Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol87.Issue3.2001:
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 chlorophyllde®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 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 6 6 - 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 overcollection. 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 handpollinated.
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 mÿ2 sÿ1. The low and high
average mean temperature was 238C and 3228C, 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 hÿ1. 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 hÿ1 accompanied by 824.5 rad hÿ1
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% EMS5 h (control),
1.2% EMS3 h and 0.4% EMS5 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 mm94 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 lÿ1 sucrose, 8 g lÿ1 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 mm20 mm plastic petri
dishes containing 40 ml of medium with half-strength MS salts and vitamins,
15 g lÿ1 sucrose, 8 g lÿ1 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 mM BA
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 mm94 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) whitemargined 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)
Significance
Yellow seedlings
per treatment (%)
Yellowish-green
seedlings per
treatment (%)
Variegated
seedlings per
treatment (%)
Total chlorophylldeficient seedlings
per treatment (%)b
4.01.0c,d
5.21.0
6.80.9
11.22.4
0.80.9
0.80.9
4.42.6
26.81.8
100.00
00
00
00
00
00
00
00
0.80.5
00
0
0
0
0
0
0.8
0.8
0
0
00
00
0.40.4
0.80.5
00
00
1.60.6
3.21.0
00
00
00
00
1.20.6
1.60.9
4.41.1
2.40.6
4.40.9
00
00
00
0.80.5
2.00.9
1.60.9
5.20.9
4.81.0
8.41.5
00
***
*
NSe
***
***
***
0
0
0.4
0
0.4
0.4
0
0
0
0
0
0
0
0.4
0.4
0.4
0.8
1.2
1.2
1.6
1.2
2.0
2.4
1.2
00
0.40.4
1.20.6
0.40.4
00
0.40.4
0.40.4
1.60.7
1.20.6
0.80.5
1.20.6
1.60.6
0
0.8
2.0
0.8
2.0
2.8
2.0
3.2
2.8
2.8
3.6
2.8
Gamma and thermal neutron radiations (kR)
0 (control)
4.41.3
0
0.1f (0.9)g
8.81.2
0
0.3 (2.8)
11.21.8
0
0.7 (5.6)
14.02.3
0
1.1 (9.3)
14.81.7
0.8
1.3 (11.2)
12.02.6
0.8
1.7 (14.0)
8.82.4
0.4
2 (16.8)
12.82.3
0
2.3 (19.6)
11.62.1
0.4
2.7 (22.4)
30.83.0
0
3 (25.2)
34.01.4
0
3.1 (26.1)
24.01.9
0
231
Albino seedlings
per treatment (%)
Y.C. Koh, F.T. Davies Jr. / Scientia Horticulturae 87 (2001) 225±240
Gamma radiation
0 (control)
10
12
15
18
21
24
27
29
Mortality per
treatment (%)a
232
Table 1 (Continued )
Mortality per
treatment (%)a
Albino seedlings
per treatment (%)
Yellow seedlings
per treatment (%)
Yellowish-green
seedlings per
treatment (%)
Variegated
seedlings per
treatment (%)
Total chlorophylldeficient seedlings
per treatment (%)b
3.2 (27.1)
29.63.3
0
0
2.0
2.40.7
4.4
Significance
***
NS
NS
NS
*
NS
84.20.6h
93.50.4
89.10.5
0
0
0
0
0.5
0.5
00
15.01.5
11.00.9
0
0.3
0.2
00
15.81.5
11.60.9
***
NS
NS
***
NS
***
%EMSh
0%5 h
1.2%3 h
0.4%5 h
Significance
a
Percent mortality(No. of dead seeds/No. of initial seeds)100.
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
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 chlorophyllde®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 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 6 6 - 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 overcollection. 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 handpollinated.
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 mÿ2 sÿ1. The low and high
average mean temperature was 238C and 3228C, 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 hÿ1. 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 hÿ1 accompanied by 824.5 rad hÿ1
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% EMS5 h (control),
1.2% EMS3 h and 0.4% EMS5 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 mm94 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 lÿ1 sucrose, 8 g lÿ1 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 mm20 mm plastic petri
dishes containing 40 ml of medium with half-strength MS salts and vitamins,
15 g lÿ1 sucrose, 8 g lÿ1 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 mM BA
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 mm94 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) whitemargined 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)
Significance
Yellow seedlings
per treatment (%)
Yellowish-green
seedlings per
treatment (%)
Variegated
seedlings per
treatment (%)
Total chlorophylldeficient seedlings
per treatment (%)b
4.01.0c,d
5.21.0
6.80.9
11.22.4
0.80.9
0.80.9
4.42.6
26.81.8
100.00
00
00
00
00
00
00
00
0.80.5
00
0
0
0
0
0
0.8
0.8
0
0
00
00
0.40.4
0.80.5
00
00
1.60.6
3.21.0
00
00
00
00
1.20.6
1.60.9
4.41.1
2.40.6
4.40.9
00
00
00
0.80.5
2.00.9
1.60.9
5.20.9
4.81.0
8.41.5
00
***
*
NSe
***
***
***
0
0
0.4
0
0.4
0.4
0
0
0
0
0
0
0
0.4
0.4
0.4
0.8
1.2
1.2
1.6
1.2
2.0
2.4
1.2
00
0.40.4
1.20.6
0.40.4
00
0.40.4
0.40.4
1.60.7
1.20.6
0.80.5
1.20.6
1.60.6
0
0.8
2.0
0.8
2.0
2.8
2.0
3.2
2.8
2.8
3.6
2.8
Gamma and thermal neutron radiations (kR)
0 (control)
4.41.3
0
0.1f (0.9)g
8.81.2
0
0.3 (2.8)
11.21.8
0
0.7 (5.6)
14.02.3
0
1.1 (9.3)
14.81.7
0.8
1.3 (11.2)
12.02.6
0.8
1.7 (14.0)
8.82.4
0.4
2 (16.8)
12.82.3
0
2.3 (19.6)
11.62.1
0.4
2.7 (22.4)
30.83.0
0
3 (25.2)
34.01.4
0
3.1 (26.1)
24.01.9
0
231
Albino seedlings
per treatment (%)
Y.C. Koh, F.T. Davies Jr. / Scientia Horticulturae 87 (2001) 225±240
Gamma radiation
0 (control)
10
12
15
18
21
24
27
29
Mortality per
treatment (%)a
232
Table 1 (Continued )
Mortality per
treatment (%)a
Albino seedlings
per treatment (%)
Yellow seedlings
per treatment (%)
Yellowish-green
seedlings per
treatment (%)
Variegated
seedlings per
treatment (%)
Total chlorophylldeficient seedlings
per treatment (%)b
3.2 (27.1)
29.63.3
0
0
2.0
2.40.7
4.4
Significance
***
NS
NS
NS
*
NS
84.20.6h
93.50.4
89.10.5
0
0
0
0
0.5
0.5
00
15.01.5
11.00.9
0
0.3
0.2
00
15.81.5
11.60.9
***
NS
NS
***
NS
***
%EMSh
0%5 h
1.2%3 h
0.4%5 h
Significance
a
Percent mortality(No. of dead seeds/No. of initial seeds)100.
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