TOMATO TRANSFORMATION

Thursday, December 04, 2003

Description
TOMATO TRANSFORMATION

Procedure
Sterilize dry seeds in 70% ethanol - 2 min. followed by 3.3% NaOCl (1:3 dilution of 10%) with 0.1% Tween 20 for 20 min., in an 250 ml Erlenmeyer. Stir fast with magnetic stirrer and then rinse 3 times with sterile double distilled (dd) water.
Germinate seeds on Nitsch medium in sterile boxes (Magenta). Density of seeds depends on the variety (VF36 & Mottle 100 seeds/Magenta box, Money-Maker 25 seeds/Magenta box).
Cotyledons can be used for transformation when there are none , or only very small true leaves present on seedlings. The tops of the seedlings are cut off in batches and immersed immediately in Jones (1) liquid medium. Cutting the cotyledon explants is done in a Petri dish (100x15 mm). Do not leave the Magenta box with the seedlings open for a long time. Seedlings are held upside down by the stem and cotyledons are cut at the proximal (wide) end and at the distal end (about 2/3 of the cotyledon remains.), when they are submerged in the medium. They are placed upside down on 100x15 mm feeder plates containing MS medium with 1 mg/l 2.4.D + 100 M Aceto syringone, on top of it a layer of suspension cells (carrot or tobacco at logarithmic phase) and on them sterile filter paper. The cotyledons are placed very densely (60-100 cotyledons on 7 cm diameter filter paper, Whatman # 1).
Incubation of cotyledons in growth room (25 C, dark) for 24 hours during which Agrobacterium is grown. (10 ml 2YT (2) medium, with the appropriate antibiotics. in 50 ml tube at 28 C, 230 rpm, in the dark).
The Agrobacterium is diluted to O.D.(600nm)=0.25-0.35 with Jones medium with 100-200 mM Aceto Syringone, pH 5.2, and aliquots of 7-10 ml are pipette into 50 x 15 mm Petri dishes.
Cotyledons from one plate are submerged in this Petri dish which contains 10 ml of Agrobacterium suspension and incubated for 15- 30 min at R.T.
Cotyledons are then blotted on sterile paper Whatman # 1(to absorb the excess of the bacteria suspension), and are placed back on the feeder plates (now less condensed: 30-50 cotyledons on each feeder plate.
After 40-48 hours of co-cultivation the cotyledons are transferred to 100x15 mm Petri dishes which contain SL.1 medium with 0.8% agar. The cotyledons are laid on the agar upside down. Plates are sealed with saran wrap and returned to the growth room for incubation. Callus, green bumps, or shoots usually will be seen at the proximal edge within 10-15 days.
Cotyledons 14 days old are transferred to SL.2 medium in 100x20 mm plates with0.8% agar for further shoot organogenesis.
Shoots or calli are excised away from the dying cotyledon and transferred again to SL.2 medium plates, for another period of 2 weeks.
Shoots are transferred again to SL.3 medium plates (3-4 weeks).
Large, normal looking shoots are transferred to rooting medium (Nitsch medium with carbenicillin 150 mg/l, kanamycin 50 mg/l and IBA 1-2 mg/l). Shoots will root during 7-15 day period.
Shoots that have rooted can be transferred into very wet soil, under law light conditions and in small pots within a closed transparent container to keep humidity. Any residual agar should be rinsed from the roots before transplanting. Lid of the container is gradually opened over a period of 5 days. After hardening of the plant it can be transferred into a bigger pot in the greenhouse.
R1 seeds are scored for kanamycin resistance by germination on Nitsch medium with 50 mg/l kanamycin, in Magentas. Resistant seedlings will be taller, will have branched roots and reduced or no anthocyanin pigmentation in the hypocotyls. Sensitive seedlings will germinate but will be short and stunted, will have stubby unbranched roots, and often have enhanced anthocyanin pigmentation in the hypocotyls. Scoring is most reliable at approximately 2-4 weeks after seeds are set out.

Recipes
Jones medium : micro & macro minerals of Murashige & Skoog (MS) medium with vitamins of Nitsch medium.
2YT medium: Bactotryptone 16 gr, yeast extract 10 gr, NaCl 5 gr /l.
Nitsch : micro & macro minerals of Murashige & Skoog, M-inositol 100 mg/l, Thiamine HCl 0.5 mg/l, Glycine 2 mg/l, Nicotinic acid 5 mg/l, Folic acid 0.5 mg/l, Biotin 0.05 mg/l, Sucrose 20 gr/l, agar 0.8%. pH 5.8.
SL.1 Jones medium with carbenicillin 400 mg/l (or 400mg/l claforan), 1 mg/l zeatin, 100 mg/l kanamycin (regeneration medium).
SL.2 Jones medium with carbenicillin 250 mg/l (or 300mg/l claforan), kanamycin 100 mg/l, zeatine 1 mg/l, zeatin riboside 0.5 mg/l.
SL.3 Jones medium with carbenicillin 250 mg/l (or 300mg/l claforan), kanamycin 100 mg/l, zeatin 0.1-0.2 mg/l. (regenerants elongation medium).
Rooting: Nitsch medium with 150 mg/l carbenicillin (200 mg/l claforan), 50 mg/l kanamycin, 1mg/l IBA.

Supplies

Tips

(责任编辑：泉水)

搜索

热门标签:

TOMATO TRANSFORMATION