12 조직배양에 의한 신품종육성

Contents

bull Embryo Rescue bull Eodosperm Culture bull Somaclonal Variation bull Somatic Hybrids bull True Breeding Organism(pure lines)

Embryo rescue bull Embryo rescue is one of the earliest and successful forms

of in-vitro culture techniques that is used to assist in the development of plant embryos that might not survive to become viable plants[1] Embryo rescue plays an important role in modern plant breeding allowing the development of many interspecific and intergeneric food and ornamental plant crop hybrids This technique nurtures the immature or weak embryo thus allowing it the chance to survive Plant embryos are multicellular structures that have the potential to develop into a new plant The most widely used embryo rescue procedure is referred to as embryo culture and involves excising plant embryos and placing them onto media culture[2]

Contents

bull Embryo Rescue bull Eodosperm Culture bull Somaclonal Variation bull Somatic Hybrids bull True Breeding Organism(pure lines)

Embryo rescue bull Embryo rescue is one of the earliest and successful forms

of in-vitro culture techniques that is used to assist in the development of plant embryos that might not survive to become viable plants[1] Embryo rescue plays an important role in modern plant breeding allowing the development of many interspecific and intergeneric food and ornamental plant crop hybrids This technique nurtures the immature or weak embryo thus allowing it the chance to survive Plant embryos are multicellular structures that have the potential to develop into a new plant The most widely used embryo rescue procedure is referred to as embryo culture and involves excising plant embryos and placing them onto media culture[2]

Embryo rescue bull Embryo rescue is one of the earliest and successful forms

of in-vitro culture techniques that is used to assist in the development of plant embryos that might not survive to become viable plants[1] Embryo rescue plays an important role in modern plant breeding allowing the development of many interspecific and intergeneric food and ornamental plant crop hybrids This technique nurtures the immature or weak embryo thus allowing it the chance to survive Plant embryos are multicellular structures that have the potential to develop into a new plant The most widely used embryo rescue procedure is referred to as embryo culture and involves excising plant embryos and placing them onto media culture[2]

bull Embryo rescue is most often used to create interspecific and intergeneric crosses that would normally produce seeds which are aborted Interspecific incompatibility in plants can occur for many reasons but most often embryo abortion occurs[3] In plant breeding wide hybridization crosses can result in small shrunken seeds which indicate that fertilization has occurred however the seed fails to develop Many times remote hybridizations will fail to undergo normal sexual reproduction thus embryo rescue can assist in circumventing this problem[4]

History bull Embryo rescue was first documented in the 18th

century when Charles Bonnet excised Phaseolus and Fagopyrum embryos planted them in soil and the cross resulted in dwarf plants[5] Soon after this scientists began placing the embryos in various nutrient media During the period of 1890 to 1904 systems for embryo rescue became systematic by applying nutrient solutions that contained salts and sugars and applying aseptic technique[6] The first successful in vitro embryo culture was performed by Hanning in 1904 he however described problems with precocious embryos that resulted in small weak and often inviable plantlets

Applications

bull Breeding of incompatible interspecific and intergeneric species

bull Overcoming seed dormancy bull Determination of seed viability bull Recovery of maternal haploids that develop

as a result of chromosome elimination following interspecific hybridization

bull Used in studies on the physiology of seed germination and development

Techniques bull Depending on the organ cultured it may be referred

to as either embryo ovule or ovary culture Ovule culture or in ovulo embryo culture is a modified technique of embryo rescue whereby embryos are cultured while still inside their ovules to prevent damaging them during the excision process[8] Ovary or pod culture on the other hand employs the use of an entire ovary into culture It becomes necessary to excise the entire small embryo to prevent early embryo abortion However it is technically difficult to isolate the tiny intact embryos so often ovaries with young embryos or entire fertilized ovules will be used

