Mutagenesis and in Vitro Culture of Tillandsia Fasciculata Swartz Var. Fasciculata (Bromeliaceae) Yong Cheong Koh, Fred T

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.).

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.

2.4.2. Micropropagation of irradiated and EMS-treated seeds After the irradiation treatments, seeds were disinfested in 10% Clorox for 10 min and transferred to the laminar ¯ow hood where they were rinsed three times in sterile distilled water before being put on 100 mmÂ20 mm plastic petri dishes containing 40 ml of medium with half-strength MS salts and vitamins, 15 g l1 sucrose, 8 g l1 Difco±Bacto agar and equimolar 0.15 mM BA and IBA. This particular BA and IBA concentration was chosen because it did not produce shoot proliferation in the wild type seedlings of T. fasciculata var. fasciculata.If mutagenized seedlings were to start shoot proliferation too early, it would have made identi®cation of variegated seedlings very dif®cult. The pH of the medium was 5.7. Twenty-®ve seeds were put in each petri dish and ten petri dishes were used per treatment; therefore, n250 per treatment for the irradiated seeds. The disinfestation procedures for the EMS-treated seeds were the same as those for the irradiated seeds. One hundred seeds were put into each petri dish. There Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240 229 were ten petri dishes per treatment (n1000). The irradiated and EMS-treated seeds were grown for 2 months before data were gathered.

2.4.3. Micropropagation of mutant chlorophyll-de®cient T. fasciculata var. fasciculata phenotypes After growing for 2 months in vitro in a medium with equimolar 0.15 mMBA and IBA, the mutagenized seedlings were examined visually for leaf variegation or non-wild type leaf color. Selected mutant seedlings were subcultured individually in 12 mmÂ94 mm test tubes containing 13 ml of solid medium containing half-strength MS salts and vitamins, and equimolar 0.3 mM BA and IBA. If no shoot proliferation occurred within 2 months, the seedlings were transferred to a medium with equimolar 0.5 mM BA and IBA.

2.5. Chlorophyll content and chlorophyll a/b ratio

Five samples of fresh leaf tissues from 14-month old in vitro seedlings were taken from the wild-type, yellowish-green and yellow phenotypes. Each sample weighed 10 mg and was cut into small pieces before being put into an Eppendorf tube with 1.2 ml of DMF. The Eppendorf tubes were stored in the refrigerator for 24 h for chlorophyll extraction. Five samples of 0.5 ml DMF from each phenotype were read at 647 and 664 nm and the respective absorbances were recorded with a Bausch and Lomb spectrophotometer (model Spectronic 21). All readings were done in the minimum amount of light and in the shortest time possible. Calculations of chlorophyll a and b contents were done by using the formulae published by Moran (1982). All treatments were analyzed by ANOVA (SAS, 1988). Unless described differently in the experimental protocol, a completely randomized design was used. The sample size (n) varied with each experiment and is described in the previous sections.

3. Results and discussion

3.1. Radiation treatments

After 2 months of growth in vitro, the surviving seedlings in each experiment were examined for the percentage of lethality and the following mutant phenotypes: albino, yellow, yellowish-green, and variegated (Fig. 1). For the purpose of phenotype classi®cation, a seedling was considered variegated if it had one or more leaves that were variegated. Whether the leaf variegation was preserved in subsequent leaves or lateral shoots was not a criterion. 230 Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240

Fig. 1. Wild type and chlorophyll-de®cient phenotypes of T. fasciculata var. fasciculata grown in vitro. These phenotypes are from the gamma radiation experiment and the combined gamma and thermal neutron radiations experiment: (a) wild type, (b) yellowish-green, (c) yellow, (d) white- margined variegated, (e) white-centered variegated, and (f) albino.

