CHAPTER 12
APPLICATION OF BIOTECHNOLOGY
IN PLANT BREEDING
生物技术在育种中的应用
BY
Niu Yingze
（牛应泽）
Dept,of Plant Breeding and Seed Engineering
§ 1 Application of Cell and Tissue Culture
in Plant Breeding
细胞组织培养在植物育种的应用
PLANT TISSUE CULTURE is a blanket term for the in vitro
cultivation of all plant parts under aseptic conditions,
Tissue culture is a technique of growing plant tissues on
synthetic medium under controlled and aseptic conditions,
1,Types of Explants in Plant Tissue Culture
植物组织培养中的外植体类型
? Plantlet
? Organs (Roots,hypocotyls,cotyledons,leaves,
ovary,ovule,anther,pistils,etc.)
? Embryos (Incl,immature seeds)
? Tissue (Shoot tip,Root tip,Meristem,callus,
? stem (nodal) segment)
? Cell (Cell suspension)
? Protoplast
? Microspore (Young pollens)
Ovary Culture
Immature Embryo Culture
Stem Segment Culture
Hypocotyl Culture
Cotyledon Culture
Microspore Culture
2,Application of In vitro Culture in Plant
Breeding 植物离体培养在育种中的应用
(1) Micropropagation
(2) Utilization of Somaclonal Variation
(3) Protoplast Culture and Somatic Hybridization
(4) In vitro Production of Haploids
(5) Embryo Rescue in Wild Crosses
(6) Transgenic Approach for Plant breeding
(7) Germplasm preservation
3,Utilization of Somaclonal Variation in
Plants 植物无性系变异的利用
Somaclonal Variation is the variation among the tissues
or plants derived from the in vitro somatic cell cultures,i.e,
callus and suspension cultures
Somaclonal Variation may be genetic or it may also result
from culture induced epigenetic(外成的） changes,
(1) Types of Somatic Mutation p,201
? Resistance
? Morphological mutations
? Characteristics mutations
? Other types of mutations
(2) Causes of Somaclonal Variation
A,Chromosomal changes 染色体变异
B,Mitotic crossing over 有丝分裂交换
C,Apparent,point” mutation 外显“点”突变
D,Cytoplasmic genetic changes 核质变化
E,Deamplification and amplification 衰减和放大
F,Transposable element activation 跳跃因子激活
G,Virus elimination 脱毒
H,Methylation or demethylation 甲基化或脱甲基化
I,Altered expression of multigene families 多基因家族
变异表达
? Positive Selection p,202
Selective media are used and the survived are
selected,
? Negative Selection
In another direction of selection,P,202
4,Selection of Somatic Mutants
体细胞突变的选择
§ 2 Protoplast Culture and Somatic
Hybridization in Plants
植物原生质体培养与体细胞杂交
1,Isolation of Protoplast
Protoplast (cytoplasm+nucleus)
Plant Cell
Cell Wall
Enzyme
treatment
Enzymes,Cellulase,hemicellulase,pectinase
(commercially available)
2,Protoplast Fusion
Fusagens,
? PEG (polyethylene glycol,聚乙二醇 )
? High pH,high Ca++
? Electrofusion
3,Selection of the Fused Cells
? Complementation of biochemistry and nutrition
? Mechanical isolation (color,fluorescence)
4,Application of Protoplast Fusion in Plant
Breeding
Overcome the distant cross barriers to introduce alien
genes,
Interspecific Cross
Intergeneric Cross
§ 3 Application of Gene Engineering in
Plant Breeding
基因工程在植物育种中的应用
1,Three basic elements in gene engineering
? Target genes 目的基因
A,Map cloning
B,Use of transposon or T-DNA tagging
C,PCR amplification
D,Gene cloning based on mRNA
? Gene vectors 基因载体
Plasmids (质粒，如 Ti质粒）
Bacteriophages 噬菌体
Cosmid 粘粒
YAC (yeast artificial chromosome) 酵母人工染色体
……
? Tool enzymes 工具酶
Restriction enzymes
Polymerase
These enzymes are used for gene isolation,amplification
and recombination
2,Genetic Transformation in Plants
