Introduction
Roses are one of the most important floral crops in the world.  However, most of the modern cultivars are susceptible to black spot and various other microbial and insect pests.  This necessitates the regular use of fungicides and insecticides by rose growers and substantially adds to their production expenses.  Molecular biology techniques are now available for genetic modification of roses in order to obtain better resistance to pathogens.  While several researchers have reported successful transformation of rose plants (Derks et al., 1995; Firoozabady et al., 1994; Marchant et al., 1998; Souq et al., 1996; van der Salm et al., 1997), there are been no commercial introductions of genetically modified cultivars with increased pathogen resistance.  Part of the difficulty associated with transformation of roses is developing an efficient regeneration system, applicable to many rose cultivars, that allows large numbers of plants to be recovered from tissue cultures.

The details of the research presented here focuses on the careful capturing of very young rose embryos (in their globular-stage) from purposely wounded rose tissue, then testing various carbohydrates and other substances to see if they will encourage greater percentages of regeneration among these embryos. Regeneration, by definition, is the development of whole plants from undifferentiated cells in tissue culture. A single rose cell can then be manipulated in tissue culture to form an embryo that germinates to form roots and leaves. The higher regeneration rates reported here offers addtional help to bioengineers and breeders as they continue to strive to genetically engineer disease and pest resistance in roses. The results from this research were significant enough for a U.S. Patent Appliation entitled "Regeneration of rose plants from embryogenic callus".

Summary of Results
Embryogenic callus cultures of three genetically diverse cultivars of rose (Rosa hybrida L.), the floribunda cv 'Trumpeter', the multiflora cv 'Dr. Huey' and the hybrid tea cv 'Tineké', were developed and used to study the effect of various carbohydrates and osmotically active compounds on somatic embryo maturation and germination.  Large numbers of cotyledonary-stage embryos are needed for quantitative experiments to identify factors that effect somatic embryogenesis of rose.  This was accomplished by dispersing the callus in liquid medium followed by filtration to isolate globular-stage embryos.  {See Figures 1, 2 & 3 below}.

Quantitative experiments were conducted to determine the maturation and germination rates of the three rose cultivars in response to media with sucrose, glucose, fructose or maltose as the primary carbon source and also in response to various concentrations of either myo-inositol, polyethylene glycol, or mannitol in combination with sucrose.  Taking into consideration both the relative number of embryos which matured on either the various carbohydrates or osmotica and the germination rate, the best treatments for regeneration of plants were 3% fructose for 'Trumpeter' (58 mature embryos/plate, 27% germination), 2.5% mannitol (with 3% sucrose) for 'Dr. Huey' (102 mature embryos/plate, 36% germination), and either 3% sucrose or 3% glucose alone for 'Tineké' (47 mature embryos/plate, 13% germination).  {See Graphs below}.

Plants were then transferred to Magenta jars containing germination medium without charcoal for further root development for 2 months and then to soil for growth in the greenhouse.  Flowers observed after growth in the greenhouse for 1.5 yr have appeared to be phenotypically normal.    {See Figures 5 & 6 below}.

1. Establishment of embryogenic callus. Embryogenic callus cultures (left) were established using the procedure of Marchant et al. (1996) for 'Trumpeter' and the procedure of Derks et al. (1995) for 'Tineké' and 'Dr. Huey'.

2. Preparation of inoculum. Suspensions were prepared from embryogenic callus by shaking and then sequentially filtering through stainless steel sieves with mesh sizes of 2, 1.2 and 0.8 mm.  This eliminated cotyledonary-stage embryos and provided a mixture of globular-stage embryos, proembryogenic masses and nonembryogenic callus cells.  Plates were inoculated (right) using 2 ml of the well mixed suspension (three plates per treatment) and then placed in the dark at 25 C.

3. Maturation of embryos The base medium for testing various carbohydrates and osmotica consisted of MS salts and vitamins, 100 mg/L myo-inositol, 2 mg/L glycine, 0.25% Phytagel, 0.5% activated charcoal, and 500 mg/L MES.  The carbohydrates tested included 3% (w/v) of either sucrose, glucose, fructose or maltose.  The osmotically active compounds were either 2.5, 5.0 or 7.5% myo-inositol, PEG (m.w. 3350) or mannitol. {See graphs below}.

4. Germination of cotyledonary-stage embryos Cotyledonary-stage embryos were cultured 10 to 11 wk on each maturation medium containing various carbohydrates and osmoticum and then transferred to germination medium (MS salts and vitamins, 100 mg/L myo-inositol, 2 mg/L glycine, 0.5% AC, 0.25% Phytagel, 3% sucrose).  Cotyledonary-stage embryos that had been transferred to germination medium were kept in the dark for 1 wk and then placed under a 12 hour light photoperiod.

5. Growth in the greenhouse Plants were transferred to Magenta jars containing germination medium without charcoal for further root development for 2 months and then to soil for growth in the greenhouse.

6. Flowering All flowers observed after growth in the greenhouse for 1.5 yr have appeared to be phenotypically normal.  Shown here are the cultivars Tineke (left) and Trumpeter (right).

References cited
Firoozabady et al. 1994.  Bio/Tech. 12: 609-613.
Derks et al. 1995.  Acta Hort. 405:205-209.
Souq et al. 1996.  Acta Hort. 424:381-388.
Van de Salm et al. 1997.  Mol Breed. 3:39-47.
Marchant et al. 1996.  Plant Sci. 120:95-105.
Marchant et al. 1998.  Ann. Bot. 81:109-114.
 

Note: This article was edited and reformatted for this web page by Ramon Jordan from a Poster prepared and presented by Kathy Kamo at a recent national Society of In Vitro Biology annual meeting.

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Last Updated   January 18, 2005 4:34 PM
URL = http://www.usna.usda.gov/Research/KamoRose.html