Characterization and Flowering Behavior of Eleven Philippine ......Phalaenopsis species; ii) assess their flowering behavior, self-compatibility, and capsule setting under ambient

Keywords: breeding, butterfly orchids, irradiation, LD50, protocorms

Characterization and Flowering Behavior of Eleven Philippine Native Phalaenopsis Species and Gamma

Irradiation Effects on Phalaenopsis aphrodite

1Institute of Crop Science, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna 4031 Philippines

2Institute of Plant Breeding, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna 4031 Philippines

3National Academy of Science and Technology, 3rd Level, Science Heritage Building, DOST Complex, Gen. Santos Avenue, Bicutan, 1631 Taguig City, Metro Manila Philippines

*Corresponding author: [email protected] [email protected]

Pablito M. Magdalita1,2*, Alangelico O. San Pascual2, and Ruben L. Villareal3

Eleven species of Phalaenopsis or butterfly orchids collected from different places in the country were characterized for flower traits, leaf characters, growth habit, and capsule maturity, length, and width; evaluated for flowering behavior; and tested for self-compatibility and capsule setting under ambient conditions in Los Baños, Laguna, Philippines. Also, Phalaenopsis aphrodite was used for mutation breeding via gamma irradiation. Subjecting self-fertilized progenies to irradiation will generate mutants with potential for breeding and selection. The eleven Phalaenopsis species studied flowered consistently under ambient conditions for two years with degrees of self-compatibility and capsule setting varying from 3.8 to 50%. P. aphrodite and P. hieroglyphica embryos cultured in vitro germinated successfully at 80–90% within 3–4 wk after explanting. Germinating embryos of P. aphrodite subjected to different levels of gamma irradiation at 10, 15, 20, and 25 Gy responded differently to the treatments. The number of complete regenerants, and those regenerants with shoot only, also differed significantly among the treatments. In addition, leaf length, width, and thickness differed significantly among the treatments after 2 yr of growth. Early flowering was observed in two plants of P. aphrodite irradiated using 15 Gy. Normally, tissue culture-derived P. aphrodite seedlings flower 3 yr after potting out, but one plant flowered at 1 yr and 8 mo while the other did at 2 yr after potting out.

Philippine Journal of Science149 (S1): 1-10, Special Issue on Nuclear S&TISSN 0031 - 7683Date Received: 18 Mar 2019

INTRODUCTIONPhalaenopsis or butterfly orchid is a genus of orchids consisting of 63 species that occur primarily as epiphytes throughout South East Asia and the Pacific Islands (Padolina 2006). It is also found in India and in the north of Australia. Their showy, exotic flowers and ease

of cultivation have made Phalaenopsis one of the most widely traded groups of horticultural plants throughout the world. The center of distribution is in the Philippines and Indonesia. Phalaenopsis thrives in low elevations up to 1,500 m asl. Normally, they grow as epiphyte near running water (Cootes 2011). With the Philippines as a primary center of distribution of the genus, it was believed that 14 species and 17 varieties are endemic or native to the Philippines (Valmayor 1984). Endemic Phalaenopsis

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species have a restricted distribution and are confined to certain localities. For instance, the purple flower group P. schilleriana is found in Quezon, Laguna, Cavite, Bicol Region, Marinduque, and Eastern Visayas. Phalaenopsis sanderiana is localized in the provinces in Mindanao. Phalaenopsis stuartiana flourishes in Surigao, Agusan, Misamis, and Bukidnon. Phalaenopsis lindenii thrives in high elevation like Mt. Province and Nueva Vizcaya, while P. lueddemanniana is widely distributed in the islands (Valmayor 1984).

The greatest number of orchid species is found in Luzon – followed by Mindanao, Visayas, and Palawan. Apparently, the larger landmasses have a much wider area for catching minute wind-borne orchid seeds. Once an orchid becomes established on a landmass, small-range dispersal becomes quite easy and so does evolution to form new species by the process of mutation, hybridization, and selection by environmental differences (Valmayor 1984).

