강의자료실 - 자료실 - 언장광장(삼척) - KNU광장 - 강원대학교

Plant Biotehcnology and Tissue Culture

강좌개요 및 목표

식물생명공학?

What is tissue culture?

• Tissue culture is the term used for “the process of

growing cells artificially in the laboratory”

(OSMS.otago.ac.nz/main/bursary)

• Tissue culture involves both plant and animal cells

• Tissue culture produces

clones

, in which all

product cells have the same genotype (unless

affected by mutation during culture)

What’s the Background?

• Tissue culture had its origins at the beginning of the 20th century with the work of Gottleib Haberlandt (plants) and Alexis Carrel (animals)

Haberlandt Carrel •전체형성능 (全體形成能, Totipotency) - Harberlandt (1902) - 한 개의 세포가 한개체를 형성 •Nobecourt (1937) : 당근유세포에 IAA를 첨가하여 새로운 세포분열 관찰 •Gautheret , White : 당근, 토마토 캘러스 형성 •Steward, Skoog (1950) : 캘러스로부터 기관분화 •Cocking (1960) : 원형질체 (Protoplast) 분리 및 배양, 융합 •1980년 이후 - 세포배양 기술확립 - 유전자조작 도입 기술 확립 - Biotech. 기술을 이용하여 실용화에 촛점

The Background, II

• The first commercial use of plant clonal propagation on artificial media was in the germination and growth of orchid plants, in the 1920’s • In the 1950’s and 60’s there

was a great deal of research, but it was only after the

development of a reliable

artificial medium

(Murashige & Skoog, 1962) that plant tissue culture really ‘took off’ commercially

The Background, III

• A more recent advance is the use of plant

and animal tissue culture along with genetic

modification using viral and bacterial

vectors

and

gene guns

to create genetically

engineered organisms

• Plant Cell and Tissue culture : 시험관내에서 무균적으로 잎이나 가지의 절편을 인공적으로 만든 배지(영양분을 공급하는 액체 혹은 액체를 한천과 같은 것으로 고형화한 것)에 놓고 인공광(光)으로 적정한 온도를 유지하며 배양하여 완전한 개체로 발달시키거나 육종 및 유용물질 생산을 이루고자 하는 생물공학의 한 연구 분야 • 연구 목적 – 첫째, 기존 우수 개체와 유전적으로 동일한 개체의 대량 복제 – 둘째, 세포수준에서 유전적 변이체 선발 및 효용이 높은 식물체를 개발 – 셋째, 세포배양을 통한 고부가가치의 이차대사산물의 생산

What is needed?

Tissue culture, both plant and animal has several

critical requirements:

• Appropriate tissue

(some tissues culture better

than others)

• A suitable growth medium

containing energy

sources and inorganic salts to supply cell growth

needs. This can be liquid or semisolid

• Aseptic (sterile) conditions

, as microorganisms

grow much more quickly than plant and animal

tissue and can over run a culture

What is Needed, II

• Growth regulators

- in plants, both auxins &

cytokinins. In animals, this is not as well

defined and the growth substances are

provided in serum from the cell types of

interest

• Frequent subculturing

to ensure adequate

nutrition and to avoid the build up of waste

metabolites

Culturing (micropropagating) Plant

Tissue - the steps

• Selection of the plant tissue (explant) from a healthy vigorous ‘mother plant’ - this is often the apical bud, but can be other tissue

• This tissue must be sterilized to remove microbial contaminants

The Steps, II

• Establishment of the explant in a culture medium. The medium

sustains the plant cells and encourages cell division. It can be solid or liquid

• Each plant species (and sometimes the variety within a species) has particular medium

requirements that must be established by trial and error

The Steps, III

• Multiplication- The explant gives rise to a callus (a mass of loosely arranged cells) which is

manipulated by varying sugar concentrations and the auxin

(low): cytokinin (high) ratios to form multiple shoots

• The callus may be subdivided a number of times

Dividing shoots

The Steps, IV

• Root formation

- The

shoots are transferred

to a growth medium

with relatively

higher

auxin: cytokinin ratios

The pottles on these racks are young banana plants and are growing roots

Tissue culture plants sold to a nursery & then potted up

The Steps, V

• The rooted shoots are

potted up (deflasked) and ‘hardened off’ by

gradually decreasing the humidity

• This is necessary as many young tissue culture plants have no waxy cuticle to prevent water loss

Why do Plant Tissue Culture?

