1. Methodology
1.1. Plant materials
1.1.1. (A) B, rapa (Orange queen, B-flash and BRP-K-42) crossed with B. olearacea (ASC58, ASC61, ASC82, ASC47, ASC117, J177, J129)
1.1.2. (B) Chinese cabbage (anticancer, CR-langgawang and CR-jungumi) crossed with B,juncea and B.napus
1.2. Methods
1.2.1. Inter-specific Hybridization and Hand Emascukation
1.2.1.1. (A) - female and (B) - pollen parent are crossed together
1.2.1.1.1. Emasculation of mature flower bud
1.2.1.2. Collection of siliquae 10,15,20,25, and 30 days after pollination
1.2.1.2.1. Surface sterilization with ethanol and Tween20
1.2.2. Embryo Rescue and Plant Generation
1.2.3. Chromosome Doubling, Selection F1 and Amphidiploids Plants
1.2.3.1. Detect the amplification of COS and OR mutant - OR mutant specific primer
1.2.3.1.1. Use 20ul of PCR mix contain :-
1.2.3.1.2. 35 cycles denaturation
1.2.3.1.3. PCR protocol
1.2.3.1.4. OR mutant
1.2.3.2. Identify genotype of allopolyploid plants with diploid chromosome
1.2.3.2.1. CyFlow Ploidy Analyzer
1.2.3.2.2. DAPI solution
1.2.4. Cytological Observation
1.2.4.1. Use anthers of embryo-rescued plants
1.2.4.1.1. Fix inflorescence immature flower bud (Carnoy's Solution) - 3h, 15dc
1.2.4.2. Pollen sterility test
1.2.4.2.1. Dissect anthers from flower buds
1.2.5. Estimation of Total Anthocyanin and Statistical Analysis
1.2.5.1. Three leaves of each genotype were froze in liquid nitrogen and grind into powder.
1.2.5.1.1. Each 100mg of samples were added into acidic methanol containing tubes
2. Introduction
2.1. Brassicaceaea family
2.1.1. Genus- Brassica
2.1.2. Cultivated vegetables and oilseed species
2.1.3. Taxonomically closely related but morphologically diverse
2.2. Species involved
2.2.1. B. rapa (AA)
2.2.2. B. nigra (BB)
2.2.3. B. oleracea (CC)
2.2.4. B. juncea (AABB)
2.2.5. B. napus (AACC)
2.2.6. B. carinata (BBCC)
2.3. Interspecific hybridization
2.3.1. FUNCTION
2.3.1.1. Trasnfer valuable traits between species
2.3.2. PROBLEM
2.3.2.1. Pre- and post- fertilization barries
2.3.2.2. Abortion of hybrid embryo
2.3.3. SOLUTION
2.3.3.1. Embryo rescue
2.4. Embryo rescue
2.4.1. FUNCTION
2.4.1.1. Modify disease and insect tolerance
2.4.1.2. Introgressing beneficial agronomic traits from wild species
2.4.1.3. Regenerate haploid plants to reduced bredding cycle
2.4.2. FACTORS TO SUCCEED
2.4.2.1. Maturation stage of embryos
2.4.2.1.1. Composition of medium
2.4.2.2. Genotype
2.4.3. GROWTH STAGES
2.4.3.1. Heterotrophic
2.4.3.1.1. Dependent on nutrient supply
2.4.3.2. Autotrophic
2.4.3.2.1. Not dependent on nutrient supply
2.5. Secondary metabolites
2.5.1. EXAMPLE
2.5.1.1. Anthocyanin
2.5.1.1.1. Present in red cabbage
2.5.1.1.2. Absent in Chinese cabbage
2.5.1.2. B-carotene
2.5.2. ADVANTAGES
2.5.2.1. Reduce inflammation
2.5.2.2. Protect against cancers
3. B. juncea x CR-langgawang
3.1. 43.5%
4. Determination of Orange Colour and Anthocyanin in BC1 and BC2
4.1. Types of crosses
4.1.1. . BC1
4.1.1.1. All BC1 plants fertile
4.1.1.1.1. EXCEPT
4.1.1.2. Variation in Anthocyanin content
4.1.1.2.1. FOR
4.1.1.3. Showed wide range of variation
4.1.1.3.1. BECAUSE
4.1.2. . BC2
4.1.2.1. Stronger resemblance to recipient parents
4.1.2.1.1. EXAMPLE
4.1.2.2. Beta flash (CMS) x cabbage
4.1.2.3. Successsful introgression of target traits
4.1.2.3.1. SUCH AS
4.1.2.3.2. BUT
4.1.2.4. BC2 progenies had less variation compared to BC1
4.1.2.4.1. BECAUSE OF
5. Segregated for Orange Colour mutant
5.1. FOR
5.1.1. Beta flash x Cabbage
6. Results
6.1. Genotypes
6.1.1. Embryo germination
6.1.1.1. B. juncea (Rogusa) X CR-langgawang
6.1.1.1.1. 10.73%
6.1.1.2. B. juncea (Rogusa) x Anticancer
6.1.1.2.1. AND
6.1.1.3. BRP-K-42 x Cabbage
6.1.1.3.1. 4.23%
6.1.2. Crosses with
6.1.2.1. Amphiploid
6.1.2.1.1. High rates of germination
6.1.2.1.2. Polyploids with chromosomes
6.1.2.2. Diploid
6.1.2.2.1. Low rates of germination
6.2. Confirmation of Embryo-Rescued Hybrid Plants
6.2.1. Silique harvested
6.2.1.1. sterilized
6.2.1.1.1. ovule dissected
6.2.2. Different species
6.2.3. PCR amplification
6.2.3.1. COS1078 marker
6.2.4. Intermediate leaf morphology
6.2.4.1. colchiploid of B . rapa X B . oleracea
6.2.4.2. confirms
6.2.4.2.1. B.rapa x B.oleracea
6.2.4.2.2. B.juncea x B rapa
6.2.4.2.3. B.napusx B.rapa
6.2.5. flowcytometry analysis
6.2.5.1. Peaks
6.2.5.1.1. tetraploid position
6.2.5.1.2. triploid position
6.2.6. Before colchicine treatment
6.2.7. Inflorescenes in hybrid plant
6.2.7.1. male sterility
6.2.7.1.1. Sterile
6.2.7.1.2. Partial sterile
6.2.7.1.3. Sterillity between
6.2.8. From
6.2.8.1. B. juncea
6.2.8.1.1. AND
6.2.9. Chromosome analysis
6.2.9.1. flower bud cells
6.2.9.1.1. contained
6.3. Days of Pollination (DAP)
6.3.1. 10 DAP
6.3.1.1. Difficult
6.3.1.1.1. The ovaries is too small
6.3.2. 15 DAP
6.3.2.1. Good for embryo germination
6.3.2.1.1. Suits for diploid-diploid crosses
6.3.3. 20 DAP
6.3.3.1. Highest number
6.3.3.1.1. Germinated embryo
6.3.4. 25 DAP
6.3.4.1. Ovaries dried out,too old
6.3.4.1.1. Small amount can be isolated
6.3.5. 30 DAP
6.3.5.1. Mostly no embryo germination
6.3.5.1.1. EXCEPT
6.4. Effect of Embryo Shape on Plant Regeneration from Rescued Embryo
6.4.1. Types of embryo shape
6.4.1.1. . Torpedo
6.4.1.2. . Irregular
6.4.1.3. . Cotyledonary (most common)
6.4.1.3.1. Higher rates in crosses between two parents of diploid species
6.4.1.4. . Globular
6.4.1.4.1. Higher rates in crosses between parents of amphidiploid and diploid species