Outstanding endemic species that contributed to the improvement of the genus through hybridization are as follows: P. lueddemanniana, a potential donor for flower color markings such as bars and blotches as well as fragrance in the hybrids; P. schilleriana for its pale purple color and large number of flowers per stalk; and P. stuartiana for the red lips of hybrid flowers. These orchids have contributed greatly to the improvement and diversification of Phalaenopsis hybrids (Vergara 1997).

The chromosome number of orchid species ranges from 2n = 10 to as high as 2n = 200, with Phalaenopsis species having 2n = 38 chromosomes. Pollination in orchids is done artificially by transferring pollinium with the aid of tweezers or toothpick to the stigmatic surface. Shortly after pollination, lobes of the column often close around the stigma and then the flower wilts. Later, the peduncle expands and the ovary develops. The union of egg and sperm nuclei results to the formation of the embryo and, depending on the species, this fertilization may occur in several days to months from pollination. This embryo/seed has no endosperm. They need an artificial medium to grow and germinate; hence, tissue culture is necessary to continue their growth (PCARRD 1994).

The continuous destruction of all various types of forest and other areas for mining probably caused the extinction of narrowly endemic species, which will never be known to the scientific world or nature-lovers in general. Approximately 85% of the orchid species found in the Philippines are endemic and found nowhere else in the world, but they are being destroyed due to forest degradation, particularly the lowland rainforests. Hence, collecting and maintaining them will save their biodiversity in the country. Further improvement will enhance their breeding and commercial values. For instance, mutation breeding of Phalaenopsis

schilleriana, Vanda sanderiana, and Dendrobium was conducted at the Philippine Nuclear Research Institute (PNRI) in Quezon City (Lapade et al. 2001). Immature embryos derived from artificial pollination and cultured in an artificial medium (Knudson 1946) were used. Protocorms that developed were cultured on the same medium added with tomato and used as explants for irradiation studies. Irradiation of immature embryo with gamma rays at doses ranging 5–10 Gy increased the percent germination of P. schilleriana and Dendrobium. The protocorms of V. sanderiana irradiated at 10 Gy and grown in artificial culture medium developed plantlets that are bigger and more vigorous than those irradiated at 20 Gy, as well as the control plants. A decrease in seedling height was observed with increasing dose of gamma radiation (Lapade et al. 2001). This study aimed to i) characterize the flower traits, leaf characters, and growth habit of different Phalaenopsis species; ii) assess their flowering behavior, self-compatibility, and capsule setting under ambient conditions; and iii) assess their effects of irradiation on the in vitro regeneration, growth, and flowering of P. aphrodite.

MATERIALS AND METHODS

Characterization of 11 Phalaenopsis SpeciesEleven Phalaenopsis species previously established and grown at the Institute of Plant Breeding, College of Agriculture and Food Science, University of the Philippines Los Baños (UPLB), College, Laguna were utilized in the phenotypic characterization of the flowers, leaves, and growth habit. Identification of the different Phalaenopsis species was carried out based on the books “Orchidiana Philippiniana Vol. I” of Dr. Helen L. Valmayor (1984), “Philippine Orchid Species” by Mr. Jim Cootes (2011), and Co’s Digital Flora of the Philippines (Pelser et al. 2011 onwards). Flower color and labellum including the size were assessed. Ten (10) fully developed flowers from 5 to 10 plants were used. First two to three uppermost flowers per plant were measured. Leaf length was measured from the base to the tip of the leaf, while leaf width was measured on the middle portion of the leaf. The texture of the leaf was described. Two fully expanded, uppermost leaves from five plants were measured. The growth habit was also described accordingly. In this study, phytography was used to deal with the descriptive terminology of plants and their component parts for the purpose of providing an accurate and complete vocabulary for description and identification. Phytographic studies in this research provide the user with a vocabulary for intelligent communication about orchids and an understanding of the use of relative terms – and help the user observe the plants more critically and describe more precisely (Radford et al. 1974).