• A single explant can be multiplied into

several thousand plants in less than a year

-this allows fast commercial propagation of

new cultivars

• Taking an explant does not usually destroy

the mother plant, so rare and endangered

plants can be cloned safely

• Once established, a plant tissue culture line

can give a continuous supply of young

Why do Plant Tissue Culture, II

• In plants prone to virus diseases, virus free

explants (new meristem tissue is usually

virus free) can be cultivated to provide virus

free plants

• Plant ‘tissue banks’ can be frozen, then

regenerated through tissue culture

• Plant cultures in approved media are easier to

export than are soil-grown plants, as they are

pathogen free and take up little space (

most

current plant export is now done in this

Why do Plant Tissue Culture, III

• Tissue culture allows fast selection for crop

improvement - explants are chosen from

superior plants, then cloned

• Tissue culture clones are ‘true to type’ as

compared with seedlings, which show greater

variability

Culturing Animal

Tissue-the Steps

• Animal tissue is obtained either from a particular

specimen, or from a ‘tissue bank’ of cryo-preserved

(cryo = frozen at very low temperatures in a special medium)

• Establishment of the tissue is accomplished in the

required medium under aseptic conditions

Culture vessels and medium for animal cell culture

Culturing Animal Tissue, II

• Growing the cells /

tissue requires an

optimum temperature,

and subculturing when

required

• Human cells, for

example are grown at

37degrees and 5%

CO2

Animal tissue/cell culture - differences

from plant tissue culture

• Animal cell lines have limited numbers of cell

cycles before they begin to degrade

• Animal cells need frequent subculturing to remain

viable

• Tissue culture media is not as fully defined as that

of plants - in addition to inorganic salts, energy

sources, amino acids, vitamins, etc., they require

the addition of serum (bovine serum is very

Animal tissue/cell culture - differences

from plant tissue culture II

• Animal tissue cultures can pose biohazard concerns, and cultures require

special inactivation with hypochlorite (e.g.

Janola,Chlorox, etc.) and then incineration

The pipettes are disposable Gloves and labcoat are

Uses of Animal Tissue Culture

• Growing viruses - these require living host cells • Making monoclonal

antibodies, used for diagnosis and research • Studying basic cell

processes

• Genetic modification & analysis

Uses of Animal Tissue Culture II

• ‘Knockout’ technology - inactivating

certain genes and tracing their effects

• Providing DNA for the

Human Genome

Genetic modification

<gene transformation>

Explants

• Pieces of organs

– Leaves

– Stems

– Roots

• Specific cell types

– Pollen

– Endosperm

– Nucellus

Nutrient Media for Plant Tissue

Cultures

Functions of medium

Provide water

Provide mineral nutritional needs

Provide vitamins

Provide growth regulators

Access to atmosphere for gas exchange

Removal of plant metabolite waste

Functions of medium

• Provide water

• Provide mineral nutritional needs

• Provide vitamins

• Provide growth regulators

• Access to atmosphere for gas exchange

• Removal of plant metabolite waste

Macroelements

• Nitrogen (N) : 아미노산, 단백질, 핵산의 구성성분 – nitrate ion (NO3- oxidized)

– ammonium ion (NH4+ _reduced) – 25-60 mM – organic

•

Potassium (K) 20 -30 mM

•

Phosphorous (P) 1-3 mM

•

Calcium (Ca) 1-3 mM

•

Magnesium (Mg) 1-3 mM

•

Sulfur (S) 1-3 mM

Micronutrients

– Iron (Fe) 1 μ M

– Manganese (Mn) 5-30 μ M

– Zinc (Zn)

– Boron (B)

– Copper (Cu) 0.1 μ M

– Molybdenum (Mo) 1 μ M

– Cobalt (Co) 0.1 μ M

– Iodine (I) Nickel (Ni), aluminum (Al), and silicon (Si)

Organic Compounds

• Sugar (carbon source, 인공광의 조도는 광합성에 불충분 ) – sucrose – others – 20 to 40 g/l • 에너지원 및 삼투압제, 농도에 따라 식물체 형태발생 반응 다양 예) 백합: 3%-자구형성, 9%-자구형성율 극히 저조 • Vitamins – thiamine (vitamin B1)

– nicotinic acid (niacin) and pyridoxine (B6) – myo-inositol

– 완전개체에서는 어느 부위에서 만들어져서 식물의 다른 부위에 공급되지만 조직배양 경우 배지에 첨가

Other Organic Material

아미노산, Urea- 질소원, 형태형성 및 체세포배 형성 카제인-아미노산, 캴슘 및 인 풍부

Other Compounds

– Activated charcoal

Support Systems

• Agar (from seaweed)

• Agarose

• Gelrite (Phytagel) (from bacteria)

• Mixtures (Phytagar)

• Mechanical (bridges, rafts)

