University of Georgia

A website from the College of Agricultural and Environmental Sciences

Parrott Lab

Genetics & Biotechnology of Crop Plants

Peanut somatic embryogenesis and transformation

Last modified on September 2, 1999; still current as of February 2007

1. Induction:

  • Explants: Axis epicotyls from mature, dry seed are placed on MSP30 for 6 weeks to induce embryogenesis.
  • Medium (“MSP30”):
    • Murashige and Skoog salts
    • B5 vitamins
    • 3% sucrose
    • pH 5.8
    • 30 mg/L of picloram
    • 0.8% Sigma agar as a solidifying agent

Comments: The embryos to the left formed from an axis on MSP30 medium. Induction efficiency is very genotype-specific.

2. Proliferation: 

The primary embryos obtained from step 1 above are transferred to MSP30 medium at monthly intervals, to obtain highly receptive cultures, as seen at right. These can usually be obtained after just a couple of transfers, with stringent selection for small, compact embryo morphology. These repetitive embryos are then transferred to liquid medium, as described below.

  • Liquid medium (FNS4P5):
    1. Finer and Nagasawa salts
    2. B5 vitamins
    3. 4% sucrose
    4. pH 5.8
    5. 5 mM asparagine
    6. 5 mg/L of picloran

Comments: Once they have gone repetitive on MSP30 medium, they may be transferred to FNS4P5 liquid medium, whereby they require biweekly subculture to fresh medium. The selection of embryogenic tissue during each subculture is essential.

3. Transformation: 

Shooting: A transformation system has been developed by using microprojectile bombardment. Two plasmids can simultaneously introduced into the target tissue. This allows transformation with any gene of interest without the need for subcloning. Over 10,000 GUS-expressing foci were typically obtained after bombarding 500 mg of tissue. Evidence of transient GUS expression can be see on the picture on the right.

Selection: Hygromycin selection (10 mg/L) is applied to suspension cultures one week after bombardment. The hygromycin concentration is increased to 20 mg/L after one week. The selection medium is changed after one week and on a weekly basis thereafter, by pipetting out the spent medium and replacing it with fresh medium.

4. Embryo histodifferentiation:

  • Medium (“MS0M3 Liquid”):
    1. Murashige and Skoog salts
    2. B5 vitamins
    3. 3% maltose
    4. pH 5.8

Comments: Globular-stage embryos give rise to cotyledonary-stage embryos upon removal of the auxin. The peanut embryos remain in this medium for three weeks (four weeks on solid MSM6ac).

5. Maturation:

  • Medium (“MS0M6 Liquid”):
    1. Murashige and Skoog salts
    2. B5 vitamins
    3. 6% maltose
    4. pH 5.8

Comments: Cotyledon-stage embryos from step 4 are broken into smaller pieces and transfered to MS0M6 liquid medium for their maturation. Physiological development at this stage is not apparent. Peanut embryos are kept on this medium for 2 weeks (4 weeks if kept on MSM6).

6. Desiccation:

Comments: Desiccation may be as simple as placing several embryos in an empty Petri dish, sealed with a micropore tape, for at least 7 days or until dry, depending on genotype and the amount of tissue in plate. 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.

7. Germination & Conversion:

  • Medium (“MSS3GB”):
    1. MS salts
    2. B5 vitamins
    3. 3% sucrose
    4. 3 mg/L BA
    5. 0.5 mg/L GA3
    6. pH 5.8
    7. 0.8% Sigma agar as a solidifying agent

Comments: Embryos stay on this medium for 6-8 weeks, this is when roots and shoots should develop. Once seedlings have visible shoot and root formation, they may be transferred to MSS3 + .2 mg/L NAA in culture tubes for root development (top left), then transplanted into soil, hardened off, and taken to a greenhouse (top center), where the plants may flower and set seed (top right).

8. References:

  • Magbanua, Z.V., H.D. Wilde, J.K. Roberts, K. Chowdhury, J. Abad, J.W. Moyer, H.Y. Wetzstein, and W.A. Parrott. 2000. Field resistance to tomato spotted wilt virus in transgenic peanut (Arachis hypogaea L.) expressing an antisense nucleocapsid gene sequence. Mol. Breed. 6:227-236.
  • Little, E.L., Z.V. Magbanua, and W.A. Parrott. 2000. A protocol for repetitive somatic embryogenesis from mature epicotyls of peanut. Plant Cell Rep. 19:351-357
  • Baker, C.M. R.E. Durham, J.A. Burns, W.A. Parrott and H.Y. Wetzstein. 1995. High frequency somatic embryogenesis in peanut (Arachis hypogaea L.) using mature, dry seed. Plant Cell Rep. 15:38-42.

According to the Peanut Advisory Council, some places that go by the name of Peanut:

  1. Upper Peanut, PA, population 14
  2. Lower Peanut, PA, — 15
  3. Peanut, CA — 35
  4. Peanut, TN — 102
  5. Peanut, PA — 200
  6. Peanut, WV — 3,036