Special Issue on Nuclear Science and Technology Magdalita et al.: Phalaenopsis Characterization, Flowering, and γ Irradiation

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Flowering Behavior of Different Phalaenopsis SpeciesThe flowering behavior of the different Phalaenopsis species was monitored for 2 yr and the peak of flowering was recorded. Self-pollination of the 11 Phalaenopsis species was done to assess self-compatibility and capsule setting. A varying number of flowers (5–8) from each species were self-pollinated and the pollinated flower was bagged and labeled accordingly. The percent success of self-fertilization was assessed 3–4 wk after pollination using the following formula:

% success of self-fertilization = (number of self-pollinated flowers that developed into capsules / total number of self-pollinated flowers) x 100

The duration of maturity of capsules was determined by monitoring the yellowing of the capsules and abscission of the petal remnant at the tip of the capsules. Capsule length was measured using a ruler from the base to the tip of the capsule, while capsule width was taken from the middle of the capsule. The number of self-pollinated capsules that reached maturity was counted for each Phalaenopsis species.

Embryo Culture of 11 Phalaenopsis SpeciesMature self-pollinated capsule harvested from each Phalaenopsis species was washed with soap and water and dried in a Petri dish lined with sterile tissue paper inside the laminar flow hood. Capsules were sterilized by dipping in 70% (v/v) ethanol followed by flaming two to three times for 10 s. The dust-like seeds embedded on cottony-like structures were scraped off using sterile scalpel and were inoculated into an artificial culture medium based on Knudson (1946) with coconut water (150 mL L–1), tomato puree (5 g L–1), and sucrose (2.0%, w/v) as organic additives, and Bacto-agar (0.8%, w/v; Difco Laboratories, Detroit, MI, USA) to solidify the medium. The pH of the medium was adjusted to pH 5.65 using 0.1 M NaOH or HCl before autoclaving at 15 psi for 20 min. After inoculation, culture vessels were covered immediately with sterile autoclavable plastic secured with a rubber band. The cultures were incubated for 3–4 mo and then subcultured on the same medium under 8 hr light and 16 hr darkness. They were incubated in the culture shelf with a fluorescent tube as a light source at photosynthetic photon flux density (PPFD) of 120 µmol m–2s–1 at a prevailing temperature of 25 ± 1 °C. The number of regenerants from each Phalaenopsis species was counted 6–8 mo from initial inoculation onto the artificial culture medium.

Irradiation Studies on Phalaenopsis aphroditeSix-month-old germinating embryos of P. aphrodite were exposed to four doses of gamma radiation – namely

10, 15, 20, and 25 Gy serving as treatments. They were irradiated at the PNRI. Untreated germinated embryos were sub-cultured and incubated in a similar manner serve as the control. After irradiation, sub-culturing in a fresh artificial culture medium followed and then incubated in the culture shelf. The cultures were grown for 6–8 mo from initial culture and assessed for different growth parameters such as the number of regenerants with complete and incomplete (shoot only) regeneration, and length and width of the fully expanded leaf.

Seedlings were potted out in 12-cm diameter clay pots containing chopped tree fern bark and acclimatized inside the net house provided with 60% shade using a black net with plastic roofing. The seedlings were not watered for 2–3 d, after which the seedlings were misted with a dilute solution of fungicide and fertilized weekly with a water-soluble fertilizer. After 2 yr, growth of seedlings inside the net house was assessed. Leaf length and leaf width were determined, while leaf thickness was measured using a micrometer caliper.

