Scientia Horticulturae 87 (2001) 225±240 Mutagenesis and in vitro culture of Tillandsia fasciculata Swartz var. fasciculata (Bromeliaceae) Yong Cheong Koh, Fred T. Davies Jr.* Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843-2133, USA Accepted 12 April 2000 Abstract The genus Tillandsia (Bromeliaceae) has very few variegated species, and cultivars with chlorophyll-de®cient variegation are especially rare. With the objective of inducing chlorophyll- de®cient leaf variegation, seeds of Tillandsia fasciculata var. fasciculata were treated with gamma radiation, combined gamma and thermal neutron radiation or by chemical mutagenesis with ethyl methanesulfonate (EMS). Wild type, albino, yellow, yellowish-green and variegated phenotypes were obtained in the subsequent M0 generation. These variegated seedlings were either sectorial or mericlinal chimeras, consequently the variegation of these seedlings was lost as they grew older. Gamma radiation at 21 kR and 27 kR produced the highest percentage of variegated seedlings (4.4%). The highest percentage of seedlings with chlorophyll-de®cient leaves was 8.4% with 27 kR gamma radiation, and 15.8% with 1.2% EMS. Radiation and chemical mutagenesis caused chlorophyll-de®ciency mutations in one or more of the histogenic layers: LI, LII, LIII. Wild types had greater total chlorophyll a, b and total chlorophyll than mutant phenotypes. Most of the yellow and yellowish-green seedlings multiplied in a solid half-strength MS medium with equimolar 0.3 or 0.5 mM BA and IBA. The yellowish-green seedlings were able to grow photoautotrophically while the yellow ones were not. This is one of the ®rst reports on the mutagenesis of a Tillandsia species. Stable periclinal chlorophyll-de®cient chimeras of Tillandsia species can likely be obtained via mutagenesis if large numbers of seeds are treated with a suitable mutagen. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Bromeliads; Chimeras; Chlorophyll a/b ratios; Micropropagation; Mutagenesis; Tillandsia; Variegation Abbreviations: BA, 6-benzyladenine; Chl, chlorophyll; DMS, N, N-dementhyl formamide; EMS, ethyl methanesulfonate; IBA, indole-3-butyric acid; kR, kilorad; M0, ®rst mutanized generation; MS, Murashige and Skoog; PPF, photosynthetic photon ¯ux * Corresponding author. Tel.: 1-409-845-5341; fax: 1-409-845-0627. E-mail address: [email protected] (F.T. Davies Jr.). 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0304-4238(00)00166-7 226 Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240 1. Introduction Induced mutations have produced many plants with improved economic value (Broertjes and van Harten, 1988; Anon, 1995). Besides the economic bene®ts, some mutants also play an important role in the study of genetics and plant development (van den Bulk et al., 1990; Bretagne-Sagnard et al., 1996). The production of chlorophyll-de®cient mutants is a common phenomenon in mutagenesis experiments and it has been reported in monocots (Khalatkar and Bhargava, 1982) and dicots (Miller et al., 1984; Aviv and Galum, 1985; Alcantara et al., 1996), but these mutants usually have not been the subject of interest. Leaf variegation is an important factor in¯uencing the popularity of ornamental plants. Whereas many variegated cultivars exist in other bromeliad genera such as Ananas, Billbergia, Cryptanthus, Guzmania, Neoregelia and Vriesia, only one chlorophyll-de®cient variegated Tillandsia is commercially available on a limited basis Ð T. cyanea `Variegata'. Prior to this report, no literature was available on the mutagenesis of Tillandsia spp. When a desirable new phenotype has been obtained through mutagenesis, the next logical step is to try to produce more of this new plant vegetatively to increase its population and preserve its unique characteristics. Researchers have attempted to determine the correlation between chlorophyll-de®cient phenotypes and the ultrastructure of chloroplasts of several chlorophyll-de®cient mutants and their respective wild types (Vaughn et al., 1978, 1980; Kirchhoff et al., 1989; Lee et al., 1989). In general, chlorophyll-de®ciency is correlated with deformed thylakoids and/or presence of fewer normal thylakoids. Although the collection and sale of wild Tillandsia spp. is still permitted in some countries, this practice is likely to be curtailed or stopped in the future as more bromeliads become endangered as a result of habitat loss and over- collection. This problem further reinforces the need to develop new cultivars through mutagenetic techniques. The aims of this research were: (1) to determine and compare the ef®cacy of gamma radiation, combined gamma and thermal neutron radiations and chemical mutagenesis with EMS in producing variegated phenotypes in Tillandsia fasciculata var. fasciculata, (2) to ascertain chlorophyll (Chl) a and b concentrations and the Chl a/b ratio in the wild type and chlorophyll-de®cient phenotypes of T. fasciculata var. fasciculata to better determine the seedling's ability to survive photoautotrophically ex vitro, and (3) to ®nd an ef®cient way of micropropagating T. fasciculata var. fasciculata so that the same protocol may be used on other Tillandsia spp. This plant was chosen because it is self-fertile and can produce thousands of seeds per plant when hand- pollinated. Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240 227 2. Experimental methods 2.1. Stock plant and seed production Forty T. fasciculata var. fasciculata plants with immature in¯orescences were purchased from a commercial producer (Tropi¯ora, Sarasota, FL), who imported them from Honduras. The stock plants were grown in a glass greenhouse with a maximum PPF of 400 mmol m2 s1. The low and high average mean temperature was 238C and 32Æ28C, respectively. The average mean low and high humidity was 65 and 98%, respectively. At anthesis the plants were self-pollinated by hand. Each pollinated plant produced 15±30 capsules that took about a year to mature. Mature capsules were brown. They were harvested before they dehisced. Harvested capsules were cleaned and disinfested in 10% Clorox for 10 min, rinsed in tap water, air-dried and kept in open plastic containers to let them dehisce. Each capsule contained one to two hundred seeds. 2.2. Radiation treatments Seeds were irradiated with either gamma radiation or combined gamma and thermal neutron radiation at the Nuclear Science Center at Texas A&M University. The radiation treatments were not replicated because of their high cost. The gamma radiation was derived from a lanthanum source. The dosage was 1.35 kR h1. A completely randomized design was used. There were eight treatments with 250 seeds per treatment. The treatments for this experiment were gamma radiation at 0, 10, 12, 15, 18, 21, 24, 27, and 29 kR. The combined gamma and thermal neutron radiation were derived from a beam port which delivered a thermal neutron dose of 111.6 rad h1 accompanied by 824.5 rad h1 of gamma radiation. Therefore, the ratio of thermal neutron to gamma radiation was 1±7.5. A completely randomized design was used. There were thirteen treatments with 250 seeds per treatment (n250). The treatments for this experiment were thermal neutron radiation at 0, 0.1, 0.3, 0.7, 1.1, 1.3, 1.7, 2, 2.3, 2.7, 3, 3.1 and 3.2 kR. 2.3. EMS treatment Each batch of seeds with trimmed trichomes (coma) was put in a piece of ®nely woven cheesecloth and the cheesecloth was tied into a bundle. The bundles of seeds were disinfested in 10% Clorox for 10 min, transferred to a laminar ¯ow hood, rinsed three times with sterile distilled water, and left to imbibe in sterile distilled water in the laminar ¯ow hood for about 22 h. 228 Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240 Imbibed seeds were used in the following treatments: 0% EMSÂ5 h (control), 1.2% EMSÂ3 h and 0.4% EMSÂ5 h. Each treatment was replicated three times. There were 1000 seeds per treatment (n1000). All treatments were carried out in a sterile 0.1 M phosphate buffer with a pH of 7.2 in a fume hood. Glass beakers and magnetic stirrers were surface sterilized with 70% ethanol before use. For every treatment, a measured amount of EMS was pipetted into the beaker containing a magnetic stirrer and an amount of phosphate buffer that would give the correct ®nal EMS concentration. The ®nal EMS/buffer mixture was 30 ml for each treatment. A bundle of imbibed seeds was lowered into a beaker and placed on the magnetic-stirrer hot plate. The EMS solution was agitated throughout the entire treatment period. When the treatment period was completed, the EMS solution was decanted and 30 ml of sterile distilled water was put into beakers containing the bundle of seeds and stirred. The sterile distilled water was changed every 15 min during the 2 h rinse. 2.4. Micropropagation 2.4.1. Micropropagation of wild type T. fasciculata var. fasciculata The trichomes of each seed were trimmed, then seeds were disinfested in 10% Clorox for 10 min, transferred to the laminar ¯ow hood where they were rinsed three times with sterile distilled water before being placed in test tubes containing basal media. Twenty seeds were used in each treatment (n20). Two seeds were put in each 12 mmÂ94 mm test-tube with 13 ml of medium. The basal medium consisted of half strength MS salts and vitamins (Murashige and Skoog, 1962), 15 g l1 sucrose, 8 g l1 Difco±Bacto agar and a pH of 5.7. The treatments differed in their equimolar concentrations of BA and IBA, which were 0 (control), 0.01, 0.03, 0.1, 0.15, 0.20, 0.25, 0.30, 0.50, 1.0, 3.0 and 10.0 mM BA and IBA.