g fresh weight

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 Medical hypothesis, 2006

Tomatoes comes in many varieties,

colors and shapes

Transgenic tomatoes –
expressing
different malarial
antigens

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Delivery of a corn-based edible
vaccine

Transgenic corn kernels (a)

Corn snack (b) or

Embryo or germ cells (c)

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 Tearless Onion

Dr Eady
Crop & Food Research in New Zealand
and his collaborators in Japan
As onions are sliced, cells are broken,
- generate sulphenic acids - unstable –
rearrange into a volatile gas - syn-propanethial-S-oxide – diffuses by air –

reaches the eye - reacts with the water to form a diluted solution of sulphuric acid –

Tear glands produce tears to dilute and flush out the irritant
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COLORED FRIUTS/FLOWERS/VEGETABLES

The-orange-purple-green-cauliflowers

karthikumarbt@kcetvnr.org 16
 Purple tomatoes high in anthocyanins

High anthocyanin purple tomato and red

wild-type tomato

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 World's First Blue Roses On Display In Japan

Tokyo, Japan –

World's first blue roses have been unveiled to the public

for the first time at an international flower fair in Japan,
following nearly two decades of scientific research.

The blue-hued blooms are genetically modified and have been

implanted with a gene that simulates the synthesis of
blue pigment in pansies.

Its scientists successfully pioneered implanting into the

flowers the gene that produces Delphinidin,
the primary plant pigment that produces a blue hue The Blue Rose was
but is not found naturally in roses. developed by Suntory
Flowers

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Transgenic plants and animals

 Transgenic plants are plants that have been

genetically engineered, a breeding approach
that uses recombinant DNA techniques to
create plants with new characteristics.
Advantages
 In plants:
 Increased and improved nutrients
 Longer shelf life, less waste
 Enhanced taste and quality
 Reduced maturation time
 Higher yielding crops, more efficient use of land
 Higher yielding crops, more efficient use of land
 Improved resistance to disease or illness
 Increased food security for growing populations and growth
challenges
 Transgenics:
1. Direct DNA Transfer
2. Agrobacterium Mediated Transformation

 Introduce naked DNA into cells (plant or animal)

 Can assay expression of the gene immediately,
or select cells that are permanently transformed
cells
 DNA introduction via direct methods:
 Chemical : using lipid
 Microinjection
 Electroporation
 Particle bombardment (Biolistics)
 needle

Microinjection of
DNA into the
pronucleus of a
newly fertilized ~ 1-2
picoliter
egg.
vol is
injected.
Injection is usually
into the sperm’s ~5-40% of
pronucleus animals
because its larger. will
contain
transgene.

From Primrose, Molec. Biotechnology

 MICROINJECTION:-
The microinjection is the process of transferring the desirable
DNA into the living cell ,through the use of glass
micropipette .

 This glass micropipette is usually of 0.5 to 5

micrometer,that easily penetrates into the cell membrane
and nuclear envelope.

 The desired gene is then injected into the sub cellular

compartment and needle is removed.
Microinjection
 Electroporation
 Use on cells without walls
(plant protoplasts or animal
cells )
 Used on monocots (maize,
rice, etc.)
 High-voltage pulses cause
pores to form transiently in
cell membrane, DNA slips in
 Drawback - its more
cumbersome to regenerate
plants from single
protoplasts than from the
tissue transformations with
Agrobacterium
 Original biolistic gun, a modified .22

DNA is bound to the microprojectiles, which are

accelerated by the macroprojectile and impact the
tissue or immobilized cells at high speeds.
J. Sanford and T. Klein, Cornell
 An Air Rifle for a DNA Gun –
Circa 1989

A.J. Thompson and D. Herrin

Repairing an organellar gene: ~ 1 x 107 cells of a
mutant of Chlamydomonas that had a deletion in the atpB
gene for photosynthesis was bombarded with the intact atpB
gene. Then, the cells were transferred to minimal medium so
that only photosynthetically competent cells could grow.

Control plate – cells were shot with tungsten

particles without DNA
The Helium Gas Gun – Circa 2000
The Hand-Held Gas Gun

Purpose:
Introduce DNA into cells that
are below the top surface
layer of tissues (penetrate
into lower layers of a tissue)

One interesting use:

Making DNA Vaccines in
whole animals.
PROSES CGE DENGAN TEKNIK BIOLISTIC

Skema Proses Chloroplast Genetic Engineering

(Wani, 2010)
KLOROPLAS

PLANT CELL CHLOROPLAST

KLOROPLAS
• Material genetik di dalam kloroplas
pertama kali dilaporkan oleh
Iwamura pada tahun 1960 (Bogorad
dan Indra, 2012).

