ARS | Publication request: Mixture Screening and Mixture-Amount Designs to Determine Plant Growth Regulator Effects on Shoot Regeneration from Grapefruit (Citrus paradisi Macf.) Epicotyls

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 14, 2011
Publication Date: N/A

Interpretive Summary: Plant tissue culture is essential for applying certain techniques to enhance plant variety development. One type of tissue culture is the regeneration of shoots from stem pieces, an important in vitro method required for the most common genetic engineering technique that uses Agrobacterium. One application of genetic engineering is to make plants resistant to diseases. In this study, we tested eight plant growth regulators to identify the best combinations for regenerating shoots from grapefruit tissue cultures. Grapefruit was selected as it is 1) susceptible to citrus canker and greening diseases; 2) has no native resistance to these diseases and; 3) is critical to Florida’s citrus economy. We discovered that the highest quality and greatest number shoots regenerated occurred with various combinations of the two plant growth regulators zeatin riboside and IAA.

Technical Abstract: The objective of this study was to improve shoot regeneration from grapefruit epicotyl explants since some important in vitro applications in citrus, such as Agrobacterium-mediated transformation, commonly use epicotyl segments from in vitro seedlings; adequate adventitious shoot production is thus an important prerequisite. Because shoot regeneration in citrus requires the addition of a cytokinin to the culture medium, we 1) screened six cytokinins (6-benzylaminopurine, kinetin, zeatin trans-isomer, 6-(y,y-dimethylallylamino)purine, zeatin riboside trans-isomer, and meta-topolin) to identify those cytokinins where shoot regeneration was greatest; 2) examined the effects of two cytokinins (zeatin riboside trans-isomer and 6-(y,y-dimethylallylamino)purine) and two auxins (indole-3-acetic acid and 1-naphthaleneacetic acid) to determine the most effective combinations for shoot regeneration; and 3) examined the effects of zeatin riboside trans-isomer and indole-3-acetic acid on shoot regeneration. In each of these experiments three responses were measured – 1) number of explants that formed buds/shoots; 2) the number of shoots greater than 2 mm; and 3) an overall quality. The resulting ANOVAs for each response were significant at p < 0.0001 (p > F). Results included 1) 6-benzylaminopurine and zeatin riboside trans-isomer were the most effective cytokinins for inducing shoot regeneration in citrus; 2) zeatin riboside trans-isomer in combination with IAA resulted in the highest quality, greatest number of explants with buds/shoots, and greatest number of shoots > 2 mm.