植物遗传转化
(1) Gene Transfer Methods
a,Vector mediated transfer 载体介导转化
Agrobacterium tumefaciens 根癌农杆菌
Agrobacterium rhizogenes 发根农杆菌
Virial vectors 病毒载体
b,Direct Gene transfer (DGT) 直接基因转化
? Physico-chemical uptake of DNA
? Liposome encapsulation
? Electroporation of Protoplasts
? Microinjection
? DNA injection into intact plants
? Incubation of seeds with DNA
? Pollen tube pathway
? Laser microbeam
? Electroporation into tissues/embryos
? Silicon carbide fiber
? Particle bombardment (Gene gun)
§ 4 Molecular Markers and Plant
Breeding
分子标记与植物育种
1,Classes of Markers
(1) Morphological markers
Major mutant loci morphological characters of
plants (conventional marker characters)
(2) Protein markers
Isozymes Biochemical markers
(3) DNA markers P,221
? RFLP – Restriction Fragment Length Polymorphisms
? RAPD – Randomly Amplified Polymorphic DNA
? SSR – Single Sequence Repeat
? AFLP - Amplified Fragment Length Polymorphisms
? SCAR – Sequence Characterized Amplified Region Markers
? SNP – Single Nuleotide Polymorphism
… …
2,Application of DNA Markers in Plant
Breeding
(1) Construction of genetic maps
(2) Marker assisted selection
(3) Mapping of polygenes (QTLs mapping)
(4) Gene pyramiding (Assembling of QTLs)
(5) DNA finger printing in crops
(6) Map-based cloning of genes
(7) Studies of genetic diversity of germplasm
Limitations in use of molecular markers
Cost for genetic analyses
Small polymorphism in many plants (Unsaturated map)
Diversity of Resynthesized Rapeseed Lins Revealed by RAPD Markers
Diversity of Resynthesized Rapeseed Lins Revealed by SSR Markers
Cluster of Resynthesized Rapeseed
Line by RAPD Markers
Till Next Chapter
APPLICATION OF BIOTECHNOLOGY
IN PLANT BREEDING
生物技术在育种中的应用
BY
Niu Yingze
（牛应泽）
Dept,of Plant Breeding and Seed Engineering
§ 1 Application of Cell and Tissue Culture
in Plant Breeding
细胞组织培养在植物育种的应用
PLANT TISSUE CULTURE is a blanket term for the in vitro
cultivation of all plant parts under aseptic conditions,
Tissue culture is a technique of growing plant tissues on
synthetic medium under controlled and aseptic conditions,
1,Types of Explants in Plant Tissue Culture
植物组织培养中的外植体类型
? Plantlet
? Organs (Roots,hypocotyls,cotyledons,leaves,
ovary,ovule,anther,pistils,etc.)
? Embryos (Incl,immature seeds)
? Tissue (Shoot tip,Root tip,Meristem,callus,
? stem (nodal) segment)
? Cell (Cell suspension)
? Protoplast
? Microspore (Young pollens)
Ovary Culture
Immature Embryo Culture
Stem Segment Culture
Hypocotyl Culture
Cotyledon Culture
Microspore Culture
2,Application of In vitro Culture in Plant
Breeding 植物离体培养在育种中的应用
(1) Micropropagation
(2) Utilization of Somaclonal Variation
(3) Protoplast Culture and Somatic Hybridization
(4) In vitro Production of Haploids
(5) Embryo Rescue in Wild Crosses
(6) Transgenic Approach for Plant breeding
(7) Germplasm preservation
3,Utilization of Somaclonal Variation in
Plants 植物无性系变异的利用
Somaclonal Variation is the variation among the tissues
or plants derived from the in vitro somatic cell cultures,i.e,
callus and suspension cultures
Somaclonal Variation may be genetic or it may also result
from culture induced epigenetic(外成的） changes,
(1) Types of Somatic Mutation p,201
? Resistance
? Morphological mutations
? Characteristics mutations
? Other types of mutations
(2) Causes of Somaclonal Variation
