Cybrids
Project
Strategy
- Requierements:
- Inbred lines of N glauca and N.langsdorffii-
- Sowing: glass house
- Protoplast isolation:
- 1. Young leaves,----surface sterilize with 70% alcohol, leaf cut into strips-- 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.