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PROTOPLAST CULTURE
Agustina Monalisa Tangapo
Protoplas
Sel tumbuhan yang telah
dihilangkan bagian dinding
selnya sel tumbuhan
telanjang tanpa dibungkus
oleh dinding sel
1. Individual cells
2. Can be fused
3. Can be transformed
4. Can be used for organelle studies
Protoplas
Explant
Isolated protoplast are capable of ingesting "foreign" material into the cytoplasm. This
material includes the introduction of nuclei, chloroplasts, mitochondria, DNA, plasmids,
bacteria and viruses.
Protoplast can be used to study wall synthesis and deposition
Protoplasts can be studied as single cell systems
Protoplasts can be induced to fuse to produce a hybrid plant, which
cannot be produced by conventional plant breeding due to
incompatibility.
Genetic transformation
ISOLASI PROTOPLAS
PURIFIKASI
FUSI PROTOPLAS
REGENERASI
Isolasi Protoplas
Enzimatik pektinase, selulose dan hemiselulose
Mekanik Klercker pada 1892 mengisolasi protoplas dari daun tanaman Stratiotes aloides memplasmolisis daun, kemudian diiris tipis-tipis dan melarutkannya
pada medium cair (Torrey dan Landgren, 1977)
Pada tahun 1960 Cocking berhasil mengisolasi protoplas yang
hidup (viable) dari jaringan akar tomat melalui perlakuan dalam
larutan enzim selulase yang diperoleh dari jamur Myrothecium
verrucaria. Pada tahun 1968, preparasi isolasi dan purifikasi
protoplas dari jaringan tanaman mulai dilakukan secara
komersial menggunakan larutan enzim sellulase dan maserozim.
Methods of isolation of protoplast
obtain sterile plant
material
rinsing in a suitable
osmoticum
facilitating enzyme
penetration
purification of the isolated
protoplasts (removal of
enzymes and cellular
debris)
transfer to a suitable
medium
Methods of isolation of
protoplast
Methods of isolation of protoplast
The chief function of the cell wall is to exert wall pressure on the protoplast preventing excessive water uptake and bursting of the cell. Before the cell wall is removed, the cell must be bathed in an isotonic plasmolyticum (mannitol or sorbitol 13 %, these sugar alcohols are less readily metabolised by plant cells). It may be advantageous to test a range of mannitol concentrations varying from 8 -15% (w/v)
Notes
Jenis dan konsentrasi enzim yang dapat
dipergunakan untuk isolasi protoplas bervariasi : Pektin glikosidase, pektinase, selulase R-10, silanase, maserozim, meiselase,
rohamen P, selulase onozuka RS, driselase, pektioliase Y-23, hemiselulase,
selulisin, naserase dan rozim. Sterilisasi dengan milipore
filter.
Setiap jenis tanaman, jenis jaringan sebagai sumber
protoplas respon yang berbeda-beda terhadap enzim tersebut.
Purifikasi Protoplas Protoplasts are filtered through a nylon
mesh (64micrometer) to remove
undigested tissue, cell clumps, and cell
wall debris.
Transfer filtrate to centrifuge tube and
spin at + 75 x g (5 min).
Debris (in supernatant) is carefully
removed (protoplasts have formed a
pellet at the base of the tube).
Protoplasts are carefully resuspended in
culture medium (plus 13% mannitol), and
the process is repeated three times.
Protoplasts are examined for density and
viability.
Protoplas hasil isolasi perlu dimurnikan
atau dipurifikasi karena protoplas hasil
isolasi masih bercampur dengan enzim,
protoplas yang pecah dan debris (sisa
jaringan yang tidak terdegradasi).
Densitas protoplas
Protoplas utuh yang telah
dimurnikan dapat digunakan
untuk budidaya, diinduksi ke arah
fusi atau untuk transfer organel
atau materi genetik harus dalam jumlah cukup dan
mempunyai viabilitas.
Jumlah optimum protoplas yang
viabel 104 105 protoplas/ml
Densitas protoplas dihitung
dengan alat haemocytometer
Figure 4.2: Cell counting using a
haemocytometer (based on Freshney)
Viabillitas protoplas
Viabilitas protoplas dapat dijui
dengan pewarnaan menggunakan
FDA (Fluorescein diacetate), PI
(Propidium Iodin), Phenosafranin, Evan
Blue, Netral Red, Rhodamin 123,
Fluorescein isotiosinat (FITC).
