Callus Culture and Induce Differentiation n Callus Explants

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第三章 植物愈伤组织的诱导及分化途径 Callus Culture and Induce Differentiation

第三章 植物愈伤组织的诱导及分化途径 Callus Culture and Induce Differentiation

n Callus Explants from Root Pieces of Cymbidium ensifolium. 建兰

n Callus Explants from Root Pieces of Cymbidium ensifolium. 建兰

n Callus cultures derived from onion

n Callus cultures derived from onion

n Induction of somatic embryos in apical leaves of Cassava木薯

n Induction of somatic embryos in apical leaves of Cassava木薯

The alfalfa tissue culture system used to produce synthetic seeds

The alfalfa tissue culture system used to produce synthetic seeds

实例:大豆体胚发生 Soybean Embryogenesis 1. Induction n Explant: Immature zygotic embryos no more then 5

实例:大豆体胚发生 Soybean Embryogenesis 1. Induction n Explant: Immature zygotic embryos no more then 5 mm long n Excise the cotyledons, and remove the end with the embryonic axis n Place on the medium with the FLAT side up n

n Medium ("MSD 40"): Murashige and Skoog salts n B 5 vitamins n 3%

n Medium ("MSD 40"): Murashige and Skoog salts n B 5 vitamins n 3% sucrose n p. H 7. 0 n 40 mg/L of 2, 4 -D n 0. 2% gellan gum as a solidifying agent n

n Culture conditions: • At the current time, the main factor of which we

n Culture conditions: • At the current time, the main factor of which we are aware is light intensity. Optimal results are obtained at 5 -10 µE m-2 s-1. Higher intensities are detrimental, as long as light comes from cool-white fluorescent tubes. Results may differ with different light sources.

n Comments: • The exact size of the immature seed depends somewhat on the

n Comments: • The exact size of the immature seed depends somewhat on the final size of the seed. • Soybean genotypes vary widely in seed size. A good rule of thumb is to use immature seed in which the embryo is still a light, translucent green. Once the embryo changes to a darker, more opaque green, it is no longer embryogenic. • Embryos should begin to appear after the second week. The explants can remain on this medium up to 6 weeks, by which time the embryos become repetitive and may be successfully transferred to the next set of media.

2. Proliferation: n Solid medium ("MSD 20"): • • • Murashige and Skoog salts

2. Proliferation: n Solid medium ("MSD 20"): • • • Murashige and Skoog salts B 5 vitamins 3% sucrose p. H 5. 8 20 mg/L of 2, 4 -D 0. 2% gellan gum as a solidifying agent

n Liquid medium ("FN Lite"): • • Finer & Nagasawa Lite macro salts Murashige

n Liquid medium ("FN Lite"): • • Finer & Nagasawa Lite macro salts Murashige & Skoog micro salts B 5 vitamins 1 g/L asparagine 5 mg/L 2, 4 -D 1% sucrose p. H 5. 8

n Comments: • Embryos which have gone repetitive on MSD 40 medium can be

n Comments: • Embryos which have gone repetitive on MSD 40 medium can be transferred successfully to MSD 20 medium where by they proliferate successfully with monthly subculture. • After a 1 -month passage on MSD 20 medium, they may be successfully transferred to FNLite medium, whereby they require biweekly subculture to fresh medium. Embryos can also be transferred from FNLite medium to MSD 20 medium with no problem. • The selection of embryogenic tissue during each subculture is essential. For best results, select compact masses of globular-stage embryos, with a raspberry appearance.

