11. 기내증식

미소증식(微小增殖) 기내증식(器內增殖)

In vitro culture

Stages of Micropropagation

• Micropropagation procedure is divided in stages for the sake of understanding. Murashige proposed three (I to III) stages, Debergh and Maene added stage ’0′. Currently we have accepted five stages procedure (0 to IV).

• Stage 0 Stage I Stage II • Stage III Stage IV

Stage 0 Selection and maintenance of stock

plants for culture initiation • This stage was basically introduced to

overcome the problem of contamination. Stock plants are grown under more hygienic conditions to reduce the risk of contamination.

Stage I

Initiation and establishment of aseptic culture

• Explant isolation – Virtually any part of the plant can be used as explant like vegetative parts (Shoot tip, meristem, leaves, stems, roots) or reproductive parts (Anthers, pollen, ovules, embryo, seed, spores). Shoot tip and auxiliary buds are most often used. Size of explant, age of the stock plant, physiological age of explant, developmental age of explant these are some of the factors which decide the success rate of stage I.

• Surface sterilization – Explants are surface sterilized by treating it with disinfectant solution of suitable concentration for a specific period. Ethyl alcohol, bromine water, mercuric chloride, silver nitrate, sodium hypochlorite, calcium hypochlorite etc. can be used as disinfectant.

• Washing – Washed with water. • Establishment of explant on

appropriate medium – There is no one universal culture medium; however modifications of Murashige and Skoog basal medium (Murashige and Skoog, 1962) are most frequently used.

Stage II Multiplication of shoots or

somatic embryo formation (rapid) using a defined culture medium.

• In this stage, rapid multiplication of the

regenerative system is carried out for obtaining large number of shoots. About 4.3 X 107 shoots can be produced from a single starting explant in a year.

• Cultures obtained from stage I are placed on a suitable medium. Normally, medium for stage I and II is same, but cytokinin proportion is increased for stage II to produce numerous shoots. This stage can be repeated a few cycles until an desired number of shoots are developed to carry out for rooting. Factors which can affect shoot multiplication are physiological status of plant material, culture media, culture environment.

Stage III Rooting of regenerated shoots or

germination of somatic embryos in vitro. • In this stage, shoots or shoot clusters from

stage II are prepared to transfer to soil. Shoots are separated manually from clusters and transferred on a rooting medium containing an auxin. Elongation of shoots prior to rooting, rooting of shoots (individual or clumps), and prehardening cultures to improve survival are some of the activities carried under this stage. Sometimes, shoots are directly established in soil as micro-cuttings to develop roots.

Stage IV Hardening

= acclimatization

• Transfer of plantlets to sterilized soil for hardening under greenhouse environment.

Rejuvenation (aging)

• Rejuvenation is a medical discipline focused on the practical reversal of the aging process.[1]

• Rejuvenation is distinct from life extension. Life extension strategies often study the causes of aging and try to oppose those causes in order to slow aging. Rejuvenation is the reversal of aging and thus requires a different strategy, namely repair of the damage that is associated with aging or replacement of damaged tissue with new tissue. Rejuvenation can be a means of life extension, but most life extension strategies do not involve rejuvenation.

재유령화-임목에서 연구현황

• 조직배양 재료 • 미숙배- 성숙배- 유묘- 성숙묘-노령목 순으로

재생력이 떨어짐 • 한 개체내에서도 생리적 나이는 다름 • 지상에서 낮은 위치에 있는 부위가 재생력이

높음(맹아지) • 연속적인 접목으로 재생력 높일 수 있음 • 왕버들 300년생에서 증식 성공 체세포배유도는 미숙배 사용이 유리

조직배양재료

• 야외재료- 오염율 높음 • 온실재료- 덜함 • 배양중인 재료- 안전하게 사용가능 • ( 배양 중인 식물체에서 잎을 따서 원형질

체를 분리하면 오염으로 인한 실패 피할 수 있음)

침엽수

• 1975년 기내배양기술에 의한 침엽수의 클론 증식 최초보고(Sommer et al., 1975)

소나무-배배양

• 종자를 발아시켜 유근의 길이가 약 5mm 자랐을 때 배와 자엽을 시료로 채취하여 10mg/l BAP와 0.01mg/l의 NAA를 첨가한 GD배지에 배양하여 부정아를 유도

- 유도된 부정아를 호르몬 무첨가 배지에 1/2GD배지에 계대배양하여 줄기로 신장 촉진.

- 0.5mg/l NAA, 1.0mg/l의 IBA, 2% sucrose가 첨가된 1/2GD배지에서 기내 발근처리함.

소나무 엽속배양

• 엽속(침엽에 줄기를 약간 붙인 부분)을 배양하는 방법

• 배지: 10mg/l BAP와 0.01mg/l의 NAA를 첨가한 GD배지및 1/2LP 배지에서 줄기(침엽원기(엽속사이)에서 나옴)를 유도함

• 호르몬 무첨가 배지에서 줄기를 신장시킴

소나무 성숙목의 눈배양

• 성숙목으로 부터 눈을 채취하여 절단부위를 바세린을 바르고 표면을 살균한 후 절단하여 배지에 치상함.