Statistical Design and AnalysisRadiation experiments and growth of the irradiated seedlings of P. aphrodite was conducted in completely randomized design (CRD) with five replications and five experimental culture vessels in each replicate. Four gamma irradiation doses (10, 15, 20, and 25 Gy) doses and an untreated as control served as treatments. In assessing the regenerated plants of P. aphrodite after 2 yr of growth in the net house, CRD with 5 replications and 20 sample plants in each replicate was used. The data gathered were subjected to one-way ANOVA using F-test. Significant differences between treatment means were detected using the least significant difference (LSD) at 0.05 level of significance. Descriptive statistics such as the percent, frequency, or count data for the different Phalaenopsis species were used.

RESULTS AND DISCUSSION

Characterization of Flowers, Leaves, and Plant HabitCharacterization of different Phalaenopsis orchids was done, and morphological descriptions of each Phalaenopsis species were described in Table 1 and their flowers were presented in Figure 1.

Flowering Behavior of Different Phalaenopsis SpeciesEleven Phalaenopsis species flowered under ambient conditions at different times of the year for two years


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(Figure 2). Year-round flowering was observed on P. equestris. This characteristic makes P. equestris a good candidate parent in developing a variety or hybrid that flowers year-round. The success of self-compatibility on the different Phalaenopsis species ranged 0–50% (Table 2). Ten different Phalaenopsis species successfully self-fertilized, indicating that they were self-compatible (Table 2). The highest percent success of self-fertilization was obtained with P. equestris and P. hieroglyphica at 50% – followed by P. schilleriana, P. lindenii, and P. stuartiana at 30%. However, the lowest percent success of self-fertilization was obtained with P. amabilis at 3.8%. This is the first report on the assessment of self-compatibility by hand pollination among different Phalaenopsis species

in the Philippines. Under natural conditions, P. equestris is self-compatible since it produced capsules throughout the year under ambient conditions. Successfully self-fertilized Phalaenopsis flower had its ovary enlarged and became yellowish green for the first two weeks after successful self-pollination. This ovary started to enlarge and turned green, while petals and sepals started to become brown (Figure 2).

Characterization of Capsule A mature Phalaenopsis capsule is yellowish and plump with its petal remaining, which eventually turns brown and later starts to disappear. However, P. pulchra did not set

Table 1. Phenotypic characteristics of the different Phalaenopsis species that flowered under ambient conditions in Los Baños, Laguna, Philippines.

Phalaenopsis species Flower characters Leaf characters Growth habit

P. amabilis (L.) Blume Milky white, 7.5 cm diameter, labellum is white with yellow and brown markings

Green, Leathery, 35 cm long, 10 cm wide

Pendent and repent

P. aphrodite Rchb.f. Milky-white, 6 cm diameter, labellum is white with yellow and red markings

Leathery, 30 cm long, 7 cm wide, Green in the adaxial and green with purplish tinge on abaxial side of leaves

Pendent and repent

P. equestris (Schauer) Rchb.f. Light violet to pinkish, star-shaped, 2.5 cm diameter

Green, Very leathery, 20 cm long, 4 cm wide

Pendent and repent

P. fasciata Rchb.f. Flower has creamy yellow background and cinnamon brown barring, star-shaped, 4 cm diameter

Light green, Leathery, Bright green, 20 cm long, 4 cm wide

Upright, monopodial

P. hieroglyphica (Rchb.f.) Sweet Creamy white flower with hieroglyphic-like maroon markings, 4 cm diameter

Green, Leathery, Plain green and shiny, 25 cm long, 5 cm wide

Upright, monopodial

P. x intermedia Lindl. White flower with reddish-pink labellum, 3 cm diameter

Green above and purplish underneath, Leathery, Dark green with purple coloration on the underside

Upright, monopodial

P. lindenii Loher White flower with pale pink suffusion, labellum is darker pink at apex with a number of radiating darker rose-colored lines at base, 3 cm diameter

Dull green to mottled silvery green, Leathery, Dark green with silvery-white mottling

