1. B.napus
1.1. resynthesize successfully by
1.1.1. B.oleracea
1.1.2. B.rapa
1.1.3. -interspecific hybridization&embryo rescue
2. Genotype
2.1. Amphidiploid x diploid
2.1.1. Higher rate of germination
2.1.2. example
2.1.2.1. Rys. B. Napus x B. rapa
2.1.2.2. B. rapa x B. juncea
2.1.2.2.1. presence of chromosomal brach in meiosis I
2.1.2.3. B. juncae x B. oleracea
2.2. Diploid x diploid
2.2.1. B. rapa x B. oleracea
3. 9 uL ultra pure H2O
4. vegetables
5. Determine chromosome number
5.1. in B. rapa x B. oleracea
5.1.1. Presence of chromosome doubling
5.1.2. Regular meiosis
5.1.2.1. Absence of pollen sterility
6. tested in
6.1. PCR amplication
6.1.1. COS1078 marker
6.2. Cytological analysis
6.2.1. Chromosome number
6.3. Flow cytometry peaks
6.4. Inflorescence
6.5. Leaf morphology
7. INTRODUCTION
7.1. Brassica(genus)
7.1.1. Brassicaceae(family)
7.1.2. 38 different species
7.1.3. one of the world's most commonly cultivated vegetable & oilseed
7.1.4. Brassica vegetable
7.1.4.1. large taxo-closely related
7.1.4.1.1. but, morphological-diverse
7.1.5. Cultivated over centuries
7.1.5.1. crossed&hybridize
7.2. transfer traits between species
7.2.1. commercial interest
7.2.2. role- speciation&morphotype diversification
7.3. genome structure
7.3.1. Shaped by
7.3.1.1. whole genome triplication
7.3.1.1.1. extensive diploidization
7.3.2. U triangle
7.3.2.1. 6 species
7.3.2.1.1. B.rapa
7.3.2.1.2. B.nigra
7.3.2.1.3. B.oleracea
7.3.2.1.4. B.juncea
7.3.2.1.5. B.napus
7.3.2.1.6. B.carinta
7.3.2.2. economically important
7.3.2.2.1. oil seed
7.3.2.2.2. condiments
7.3.3. interspecies hybridization
7.3.3.1. amphiphoid species
7.3.4. Improvement in Brassica
7.3.4.1. naturally occuring genetic variations
7.3.4.1.1. difficult
7.3.4.2. utilizes in
7.3.4.2.1. hybridization programs
7.4. Interspecific hybridization
7.4.1. scientist
7.4.1.1. trying to produce new morphotype
7.4.1.2. transfer traits of interest
7.4.2. role
7.4.3. Breeders
7.4.3.1. attempt to hybridize closely related
7.4.3.1.1. new improved varities
7.4.4. isolating & growing
7.4.4.1. immature/mature zygotic embryo
7.4.5. new biotechnology tools
7.4.5.1. hybridization achieved
7.4.5.2. abortion of hybrid embryo
7.4.5.3. rescuing
7.4.5.3.1. inherent weak
7.4.5.3.2. immature hyrbids
7.4.5.3.3. to
7.4.6. In Brassicaceae
7.4.6.1. limited avaibility of variations
7.4.6.1.1. species level
7.4.6.2. conducted for
7.4.6.2.1. disease tolerance
7.4.6.2.2. insect tolerance
7.4.6.2.3. alter nutritional quality
7.4.7. depends on
7.4.7.1. maturation stage of embryo
7.4.7.2. composition of medium
7.4.8. Interspecies hybridiation
7.4.8.1. limitated by
7.4.8.1.1. pre- and post-fertilization barriers
7.4.8.1.2. abortion of hybrid embryos
7.5. Embryo rescue
7.5.1. followed by
7.5.1.1. interspecific hybridization
7.5.1.1.1. for cultivation
7.5.2. definition
7.5.2.1. sterile condition
7.5.2.2. aseptic nutrient
7.5.2.3. role
7.5.2.3.1. to obtain viable plant
7.5.2.3.2. genotype
7.5.3. techniques
7.5.3.1. for
7.6. Brassica sp.
7.6.1. interspecific hybrid
7.6.1.1. embryo&ovary culture
7.6.1.2. B.juncea
7.6.1.2.1. and
7.6.1.3. B.juncea
7.6.1.3.1. and
7.6.2. embryo rescue
7.6.2.1. success rate
8. METHODOLOGY
8.1. Plant Materials
8.1.1. Aim
8.1.1.1. Transfer B-carotene genes from cabbage to cabbage
8.1.2. Cross
8.1.2.1. B-carotene-enriched Chinese cabbage X 8 white cabbage lines
8.1.3. To enrich anthocyanin, cross
8.1.3.1. Chinese cabbage cultivars X Anthocyanin-enriched amphiploid and resynthesized B-napus line
