PLANT IN VITRO CULTURE

Oleh:
Gieks Sugiyono, S.Si., Ph.D

JENDERAL SOEDIRMAN UNIVERSITY

FACULTY OF BIOLOGY
•8/7/2019 PURWOKERTO •1
 Competence

Understanding both theoretical basis and principles

of plant in vitro culture, having the ability and skills
to carry out plant in vitro culture both individually
and team work, understanding the use and
application of plant in vitro culture, skillful in
managing and disseminating idea and information
about plant in vitro culture

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 Deskripsi Singkat MK (1-2)
Mata kuliah ini membahas tentang:
1. Sejarah, teori dasar, ciri-ciri, dan tipe-tipe kultur
in vitro tumbuhan (KIVT);
2. Manfaat KIVT, faktor-faktor yang mempengaruhi
keberhasilan KIVT;
3. Beberapa teknik dasar KIVT (teknik sterilisasi,
pembuatan media, inokulasi dan sub kultur, serta
teknik pemeliharaan kultur);
4. Kultur embryo;
5. Kultur organ (anther dan meristem);

•8/7/2019 •3
 Deskripsi Singkat MK (2-2)
Mata kuliah ini membahas tentang:
6. Teknik perbaikan kualitas tanaman (variasi
somaklonal, embryo rescue, seleksi in vitro, dan fusi
protoplas);
7. Pembuatan tanaman bebas penyakit;
8. Mikropropagasi vegetatif;
9. Peran kultur in vitro dalam rekayasa genetika
tumbuhan;
10. Aklimatisasi tanaman hasil kultur;
11. Troubleshooting (kontaminasi, vitrifikasi,
browning).
•8/7/2019 •4
 Outcomes
1. Mahasiswa terampil berkomunikasi ilmiah
2. Mahasiswa mampu bekerja dalam tim
3. Mahasiswa memiliki bekal kewirausahaan

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 Evaluation Components
1. Weekly quiz : 20 %
2. Structured Assignment : 10 %
3. Laboratory Work : 30 %
4. Mid Test : 20 %
5. Final Test : 20 %

• Contact:
1. e-mail: gieks_sugiyono@hotmail.com
2. Mobile : 0813 273 96 412

•8/7/2019 •Gieks •6
 Kunci Sukses
1. Banyak belajar
2. Visi jangka panjang yang diwujudkan
3. Bertindak segera
4. Menerima kritikan
5. Mempelajari teknologi
(Bill Gates)
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 DEFINITIONS

Tissue culture:

is a technique which involves the establishment

of a more or less dedifferentiated cell or tissue
culture, proliferation for a number of cell
generations and subsequent regeneration of
plants (Larkin and Scowcroft, 1981).

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 What is Plant Tissue Culture?

“… the aseptic culture of plant protoplasts, cells, tissues

or organs under conditions which lead to cell
multiplication or regeneration of organs or whole
plants “

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Pierik, (1987) and Thorpe (2000) used the term
“in vitro culture” to describe a culture of plants,
seeds, embryos, organs, explants, tissues, cells
and protoplast, on nutrient media under sterile
conditions.

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 Basic in vitro propagation ...

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 THEORY

The cell theory of Schwann and

Schleiden (1838-1839)

“ The cell was described as the smallest

biological unit that could be considered
totipotent”

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 “Totipotency theory”

“Cells are autonomic, and in principle,

are capable of regenerating to give a
complete plant” (Haberlandt, 1902)

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Totipotency ….
… each livinggenetic
complete cell has a complete
blue print genetic
blueprint and therefore has the potential to
develop into an entire plant.
……cells
cellslines
differentiate
differentiate to form specialised
tissues and organs

unlikeplant
… living animal cells,
cells canliving plant cells can
re-differentiate
de-differentiate and then re-differentiate to
form different cell types
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 HISTORY

•8/7/2019 •15
Early Cell Culture ….

…Haberlandt .. early 1900’s

… proposed concept of totipotency
… cells cultured under right conditions

Callus cultured from tree cambium

(Gautheret, Nobecourt, Whire in the 1930s.

