Cybrids
Project
Cybrids
CMS interspecific hybrids
- Idenitification of CMS Sources:
DNA sequence which encodes CMS and in fertile line
- Segregation pattern of mtDNA (Halfsib and parent with same sequence maternal inheritance)
- protoplast fusion breaks coinheritance of chDNA and recombinant mtDNA, so mt DNA as a molecular marker in somatic hybrids.
- Identification of CMS associated proteins and cms and fertile lines.
- Generally
Compare mitochondrial genes, transcript profiles or genomes in fertile and CMS lines.Then assessement of nuclear fertility restorers on expression of abnormal genes. B'coz it may affect both CMS and also normal gene.
Use of other wild sources for CMS Diversification
1.N.undulata---------Rattle virus,tobacco etch virus, tmv, wild fire 2.N.plumbaginifolia--black shank,powdary mildew, wild fire 3.N.gossei-----------root knot nematodes, pm, tmv, tobacco streak
Cybrids:
Cybrids/ Cytoplasmic hybrids are the cells or plants, containing nucleus of one species and cytoplasm fromboth the parents produced from protoplasm fusion. 1.High frequency of cybrids obtained by X-ray or gamma irradiation. 2.Plasma/cytoplasmic genes of only one parent will be recovered in the cybrids.
Advantages:
1.Transfer of male sterile cyotplasm to the cultivated species in a
single generation (transfer of plasma genes of one species into the nuclear background of another species)
2.Sexually incompatible combinations, 3.Recovery of recombinants between the parental mitochondrial or
chloroplast DNAs
4.Wide variety of combination
Cybrid: 1.CMS through Backcross needs 5-7 generations and protoplastfusion needs
few months, Eg: tobacco, brassica,citrus and recently in rice
2. Protoplasts of CMS donars exposed to high doses of irradiations
(Eletrofussing gamma irradiation) to inactivate the nucleus
3. Fuse to the recipient parent treated with iodoacetamide (Prevents cell
division)
a. Cybridity confirmation: 4. mtDNA restriction patterns, morphological traits, isozymes
andcytological tests.
5. eg. CMS into 35 japonica cultivars.
Strategy
Protocol
- Inbred lines of N glauca and N.langsdorffii-
- Sowing: glass house
- Protoplast isolation:
- 1. surface sterilize young leaves with 70% alcohol, cut leaf into trips-- placed in an enzyme solution containing 1:1 mixture of M2 culture medium and E1 enzyme solution (2% cullulase, 2% Hemicellulase Rhozyme and 1% pectinase) incubate for 4hrs @ lab conditions (23degree)--- initial obtained protoplast will be inferion in quality so discard----and add Enzyme M2 medium and incubate for 30min to the remaining leaf fragments.
--newly released protoplast are mixed (Both male and female) and filter through 80um stainless steel mesh or miracloth to a centrifuge tube---- wash that in solution D (3.5 mM CaCl2, 0.7 mM KH2PO4, 0.5M glucose and 1lt of dist. water)
-- pipette 150ul of protoplast suspension on to a 22x22mm coverslip, place this in petri dish to reduce evaporation. -- A tiny drop of silicone 200 fluid 100cs wil be used to hold the cover slip in position. when protoplast settled onto the cover slip,
--add 450 ul of PEG solution of P2 (PEG 0.33M (1540), CaCl2.2H2O-10.5 mM, Kh2(PO4).H2O-0.7mM in a lt of water (pH 5.5, Molality 0.34)) to the protoplast culture.
--incubate at room temp and ordinary light conditions in labfor 30 mins.
-- Adhesion of membranes began immediately.
--add 500ul of M3 medium, incubate for 10 mins, and wash the protoplast 5 times with 10ml of M3 medium. -- culture containing fusion products were maintained with low light (50-100 lux) warm temp with minimum vibration.
-- after few days, culture medium being used up and colonies began to grow,additional drops of M3 medium added in to suspension
-- when visible colonies were formed (2-3mm dia), transfer it to agar medium containing sucrose, mineral salt and iron as in MS formulation, vitamins of B5 medium and no phytohormones in 60ml specimen container.
--- Cultures were maintained under 26 degree temp, light (1000lux) for 16 hrs and 22degree in the dark for 8 hrs until calli formed as peanut size.
---Protoplast preparation, fused and suspended in M3 medium---15 days ---Calli sub culturing on hormoneless media-------------------------20days--- (8/culture) ---differentiation, analyse cytologically---------------------------------7 weeks after fusion
second sub culturing------------------------------------------------------10weeks after fusion
---all subsequent subcultures were grown on MS media containing Major and minor salts, 30 g/l sucrose, 27.8 mg/l iron and no vitamins, glycine,inositol or hormones. --- Medium was solidified with Bacto-agar at 0.8% with a pH of 5.5.
sub cultures were grown at a 25 degree temp, 16 hrs of light at 5000 lux ot 8hrs dark.
12 weeks after fusion, some cultures had differentiated as leaf and stem (no roots)
--- so they grafted on to the young plants of N. langsdorffii or N.gluaca.
4 months after fusion first roots were produced on calli. these could then be transplanted into soil in pots ingreen house after initial few weeks of intensive care in mist section.
7 months after fusion, first para sexual hybrid began to flower.
somatic chromosome no. were determined from acetocarmine smears of bud stage corollas,
pairing observed in metaphase-I, in pollen mother cells. ---% viable pollen was counted in mounts prepared with lactophenol aniline blue, ---all parasexual hybrids were selfed to obtain progeny.
Presentation
- Slide 1 : Introduction
- CMS diversification
- Introduction
- Slide 2 : CMS Diversification : Searching Black cat in Dark ?
- Risk factors
- Our strategies
- Slide 3 Cybrids : By definition
- Principle of making a cybrid
- Slide 4 Cybrids :: the very purpose!
- How it is going to be useful in CMS diversification ?
- Slide 5 Cybrids:: Resources
- The resources planned
- Cytoplasmic resources
- The available species to work on
- eg with case studies
- Slide 6 Making cybrids (Protocol)
- Flow chart
- Slide 7 Cybrids:: The Risk factors
- Disadvantages
- But what are the advantages"
- Slide 8 Project:: Cybrids (Proposed timelines)
- Feasibilities and timelines
- Slide 9 Possible outcome to business
- Questioning the audience ?