A filter paper-based liquid culture system for citrus shoot organogenesis - a mixture-amount experiment

Authors: Niedz, Randall; Hert, Mizuri

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/29/2018
Publication Date: 10/12/2018
Citation: Niedz, R.P., Hert, M.M. 2018. A filter paper-based liquid culture system for citrus shoot organogenesis - a mixture-amount plant growth regular experiment. In Vitro Cellular & Developmental Biology - Plants. 54(6):658-671.

Interpretive Summary: Applying plant tissue culture methods to plant breeding and horticulture applications is used when the application is difficult or impossible through conventional approaches. One application important in citrus breeding is genetic engineering, and genetic engineering requires plant tissue culture to produce the engineered trees. However, genetic engineering is very inefficient in many of the most important commercial citrus types such as sweet orange. The plant tissue culture method used to produce genetically engineered trees is adventitious shoot regeneration, which is the regeneration of shoots from plant parts that contain no buds, such as a stem piece. The shoot regeneration system used in citrus uses a gelling agent, often agar, in the culture medium. A gelling agent is used to support the cultured plant part; otherwise, the plant part would be covered in liquid and would not grow well or die due to low oxygen. This study examined the feasibility of using a liquid-based system for shoot regeneration. The results showed that a liquid-based system composed of filter paper disks in a Petri dish and soaked with culture medium is sufficient to produce regenerate high quality shoots. The system was tested in two citrus types, a rootstock type, Carrizo, and a sweet orange, Ridge Pineapple. Because high quality shoots were regenerated, a liquid-based system may be useful as a plant tissue culture method in the genetic engineering of citrus.

Technical Abstract: The objective of this study was to determine the effects of a static liquid culture system on shoot regeneration from citrus epicotyl explants. Two citrus types were used, Carrizo citrange and Ridge Pineapple sweet orange. A liquid culture system comprised of a Petri dish, cellulose filter paper discs, and liquid culture medium was used. Shoot regeneration experiments were conducted over 6 weeks that included 2 weeks in the dark followed by 4 weeks in the light. Three responses were measured – the number of explants that formed shoots/buds, the number of explants with shoots > 2 mm, and the overall quality of the explant and shoots. The effects of the number of paper discs, volume of liquid medium, and explant size on shoot regeneration were determined. High quality shoots were produced from explants cultured in a wide range, 5.25 to 12 mls, of medium volumes and explant sizes, 2 to 15 mm. The effects of zeatin riboside (ZR), 6-benzylaminopurine (BA), and indole-3-acetic acid (IAA) on shoot regeneration were determined using a mixture-amount design. Cytokinins and auxins interact both by proportion and amount and mixture-amount designs are the only designs that can determine these effects. The effects were similar for the two citrus types and were as follows: 1) ZR or BA both produced high quality shoots; 2) ZR and BA were not synergistic; 3) 20 µM ZR produced the most shoots; 4) BA and IAA were strongly synergistic, which resulted BA producing the greatest number of shoots when IAA was included in the mixture; 5) ZR and IAA were antagonistic, particularly with Ridge Pineapple. Though ZR resulted in the most shoots (twelve 2 mm shoots/explant), the difference compared to BA and IAA (ten 2 mm shoots/explant) may not be sufficiently large to justify its use in routine applications considering its extremely high cost.