Fluorescein diacetate (FDA): accumulates
only inside the plasmalemma of viable
protoplasts, can be detected with
fluorescence/UV microscopy
Evans blue: Intact viable protoplasts, exclude
the Evans blue stain. Impermeability of the cell
to Evans blue indicates a living cell.
Cyclosis or protoplasmic streaming can be a
measure of viability.
Isolation of protoplasts from green tissue.
Protoplasts were prepared from 2-week-old kitaake plants and transformed as described (Methods). Picture was taken under bright field light using a Zeiss Axiovert 25 microscope with a 40 objective. Larger clear cells are derived from stem tissue whereas smaller chloroplast-filled cells were derived from green leaf tissue.
Bart et al. Plant Methods 2006 2:13 doi:10.1186/1746-4811-2-13
Protoplast culture and regeneration
Protoplas yang telah dimurnikan dikultur pada medium agar semisolid dan
liquid.
Protoplas sering dikultur pada media liquid untuk meregenerasi dinding sel
terlebih dahulu, sebelum dikultur ke media agar.
Media agar semisolid gel agar lunak (0.75 % w/v) agarose
Protoplas yang sudah berhasil didapat apabila tidak akan difusikan dapat
langsung diregenerasikan.
Protoplas dapat dimanfaatkan untuk mendukung
penelitian dasar biologi tanaman dan di bidang rekayasa genetik, misalnya hibridisasi somatik untuk meningkatkan kualitas tanaman.
Protoplast culture and regeneration
From the protoplast solution of known density (about
105 protoplast/ml) about 1 ml suspension is poured on
sterile and cooled down nutrient medium in Petri dishes.
The plates are incubated at 25C in a dim white light.
The protoplasts regenerate a cell wall, undergo cell
division and form callus. The callus can also be
subcultured. Embryogenesis begins from callus when it is
placed on nutrient medium lacking mannitol and auxin.
The embryo develops into seedlings and finally mature
plants.
SWEET ORANGE SUSPENSION CULTURE PROTOPLASTS LEAF-DERIVED CITRUS PROTOPLASTS
Pembentukan dinding sel
dan proses pembelahan
sel (A=protoplas yang
telah meregenerasi
dinding selnya secara
sempurna, B= awal
pembelahan sel, C=akhir
pembelahan sel dan
D=Sel baru hasil
pembelahan
A. A terminal bud was placed in an enzyme solution consisting of 1% Cellulase Onozuka R-10, 0.05%
Macerozyme Onozuka R-10, 1mM CaCl2, 1 mM NaCl, 0.1 mM KCl, and an appropriate osmotic
concentration of sorbitol (pH 5.5).
B. 1 hr after incubation with the enzyme solution.
C. 2 hr after incubation with the enzyme solution. Only debris was left over the mesh, and isolated
protoplasts were on the bottom of the vessel
D. These freshly isolated protoplasts often have irregular shapes, and the cytoplasmic streaming has
generally ceased.
Once the protoplasts have regenerated a cell wall, they undergo cell division and form a callus.This callus can be subcultured. The callus may undergo embryogenesis or organogenesis after about 3-4 weeks, in the correct culture conditions. The embryoids/organs can be grown up in the same manner as for most cultured plantlets .
The first division
of a rice protoplast
four days after
isolation
Protoplast derived
plantlet of rice
growing in a test tube
Fusi Protoplas
Proses peleburan (fusi) antara protoplas dengan
latar belakang genetik yang berbeda (beda
spesies atau genus) dan dapat diregenerasikan
menjadi tanaman utuh dan mempunyai karakter
gabungan antara kedua induknya (asal protoplas).
Fusi protoplas intra-spesies; inter-spesies; inter-genus.
Fusi Protoplas
The nuclei of two protoplasts may or may not fuse together even after fusion of cytoplasms.
The binucleate cells are known as heterokaryon or heterocyte
When nuclei are fused the cells are known as hybrid or synkaryocyte (Constabel, 1978), and when only cytoplasms fuse and genetic information from one of the two nuclei is lost is known as cybrid i.e. cytoplasmic hybrid or heteroplast (Doods and Roberts, 1985).
