The physiology and structure of cereal leaves produced by vitrification

Abstract

The vast commercial potential of plant tissue culture is often limited by the failure of a large percentage of cultured plants to survive transfer to external conditions. This study aimed to determine changes caused by tissue culture conditions and the implications those changes may have on plant survival when transferred out of culture.

Seedlings of wheat (Triticum aestivum 'ND 7532') were grown in closed conditions and open conditons within a growth cabinet and comparisons of anatomy, morphology and epicuticular wax covering made at various stages of development. Photosynthesis rate was recorded in seedlings when 12 days old.

A reduction in leaf thickness and changes in epicuticular wax structure seen in seedlings grown in vitro was attributed to high humidity and low light levels. Stomatal indices were lower in tissue cultured seedlings with the most significant effect on the adaxial surface of the leaf. Abnormal shaped stomata were noted on leaves grown in high humidity. Stomata were continually open in vitro with apertures twice as wide as seen in outside or open cabinet grown seedlings. The stomata failed to close in response to an 18% solution of mannitol or 10⁻⁵ M ABA added to the medium. However, addition of a higher concentration of ABA (10⁻⁴ M) produced partial stomatal closure in light grown tissue cultured seedlings. Dark grown seedlings in vitro did not respond to mannitol or ABA.

Epicuticular wax covering on leaves diminished as humidity levels increased; the greatest amount being recorded on leaves of seedlings grown in the open cabinet. Twelve day old tissue cultured seedlings exposed to growth cabinet conditions were shown to have functional stomata. Seedlings grown on a salts only agar medium responded to the changed environment by photosynthesising at a rate similar to outside grown seedlings, acclimatised to cabinet conditions. The inclusion of sucrose in the medium restricted photosynthesis in cultured seedlings, as after 24 h such treatments displayed no net photosynthesis. Photosynthesis rates for seedlings grown within the cabinet were consistently lower than for those recorded on seedlings grown outside. This was almost certainly due to reduced radiant energy levels.

There were indications that a reduction in sucrose concentration in the medium prior to transfer out of culture may improve the plants' autotrophic abilities. A reduction in humidity within the culture vessel may improve epicuticular wax deposition and further increase survival chances. However, the impracticality of reducing humidity makes reduction of sucrose in the medium a more viable option.