8.1.4. BC1 and BC2 developed by
8.1.4.1. backcrossing
8.1.4.1.1. Colchiploid F1 X Cabbage lines X anthocyanin enrichment
8.2. Interspecific Hybridization Followed by Hand Emasculation
8.2.1. Chinese cabbage cultivars X Amphiploid sp.
8.2.2. Steps
8.2.2.1. Emasculation (mature flower buds)
8.2.2.1.1. Dusting method
8.2.2.2. Avoid pollination on pollinated buds
8.2.2.3. Remove 5-7 days after pollination
8.3. Embryo Rescue And Plant Regeneration
8.3.1. After pollination
8.3.1.1. Steps
8.3.1.1.1. Collect siliquae
8.3.1.1.2. Excised ovules containing embryo
8.3.1.1.3. culture isolated ovules on MS medium
8.3.1.1.4. Generate different shaped embryo
8.3.1.1.5. Germinate the embryo
8.3.1.1.6. Achieve true to type plant
8.4. Chromosome Doubling, Selection Of F1 And Amphidiploids Plants
8.4.1. Plant root
8.4.1.1. remove MS medium - washed
8.4.1.2. Submerged in 0.1% colchicine
8.4.1.3. Kept for 4H
8.4.1.3.1. Fluorescent light - 1000 lux
8.4.1.4. Washed 3X
8.4.1.4.1. Tap water
8.4.1.5. Planted in multi-hole trays
8.4.1.5.1. coco-peat soil
8.4.1.5.2. hardened
8.4.1.5.3. Transferred - greenhouse
8.4.2. Successfully crossed hybrid plants
8.4.2.1. identify by
8.4.2.1.1. PCR amplification
8.4.3. DNA isolated
8.4.3.1. 1 g leaves
8.4.3.1.1. parental lines
8.4.3.1.2. Interspecies hybrids plants
8.4.3.2. protocol by Ishikawa et al.
8.4.3.2.1. slight modifications
8.4.3.3. PCR
8.4.3.3.1. detecting amplification
8.4.3.3.2. 20 uL PCR mix
8.4.3.3.3. Steps
8.4.3.3.4. OR mutants
8.4.4. Genotype
8.4.4.1. Steps
8.4.4.1.1. 1 cm2 young leaf
8.4.4.1.2. DAPI solution + leaf cells
8.4.4.2. Allopolyploidy plants
8.4.4.2.1. diploid chromosome
8.5. Cytological Observation
8.5.1. Anthers
8.5.1.1. flower buds - embryo rescued
8.5.1.1.1. cytological study
8.5.1.2. Inflorescence w/ immature flower buds
8.5.1.2.1. fixed in Carnoy's solution
8.5.1.2.2. maintained at 15oC, 3H
8.5.1.3. Fixation
8.5.1.3.1. 1 flower bud
8.5.1.4. visible anther wall
8.5.1.4.1. removed
8.5.1.4.2. coverslip ( 1.5 X 1.5 cm)
8.5.1.4.3. chromosome
8.5.1.5. Pollen sterility test
8.5.1.5.1. anther - dissected
8.6. Estimation Of Total Anthocyanin And Statistical Analysis
8.6.1. Total anthocyanin content
8.6.1.1. 3 leaves - each genotype
8.6.1.1.1. frozen in liquid N
8.6.1.1.2. ground into powder
8.6.1.1.3. 100 mg - sample
8.6.1.2. quantify
8.6.1.2.1. 3 replicates from each biological sample
8.6.1.3. analyzed
8.6.1.3.1. 1-way ANOVA
8.6.1.3.2. Tukey's pairwise comparison
9. RESULTS
9.1. Most suitable for embryo development
9.2. Days after pollination (DAP)
9.2.1. 10
9.2.1.1. Ovary tedious
9.2.2. 15
9.2.3. 20
9.2.3.1. Amphidiploid species
9.2.3.2. Higher number of embryos
9.2.4. 25
9.2.4.1. Ovary dried out
9.2.5. 30
9.2.5.1. Embryos could not be formed
9.3. Embryo shape
9.3.1. Embryo culture
9.3.1.1. Cotyledonary
9.3.1.1.1. Higher rate of plant regeneration
9.3.1.2. Torpedo
9.3.1.3. Irregular
9.3.1.4. Globular
9.3.2. Plant regeneration
9.3.2.1. Globular (Amphidiploid x diploid)
9.3.2.2. Cotyledonary (Diploid x diploid)
9.4. Hybridity
9.4.1. SUCCESS
9.5. Variation in trait
9.5.1. orange/yellow
9.5.2. anthocyanin
9.5.3. BC2 successful introgression (both trait) compare to cabbage
10. DISCUSSIONS
10.1. factors to success
10.1.1. 1) age of embryo
10.1.2. 2) shape of embryo
10.1.3. 3) genotypes
10.1.4. 4) media composition
10.2. rate of plant regeneration
10.2.1. cross combination
10.2.2. emryo isolation time
10.2.3. embryo shape
10.2.4. genotypes