• Sand

Media Formulations

• Many available

• Differ in salt concentrations

• 고체배양과 액체배양

– 고체배양 (solid media culture): 절편을 약 0.8% 한천으로 고형화한 배지 위에 놓고 배양 – 액체배양 (suspension culture) : 캘러스를 300ml 플라스크에 든 50ml 액체배지에서 100rpm의 shaker에서 배양 – 계대배양 : 고체배지는 1회/4주, 세포현탁배양은 1∼2회/주

• 인공광 조사 목적

• 대부분 식물이 광주기에 반응

• 조직배양에서의 광주기

Light

• 자연상태에서는 계절적인 온도변화와 하루 중

시간대에 따른 온도 변화가 뚜렷함

• 조직배양에서는 생장에 적절한 항온유지

• 조직배양시 일반적인 온도관리

Temperature

• 미세증식의 대상 재료는 표백제(락스)를 희석한 용액에 10분 정도 담근 후 멸균수로 세척하여 사용 • 조직배양은 무균상(clean bench)에서 작업 – 외부 공기를 흡입하여 두터운 필터(5㎛ 이하)를 통과시킴으로써 미생물을 포함한 작은 입자를 거른 후 배기시키는 장치에서 작업 • 무균상 소독 : 자외선 등 조사, 70% 에탄올로 소독 • 배지&용기 : 고온고압 멸균기로 멸균 후 사용 • 작업 소도구 : 외과수술용 핀셋, 메스 등 – 한 동작 후 에탄올에 담근 다음 알코올 램프로 표면을 태워 멸균하고 이를 식힌 후 식물재료를 자르거나 하는 동작에 사용

callus callus shooting shooting rooting rooting plant plant plant plant Auxin Auxin Auxin Auxin cytokinin cytokinin cytokinin cytokinin •오옥신과 시토키닌의 농도의 비율 기관발생을 결정하는 주요한 요인으로 작용, 오옥신의 시토키닌에 대한 비율이 낮으면 줄기가 발생하고, 비율이 높으면 뿌리가 발생하며 그 중간 비율에서 캘러스가 형성 1. 1. CytokininCytokinin : : 생장을생장을 조절조절, , 세포분열세포분열 촉진촉진 --> > 잎과잎과 곁눈곁눈 생장생장 촉진촉진, , 개화촉진 개화촉진 2.

2. AuxinAuxin ((옥신옥신, IAA) : , IAA) : 빛의빛의 반대방향으로반대방향으로 이동이동, , 신장촉진신장촉진, , 뿌리형성촉진뿌리형성촉진, , 측아생장저해

측아생장저해

3.

3. GiberellinGiberellin ((지베렐린지베렐린) : ) : 생장생장 촉진제촉진제, , 휴면타파제휴면타파제

Plant hormones are endogenous organic compounds active at very low concentration, produced in one tissue, and translocated to

another point in the plant where their effects on growth and development are manifested.

• auxin (indoleacetic acid)

• cytokinins (zeatin, zeatin riboside, isopentenyl adenine) • gibberellins (GA_x...125)

• abscisic acid (ABA) • ethylene

• others (real and fabled; jasmonic acid, brassinolide, florigen, juvenone)

Class Endogenous Hormone Growth Regulators auxin indoleacetic acid IBA, NAA, 2,4-D, others cytokinin zeatin, zeatin riboside kinetin, BA, 2iP, TDZ gibberellin GA_x...125 GA₃, GA₄₊₇

abscisic acid abscisic acid (ABA)

ethylene ethylene Ethephon, Ethrel

Functions of Auxin

9Stimulates cell division in the cambium and, in combination with cytokinins in tissue culture

9Stimulates differentiation of phloem and xylem

9Stimulates root initiation on stem cuttings and lateral root development in tissue culture

9Mediates the tropistic response of bending in response to gravity and light

9The auxin supply from the apical bud suppresses growth of lateral buds

9Delays leaf senescence

9Can inhibit or promote (via ethylene stimulation) leaf and fruit abscission

9Can induce fruit setting and growth in some plants

9Involved in assimilate movement toward auxin possibly by an effect on phloem transport

9Delays fruit ripening

9Promotes flowering in Bromeliads

9Stimulates growth of flower parts

9Promotes (via ethylene production) femaleness in dioecious flowers

9Stimulates the production of ethylene at high concentrations

Auxin (indoleacetic acid)

Produced in apical and root meristems, young leaves, seeds in developing fruits.

• cell elongation and expansion

• suppression of lateral bud growth

• initiation of adventitious roots and callus

• stimulation of abscission (young fruits) or delay of abscission • hormone implicated in tropisms (photo-, gravi-, thigmo-)

Auxin 적용 실

지 예