Upright, monopodial

P..lueddemanniana Rchb.f. Ivory white flower traversed with magenta bars, labellum is carmine and yellow, 4 cm diameter

Pale green, Leathery, Green leaves, 25 cm long, 6 cm wide

Upright, monopodial

P. pulchra (Rchb.f.) Sweet Solid purple, star-shaped, labellum is purple with bright yellow lateral lobes, 3 cm diameter

Green, Leathery, 26 cm long, 7 cm wide

Upright, monopodial

P. schilleriana Rchb.f. Violet to purplish pink, labellum lateral lobes upright but flaring outside, mid-lobe is obovate with two hair-like appendages on its tip, 5 cm diameter

Dark green and marbled with silvery gray upper surface and purplish-green underneath, Very leathery, Very attractively mottled or has tiger-like patterns, 40 cm long, 8 cm wide

Pendent and repent

P. stuartiana Rchb.f. Creamy white, inner half of the lateral sepals are beautifully spotted with dark cinnamon blotches over a base color of sulfur yellow, 4 cm diameter

Green, Leathery, Mottled or has tiger-like patterns, 25 cm long, 7 cm wide

Upright, monopodial


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any capsule. This may suggest self-incompatibility or may require lower temperatures in higher elevation or may need natural pollinating agents for capsule setting. Except for naturally self-fertilized orchids, all others are pollinated by animals including bees, butterflies and moth, birds, and ants – each of which is attracted in different ways (Pridgeon 1992). Ten out of the 11 (90.91%) Phalaenopsis species that flowered under ambient conditions produced self-fertilized capsules after assisted pollination.

Capsule MaturityCapsules of 10 Phalaenopsis species matured 3–5 mo after self-fertilization (Table 3). These species included P. schilleriana, P. equestris, P. aphrodite, P. lueddemanniana, P. amabilis, P. x intermedia, and P. stuartiana. The capsules of P. fasciata matured in 4–5 mo

after self-fertilization. This result jibed with the previous report that capsules of P. amabilis matured in 2–4 mo (Lapade et al. 1996). It has been reported that the maturity of an orchid capsule may take several months (PCARRD 1994). In contrast, capsules of P. hieroglyphica matured in 8–9 mo, while capsules of P. lindenii matured in 9–10 mo – indicating that P. hieroglyphica and P. lindenii had the longest capsule maturity period among the 10 Phalaenopsis species. In general, mature capsules were in full-balloon stage, yellowish in color, and with petals and sepals completely dried, turned brown, and then disappeared gradually.

Length and Width of CapsuleThe 10 different Phalaenopsis species produced capsules after assisted pollination with varying length and width

Figure 1. The flowers of the different Phalaenopsis species that bloomed under ambient conditions in Los Baños, Laguna, Philippines.

Figure 2. Flowering months of 11 Phalaenopsis species grown under ambient conditions in Los Baños, Laguna, Philippines.


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(Table 3). The longest self-pollinated capsules among the 10 Phalaenopsis species were shown by P. schilleriana, P. amabilis, and P. aphrodite. On the other hand, P. lindenii had the shortest self-pollinated capsule. Eight Phalaenopsis species had an average capsule width (5.0–5.6 mm) – including P. lindenii, P. fasciata, P. stuartiana, P. schilleriana, P. leuddemanniana, P. x intermedia, P. aphrodite, and P. amabilis. P. hieroglyphica produced the widest self-pollinated capsule, while P. equestris had the narrowest capsule among the 10 Phalaenopsis species evaluated. These basic data on characteristics and maturity of self-pollinated capsules of different Phalaenopsis species provide useful information for orchid scientists and growers as this may serve as a guide in the harvesting of self-pollinated capsules for embryo culture. It has been reported that the most efficient way of multiplying orchids

is by using tissue culture via embryo culture of seeds (Valmayor 1984, PCARRD 1994, Nayak et al. 2006).