• Genom kloroplas berbentuk sirkuler

dan double-stranded DNA

• Pada tanaman tingkat tinggi, genom

kloroplas berukuran sekitar 120-160
kb dan memiliki sekitar130 gen.

lh3.googleusercontent.com
 PERBANDINGAN REKAYASA GENOM INTI DENGAN
REKAYASA GENOM KLOROPLAS
Nucleus Engineering Chloroplast Engineering
Level ekspresi gen lebih rendah Level ekspresi transgen tinggi
1.Ekspresi gen
sehingga akumulasi protein asing sehingga akumulasi protein asing
rendah. tinggi.

Salinan transgen per sel sedikit Salinan transgen per sel banyak,
2.Jumlah kopian
karena dalam 1 sel hanya ada 1 inti sel. dapat mencapai lebih dari 10.000
transgen kopian.

Pewarisan transgen secara paternal Pewarisan transgen secara maternal,

3.Polusi gen
melalui polen, memungkinkan transgen tidak terdapat pada polen.
terjadinya gene flow dengan wild-
type.
Pernah terjadi, dan mengakibatkan Belum pernah dilaporkan.
4.Gene silencing
penurunan ekspresi transgen.
Agrobacterium tumefaciens
 Characteristics
 Plant parasite that causes Crown Gall Disease
 Encodes a large (~250kbp) plasmid called
Tumor-inducing (Ti) plasmid
 Portion of the Ti plasmid is transferred between
bacterial cells and plant cells  T-DNA (Tumor
DNA)
 Agrobacterium tumefaciens
 Tumor formation = hyperplasia
 Hormone imbalance
 Caused by A. tumefaciens

Lives in intercellular spaces of the plant

 Plasmid contains genes responsible for the
disease
 Part of plasmid is inserted into plant
DNA
 Wound = entry point  10-14 days
later, tumor forms
Ti plasmid of A. tumefaciens
1. Auxin, cytokinin, opine
synthetic genes
transferred to plant
2. Plant makes all 3
compounds
3. Auxins and cytokines
cause gall formation
4. Opines provide unique
carbon/nitrogen source
only A. tumefaciens can
use!
  How is T-DNA modified to allow genes of
interest to be inserted?
 In vitro modification of Ti plasmid
 T-DNA tumor causing genes are deleted and replaced
with desirable genes (under proper regulatory control)
 Insertion genes are retained (vir genes)
 Selectable marker gene added to track plant cells
successfully rendered transgenic [antibiotic resistance
gene  geneticin (G418) or hygromycin]
 Ti plasmid is reintroduced into A. tumefaciens
 A. tumefaciens is co-cultured with plant leaf disks under
hormone conditions favoring callus development
(undifferentiated)
 Antibacterial agents (e.g. chloramphenicol) added to kill
A. tumefaciens
 G418 or hygromycin added to kill non-transgenic plant
cells  Surviving cells = transgenic plant cells
Agrobacterium and genetic engineering:
Engineering the Ti plasmid
Tanaman Transgenik
 Tanaman yang padanya DNA sebagai
bahan genetik yang berasal dari
spesies yang lain dimasukkan dan
mampu disandikan
 Tanaman yang dipaksa mengandung
gen yang berasal dari spesies yang
tidak sekerabat (virus, bakteri,
hewan, manusia dan tanaman yang
lain)
 Agrobacterium-mediated transformation

Agrobacterium tumefaciens

cause ‘Crown gall’ disease

Agrobacterium is a ‘natural genetic engineer’

i.e. it transfers some of its DNA to plants
karthikumarbt@kcetvnr.org 47
karthikumarbt@kcetvnr.org 48
VirE2 may get DNA-protein complex across host PM

Dumas et al.,karthikumarbt@kcetvnr.org
(2001), Proc. Natl. Acad. Sci. USA, 98:485 49
 Production of transgenic plants

Isolate and clone gene of interest

Add DNA segments to initiate or enhance gene

expression

Add selectable markers

Introduce gene construct into plant cells

(transformation)

Select transformed cells or tissues

Regenerate whole plants

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Tanaman Transgenik