Glycine max (soybean) and Zea mays (corn) in tissue culture; isolation, growth, and morphogenesis of explants, cells, and protoplasts

Maize and soybean tissues have been maintained in culture under different growth conditions. Primary emphasis was placed on tissue from inbred varieties proven to give some degree of morphogenesis in culture. Tissues from actively growing sites of developed corn plants typically formed rhizogenic callus masses in culture. This tissue did not differentiate beyond the root forming stage. However, embryonic scutellar tissue from inbred strain Al88 formed meristematic centers which developed into whole plantlets when the exogenous 2,4-D concentration was reduced. Protoplasts isolated from the leaves of plantlets regenerated and formed cell walls in culture and could be maintained for extended periods of time, but failed to divide and form calli;No plantlet regenerating culture system is known for soybeans. Work with wild varieties showed that whole plants could be regenerated from apical stem cuttings. This led to further experimentation using apical cuttings from seedlings of cultivated Glycine max varieties. Best growth in terms of plant regeneration occurred when low concentrations of 2,4-D and kinetin were present in the medium. High kinetin to 2,4-D concentrations encouraged shoot development, while high concentrations of 2,4-D favored callus tissue growth;Attempts to enzymatically produce protoplasts from soybean leaf cells were unsuccessful. Chemical extractions of leaf cells with organic solvents and other agents that alter the molecular structure of the cell wall also failed to increase enzymatic digestibility. A crude enzyme prepared from culture extracts of saprophytic organisms aided in the digestion of cell walls, but proved to be inefficient for protoplast isolation;A variety of tissues, beside leaf tissue, were treated with commercially available enzymes. Many single cells were produced, but protoplasts were rare (1/10,000 cells). When young pod tissue was treated with pectinase and cellulase a consistant yield of protoplasts (5 x 10(\u276)/gram of tissue) resulted. These protoplasts regenerated cell walls and underwent divisions forming callus masses and adventitious roots;Intact soybean mesophyll cells were also isolated by grinding leaf tissue in a mortar or by beating the tissue with a magnetically stirred smooth Teflon bar. Good separation of predominantly palisade and spongy mesophyll cells occurred using both mechanical methods. Only those cells separated into buffered sterile salt solution using the Teflon bar survived and grew in B5 medium preconditioned by actively growing suspension cultures. Cultivation of the resulting callus tissue on Murashige-Skoog agar medium containing reduced concentrations of growth regulators resulted in adventitious root formation. This provides evidence that the organogenic capacity of the tissue is maintained in the separated cells