In the gamma radiation experiment, the appearance of yellowish-green and total chlorophyll-de®cient phenotypes was ®rst observed at 12 kR (Table 1). In the combined gamma and thermal neutron radiations experiment, the same phenotypes were observed at the combined dose of 0.9 kR (Table 1). Compared to gamma radiation, a much lower dose of combined gamma and thermal neutron radiations was adequate for the ®rst appearance of the yellow, albino and variegated phenotypes, e.g. 21 kR gamma radiation vs. 2.8 kR combined gamma and thermal neutron radiations for the ®rst appearance of yellow phenotypes. Table 1 Response of T. fasciculata var. fasciculata seeds to gamma, combined gamma and thermal neutron radiations, and ethyl methanesulfonate (EMS). Seedlings were cultured in vitro at equimolar 0.15 mM BA and IBA (data recorded after 2 months of in vitro culture) Dosage (kR) Mortality per Albino seedlings Yellow seedlings Yellowish-green Variegated Total chlorophyll- treatment (%)a per treatment (%) per treatment (%) seedlings per seedlings per deficient seedlings

treatment (%) treatment (%) per treatment (%) 225±240 (2001) 87 b Horticulturae Scientia / Jr. Davies F.T. Koh, Y.C. Gamma radiation 0 (control) 4.0Æ1.0c,d 0Æ00 0Æ00Æ00Æ0 10 5.2Æ1.0 0Æ00 0Æ00Æ00Æ0 12 6.8Æ0.9 0Æ0 0 0.4Æ0.4 0Æ0 0.8Æ0.5 15 11.2Æ2.4 0Æ0 0 0.8Æ0.5 1.2Æ0.6 2.0Æ0.9 18 0.8Æ0.9 0Æ00 0Æ0 1.6Æ0.9 1.6Æ0.9 21 0.8Æ0.9 0Æ0 0.8 0Æ0 4.4Æ1.1 5.2Æ0.9 24 4.4Æ2.6 0Æ0 0.8 1.6Æ0.6 2.4Æ0.6 4.8Æ1.0 27 26.8Æ1.8 0.8Æ0.5 0 3.2Æ1.0 4.4Æ0.9 8.4Æ1.5 29 100.0Æ00Æ00 0Æ00Æ00Æ0 Significance *** * NSe *** *** *** Gamma and thermal neutron radiations (kR) 0 (control) 4.4Æ1.3 0 0 0 0Æ00 0.1f (0.9)g 8.8Æ1.2 0 0 0.4 0.4Æ0.4 0.8 0.3 (2.8) 11.2Æ1.8 0 0.4 0.4 1.2Æ0.6 2.0 0.7 (5.6) 14.0Æ2.3 0 0 0.4 0.4Æ0.4 0.8 1.1 (9.3) 14.8Æ1.7 0.8 0.4 0.8 0Æ0 2.0 1.3 (11.2) 12.0Æ2.6 0.8 0.4 1.2 0.4Æ0.4 2.8 1.7 (14.0) 8.8Æ2.4 0.4 0 1.2 0.4Æ0.4 2.0 2 (16.8) 12.8Æ2.3 0 0 1.6 1.6Æ0.7 3.2 2.3 (19.6) 11.6Æ2.1 0.4 0 1.2 1.2Æ0.6 2.8 2.7 (22.4) 30.8Æ3.0 0 0 2.0 0.8Æ0.5 2.8 3 (25.2) 34.0Æ1.4 0 0 2.4 1.2Æ0.6 3.6

3.1 (26.1) 24.0Æ1.9 0 0 1.2 1.6Æ0.6 2.8 231 232

Table 1 (Continued )

Dosage (kR) Mortality per Albino seedlings Yellow seedlings Yellowish-green Variegated Total chlorophyll- 225±240 (2001) 87 Horticulturae Scientia / Jr. Davies F.T. Koh, Y.C. treatment (%)a per treatment (%) per treatment (%) seedlings per seedlings per deficient seedlings treatment (%) treatment (%) per treatment (%)b 3.2 (27.1) 29.6Æ3.3 0 0 2.0 2.4Æ0.7 4.4 Significance *** NS NS NS * NS %EMSÂh 0%Â5 h 84.2Æ0.6h 00 0Æ00 0Æ0 1.2%Â3 h 93.5Æ0.4 0 0.5 15.0Æ1.5 0.3 15.8Æ1.5 0.4%Â5 h 89.1Æ0.5 0 0.5 11.0Æ0.9 0.2 11.6Æ0.9 Significance *** NS NS *** NS *** Percent mortalitya (No. of dead seeds/No. of initial seeds)Â100. b Percent total chlorophyll-de®cient is the sum of albino, yellow, yellowish-green and variegated phenotypes. c Each treatment contained 1000 seeds of which an average of 159 were viable before irradiation (n240). d Standard error. e NS: nonsigni®cant. * Signi®cant at p<0.03. *** Signi®cant at p<0.0001. f Thermal neutron dosage alone. g Combined dosage of thermal neutron and gamma radiations at the ratio of 1:7.5. h Each treatment contained 250 seeds of which an average of 240 were viable before irradiation (n240). Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240 233

