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.