EMBRYO GENESIS IN LIQUID         

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1. Induction:    

      Explant:
          o Immature zygotic embryos no more than 5 mm long
          o Excise the cotyledons, and remove the end with the embryonic axis
          o Place on the medium with the FLAT side up

   
      Medium ("MSD40"):                                                        Medium VB+1%

          o Murashige and Skoog salts
          o B5 vitamins
          o 3% sucrose                                                                 o 1% glucose             
          o pH 7.0                                                                          o pH 5.5
          o 40 mg/L of 2,4-D                                                         o 5 mg/L of 2,4-D + 0.5 mg/L BAP
          o 0.2% gellan gum as a solidifying agent

   
      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.

   
      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:

   
      Solid medium ("MSD20"):
          o Murashige and Skoog salts
          o B5 vitamins
          o 3% sucrose
          o pH 5.8
          o 20 mg/L of 2,4-D
          o 0.2% gellan gum as a solidifying agent

   
      Liquid medium ("FN Lite"):                                               Liquid medium VB+1.5%
          o Finer & Nagasawa Lite macro salts
          o Murashige & Skoog micro salts
          o B5 vitamins
          o 1 g/L asparagine
          o 5 mg/L 2,4-D                                                                 o 5 mg/L of 2,4-D + 0.1 mg/L BAP
          o 1% sucrose                                                                   o 1.5% glucose
          o pH 5.8                                                                            o pH 5.5

   
      Comments:
      Embryos which have gone repetitive on MSD40 medium can be transferred successfully to MSD20 medium where by they proliferate successfully with monthly subculture. After a 1-month passage on MSD20 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 MSD20 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:

There are two options: solid medium or liquid medium

Using solid medium:

   
      Histodifferentiation Medium ("MSM6AC"):
          o Murashige and Skoog salts
          o B5 vitamins
          o 6% maltose
          o pH 5.8
          o 0.5% activated charcoal
          o 0.2% gellan gum as a solidifying agent

   
      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.

   
      Maturation Medium ("MSM6"):
          o Murashige and Skoog salts
          o B5 vitamins
          o 6% maltose
          o pH 5.8
          o 0.2% gellan gum as a solidifying agent

   
      Comments:
      Cotyledon-stage embryos from the step 3 may be separated individually, and transferred to MSM6 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:

   
      Histodifferentiation and Maturation medium ("FNL0S3S3GM")             Liquid medium VB+2%
          o FN Lite macro salts
          o Murashige and Skoog micro salts
          o B5 vitamins
          o 30 mM glutamine (filter-sterilize)
          o 2 mM methionine
          o 3% sucrose                                                                                              o 2% glucose
          o 3% sorbitol
          o pH 5.8                                                                                                      o pH 5.5

   
      Comments
      One cluster of globular-stage embryos, taken from either MSD20 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. After 5 weeks, the resulting cotyledonary-stage embryos will be ready for desiccation. Use of FNL0S3S3GM permits the recovery of up to 1000 cotyledonary stage embryos per mg of tissue, within a 5-week period. As long as sorbitol is present, germination rates of about 40% can be obtained after desiccation.

4. Desiccation:

   
      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 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:

   
      Medium ("MS0"):
          o Murashige and Skoog salts
          o B5 vitamins
          o 3.0% sucrose
          o pH 5.8
          o 0.2% gellan gum as a solidifying agent

   
      Comments:
      At this point, photoperiod becomes critical to prevent the premature induction of flowering. A 23-hour photoperiod is very effective for this. 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. Once the plants have reached the desired size, the photoperiod can be reduced to permit flowering and seed set.

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