We have succeeded in modifying the tissue culture procedures originally developed by Trolinder and Goodin (Plant Cell Tiss. Org. Cult. 12:31-53, 988). Our goal was to optimize a process using culture plates rather than suspension cultures and yet maintain a high efficiency of somatic embryo induction, germination and plant recovery. Systematic study of the process of somatic embryogenesis was conducted, focusing on 4 major areas: a) tissue selection and section treatment; b) embryogenic callus initiation; c) embryoid development; and d) maturation and desiccation.

a. Seeds of Gossypium hirsutum L. cv. Coker 312 were germinated on basal media of MS salts, C85 vitamins, 3% glucose and 1.6 g/l Gelrite. Several tissues were tested for the production of callus on different types of media, including hypocotyl, cotyledon, leaf and somatic embryo. Hypocotyl discs of 1-2mm thick sliced from 4-5d. hypocotyl tissue produced good callus and gave a high percentage of somatic embryogenesis. This was especially true when NAA and IAA were used to induce somatic embryo formation.

b.Three auxins, IAA, NAA and 2, 4-D were tested for their ability to initiate embryogenic callus production when combined with Kinetin. Induced calli were scored for amount of callus, the number of embryoids produced, and ultimately for the normality of the embryoids. Explants grown on media containing NAA yielded less callus and there was a delay of 10 days for the observation of globular embryoids on semi-solid callus initiation media (MS salts, GB5 vitamins, 2.0 mg/L NAA, 1.0 mg/L Kinetin, 3% glucose and 1.6 g/L Gelrite) compared to identical tissues cultured on the same media containing the other auxins: 0/1 mg/L 2, 4-D and 2.0 mg/L IAA. Cultures grown on 2,4-D gave rise to more callus and eventually to more embryoids than those grown on IAA.

c.Somatic embryos were classified into six types based on their observed morphology: trumpet (assumed to be precociously germinated embryoids), tulip, jar, umbrella, irregular, and fused. Somatic embryos with mono and Polycotyledonary tissue were classified as irregular, but all of the other embryoid types are shaped as described in their titles. All 6 embryoid types were cultured further to test for their ability to complete development. Only the trumpet and tulip types have the potential to germinate and to produce plantlets and they are considered normal. IAA induced the production of up to six times as many normal embryoids than 2,4-D.

d.A wide range of treatments was used in an attempt to allow induced somatic embryos to mature prior to germination and plant recovery. Embryoids were allowed to develop on semi-solid Gelrite plates containing various combinations and percentages of glutamine, casein hydrolysate, glucose, sucrose, maltose, sorbitol, mannitol, gibberellic acid, kinetin, activated charcoal and abscisic acid. No apparent benefit was obtained using any of these reagents under our culture conditions. In some cases the culture treatments proved detrimental to development. For example, most embryoids either swelled and became abnormal or went back to callus on basal media containing 2% or 4% sucrose and in any of the treatments containing 1.5% maltose.

Mechanical rather than chemical desiccation proved beneficial to plant recovery. When embryoids were allowed to air dry in the laminar flow hood for five minutes before being transferred to empty culture plates and stored for 2 1/2 days, 90 percent germinated on germination media (Steward Hsu's medium with 2% glucose in 1.6 9/L Gelrite and 0.1 Mg/L IAA (pH 5.8)). 40% of the initial embryoids actually went on to plant recovery following this treatment. Seedlings were grown in 1:1, vermiculite: perlite (supplemented with germination media salts, vitamins and growth regulators), "hardened" in plastic bags in the growth chamber, then transferred to Fafard-3B soilless mix and grown to maturity in the greenhouse. 25 IAA derived mature plants were evaluated for growth characteristics. 2 (8%) were abnormal (short stature, bushy growth, curled leaves, etc.), 23 (92%) appeared to be normal and flowered and 21 (84%) set bolls which developed apparently normal seed. The stature and growth habit of the selected greenhouse cultivated regenerants were comparable to zygotic seedderived plantlets grown under identical conditions.