… cells kept alive but did not develop

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Early tissue culture ….
- dependent on discovery of
“growth regulators”

 Cell enlargement … role of auxins

 Cell division ... role of cytokinins

 Regeneration from tobacco pith ..

(Skoog and Miller) … interaction of auxin
and cytokinin gives differentiation.
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 Further development …

 GA for growth of shoots

 Aux + Cyt + sucrose

> vascular development

 Culture of ‘thin layers’

… many interacting factors eg pH

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 Carrot plants from root cells – Stewart in 1964

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[Steeves & Sussex 1972]
Tobacco plants from single cells
– Vasil & Hilderbrandt 1965

•8/7/2019 •21 1972]

[Steeves & Sussex
Plant Organ Culture ….

Murashige and Skoog 1962 - mineral media

 micropropagation

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Controlling Growth
& Development
 Physiological mechanisms
 Physical manipulation
 Chemical Plant Growth Regulators
(PGRs)
 Environment

 Genotype

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Growth Regulators
 Hormone theory
 Auxins
 Cytokinins
 Gibberelins (GA)
 Ethylene
 Abscisic Acid (ABA)
 Other PGRs

•8/7/2019 •24
 Hormone Balance
Auxin Cytokinin
High Low

Root formation on cuttings

Embryogenesis
Adventitious root formation in callus
Callus initiation
Adventitious shoot formation
Axillary shoot growth
Low High
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 Auxin : Cytokinin ratio

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 Hormone Action …..

 Application + uptake
 Endogenous + applied
 Accumulation & Habituation
 Interactions / Sequence
 Pulsing vs prolonged exposure

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 OBJECTIVES

A. Propagation of superior genotypes

B. Use of plant cultures for manufacture
of plant products
C. Genetic manipulation of plants (Plant
Improvement)
D. Genetic conservation

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 A. Plant propagation
I. 1. Cashew (Anacardium occidentale)
2. Clove (Syzigium aromaticum)
3. Papaya (Carica papaya) Cashew nut

4. Rami (Bochmeria nivea)

5. Teak (Tectona grandis)
6. Vanilla (Vanilla plamifolia)
Cengkeh

Vanilla

Sukun Pepaya
•8/7/2019
Teak
Jati •29
II. Potential and rare medicinal plants

Pranawijaya
III. Horticulture Ornamental plants

Gerbera Anggrek
Mawar
IV. Crops: Sweet potato, cassava etc ,

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Yam Gembili Kentang hitam
 7. Sukun (Artocarpus communis)
8. Abacca (Musa textilis)
9. pepper (Piper nigrum)
10. Cynamom (Cinnamomum sp.)
11. Mangosten (Garcinia
mangostana)
Abaka

Lada

Manggis

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 B. Production of Plant Product
B. Secondary metabolites production

1. Terong KB (Solanum khazianum & S. laciniatum)

2. Nilam (Pogostemon cablin)
3. Mentha (Mentha pipenta & M. arvensis)
4. Clove (Syzigium aromaticum)
5. Pegagan
6. Pule pandak (Rauvolfia serpentina)

Cengkeh
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C. Plant improvement

Methodes: Somaklonal
variasi pada
1. Somaclonal variation mawar

Vanilla (Vanilla planifolia)

Nilam (Pogostemon cablin)
Ginger (Zingiber oficinale)
Rose (Rosa hybrida)
Gerbera (Gerbera jamesonii)

•8/7/2019 Gerbera •33

 Induksi variasi somaklonal

Variasi somaklonal

Somaklon
•8/7/2019di lapangan •34
 Nilam (Pogostemon cablin)
Masalah: variasi genetik sangat sempit
radiasi

suspensi sel - somaklon di tanam di

0, 1, 2, & 3 krad 2 lokasi (Bandung &
Bogor)  3 tahun
- 5 somaklon memiliki
kandungan minyak
lebih tinggi daripada
kontrol  3% – 4%

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 Jahe (Zingiber officinale)
Masalah: penyakit yang disebabkan Ralstonia
solanacearum
tunas in vitro (periode kultur in-vitro = 24 bulan)
radiasi 0, 1, dan 1.5 krad

- somaklon ditanam di daerah endemik

- 1 somaklon (kJ 210): resisten penyakit

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Latar belakang: somaklon jahe
2. Embryo rescue
Vanilla (Vanilla planifolia X Vanilla albida)