리기테다소나무-배배양

• 발아된 배자엽을 10mg/l BAP와 0.01mg/l의 NAA를 첨가한 GD배지에 배양하여 부정아를 유도

• 활성탄이 첨가된 1/2GD배지에 계대배양하여 줄기신장 유도

• 0.5mg/l NAA, 1.0mg/l의 IBA, 2% sucrose가 첨가된 1/2GD배지에서 기내 발근처리함. 종자당 줄기형성수: 40-240개. 종자당 평균 15.5본, 최대 125본의 조직배양묘 얻음

리기테다소나무-엽속배양

• 2년생의 유령실생묘에서 시료채취 • BAP 10-20mg/l 첨가한 수정MS배지에서 엽

속이 팽대함 • 활성탄 첨가의 호르몬 무첨가의 1/2GD배지

에 계대배양하여 줄기로 신장촉진 • BAP 5.0mg/l 와 NAA 0.05mg/l 가 첨가된 GD

배지에서 줄기를 증식하여 엽속단 최고 24개, 평균 13개의 줄기 증식

• IBA 1.0mg/l 와 NAA 0.5mg/l 가 첨가된 1/2GD배지에서 효과적으로 발근

잣나무-배배양

• 종자에서 배를 추출하여 BAP 5.0mg/l와 NAA 0.01mg/l 가 첨가된 1/2LP배지에서 부정아 유도

• 활성탄 1%가 첨가된 호르몬 무첨가 1/2GD배지에 계대배양하여 줄기 신장 촉진

• 발근배지에 이식하여 조직배양묘로 양성

활엽수 아배양

아배양에 의한 수종별 증식율 (1개월간 증식율)

수종 증식율 수종 증식율

상수리나무 7 참오동나무 4

피나무 3 아까시나무 10

박달나무 20 호도나무 3

물박달나무 48 대추나무 4

거제수나무 3 산사나무 4

물오리나무 7 배롱나무 10-20

들메나무 2 미선나무 4

느티나무 4 망개나무 6

밤나무 2 포플러류 10-20

* 다경이 생긴 숫자에 따라 증식율이 달라짐

증식법이 확립된 수종

• 참나무류, 자작나무류, 피나무, 들메나무, 물오리나무, 느티나무, 흰배롱나무,미선나무, 망개나무

• 호도나무, 산사나무, 대추나무

세포배양

• 액체배지에 캘러스를 넣어 배양- 진탕이 필요함

• 삼각플라스크, 배양조를 배양용기로 사용 • 배양세포를 고체배지에 옮겨 캘러스를 유

도하고 기관형성을 통해 식물체 유도로 증식 가능

• 체세포배유도로 식물체 증식가능

뿌리배양

• 산삼배양근 대량생산에 이용 • 100년 된 산삼뿌리를 배양하여 캘러스를

유도 • 뿌리유도 작은 용기에서 큰 용기로 옮겨

배양 • 뿌리를 대량 증식 • 약용, 화장품 주조(술 만들기) 등의 용도로

사용- 상업화 됨

In Vitro Developmental Pathways

Explant - Piece of tissue put into culture -Tissue selection depends on purpose, species, many factors

Explants

• Pieces of organs – Leaves –Stems –Roots –Cotyledons –Embryos –Other

Explants

• Specific cell types – Leaf tissue – Embryo – Pollen – Endosperm – Nucellus

Callus

• Unorganized, growing mass of cells • Dedifferentiation of explant

– Loosely arranged thinned walled, outgrowths from explant

– No predictable site of organization or differentiation • Auxin + cytokinin • Often can be maintained indefinitely by

subculture, but may lose ability to redifferentiate

• Compact vs friable • Habituation

Three stages of callus culture

• Induction: Cells in explant dedifferentiate and begin to divide

• Proliferative Stage: Rapid cell division

• Differentiation stage (sometimes): organogenesis or embryogenesis

Induction

© 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS

Division

E. Sutton, UC Davis

Callus

© 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS

Differentiation

• Organogenesis • Somatic embryogenesis

Cell and Suspension Culture

• Cell Cultures? • Suspension Cultures

Suspension cultures

• Can be initiated from any part of the plant.

• Usually initiated from friable callus already growing in culture.

• Transferred into liquid medium.

Agitation

• Breakdown of cell aggregates into smaller clumps of cells

• Maintains a uniform distribution of cells and cell clumps in the medium

• Provides gas exchange

Medium

• Same as for callus culture?

• Gamborg B5 • Conditioning

Growth Curve

E. Sutton, UC Davis

Batch Cultures

• A certain number of cells is used to inoculate the culture, in a given volume

• Erlenmeyer flask: volume should be about 20% of flask capacity for aeration.