A,Chromosomal changes 染色体变异
B,Mitotic crossing over 有丝分裂交换
C,Apparent,point” mutation 外显“点”突变
D,Cytoplasmic genetic changes 核质变化
E,Deamplification and amplification 衰减和放大
F,Transposable element activation 跳跃因子激活
G,Virus elimination 脱毒
H,Methylation or demethylation 甲基化或脱甲基化
I,Altered expression of multigene families 多基因家族
变异表达
? Positive Selection p,202
Selective media are used and the survived are
selected,
? Negative Selection
In another direction of selection,P,202
4,Selection of Somatic Mutants
体细胞突变的选择
§ 2 Protoplast Culture and Somatic
Hybridization in Plants
植物原生质体培养与体细胞杂交
1,Isolation of Protoplast
Protoplast (cytoplasm+nucleus)
Plant Cell
Cell Wall
Enzyme
treatment
Enzymes,Cellulase,hemicellulase,pectinase
(commercially available)
2,Protoplast Fusion
Fusagens,
? PEG (polyethylene glycol,聚乙二醇 )
? High pH,high Ca++
? Electrofusion
3,Selection of the Fused Cells
? Complementation of biochemistry and nutrition
? Mechanical isolation (color,fluorescence)
4,Application of Protoplast Fusion in Plant
Breeding
Overcome the distant cross barriers to introduce alien
genes,
Interspecific Cross
Intergeneric Cross
§ 3 Application of Gene Engineering in
Plant Breeding
基因工程在植物育种中的应用
1,Three basic elements in gene engineering
? Target genes 目的基因
A,Map cloning
B,Use of transposon or T-DNA tagging
C,PCR amplification
D,Gene cloning based on mRNA
? Gene vectors 基因载体
Plasmids (质粒，如 Ti质粒）
Bacteriophages 噬菌体
Cosmid 粘粒
YAC (yeast artificial chromosome) 酵母人工染色体
……
? Tool enzymes 工具酶
Restriction enzymes
Polymerase
These enzymes are used for gene isolation,amplification
and recombination
2,Genetic Transformation in Plants
植物遗传转化
(1) Gene Transfer Methods
a,Vector mediated transfer 载体介导转化
Agrobacterium tumefaciens 根癌农杆菌
Agrobacterium rhizogenes 发根农杆菌
Virial vectors 病毒载体
b,Direct Gene transfer (DGT) 直接基因转化
? Physico-chemical uptake of DNA
? Liposome encapsulation
? Electroporation of Protoplasts
? Microinjection
? DNA injection into intact plants
? Incubation of seeds with DNA
? Pollen tube pathway
? Laser microbeam
? Electroporation into tissues/embryos
? Silicon carbide fiber
? Particle bombardment (Gene gun)
§ 4 Molecular Markers and Plant
Breeding
分子标记与植物育种
1,Classes of Markers
(1) Morphological markers
Major mutant loci morphological characters of
plants (conventional marker characters)
(2) Protein markers
Isozymes Biochemical markers
(3) DNA markers P,221
? RFLP – Restriction Fragment Length Polymorphisms
? RAPD – Randomly Amplified Polymorphic DNA
? SSR – Single Sequence Repeat
? AFLP - Amplified Fragment Length Polymorphisms
? SCAR – Sequence Characterized Amplified Region Markers
? SNP – Single Nuleotide Polymorphism
… …
2,Application of DNA Markers in Plant
Breeding
(1) Construction of genetic maps
(2) Marker assisted selection
(3) Mapping of polygenes (QTLs mapping)
(4) Gene pyramiding (Assembling of QTLs)
(5) DNA finger printing in crops
(6) Map-based cloning of genes
(7) Studies of genetic diversity of germplasm
Limitations in use of molecular markers
Cost for genetic analyses
Small polymorphism in many plants (Unsaturated map)
Diversity of Resynthesized Rapeseed Lins Revealed by RAPD Markers
Diversity of Resynthesized Rapeseed Lins Revealed by SSR Markers
Cluster of Resynthesized Rapeseed
Line by RAPD Markers
Till Next Chapter