Green bean (Vigna radiata X Vigna mungo)

Planlet hasil
Polong kacang hijau Biji kacang hijau penyelamatan embrio
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 Latar belakang: buah hasil tanaman fusi
3. In vitro selection
Vanilli (Vanilla planifolia)
Lada (Piper nigrum)
Kedelai (Glycine max)
Padi (Oryza sativa)
Kalus kedelai Kalus padi
dalam PEG dalam PEG
- Vanilli (Vanilla planifolia) Variation on fruits length of vanilla
somaclone
Masalah: penyakit yang disebabkan oleh Fusarium oxysporum
struktur globular (berukuran ± 1 mm)
seleksi bertahap dan seleksi silang
menggunakan asam Fusaric atau filtrat

Beberapa somaklon diuji menggunakan F. oxysporum 

terdapat somaklon yang resisten

- Lada (Piper nigrum)

Masalah: penyakit yang disebabkan oleh Phytopthora infestan
jaringan daun  kalus
Dua kali seleksi dengan P. infestan
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Beberapa somaklon Lada
 Kalus diseleksi Kalus diseleksi
menggunakan menggunakan
PEG Al dan pH 4

 beberapa somaklon
 penelitian masih
berlangsung

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4. Protoplas fussion
Solanum melongena X S. aethiopicum

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Fusi protoplas
1. Pepaya (Carica papaya)
Gen: delay ripening

2. Padi (Oryza sativa)

Gen: BT  penggerek
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batang
 D. Germplasm Conservation
Purwoceng
Bidara upas

Kunyit putih Methodes:

Temu putri
1. Normal Growth
2. Minimal Growth
3. Cryopreservation

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 Tanaman obat langka
1. Pulasari (Alyxia stellata)
2. Purwoceng (Pimpinella pruatjan)
3. Inggu (Ruta oryustifolia)
4. Bidara upas (Meremia mammosa)
5. Temu puteri (Curcuma petiolata)
6. Pule pandak (Rauvolfia serpentina)
7. Pulai (Alstonia scholaris)
8. Ki sariawan (Symplocas odoratisima)
9. Pranawijaya (Andira horsfidii)
10. Puar (Elettaria sumatrana)
•8/7/2019 •44
Potential medicinal plants
1. Ginger (Zingiber officinale)
Daun dewa
2. Kencur (Kaemfena galangan)
3. Tempuyung (Sonchus arvensis)
4. Daun encok (Plumbayo zeylonica)
5. Daun dewa (Gynura precumbens)
dan (Gynura pseudochina)
6. Som jawa (Talinum paniculatum)
7. Terong KB (Solanum khasianum &
S. laciniatum)
Anectochillus
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Potential medicinal plants
6. Temugiring (Curcuma heyneana)
7. Kolesom (Talimum paniculatum & T.
racemosum)
8. Handeleum (Graftophyllum pictum)
9. Tangguh (Pettivera alliaceae)
10.Anectochillus taiwanensis

•8/7/2019 •46
 CHARACTERISTICS

• Occur on a small scale

• All micro-organisms and pests are excluded
• The environmental growth conditions, with
regards to physical, nutritional, and
hormonal factors are optimised

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 • The normal patterns of plant development
can be changed
• The possibility to manipulate the explant’s
growth

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 TYPES

• Culture of intact plants

• Embryo culture
• Organ culture (meristem, anther)
• Callus culture
• Single cell culture
• Protoplast culture

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 De Fossard
(1977)

• Organised : The characteristic

organisational of plant on the individual organ
being maintained.
• Non-organised: Organised de-differentiated
non-organised
• Non-organised/Organised: Regeneration

•8/7/2019 •51
• Embryo culture : shortening breeding cycles,
overcoming incompatibility.

• Meristem culture: eliminating pathogens,

mass cloning of plants, germ plasm collection,
cryopreservation

• Callus culture: cloning of plants, creation of

genetic variants, elimination of pathogens,
source of protoplast, production of secondary
metabolites

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• Anther culture: production of homozygotes,
mutation induction

• Protoplast culture: somatic hybridisation,

transformation studies.

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