Somatic embryogenesis in loblolly pine (Pinus taeda) and Douglas fir (Pseudotsuga menziesii): improving culture initiation and growth with MES pH buffer, biotin, and folic acid

Abstract

Loblolly pine (Pinus taeda L.) somatic embryogenesis initiation was improved by supplementing the initiation medium with the pH buffer agent 2(n-morpholino)ethanesulphonic acid (MES) at 250 mg l⁻¹, folic acid at 0.5 mg l¹, and biotin at 0.05 mg l⁻¹. MES and vitamins increased the percentage of explants with extruded tissue that continued the initiation process to form embryogenic tissue. The increase in initiation was about 12%. Initiation of 12 open-pollinated families averaged 38.5%, which is 16% higher than initiation on medium without these additives. When tested with 18 control-pollinated families, initiation averaged 26.3%. Basal medium contained a combination of modified 1/2 P6 salts, activated carbon (AC) at 50 mg ⁻¹, Cu and Zn adjusted to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500 mg l⁻¹ casamino acids, 450 mg l⁻¹ glutamine, 2 mg l⁻¹ NAA, 0.63 mg l⁻¹ BAP, 0.61 mg l⁻¹ kinetin, 3.4 mg l⁻¹ silver nitrate, 10 μM 8-Br-cGMP, 0.1 μM brassinolide, and 2 g l⁻¹ Gelrite. Early-stage embryo growth and initiation in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) were also improved in the presence of these additives.