Factors to consider bull Embryos are manually excised and placed immediately onto

a culture media that provides the proper nutrients to support survival and growth (Miyajima 2006) While the disinfestation and explant excision processes differ for these three techniques many of the factors that contribute to the successful recovery of viable plants are similar The main factors that influence success are the time of culture the composition of the medium and temperature and light Timing mainly refers to the maturation stage of the embryo before excision The optimal time especially for the rescue of embryos involving incompatible crosses would be just prior to embryo abortion Nevertheless due to difficulties involved with the rearing of young embryos compared to those that have reached the autotrophic phase of development embryos are normally allowed to develop in vivo as long as possible

bull While in general two main types of basal media are the most commonly used for embryo rescue studies ie Murashige and Skoog medium (MS) [10] and Gamborgrsquos B-5[11] media (Bridgen 1994) the composition of the media will vary in terms of the concentrations of media supplements required This will generally depend on the stage of development of the embryo For instance young embryos would require a complex medium with high sucrose concentrations while more mature embryos can usually develop on a simple medium with low levels of sucrose The temperature and light requirement is generally species specific and thus its usually regulated to be the within the same temperature requirement as that of its parent with embryos of cool-season crops requiring lower temperatures than those of warm-season crops

References

bull Embryo rescue and plant regeneration following interspecific crosses in the genus Hylocereus (Cactaceae)

bull Embryo rescue A tool to overcome incompatible interspecific hybridization in Gossypium Linn

poplar bull In-ovary embryo culture as a tool for poplar

hybridization C Raquin L Troussard M Villar

bull Canadian Journal of Botany 1993 71(10) 1271-1275 101139b93-151

bull Abstract bull We report here the development of an easy in vitro method

for culturing Populus sp embryos Half-capsules were cultured in an agar medium with Murashige and Skoog inorganic salts and sucrose Embryos germinated in the half-capsule and were subcultured in jars before being transferred to the greenhouse Twenty-one different intra- and inter-specific crosses were successful with 89 of plantlets successfully acclimatized in greenhouse

bull 1992 Embryology and embryo rescue of interspecific hybrids in Actinidia Acta-Hort 29793-97

Somaclonal variation

bull Somaclonal variation is the variation seen in plants that have been produced by plant tissue culture Chromosomal rearrangements are an important source of this variation

bull Somaclonal variation is not restricted to but is particularly common in plants regenerated from callus The variations can be genotypic or phenotypic which in the latter case can be either genetic or epigenetic in origin Typical genetic alterations are changes in chromosome numbers (polyploidy and aneuploidy)

poplar bull In-ovary embryo culture as a tool for poplar

hybridization C Raquin L Troussard M Villar

bull Canadian Journal of Botany 1993 71(10) 1271-1275 101139b93-151

bull Abstract bull We report here the development of an easy in vitro method

for culturing Populus sp embryos Half-capsules were cultured in an agar medium with Murashige and Skoog inorganic salts and sucrose Embryos germinated in the half-capsule and were subcultured in jars before being transferred to the greenhouse Twenty-one different intra- and inter-specific crosses were successful with 89 of plantlets successfully acclimatized in greenhouse

bull 1992 Embryology and embryo rescue of interspecific hybrids in Actinidia Acta-Hort 29793-97

Somaclonal variation

bull Somaclonal variation is the variation seen in plants that have been produced by plant tissue culture Chromosomal rearrangements are an important source of this variation

bull Somaclonal variation is not restricted to but is particularly common in plants regenerated from callus The variations can be genotypic or phenotypic which in the latter case can be either genetic or epigenetic in origin Typical genetic alterations are changes in chromosome numbers (polyploidy and aneuploidy)

bull 1992 Embryology and embryo rescue of interspecific hybrids in Actinidia Acta-Hort 29793-97

Somaclonal variation

bull Somaclonal variation is the variation seen in plants that have been produced by plant tissue culture Chromosomal rearrangements are an important source of this variation

bull Somaclonal variation is not restricted to but is particularly common in plants regenerated from callus The variations can be genotypic or phenotypic which in the latter case can be either genetic or epigenetic in origin Typical genetic alterations are changes in chromosome numbers (polyploidy and aneuploidy)