Protoplast fusion
1. Somatic hybrids
2. Cybrids (cytoplasmid hybrid
atau heteroplast)
Inter-specific fusions
Datura innoxia X D. stramonium = D. straubii (O. Schieder, 1978)
Tomate X Kartoffel = Tomoffel (G. Melchers, 1978)
Lycopersicum esculentum X Solanum tuberosum
2n x 2n 4n Synkaryon
2n Heterokaryon
Fusion of haploid protoplasts (derived from anther cultures)
n + n= 2n
Somatic hybrids
Fusi Protoplas
There are some genetic factors which are carried in cytoplasmic inheritance, instead of nuclear genes, for example, male sterility in some plants. Susceptibility and resistance to some of the pathotoxins and drug are controlled by cytoplasmic genes. Therefore, production of cybrids which contain the mixture of cytoplasms but only one nuclear genome can help in transfer of cyloplasmic genetic information from one plant to another. Thus, informations of cybrid can be applicable in plant breeding experiments. (Vasil, 1982).
Fusion products - the hybrids and cybrids
Hibridisasi
Somatik
TUGAS........................(^_^)
Methods of somatic hybridization
Procedure for successful somatic hybridization is as below:
(i) isolation of protoplasts from suitable plants,
(ii) mixing of protoplasts in centrifuge tube containing fugigenic chemicals i.e.
chemicals promoting protoplast fusion, such as polyethylene glycol (PEG)
(20%, W/V), sodium nitrate (NaNO3), maintenance of high pH 10.5 and
temperature 37C (as a result of fusion of protoplasts viable
heterokaryons are produced. PEG induces fusion of plant protoplasts and
animal cells and produces heterokaryon (Davey et al, 1978),
(iii) wall regeneration by heterokaryotic cells,
(iv) fusion of nuclei of heterokaryon to produce hybrid cells,
(v) plating and production of colonies of hybrid cells,
(vi) selection of hybrid, subculture and induction of organogenesis in the hybrid
colonies, and
(vii) transfer of mature plants from the regenerated callus.
Fusion of
protoplasts
of potato
and tomato,
and
production
of hybrid
plant
(pomato).
Diagram prosedur fusi protoplas dari mesofil daun tembakau
dan daun plantlet kentang;
A. Plantlet kentang;
B. Daun dari A dipotong-potong;
C. Potongan daun C diinkubasikan dalam medium plasmolisis
selama 1-8 jam selanjutnya diinkubasikan dalam medium
enzym selama 4-16 jam;
D1. Protoplasma dalam C disaring dengang filter steril, mess
63 mikron ;
D2. Suspensi protoplasma disentrifugasi;
E. Pelet diresuspensi dalam medium pengapung (flotation) 10
ml ditambah medium pencuci 1 ml selanjutnya disentrifuge,
protoplasma akan melayang-layang diantara medium flotasi
dan medium pencuci;
F. Protoplasma diresuspensi lagi dengan medium CPW 13 M
dengan kerapatan 1x 106 per mililiter;
G. 1. Protoplasma diteteskan pada tengah-tengah cawan petri
steril ditambahkan dengan satu tetes minyak mineral; 2.
Tetesan tersebut ditutup menggunakan gelas penutup steril; 3.
Tambahkan 3 tetes dari setiap macam suspensi protoplasma;
H. Protoplas difusikan secara bertahap menggunakan medium
fusagen PEG 22,5 dengan cara meneteskannya sebanyak 6
tetes;
I. Protoplasma dicuci dengan medium pencuci Ca+. Medium
pencuci dicampur dengan medium kultur;
J. Protoplasma diinkubasikan di bawah sinar pada suhu 25oC;
K. setelah 2-3 minggu koloni multiseluler diembeding dengan
agarose;
L. Koloni seluler pada K ditanam pada medium MS basal
(sumber: Trigiano & Gray, 2000).
RMAN
NOVA + SUCCARI SOMATIC HYBRID FRUIT
(father of several hundred triploid progeny)
Cybrid of Murcott (the Honey tangerine) containing the mtDNA CMS of G1 Satsuma mandarin
Thanks a lot for u att.