Masters Theses

Date of Award

12-1978

Degree Type

Thesis

Degree Name

Master of Science

Major

Plant, Soil and Environmental Sciences

Major Professor

Bob V. Conger

Committee Members

James Caponetti, Vernon Reich

Abstract

The primary objectives of this study were to demonstrate plant regeneration from callus in tall fescue (Festuca arundinacea Schreb.) and to determine the frequency of root and shoot organogenesis over increasing time of callus maintenance. Secondary objectives focused on the influence of different auxins and auxin concentration on callus induction and growth, and changes in relative DNA contents over increasing time in callus maintenance. Studies on organogenesis in tall fescue involved two experiments. In the first, callus was initiated from mature embryos on a modified Murashige and Skoog medium containing 22.6 μM/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). Incubation was in the dark at 24°C for 30 days. The calli were divided with one portion being placed on callus main-tenance with 22.6 μM/liter of 2,4-D and the other portion on regeneration medium containing 2.26 μM/liter of 2,4-D. Cal lus maintenance was in the dark. Calli on the regeneration medium were placed in a 12 hour/12 hour D/N cycle at 24 /15°C. Light was from cool white fluorescent bulbs at 180 μ Einsteins sec-1 m-2 . After 30 days calli were scored for root and shoot formation. After the second and third 30 day periods, the calli in maintenance were again divided and tested for regeneration as described above. The second experiment was a continuation of the previous experiment except for the following modifications. Induction of calli was with 40.7 μM/liter of 2,4-D. After 28 days, the calli were placed on a medium with 22.6 μM/liter of 2,4-D. The calli were allowed to grow another 28 days before serial subculturing and testing for regeneration at this time and after subsequent periods of 28 and 56 days. To determine the influence of different auxins on callus induction and growth, eight different auxins, 2,4-D, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5- trichloropropionic acid (silvex), indoleacetic acid (lAA), napthaleneacetic acid (NAA), p-chlorophenoxyacetic acid (pCPA), 4-amino-3,5,6-trichloropicolinic acid (picloram), and 3,6-dichloro-o-anisic acid (dicamba) were tested at 5, 10, 20, 40, and 60 μM/liter. After a period of 28 days, callus fresh weight, length of first leaf, and root growth as determined qualitatively by visual observation, were recorded for individual calli. To determine relative amounts of DNA content, microspectrophotometric studies were conducted on calli at 6, 9, 12, 18, and 24 weeks of age. The calli were fixed in 3:1 ethanol:acetic acid and stained by the Feulgen method. Relative amounts of DNA in approximately 800 nuclei at each age were determined with a Zeiss microspectrophotometer. The major findings of this research indicate that while regenerated plants can be obtained in vitro from callus tissue the frequency, particularly of shoot organogenesis is low. In experiment 1, the percent calli with root regenera-tion was 39.4, 22.6, and 12.9 while that with shoots was 3.4, 1.3, and 0.4 after the first second and third subcul-tures respectively. In experiment 2, the percent calli with root regeneration was 45.8, 44.1, and 46.2 while that with shoots was 9.6, 5.5, and 2.9 after each successive subculture. Results on callus induction studies showed that Dicamba and Picloram were the most effective for producing callus growth. The optimum concentration for Dicamba was 20 μM and for Picloram 20-60 μM. Data from the microspectrophotometry showed that 86.4% of the nuclei at all ages contained a 2C amount of DNA typical of the pre DNA synthesis phase (G^) of the diploid cell cycle. Therefore, decreasing frequencies of plant regeneration from callus does not appear to be related to endoreduplication and subsequent increase in ploidy.

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