3.2. EMS treatments

The average percentage of viable seeds in the control was 16% (Table 1). This low viability was due to the old age of the seeds (about 15 months). Thus, the number of viable seeds in each EMS treatment was 159 out of 1000. In the computation of lethality of yellow, yellowish-green, variegated, and total chlorophyll-de®cient seedlings, 159 was used as the denominator. Since each treatment was repeated three times, the combined total number of viable seeds in each treatment was 159Â4636. The total number of viable seeds used were 240 for the gamma radiation treatments and combined gamma and thermal neutron radiations treatments. Therefore, despite the low initial seed viability, the data obtained from the EMS experiments still showed mutagenicity of EMS on seeds. No albino seedlings were observed. It was possible that some albino seedlings were produced but they died before the ®rst set of data was taken 2 months after the EMS treatment. The chlorophyll-de®cient phenotypes obtained from the chemical EMS experiment looked very similar to those from the radiation mutagen experiments.

3.3. Micropropagation

3.3.1. Micropropagation of wild type T. fasciculata var. fasciculata Since T. fasciculata var. fasciculata seedlings are very slow growing, it was not necessary to transfer them to fresh media during the 6 months of the experiment. No multiplication occurred in the media with equimolar BA and IBA at 0, 0.01, 0.03, 0.1 and 0.15 mM (Table 2). Each seedling in the media with equimolar BA and IBA at 0.20 and 0.25 mM formed several shoots. Equimolar BA and IBA at 0.30 and 0.50 mM, respectively, produced 68% and 75% of seedlings with spherical clumps of shoots. One hundred percent of the seedlings in the media with equimolar BA and IBA at 1.0, 3.0 and 10.0 mM formed spherical clumps of shoots. The size of these clumps of shoots increased with the BA and IBA concentrations in the medium (Table 2). Two spherical clumps of shoots were removed from the media containing equimolar BA and IBA at 0.3, 0.5, 1.0, 3.0, and 10.0 mM. Each clump of shoots was halved and cultured in the same basal medium without any phytohormones and they subsequently grew into clumps of normal shoots. Rooting occurred with in vitro culture and during ex vitro acclimatization of T. fasciculata, however, the goal of this experiment was to form clumps of shoots for subculturing and multiplication.

3.3.2. Micropropagation of mutant chlorophyll-de®cient T. fasciculata var. fasciculata phenotypes The total number of yellowish-green, yellow and variegated seedlings from the gamma radiation experiment was 15 (0.7% of viable seeds), 4 (0.2% of viable 234 Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240

Table 2 Response of wild type T. fasciculata var. fasciculata seedlings to multiplication media (data recorded after 6 months of in vitro culture) Equimolar Germination Seedlings with Seedlings with Diameter of 4-month media BA, (%)a multiple shoots spherical clumps old spherical clumps IBA (mM) (%) of shoots (%) of shoots (mm) 0 (control) 95 0Æ0b 0Æ00Æ0 0.01 100 0Æ00Æ00Æ0 0.03 100 0Æ00Æ00Æ0 0.10 100 0Æ00Æ00Æ0 0.15 100 0Æ00Æ00Æ0 0.20 100 70Æ8.2 0Æ00Æ0 0.25 95 75Æ8.3 0Æ00Æ0 0.30 100 95Æ5.0 65Æ7.6 9.0Æ0.2 0.50 100 100Æ075Æ8.3 9.9Æ0.6 1.0 100 100Æ0 100Æ0 10.9Æ0.4 3.0 100 100Æ0 100Æ0 11.7Æ0.3 10.0 100 100Æ0 100Æ0 13.3Æ0.4 Significance NSc *** *** *** a Criteria for percent germination was based on seed that germinated forming a visible shoot system in vitro (n20). b Standard error. c NS: nonsigni®cant. *** Signi®cant at p<0.0001. seeds) and 36 (1.7% of viable seeds), respectively. The total number of yellowish- green, yellow and variegated seedlings from the gamma and thermal neutron radiations experiment was 37 (1.2% of viable seeds), 3 (0.1% of viable seeds) and 28 (0.9% of viable seeds), respectively. The total number of yellowish-green, yellow and variegated seedlings from the EMS experiment was 152 (8.0% of viable seeds), 7 (0.4% of viable seeds) and 3 (0.2% of viable seeds), respectively. The respective percentages of various chlorophyll-de®cient mutant phenotypes that responded to the multiplication media are summarized in Table 3.