3. Histodifferentiation & Maturation: n There are two options: solid medium or liquid medium

3. Histodifferentiation & Maturation: n There are two options: solid medium or liquid medium n Using solid medium: • Histodifferentiation Medium ("MSM 6 AC"): – Murashige and Skoog salts – B 5 vitamins – 6% maltose(麦芽糖) – p. H 5. 8 – 0. 5% activated charcoal – 0. 2% gellan gum as a solidifying agent

n Comments: • Globular-stage embryos from either step 1 or step 2 above give

n Comments: • Globular-stage embryos from either step 1 or step 2 above give rise to cotyledonary-stage embryos upon removal of the auxin. • A week on this medium is usually necessary, and embryos may remain on this medium up to a month without detrimental effects

n Maturation Medium ("MSM 6"): Murashige and Skoog salts n B 5 vitamins n

n Maturation Medium ("MSM 6"): Murashige and Skoog salts n B 5 vitamins n 6% maltose n p. H 5. 8 n 0. 2% gellan gum as a solidifying agent n

n Comments: • Cotyledon-stage embryos from the step 3 may be separated individually, and

n Comments: • Cotyledon-stage embryos from the step 3 may be separated individually, and transferred to MSM 6 medium for their maturation. • When the embryos reach physiological maturity, they lose their green color, and acquire a creamy yellow color. This usually happens 6 -8 weeks after embryos first reach the cotyledon stage. • Embryos which have not lost their green color after this time period can also be taken on to the next stage.

Using liquid medium: n Histodifferentiation and Maturation medium ("FNL 0 S 3 S 3

Using liquid medium: n Histodifferentiation and Maturation medium ("FNL 0 S 3 S 3 GM") n • • FN Lite macro salts Murashige and Skoog micro salts B 5 vitamins 30 m. M glutamine (filter-sterilize) 2 m. M methionine 3% sucrose 3% sorbitol p. H 5. 8

n Comments One cluster of globular-stage embryos, taken from either MSD 20 or FNLite

n Comments One cluster of globular-stage embryos, taken from either MSD 20 or FNLite medium and approximately 3 mm in diameter (not to exceed 20 mg), can be broken apart and placed in flask with about 35 ml of the liquid histodifferentiation and maturation medium. n After 5 weeks, the resulting cotyledonary-stage embryos will be ready for desiccation. Use of FNL 0 S 3 S 3 GM permits the recovery of up to 1000 cotyledonary stage embryos per mg of tissue, within a 5 -week period. As long as sorbitol(山梨醇) is present, germination rates of about 40% can be obtained after desiccation. n

4. Desiccation(干燥) n Comments: • Desiccation may be as simple as placing several embryos

4. Desiccation(干燥) n Comments: • Desiccation may be as simple as placing several embryos in an empty Petri dish, sealed with a plastic wrap, for 3 to 7 days, depending on the genotype.

 • The actual number of embryos in the dish depends on the size

• The actual number of embryos in the dish depends on the size of the embryos, but enough need to be placed in the plate to maintain a high humidity. • If the embryos are small, and too few are placed in the dish, the embryos will dry out too quickly. This can be prevented by adding 1 -2 cc of solidified medium to the plate. • Alternatively, the embryos may be placed in an unsealed dish, which is in turn placed in a chamber containing a saturated KCl solution, which will provide about 85% relative humidity.

5. Germination & Conversion: n Medium ("MS 0"): n Murashige and Skoog salts n

5. Germination & Conversion: n Medium ("MS 0"): n Murashige and Skoog salts n B 5 vitamins n 3. 0% sucrose n p. H 5. 8 n 0. 2% gellan gum as a solidifying agent

n Comments: n At this point, photoperiod becomes critical to prevent the premature induction

n Comments: n At this point, photoperiod becomes critical to prevent the premature induction of flowering. A 23 -hour photoperiod is very effective for this. n Once seedlings have visible shoot and root formation, they may be transferred into Magenta boxes for further growth, then transplanted into soil, hardened off, and taken to a greenhouse. n Once the plants have reached the desired size, the photoperiod can be reduced to permit flowering and seed set.

n Bioreactor for continuous cell suspensions

n Bioreactor for continuous cell suspensions

n Controlled chamber n Shaker for suspension culture

n Controlled chamber n Shaker for suspension culture

n Haemocytometer for cell number counting n Filter apparatus to filter off cells to

n Haemocytometer for cell number counting n Filter apparatus to filter off cells to measure wet/dry weight

n Side arm flask to measure settled cell volume (note red callus in side

n Side arm flask to measure settled cell volume (note red callus in side arm) n Centrifuge to spin down cells to determine packed cell volume