The 10 Phalaenopsis species developed capsules in varying numbers via hand pollination (Table 4). P. aphrodite developed the highest number of self-pollinated capsules (Figure 2), followed by P. hieroglyphica. This result jibed with the previous observations of orchid hunters from Marinduque, Philippines that P. aphrodite is a prolific bearer of capsules even under natural conditions in the forests (Magdalita 2016). This further explains why P. aphrodite is widely available naturally in forested areas. Other Phalaenopsis species including P. x intermedia, P. stuartiana, P. fasciata, P. lindenii, P. lueddemanniana, P. equestris, and P. schilleriana produced self-fertilized capsules; however, P. amabilis produced the lowest number of self-fertilized capsules.

The mature capsule contains dust-like seeds, which are embryos devoid of endosperm that are embedded in cottony-like structures and are dirty white to creamy white (Figure 3b). Capsules were characterized for their length and width (Table 3). Different collections of Phalaenopsis species grown in the net house (Figure 3a) were used in pollination. Flowers with successful self-fertilization produced mature capsules (Figure 3b). The embryos obtained from the mature capsules germinated 1–2 mo after inoculation on the artificial culture medium. Initially, they germinate for 3–5 mo after explanting (Figure 3c). These seeds germinated and developed into plants 9–10 mo after inoculation on the culture medium. The highest number of plants was obtained from P. hieroglyphica, followed by P. aphrodite (Table 4). P. equestris was followed by P. amabilis. No plants were produced from cultures of P. lueddemanniana, P. lindenii, and P. fasciata because embryos did not germinate on the artificial culture medium used – suggesting that other media formulations may be needed to germinate these species.

Table 3. Capsule characteristics of different Phalaenopsis species.

Phalaenopsis speciesDuration of pollination to capsule maturity in Los

Baños, Laguna (mo)

Ave. length of capsules (mm)

Ave. width of capsules (mm)

P. schilleriana 3–4 85.63 ± 20.43 5.2 ± 1.58

P. equestris 3–4 43.00 ± 14.39 4.5 ± 1.22

P. aphrodite 3–4 79.00 ± 29.24 5.3 ± 1.43

P. lueddemanniana 3–4 51.5 ± 3.53 5.2 ± 1.7

P. amabilis 3–5 82.33 ± 24.83 5.6 ± 1.97

P. lindenii 9–10 31.33 ± 4.04 5.0 ± 0.06

P. fasciata 4–5 42.67 ± 7.51 5.0 ± 0.025

P. hieroglyphica 8–9 42.33 ± 8.74 8.3 ± 0.21

P. x intermedia 3–4 77.5 ± 16.58 5.3 ± 0.48

P. stuartiana 3–5 74 ± 7.81 5.1 ± 0.15

Table 2. Phalaenopsis species collected, the number of self-pollinated flowers, and the percent success of self-fertilization of the 11 Phalaenopsis species grown under greenhouse conditions in Los Baños, Laguna, Philippines.

Phalaenopsis species

No. of self-pollinated flowers

Percent success of self-fertilization

P. schilleriana 50 30

P. equestris 20 50

P. aphrodite 130 4

P. lueddemanniana 20 15

P. amabilis 30 3.8

P. lindenii 10 30

P. fasciata 6 17

P. hieroglyphica 8 50

P. x intermedia 40 13

P. stuartiana 20 30

P. pulchra 20 0


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Irradiation Study on Phalaenopsis aphroditeGerminating embryos of P. aphrodite that are 4–5 mo old and irradiated with different doses of gamma rays developed into regenerants. In preliminary experiments, LD50 was determined at 12 Gy. Significant differences were detected among the five irradiation treatments. In addition, significant differences between treatment means for the five parameters evaluated were also detected (Table 5). The number of regenerants decreased with increasing doses of radiation (Table 5). The untreated had significantly the highest mean number of regenerants followed by 10 Gy and 15 Gy, suggesting that lower doses of gamma radiation promoted the development of regenerants in P. aphrodite. This jibed with a report on irradiated callus of yam (Dioscorea alata L.) cv. 'Kinampay' that callus growth was stimulated when root explants were irradiated with lower doses of gamma rays at 5 and 10 Gy (Lapade et al. 1993).