Somaclonal variation

bull Somaclonal variation is the variation seen in plants that have been produced by plant tissue culture Chromosomal rearrangements are an important source of this variation

bull Somaclonal variation is not restricted to but is particularly common in plants regenerated from callus The variations can be genotypic or phenotypic which in the latter case can be either genetic or epigenetic in origin Typical genetic alterations are changes in chromosome numbers (polyploidy and aneuploidy)

bull chromosome structure (translocations deletions insertions and duplications) and DNA sequence (base mutations) A typical epigenetics-related event would be gene methylation

bull If no visual morphogenic changes are apparent other plant screening procedures must be applied There are both benefits and disadvantages to somaclonal variation The phenomenon of high variability in individuals from plant cell cultures or adventitious shoots has been named somaclonal variation

Somaclones

bull Somaclonal variations bull Populus X berolinensis(2013)

Benefits

bull The major likely benefit of somaclonal variation in plant is improvement Somaclonal variation leads to the creation of additional genetic variability Characteristics for which somaclonal mutants can be enriched during in vitro culture includes resistance to disease pathotoxins herbicides and tolerance to environmental or chemical stress as well as for increased production of secondary metabolites Micropropagation can be carried out throughout the year independent of the seasons and plants

Benefits

bull The major likely benefit of somaclonal variation in plant is improvement Somaclonal variation leads to the creation of additional genetic variability Characteristics for which somaclonal mutants can be enriched during in vitro culture includes resistance to disease pathotoxins herbicides and tolerance to environmental or chemical stress as well as for increased production of secondary metabolites Micropropagation can be carried out throughout the year independent of the seasons and plants

Disadvantages

bull A serious disadvantage of somaclonal variation occurs in operations which require clonal uniformity as in the horticulture and forestry industries where tissue culture is employed for rapid propagation of elite genotypes

Reducing somaclonal variation

bull Different steps can be used to reduce somaclonal variation It is well known that increasing numbers of subculture increases the likelihood of somaclonal variation so the number of subcultures in micropropagation protocols should be kept to a minimum Regular reinitiation of clones from new explants might reduce variability over time Another way of reducing somaclonal variation is to avoid 24-D in the culture medium as this hormone is known to introduce variation

Reducing somaclonal variation

bull Different steps can be used to reduce somaclonal variation It is well known that increasing numbers of subculture increases the likelihood of somaclonal variation so the number of subcultures in micropropagation protocols should be kept to a minimum Regular reinitiation of clones from new explants might reduce variability over time Another way of reducing somaclonal variation is to avoid 24-D in the culture medium as this hormone is known to introduce variation

bull Vitrification hyperhydracity may be a problem in some species[clarification needed] In case of forest trees mature elite trees can be identified and rapidly cloned by this technique[citation needed]

bull High production cost has limited the application of this technique to more valuable ornamental crops and some fruit trees

True breeding organism

bull A true breeding organism sometimes also called a pure-bred is an organism that always passes down a certain phenotypic trait to its offspring An organism is referred to as true breeding for each trait to which this applies and the term true breeding is also used to describe individual genetic traits[1] In Mendelian genetics this means that an organism must be homozygous for every trait for which it is considered true breeding Apomixis and parthenogenesis types of asexual reproduction also result in true breeding although the organisms are usually not homozygous[2]

Examples of True breeding organism

bull A pure-bred variety of cat such as Siamese only produces kittens with Siamese characteristics because their ancestors were inbred until they were homozygous for all of the genes that produce the physical characteristics and temperament associated with the Siamese breed

bull When a true-breeding plant with pink flowers is self-pollinated all its seeds will only produce plants that also have pink flowers Gregor Mendel cross-pollinated true-breeding peas in his experiments on patterns of inheritance of traits

bull Pure lines are obtained from a single ancestor and are maintained by self-pollinization and selection Individuals in pure lines repeat over several generations the same genetically fixed traits Pure lines are important in agriculture inasmuch as they are the main structural elements of plant varieties Hybridization of two pure lines often results in heterosis in the first hybrid generation certain hybrid varieties of corn are obtained in this way

bull The term ldquopure linerdquo is sometimes incorrectly applied to inbred lines which are the progeny of animals or cross-pollinated plants obtained from a single pair of ancestors and maintained over a number of generations by constant crossing of related individuals and by selection Such lines are almost always used in genetic research on higher organisms For example ldquopure linerdquo laboratory mice are used to study carcinogenesis and cancer treatment