3.4. Chlorophyll content and Chl a/b ratio

The total chlorophyll content per unit fresh weight of the yellow and the yellowish-green phenotypes were 5% and 12.6%, respectively, of the wild type (Table 4). The wild type had a Chl a/b ratio of 1.1 whereas both the yellow and yellowish-green phenotypes had a Chl a/b ratio of 2.0 (Table 4). There is a directly proportional relationship between the abundance of thylakoids and the amount of chlorophyll present in T. fasciculata var. fasciculata (Koh, 1998). The wild type and the yellowish-green seedlings were able to grow photoautotrophically and survive acclimatization ex vitro, but the yellow ones were not. Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240 235

Table 3 Response of T. fasciculata var. fasciculata mutant seedlings with chlorophyll-de®cient leaf color to multiplication mediaa Medium with Yellowish-green Yellow mutants Variegated equimolar BA, mutants that that multiplied mutants that IBA (mM) multiplied (%) (%) multiplied (%) Gamma radiation treatmentsb 0.3 53 0 36 0.5 40 100 42 Combined gamma and thermal neutron radiation treatmentsc 0.3 11 0 39 0.5 59 67 39 EMS treatmentsd 0.3 67 43 100 0.5 11 29 0 a The basal medium contained half strength MS salts and vitamins, 15 g l1 sucrose and 8 g l1 agar as in Tables 1±4. All seedlings were grown in equimolar 0.15 mM BA and IBA for 2 months before being transferred to the multiplication media. b n15, 4 and 36 for the yellowish-green, yellow and variegated phenotypes, respectively. c n37, 3 and 28 for the yellowish-green, yellow and variegated phenotypes, respectively. d n152, 7 and 3 for the yellowish-green, yellow and variegated phenotypes, respectively.

Wild-type seedlings were more vigorous than the yellowish-green ones both in vitro and ex vitro. In soybean (Keck and Dilley, 1970), the light green mutant is viable and had about 50% of the pigment content of the wild type plant. Whereas the lethal yellow mutant had only 1±2% of the pigment content of the wild type plant. Chlorophyll-de®ciency reduced the rate of plant growth.

Table 4 Chlorophyll a/b ratio of wild type and chlorophyll-de®cient mutants of T. fasciculata var. fasciculata Phenotypes Chl a Chl b Chl total Chl a/b (mgmg1)a (mgmg1)b (mgmg1)c ratio Wild type 0.63 ad 0.56 a 1.19 a 1.1 Yellowish-green 0.10 b 0.05 b 0.15 b 2.0 Yellow 0.04 b 0.02 b 0.06 b 2.0 Significance *** *** *** Nae a Chl a: micrograms of chlorophyll a per milligram of fresh leaf tissue. b Chl b: micrograms of chlorophyll b per milligram of fresh leaf tissue. c Chl totalChl aChl b. d Mean separation within columns at p<0.05 by Duncan's multiple range test. e NA: not applicable. *** Signi®cant at p<0.0001. 236 Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240

The appression of thylakoids is correlated with a decrease in the Chl a/b ratio and the appearance of the light-harvesting Chl a/b-protein complex in the thylakoids (Hoober, 1984). The ultrastructure (Koh, 1998) and the Chl a/b ratios of the wild type and the chlorophyll-de®cient phenotypes of T. fasciculata var. fasciculata concur with Hoober's observation. Plants with a higher (Niels et al., 1978) or a lower (Vaughn et al., 1978; Kirchhoff et al., 1989) Chl a/b ratio than that of their respective wild type plants have been reported to be able to survive photoautotrophically. The more important determinant in photoauto- trophism may be the absolute amount of the total Chl a and Chl b possessed by a plant.