• Roller cultures

Continuous Culture

• Bioreactors • Closed continuous cultures: Remove

some of the media and replace with fresh. Continuous removal or periodic. Terminate growth at harvest. Start over.

• Open continuous culture: Not only remove some of media, but cells too. Maintain cell density at optimal level. Can be grown for years.

Why is it possible to regenerate in vitro?

• Totipotency – Initial state – Competence – Determination – Differentiation

Only occurs in a few cells in culture. Why?

• Pre-determination prior to culture • Newly formed meristems may act as

sinks • Meristematic centers might actually

produce compounds that inhibit neighboring cells.

Organogenesis

The formation of organs (such as leaves, shoots, roots) on a plant organ, usually of a different kind.

Organogenesis

• Rule of thumb: Auxin/cytokinin 10:1-100:1 induces roots.

• 1:10-1:100 induces shoots

• Intermediate ratios around 1:1 favor callus growth.

http://users.ugent.be/~pdebergh/pri/pri4ez04.jpg

Indirect organogenesis

Explant → Callus → Meristemoid → Primordium

Indirect Organogenesis • Dedifferentiation

– Less committed, more plastic developmental state

• Induction – Cells become organogenically competent

and fully determined for primordia production

– Change in culture conditions? • Differentiation

© 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS

© 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS

© 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS

Direct Organogenesis

Somatic Embryogenesis

Parthenocarpy

Apomixis

In vitro somatic embryogenesis

Soybean – Wayne Parrot, UGA

Somatic Embryos

• Bipolar • Not connected to explant or callus

cells by vascular tissue • In most woody plants, tissue must

be juvenile or reproductive

Indirect Somatic Embryogenesis

Induction

• Auxins required for induction –Proembryogenic masses form –2,4-D most used –NAA, dicamba also used

Development

• Auxin must be removed for embryo development

• Continued use of auxin inhibits embryogenesis • Stages are similar to those of zygotic

embryogenesis – Globular – Heart – Torpedo – Cotyledonary – Germination (conversion)

Maturation

• Require complete maturation with apical meristem, radical, and cotyledons

• Often obtain repetitive embryony • Storage protein production necessary • Often require ABA for complete

maturation • ABA often required for normal embryo

morphology – Fasciation – Precocious germination

Germination

• May only obtain 3-5% germination • Sucrose (10%), mannitol (4%) may be required • Drying (desiccation)

– ABA levels decrease – Woody plants – Final moisture content 10-40%

• Chilling – Decreases ABA levels – Woody plants

Rubber tree from somatic embryo CIRAD

http://images.google.com/imgres?imgurl=http://www.ars-grin.gov/ars/SoAtlantic/fp/hb/niedz/torpedo.jpg&imgrefurl=http://www.ars-grin.gov/ars/SoAtlantic/fp/hb/niedz/niedz.html&h=216&w=160&sz=16&hl=en&sig2=q2-M_hcPj-KQs7TDxv6sdA&start=1&tbnid=_rieWcG5ypdozM:&tbnh=107&tbnw=79&ei=KiutRd6ZJJWe7QHO1YWjBw&prev=/images?q=citrus+somatic+embryos&svnum=30&hl=en&lr=&sa=X

Factors that Influence SE

• Genotype • Growth regulators • Carbon source • Nitrogen

Maturation and Germination (Conversion)

체세포배 문제와 해결방법

• 우량목으로 판명된 성숙목이 재료로 가장 바람직하나 나이가 많아지면 체세포배유도가 어려움.

• 종자로 성숙과정인 미숙배가 제일 활력 있음. • 종자는 접합자배이므로 유전적으로 검증되지

못한 것임 • 해결방법: 재료를 초저온에 보존하다가 우수

한 것으로 검정된 체세포배는 계속 사용하고 불량한 것의 체세포배는 폐기

Synthetic seed

• Potential method of clonal production • Naked embryos is fragile • Must be encapsulated • Sodium alginate+ Calcium chloride • Polyox

POLYOX™

• POLYOX™ - Water-Soluble Polymers with Added Functionality

• POLYOX™ water-soluble Poly(ethylene) Oxide polymers are non-ionic, high molecular weight water-soluble Polyethylene Oxide polymers that exhibit many properties typical of other classes of water-soluble polymers - lubricity, binding, water retention, thickening and film formation. They are available in a wide range of molecular weights, enabling formulations designed for individual specifications.

Germination

• May only obtain 3-5% germination • Sucrose (10%), mannitol(4%) may be required

• Drying(dessication) - ABA levels decrease - Final moisture content 10-40% • Chilling - Decreases ABA levels

Successful species

• Walnut Pinus strobus Picea abies

• Tulip tree • Pinus densiflora Pinus rigida * P. taeda • Larix kaemfaeri, • 헛개나무 • •