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Abstract

Large numbers of normal chrysanthemum plants were produced from tissue cultures derived from 0.5 mm high shoot-tips. The cultures, which consisted of callus with superficial meristems, doubled in weight every 3 days in liquid Murashige-Skoog medium containing 2.0 mg/1 kinetin and 0.02 mg/1 NAA. Cultures resumed growth after at least 6 months storage at 4.5°C. Plantlets formed within 6-12 weeks when small pieces of tissue were transferred to agar media containing 0.5-2.0 mg/1 kinetin. Addition of GA₃ (10 mg/1) promoted formation and elongation of leaves and shoots on the tissue. Ability to form plantlets was retained after repeated subculture; a culture started in April 1970 is still producing plantlets. One thousand plantlets were potted and grown in the greenhouse. Small (<1 cm high) plantlets initially grew more slowly than larger ones but almost all plants produced normal white flowers after 2 months short day treatment. This system could produce up to 9 × 10¹⁴ plantlets or 90 billion 15 cm plants within a year, a great increase over the number possible via conventional propagation.

Abstract

Up to 50% of bulblets generated in vitro at 30°C from bulbscale explants of Lilium longiflorum ‘Ace’ produced an elongated axis in the 14 weeks after removal from tissue culture and potting in vermiculite. None of the ‘Ace’ bulblets produced in vitro at 25° and none of the ‘Nellie White’ bulblets produced in vitro at either 25° or 30° formed an elongated axis. Increased length of time that ‘Ace’ bulbs were stored at 4° before explanting as well as immersing bulblets generated in vitro at 30° in water at 45° for 1 hour before potting increased the percentage of bulblets with an elongated axis. Axis elongation was not related to bulblet size or to position of scale used for explanting (inner vs. outer bulb scale). Exposing ‘Ace’ bulblets generated at 30° to 3 or more weeks at 4° reduced the percentage of bulblets with an elongated axis to zero. Treatment of chilled bulblets for 2 hours in water at 45° did not reverse the effect of cold.

Heterozygous multiple marker genetic stocks were synthesized by crossing three multiple genetic marker stocks to a common inbred parent PU812. The four parents and 3 F₁'s were cultured to obtain regenerants from leaf discs. Fifty four regenerants were derived from 3 F₁'s and 12 from the 4 parents, Among the regenerants, 16 plants were identified as tetraploid (24.2%); low fertility was usually associated with tetraploidy, however there were a few exceptions.

Selfed seeds, identified by cluster number, were harvested from sexual F₁'s and R₀ plants for F₂ progeny tests for the known marker genes. While there were no abnormal segregations for marker genes in the sexual progenies, 13 of 46 progenies from tissue culture derived regenerants showed significant deviations from expected normal segregations for a number of markers. Two of the abnormal progenies were identified as tetraploids by root-tip examinations; segregation ratios fit duplex random chromatid segregation for gene a on chromosome 11 and random chromosome segregation for gene c on chromosome 6. The cams of abnormal segregations in other progenies remain unknown. Results suggest that unknown genetic events arising during tissue culture may distort segregations for marker genes in the subsequent sexual progeny of tissue culture regenerants.

In Pelargonium, the plastid mutation in three independent cell layers L1, L2, and L3, can produce plastid chimeras with visible shoot colour difference such as GWG (green-white-green) and GGW (green-green-white). Chimera can be used to trace the relationship between the cell layers of different genotypes during shoot development and the effect of the mutated genes on shoot development. In this study, we have obtained different adventitious shoots with GGG, GWG, GGW, and WWW combinations of cell layers through tissue culture of petioles and internodes from GGW and GWG chimeras of Pelargonium zonale `Mrs Pollock'. Much higher percentage (14.9%) of chimeral adventitious shoots was obtained from GGW tissues than from GWG tissues (4.2%). Of the 10.8% chimeral adventitious shoots regenerated in this experiment, 8.6% are different from the original type of explants. This result indicated that cells at least in both L2 and L3 of the explants were involved in the regeneration of the adventitious shoots. The number of shoot types regenerated is likely dependent on the number and the type of cells that were in direct contact with the culture medium. It is suggested that the mixed cells can be used to produce the chimera by tissue culture. Three possible ways to form the chimeras in vitro culture were discussed. Chemical names used: TDZ =1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (Thidiazuron); IAA = Indole-3-acetic acid; PVP = polyvinylpyrrolidone.

Growing tomato fruits in tissue culture, using ovaries, could be used as a model system to study fruit development and sink strength/activity. Producing a “normal and healthy” fruit is essential in developing this system. Many factors affect the growth and development of the fruit. The objective of this study was to investigate the effect of the age of the ovary (i.e., the number of days after pollination) on growth and final fruit size. The results indicate that the fruit size, root development, and uniformity in growth of the fruit were affected by the initial age of the ovary. The older the ovary, the greater was the final fruit size and uniformity. The development of root mass was not affected by the age of the ovary until 7 days of pollination. However, root development was suppressed in ovaries that were of 9 days after pollination. The fruits from younger ovaries were more irregular in shape. All the fruits from ovaries harvested at 9 days after pollination were more uniform and round as compared to other treatments.

Effects of autoclaving volume, gelling agent (Bactoagar, Gel-gro, Phytagar), and basal salts [Murashige and Skoog (MS); Woody Plant Medium (WPM); Gamborg B5 (GB)] on gel strength and pH of tissue culture media were tested. Gel strength was significantly affected by gelling agent and basal medium. MS media were generally softer than comparable WPM or GB media. As the vessel volume during autoclaving decreased, gel strength significantly decreased with Phytagar and Bactoagar gelling agents; Gel-gro had greater gel strength at the intermediate volume of medium autoclave. In all cases, autoclaving resulted in a pH decrease of 0.2 to 0.5 pH units. Lower pH values were associated with softer gels. The type of gelling agent did not greatly affect the postautoclave pH; mean values among gelling agents were within 0.05 pH units. Postautoclave pH of MS medium was lower than that of WPM or GB. This study verifies the need to observe uniform sterilization protocols to maintain consistency in the chemical and physical properties of media.

Plants of `Northblue' blueberry, propagated in tissue culture (TC) or from softwood, single-node cuttings (ST), were evaluated in field plantings established in 1984 at Becker and Grand Rapids, in central and northern Minnesota, respectively. Plantings were observed from 1987 through 1994 to determine the persistence of such effects as increased vigor, more spreading growth habit, and higher yield observed for TC plants during the initial 3 years after planting. TC plants had significantly higher yields at Grand Rapids in 1989 and 1994. At Grand Rapids, the consistently greater plant spread (bearing area) of TC plants resulted in higher yields of TC plants over all years combined. At Becker, TC and ST plants did not differ for plant height or spread after 10 years and, in 2 of 5 years, ST plants had heavier average berry weights. At Grand Rapids, TC plants did not differ consistently in height, or subjective ratings of the amount of bloom or crop. The effects of propagation method on yield and growth habit of `Northblue' are limited to early years in warmer locations, but can be of longer-term significance in colder areas with shorter growing seasons and lower winter temperatures, where plant spread is a more important factor than plant height in determining yield.

Watermelon (Citrullus Lanatus (Thunb.) Matsum and Nakai) and muskmelon (Cucumis melo) were regenerated from immature cotyledons cultured on MS medium containing 10 μM BA. Small population of watermelon and muskmelon regenerants contained tetraploids as variants. The tetraploid individuals were recognized by morphological features including enlarged leaves, tendrils, male flowers, and variable pollen grains. After self-pollination, seed lots reflected differences in size expected from tetraploid parents.. Cytological data from root tips of R₁ populations will be presented.