3.5. Mutagenesis

This is one of the ®rst studies to show that it is possible to obtain variegated T. fasciculata var. fasciculata through mutagenesis. T. fasciculata var. fasciculata was also successfully micropropagated. Mutagenesis can be ef®cient in causing leaf color mutation. Using 2 kR of X-rays, Broertjes and Lefferring (1972) obtained, among other mutants, one dark-green and two pale-green mutants from Kalanchoe `Annette'; one red-margined and six pale-green mutants from Kalanchoe `Josine'. The percentage of each mutant phenotype was not reported. With T. fasciculata var. fasciculata, at the combined dose of 9.3 kR, the combined gamma and thermal neutron radiation treatment had a 2.0% chlorophyll-de®cient seedlings, while the 10 kR gamma radiation treatment had no chlorophyll-de®cient seedlings. However, all the gamma radiation treatments of 21, 24, and 27 kR had greater chlorophyll-de®cient seedling percentages than their corresponding treatments in the combined gamma and thermal neutron radiations experiment. Most of the longer treatments in the combined gamma and thermal neutron radiation treatments were conducted discontinuously over a period of days and the intervening radiation-free time might have caused a reduction in the mutagenicity of these treatments. A combined gamma and thermal neutron radiation of 27.1 kR produced 30.8% lethality whereas a gamma dosage of 27 kR only produced 17.2% lethality. Percent lethality100[(No. of live seedlings/No. of viable seeds)Â100]. Gamma radiation at 21 and 27 kR produced the highest percentage (4.4%) of variegated seedlings. However, only sectorial and mericlinal seedlings were obtained. The majority of these variegated seedlings had only one to two variegated leaves whose variegation was not preserved in subsequent leaf development. No periclinal variegated seedlings were recovered. However, stable variegated bromeliads from other genera have been reported by other researchers. De Loose (1966) obtained a stable variegated seedling of Guzmania peacockii (Bromeliaceae) after irradiating the seeds with 33 Gy (1 Gy100 rad) of gamma radiation. The new cultivar was named G. peacockii `Edith'. Lapade et al. (1995) Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240 237 obtained six variegated pineapple plants by recurrently irradiating axillary buds with gamma rays ranging from 5 to 50 Gy. In the combined gamma and thermal neutron radiation experiment, the highest radiation dose used only produced a mortality of 34%. Consequently, only data from the lower range of combined gamma and thermal neutron radiations were obtained. In the 1.2% EMSÂ3 h treatment (average of 59% lethality), 15.8% of viable seeds produced chlorophyll-de®cient M0 (®rst mutagenized generation) seedlings, whereas in the 0.4% EMSÂ5 h treatment (average of 32% lethality), only

10.1% of the viable seeds produced chlorophyll-de®cient M0 seedlings. The EMS data presented in this report are comparable to those obtained by other authors who used EMS on other plant species. Bretagne-Sagnard et al. (1996) obtained

10% M1 (®rst generation of progeny of M0) ¯ax seedlings with various degrees of leaf variegation. Miller et al. (1984) obtained 18% plastome mutants in carrot seedlings and recovered seven stable periclinal chimeras from EMS treated carrot seeds: 1 GGW, 2 GWG and 4 GYG. In Capsicum annuum (Alcantara et al., 1996) a wide range of chlorophyll-de®cient phenotypes was obtained after EMS treated seeds. Lower plastome mutation percentages were reported for adventitious shoots derived from African violet leaf cuttings: 0.84±1.92% (Geier, 1983). Variegated T. fasciculata var. fasciculata seedlings with a white leaf margin or a white central stripe leaves were observed in different mutated seedlings (Koh, 1998). In monocots with three histogens, the presence of leaves with white margins indicates that the LI or both the LI and LII are albino, and the presence of leaves with a white center indicates that the LIII or both the LII and LIII are albino (Stewart and Dermen, 1979; Tilney-Bassett, 1986). The existence of the pure yellow and pure yellowish-green mutants of T. fasciculata var. fasciculata and their ability to produce true to type shoots in vitro indicated that their chlorophyll-de®ciency mutation affected all three histogens; hence, every histogen could potentially be mutated by the mutagens used.

