Seminar Series Abstract

Development of Plant Regeneration and Transformation Systems in Daylilies

Jing-Tian Ling and Roger J. Sauve

Cooperative Agricultural Research Program Seminar Series
Tennessee State University, Nashville, TN
April 28, 1999

Introduction.  Daylilies (Hemerocallis spp.) are the number one perennial plant sold in the United States. They have large, attractive and showy flowers, exhibit a high resistance to pests and diseases, and are adaptable to a wide range of soils and climatic conditions. Daylilies are also planted along highways for the control of soil erosion. Conventional daylily breeding programs depend upon sexual hybridization and chromosome duplication to introduce variation. The development of plant regeneration systems and genetic transformation systems for daylilies makes it possible to introduce outstanding characteristics using somatic hybridization and biotechnology techniques.

Objectives.  1) To develop an efficient plant regeneration system for daylilies. 2) To develop a protocol for transformation of daylily cells and regenerate transgenic plants.

Methodology.  Techniques of plant tissue culture and of molecular biology were applied to develop plant regeneration systems and genetic transformation systems in daylilies.

Results.  In our studies, mesophyll protoplasts were successfully isolated from leaf tissues of a diploid daylily (Hemerocallis x 'Red Magic') by enzymatic digestion with a solution containing 0.5% Pectolyase Y-23, 0.1% Cellulase R-10, 0.1% Driselase, 0.6 M sorbitol and half-strength Murashige & Skoog (MS) inorganic salts. When cultured on MS medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA, the protoplasts underwent sustained division to produce multicellular colonies. The optimal plating density for cell division was 0.5 x 10⁵ protoplasts/ml. The highest plating efficiency was obtained in cultures grown in media solidified with 0.2% Gelrite. Under these conditions, formation of colonies occurred from 14% of cultured protoplasts. Calli were recovered from nine colonies only after the cultures were treated with a conditioned medium. Intact plants were regenerated from protoplast-derived calli through organogenesis. Protoplast-derived plants were successfully established in the greenhouse. Daylily calli were induced from immature flower buds and flower stalks on MS medium supplemented with 50 g/l sucrose, 0.5 mg/l NAA, 2 mg/l BA and 160 mg/l adenine. The calli proliferated well and maintained the regeneration capacity when they were subcultured monthly on MS medium supplemented with 50 g/l sucrose, 10 mg/l NAA, 2 mg/l BA and 160 mg/l adenine. Shoot regeneration occurred on MS medium supplemented with 0.1 mg/l NAA and 2.0 mg/l TDZ. The highest rooting frequency (88%) was obtained on half-strength MS medium. Nearly all rooted shoots (96%) survived after greenhouse acclimatization. Suspension culture cells of Hemerocallis x 'Stella de Oro' were transformed with the phosphinothricin acetyltransferase (PAT) gene under control of the CaMV 35S promoter via microprojectile bombardment. Selection of transformed calli were initiated 2-3 weeks after bombardment on a medium that contained 4 mg/l phosphinothricin (PPT). Transgenic shoots were regenerated from PPT-resistant calli on a medium that contained 2 mg/l TDZ and 0.01 mg/l NAA, and rooted on a half-strength MS medium.

Impact.  The development of efficient plant regeneration and transformation protocols for daylilies now allows for the genetic improvement of daylilies using genetic engineering techniques. This provides an opportunity to produce daylily cultivars with distinguished features such as extended flower longevity, unique flower colors and herbicide resistance.

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