However, in the present study, 25 Gy had the lowest number of regenerated plantlets – suggesting that higher doses of radiation could be inhibitory to growth. Similarly, the number of complete regenerants and incomplete regenerants (shoot only) decreases significantly with increasing amount of irradiation (Table 5). In terms of leaf growth of irradiated plants, leaf length and leaf width also decrease significantly with increasing amount of irradiation.

In general, the number of regenerants and their leaf growth decreases with increasing irradiation dose (Table 5). The present result indicates that as the dose of irradiation is increased, the development of regenerants in P. aphrodite decreases. This could be due to the lethal effects of irradiation on the developing seedling. Medina et al. (2005) reported that ionizing radiation can – either by

Table 4. The number of mature capsules that developed on different Phalaenopsis species.

Phalaenopsis species

No. of mature capsules

harvested

No. of plants produced by

embryo culture

P. schilleriana 9 10

P. equestris 4 21

P. aphrodite 47 50

P. lueddemanniana 4 0

P. amabilis 2 20

P. lindenii 4 0

P. fasciata 3 0

P. hieroglyphica 10 150

P. x intermedia 3 3

P. stuartiana 3 16

Figure 3. Different Phalaenopsis species grow inside the net house and used for pollination (A); the self-fertilized capsules of Phalaenopsis aphrodite and the mature capsule containing the seeds that are dust-like and embedded in cottony-like structures (B); and the embryo cultures being kept in the tissue culture laboratory (C).

direct or indirect action – break chromosomes leading to structural rearrangements and even loss of a part or whole chromosome that damage the cells and could bring cell death, hence prohibiting growth. The present result is like the previous finding in Vanda sanderiana where there was a decrease in seedling height as the dose of gamma radiation is increased (Lapade et al. 2001).

An important observation in the gamma-irradiated P. aphrodite is the induction of early flowering in a plant by 1 yr and 8 mo from the initial culture of germinating embryos irradiated with 15 Gy (Figure 4A). There are two plants that flowered in this treatment, but none in


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Table 5. Influence of irradiation on the regeneration and seedling characteristics of Phalaenopsis aphrodite.

Irradiation treatments (Gy)

No. of plantlets regenerated

No. of complete regenerants produced

No. of incomplete regenerants (shoot only)

Length of fully expanded leaf (mm)

Width of fully expanded leaf (mm)

Untreated 71.6 ± 19.62 a 30.0 ± 8.05 a 41.7 ± 15.51 a 19.6 ± 5.26 a 10.3 ± 3.33 a

10 29.1 ± 49.88 b 20.1 ± 4.91 b 12.7 ± 23.93 b 18.7 ± 6.59 a 10.3 ± 2.71 a

15 24.6 ± 7.35 c 16.4 ± 3.58 c 4.4 ± 4.10 c 15.9 ± 4.04 b 9.4 ± 2.13 a

20 18.8 ± 8.47 d 14.3 ± 5.41 c 4.0 ± 3.36 c 14.3 ± 3.64 b 9.1 ± 1.43 a

25 14.0 ± 5.34 d 12.6 ± 4.22 c 1.4 ± 1.81 c 14.2 ± 5.00 b 8.0 ± 3.39 a

*Values with the same letter are not significantly different at 0.05 level LSD.