3.5.1. Micropropagation of wild type T. fasciculata var. fasciculata Seed explants of T. fasciculata var. fasciculata produced spherical clumps of shoots in half-strength MS media with equimolar of BA and IBA between the range 0.3 and 10.0 mM. In terms of the number of shoots produced and the time required for multiplication, the multiplication media presented in this report compared favorably with published media for the micropropagation of bromeliads in the subfamily Tillandsioideae by seeds. A half-strength MS medium with equimolar BA and IBA at 1 and 10 mM produced more than 20 shoots per seedling in 4 months. A medium with equimolar 0.3 mM BA and IBA also proved to be excellent for the multiplication of T. ¯abellata seedlings (data not reported). Mercer and Kerbauy (1992) achieved a multiplication rate of about 22 shoots per seedling for Vriesia fosteriana after 3 months of culture in a solid 238 Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240

Knudson medium with 2.7 mMNAAand8.9mM BA. Rogers (1984) micropropagated several Tillandsia species including T. dyeriana through seeds by using different stage 1 and 2 media. The stage 1 medium contained coconut water and the stage 2 medium was solid vacin and went medium with 0.1 ppm NAA and 2 ppm BA. However, the rate of multiplication was not reported.

3.5.2. Micropropagation of mutant chlorophyll-de®cient T. fasciculata var. fasciculata phenotypes Each chlorophyll-de®cient T. fasciculata var. fasciculata seedling explant was considered to have multiplied if it produced two or more shoots. Although multiplication occurred in some of the variegated seedlings, the original variegation was not passed on to the newly formed shoots since the variegated seedlings were either sectorial or mericlinal chimeras. Each chlorophyll-de®cient phenotype from all the treatments within each experiment was treated as a group because very few chlorophyll-de®cient individuals were found in each treatment. The growth of the chlorophyll-de®cient mutants were very slow and usually each seedling produced fewer than ®ve shoots. Since the seedlings that grew in 0.5 mM BA and IBA still looked normal, it was likely that they would respond favorably to slightly higher levels of the same chemicals. To our knowledge there is no literature on the multiplication of bromeliads using seedling explants. All the yellow and yellowish-green seedlings that multiplied produced shoots that looked exactly like their respective original seedlings. The sorting out process that is typical of heteroplastic plants was not observed. Therefore, it is likely that nuclear mutations caused the chlorophyll de®ciency in these seedlings. This research shows that it is possible to obtain variegated T. fasciculata var. fasciculata through mutagenesis. Since many Tillandsia spp. produce a large number of seeds, mutagenesis of seeds from this genus is a viable option for creating new ornamental phenotypes. The production of stable variegated bromeliads in Tillandsia spp. through various mutagenesis methods should be explored since this genus rarely produces such plants naturally and there is a considerable consumer demand for such plants. An objective of this research was to produce T. fasciculata var. fasciculata seedlings with longitudinal leaf variegation. Although some variegated seedlings were produced, they were unstable sectorial or mericlinal chimeras. Sometimes such chimeras will produce lateral shoots that are periclinal chimeras, but this could take many generations of vegetative propagation and selection to achieve. It takes 6±10 years for a T. fasciculata var. fasciculata seedling to ¯ower. If time and resources were available, it would be of interest to grow some of these

M0 seedlings to maturity and observe their ¯oral morphology and physiological adaptations. Since most mutations are recessive and not visible in the M0,it would even be better to observe the M1 and M2 populations. In gamma ray and EMS-treated Papaver somniferum (Chauhan and Patra, 1993), chlorophyll- Y.C. Koh, F.T. Davies Jr./ Scientia Horticulturae 87 (2001) 225±240 239 de®cient seedlings occurred only in the M2 and not in the M1. It is likely that more chlorophyll-de®cient T. fasciculata var. fasciculata seedlings would be obtained in the M1 and M2 than in the M0.

4. Summary

1. Various mutagenic treatments generated transiently variegated seedlings of T. fasciculata var. fasciculata; 2. under the conditions employed no periclinal mutations were identi®ed; 3. wild types had greater chlorophyll a, b and total chlorophyll than mutant phenotypes; 4. mutagenically produced yellowish-green seedlings were able to grow photoautotrophically while the yellow ones were not. The procedures on the mutagenesis of T. fasciculata var. fasciculata elucidated in this research will be helpful to future researchers who are interested in the mutagenesis of Tillandsia spp., particularly since this is the ®rst report on the mutagenesis of a Tillandsia spp.

Acknowledgements

The authors wish to thank the Nuclear Science Center at Texas A&M University for providing funding (NSC Grant No. 97-0241) for irradiating the seeds used in the physical mutagenesis part of this research. We also thank R. Daniel Lineberger for his critical review of this paper.

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