Protoplast bull Protoplast from ancient Greek πρωτόπλαστος

(prōtoacuteplastos first-formed) initially referred to the first human[citation needed] or more generally to the first organized body of a species In modern biology it has several definitions

bull A protoplast is a plant bacterial or fungal cell that had its cell wall completely or partially removed using either mechanical or enzymatic means ndash Protoplasts Have their cell wall entirely removed and are

derived from gram + (gram-positive) ndash Spheroplasts Have their cell wall only partially removed and

are gram - (gram-negative) bull More generally protoplast refers to that unit of biology

which is composed of a cells nucleus and the surrounding protoplasmic materials

bull The term ldquopure linerdquo is sometimes incorrectly applied to inbred lines which are the progeny of animals or cross-pollinated plants obtained from a single pair of ancestors and maintained over a number of generations by constant crossing of related individuals and by selection Such lines are almost always used in genetic research on higher organisms For example ldquopure linerdquo laboratory mice are used to study carcinogenesis and cancer treatment

Protoplast bull Protoplast from ancient Greek πρωτόπλαστος

(prōtoacuteplastos first-formed) initially referred to the first human[citation needed] or more generally to the first organized body of a species In modern biology it has several definitions

bull A protoplast is a plant bacterial or fungal cell that had its cell wall completely or partially removed using either mechanical or enzymatic means ndash Protoplasts Have their cell wall entirely removed and are

derived from gram + (gram-positive) ndash Spheroplasts Have their cell wall only partially removed and

are gram - (gram-negative) bull More generally protoplast refers to that unit of biology

which is composed of a cells nucleus and the surrounding protoplasmic materials

Protoplast bull Protoplast from ancient Greek πρωτόπλαστος

(prōtoacuteplastos first-formed) initially referred to the first human[citation needed] or more generally to the first organized body of a species In modern biology it has several definitions

bull A protoplast is a plant bacterial or fungal cell that had its cell wall completely or partially removed using either mechanical or enzymatic means ndash Protoplasts Have their cell wall entirely removed and are

derived from gram + (gram-positive) ndash Spheroplasts Have their cell wall only partially removed and

are gram - (gram-negative) bull More generally protoplast refers to that unit of biology

which is composed of a cells nucleus and the surrounding protoplasmic materials

Protoplasts of cells from a petunias leaf

Protoplasts of Physcomitrella patens

Uses for protoplasts bull Protoplasts can be used to study membrane biology including the

uptake of macromolecules and viruses These are also used in somaclonal variation

bull Protoplasts are widely used for DNA transformation (for making genetically modified organisms) since the cell wall would otherwise block the passage of DNA into the cell[1] In the case of plant cells protoplasts may be regenerated into whole plants first by growing into a group of plant cells that develops into a callus and then by regeneration of shoots (caulogenesis) from the callus using plant tissue culture methods[2] Growth of protoplasts into callus and regeneration of shoots requires the proper balance of plant growth regulators in the tissue culture medium that must be customized for each species of plant Unlike protoplasts from vascular plants protoplasts from mosses such as Physcomitrella patens do not need phytohormones for regeneration nor do they form a callus during regeneration Instead they regenerate directly into the filamentous protonema mimicking a germinating moss spore[3]

Uses for protoplasts bull Protoplasts can be used to study membrane biology including the

uptake of macromolecules and viruses These are also used in somaclonal variation

bull Protoplasts are widely used for DNA transformation (for making genetically modified organisms) since the cell wall would otherwise block the passage of DNA into the cell[1] In the case of plant cells protoplasts may be regenerated into whole plants first by growing into a group of plant cells that develops into a callus and then by regeneration of shoots (caulogenesis) from the callus using plant tissue culture methods[2] Growth of protoplasts into callus and regeneration of shoots requires the proper balance of plant growth regulators in the tissue culture medium that must be customized for each species of plant Unlike protoplasts from vascular plants protoplasts from mosses such as Physcomitrella patens do not need phytohormones for regeneration nor do they form a callus during regeneration Instead they regenerate directly into the filamentous protonema mimicking a germinating moss spore[3]

Somatic fusion(wiki)

bull Somatic fusion also called protoplast fusion is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both a somatic hybrid Hybrids have been produced either between different varieties of the same species (eg between non-flowering potato plants and flowering potato plants) or between two different species (eg between wheat triticum and rye secale to produce Triticale)

Fused protoplast (left) with chloroplasts (from a leaf cell) and coloured vacuole (from a petal)

The somatic fusion process occurs in four steps

bull The removal of the cell wall of one cell of each type of plant using cellulase enzyme to produce a somatic cell called a protoplast

bull The cells are then fused using electric shock (electrofusion) or chemical treatment to join the cells and fuse together the nuclei The resulting fused nucleus is called heterokaryon

bull The somatic hybrid cell then has its cell wall induced to form using hormones

The somatic fusion process occurs in four steps

bull The removal of the cell wall of one cell of each type of plant using cellulase enzyme to produce a somatic cell called a protoplast

bull The cells are then fused using electric shock (electrofusion) or chemical treatment to join the cells and fuse together the nuclei The resulting fused nucleus is called heterokaryon

bull The somatic hybrid cell then has its cell wall induced to form using hormones

bull The cells are then grown into calluses which then are further grown to plantlets and finally to a full plant known as a somatic hybrid

bull Different from the procedure for seed plants describe above fusion of moss protoplasts can be initiated without electric shock but by the use of polyethylene glycol (PEG) Further moss protoplasts do not need phytohormones for regeneration and they do not form a callus[3] Instead regenerating moss protoplasts behave like germinating moss spores[4] Of further note sodium nitrate and calcium ion at high pH can be used although results are variable depending on the organism[5]

Characteristics of Somatic Hybridization and Cybridization

bull 1 Somatic cell fusion appears to be the

only means through which two different parental genomes can be recombined among plants that cannot reproduce sexually (asexual or sterile)

bull 2 Protoplasts of sexually sterile (haploid triploid and aneuploid) plants can be fused to produce fertile diploids and polyploids

bull 3 Somatic cell fusion overcomes sexual incompatibility barriers In some cases somatic hybrids between two incompatible plants have also found application in industry or agriculture

bull 4 Somatic cell fusion is useful in the study of cytoplasmic genes and their activities and this information can be applied in plant breeding experiments

Inter-specific and inter-generic fusion achievements

Cross Crossed with

Oat Maize

Brassica sinensis B oleracea

Torrentia fourneri T bailloni

Brassica oleracea B campestris

Datura innoxia Atropa belladonna

Nicotiana tabacum N glutinosa

Datura innoxia D candida

Arabidopsis thaliana Brassica campestris

Petunia hybrida Vicia faba

Somatic Hybrids in woody plants

bull 탱자 Poncirus trifoliata bull 당귤 Citrus sinensis

bull Theor Appl Genet 1985

Nov71(1)1-4 doi 101007BF00278245

bull Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata

bull Ohgawara T1 Kobayashi S Ohgawara E Uchimiya H Ishii S

Somatic Hybrids in woody plants

bull 탱자 Poncirus trifoliata bull 당귤 Citrus sinensis

bull Theor Appl Genet 1985

Nov71(1)1-4 doi 101007BF00278245

bull Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata

bull Ohgawara T1 Kobayashi S Ohgawara E Uchimiya H Ishii S

bull Abstract bull Somatic hybrid plants of Rutaceae were obtained by

protoplast fusion between Citrus sinensis Osb (Trovita orange) and Poncirus trifoliata Protoplasts isolated from embryogenic cells of C sinensis and from leaves of P trifoliata and the culture of fusion products in the presence of high concentrations of sucrose were essential requirements for the selection of hybrids Green globular embryoids derived from protoplasts resulted in the regeneration of trifoliate plants Other morphological characters of these plants were intermediate between both parents The chromosome number in one of the hybrid plants was 36 which was the sum of C sinensis (2n=18) and P trifoliata (2n=18) EcoRI restriction analysis of rDNA confirmed the presence of parental nuclear DNAs in the hybrid

Two interspecific somatic hybrid plants regenerated via protoplast electro-fusion

Sheng Wu Gong Cheng Xue Bao 2000 Mar16(2)179-82 Abstract Protoplasts isolated from cell suspension cultures of Bonnaza navel orange (Citrus sinensis L Osbeck) were electrically fused with mesophyll protoplasts of rough lemon (Citrus jambhiri Lush) and Goutou orange (Citrus aurantium L) respectively Plants regenerated from both fusion combinations Chromosome counting of randomly selected fifty two globular embryoids as well as all the regenerated seventy four plants from Bonnaza navel + rough lemon revealed that twenty six embryoids were tetraploids and the rest were diploids while 100 regenerated plants were tetraploids The results inferred that somatic hybrids were more competitive than parental genotypes in the process of plant regeneration All the regenerated 14 plants from Bonnaza navel + Goutou orange were tetraploids as revealed by chromosome counting POX isozyme and RAPD analysis verified that the plants from Bonnaza navel + rough lemon were hybrids and RAPD analysis confirmed the hybridity of those from Bonnaza navel + Goutou orange

Two interspecific somatic hybrid plants regenerated via protoplast electro-fusion

Sheng Wu Gong Cheng Xue Bao 2000 Mar16(2)179-82 Abstract Protoplasts isolated from cell suspension cultures of Bonnaza navel orange (Citrus sinensis L Osbeck) were electrically fused with mesophyll protoplasts of rough lemon (Citrus jambhiri Lush) and Goutou orange (Citrus aurantium L) respectively Plants regenerated from both fusion combinations Chromosome counting of randomly selected fifty two globular embryoids as well as all the regenerated seventy four plants from Bonnaza navel + rough lemon revealed that twenty six embryoids were tetraploids and the rest were diploids while 100 regenerated plants were tetraploids The results inferred that somatic hybrids were more competitive than parental genotypes in the process of plant regeneration All the regenerated 14 plants from Bonnaza navel + Goutou orange were tetraploids as revealed by chromosome counting POX isozyme and RAPD analysis verified that the plants from Bonnaza navel + rough lemon were hybrids and RAPD analysis confirmed the hybridity of those from Bonnaza navel + Goutou orange

Eodosperm Culture

bull 배유는 나자식물에서는 일배체 피자식물에서는 삼배체임

bull 사과 바나나 사탕무우 수박 오디 등에서 배유배양이 상업적으로 이용되고 있음

bull 완전한 3배체유기 Actinidia chinensis(참다래) Citrus grandis(문단) Pyrus malus(배) Citrus(귤속) Santalum album Putranjiva roxburgii에서 만들어 짐

• 12 조직배양에 의한 신품종육성
• Contents
• Embryo rescue
• 슬라이드 번호 4
• History
• Applications
• Techniques
• Factors to consider
• 슬라이드 번호 9
• References
• poplar
• 슬라이드 번호 12
• Somaclonal variation
• 슬라이드 번호 14
• Somaclones
• Benefits
• Disadvantages
• Reducing somaclonal variation
• 슬라이드 번호 19
• True breeding organism
• Examples of True breeding organism
• 슬라이드 번호 22
• 슬라이드 번호 23
• Protoplast
• 슬라이드 번호 25
• Uses for protoplasts
• Somatic fusion(wiki)
• Fused protoplast (left) with chloroplasts (from a leaf cell) and coloured vacuole (from a petal)
• The somatic fusion process occurs in four steps
• 슬라이드 번호 30
• Characteristics of Somatic Hybridization and Cybridization
• 슬라이드 번호 32
• Inter-specific and inter-generic fusion achievements
• Somatic Hybrids in woody plants
• 슬라이드 번호 35
• Two interspecific somatic hybrid plants regenerated via protoplast electro-fusion
• Eodosperm Culture