Figure 4. Flowering plants of Phalaenopsis aphrodite whose germinated embryos were irradiated with 15 vGy Gamma radiation. The first plant flowered 1 yr and 8 mo after protocorms were irradiated (A), while the other plant flowered 2 yr after germinated embryos were irradiated (B).

the control and in the other treatments (Figure 4B). Since orchids derived from seeds normally flower in three years’ time, the induction of early flowering in these two plants may suggest that irradiation using 15 Gy could shorten juvenility of P. aphrodite. Possibly, mutagenic radiation had some stimulating effects in plants, and this effect could be due to the destruction of inhibitors and release of activators that induces early flowering. While it is a preliminary finding, to our knowledge, this is the first report on the induction of early flowering of P. aphrodite irradiated with gamma rays at 15 Gy. In a similar experiment, induction of early flowering by 15 months was observed in gamma-irradiated Dendrocalamus strictus; however, this species normally flowers 20–40 years after planting without irradiation treatments (Kapoor and Sharma 1992). Also, induction of early maturation

in soybean (Glycine max) by 1 wk earlier than the non-irradiated plants was achieved using gamma irradiation at 20 kr (Barrida and Medina 2003). Furthermore, induction of early flowering was also achieved in chickpea (Cicer arientum) mutants exposed to lower concentrations of mutagenic chemicals such as ethyl methyl sulfonate and salicylic acid, but higher concentrations of these mutagens caused late flowering (Kashid and Moore 2016).

In terms of regeneration, the untreated germinating embryos significantly had the highest number of complete regenerants, followed by germinating embryos irradiated at 10 Gy. The lowest number of complete regenerants was obtained at 25 Gy. The same trend was observed for the number of incomplete regenerants (with shoot only) (Table 5). The mean number of incomplete regenerant (with shoot only) was also significantly highest in the untreated plants followed by 10 Gy, and the lowest number of complete regenerants was observed in 25 Gy. These results again suggest that increasing irradiation dose is inhibitory to the growth of P. aphrodite. This result corroborated with the previous finding that gamma irradiation at higher doses inhibited the growth of cashew (Anacardium occidentale L.) and mangosteen (Garcinia mangostana L.) as it caused a decrease in seedling height (Lapade et al. 2004). Furthermore, the longest and widest expanded leaves were observed in untreated plants while the shortest and narrowest expanded leaves were observed in 25 Gy, but significant differences between treatments for these characters were not detected.

Further observations on the second year of growth of irradiated P. aphrodite, indicated that lower doses of gamma radiation enhanced the growth of P. aphrodite as suggested by the growth of the leaves (Table 6). Consistently, the untreated and the plants treated with 10 Gy radiation dose had plants with significantly longer and wider leaves than those treated with higher doses of radiation. This finding corroborated with the previous report that gamma radiation at lower doses promoted growth by promoting seedling height and earliness to capsule formation in Cattleya alliances (Thammasiri 1996) and in soybeans (Glycine max L.) (Barrida and


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Medina 2003). It was purported that in gamma-irradiated plants, nutrient uptake was enhanced while the gas exchange was increased but not in non-radiated plants; moreover, there were increased activities on the key photosynthetic enzyme RuBisCO and on nitrate reductase, which is essential in nitrogen assimilation (Babu et al. 2012). In contrast, higher doses of radiation (15, 20, and 25 Gy) had plants with significantly shorter and narrower leaves, indicating growth was decreased (Table 6). One reason for the decreased performance of plants exposed to high doses of gamma radiation is the decrease in carbon and nutrient assimilation efficiency, which later translates into decreased plant health and growth (Babu et al. 2012).

Significant differences between different treatments existed for leaf thickness (Table 6). Untreated plants had significantly thicker leaves followed by those treated with 10 Gy radiation dose. However, higher doses of gamma irradiation significantly decreased leaf thickness in two-year-old P. aphrodite. The results indicated that higher radiation doses of 15–25 Gy could have some negative effects on plant growth – suggesting that as the radiation dose is increased, lethality is manifested. Since gamma radiation is a type of linear energy transfer radiation, it is dose-dependent where the effect of exposure to high dosage could cause an increased lethality to the organism. According to Mitchel (2006), the ability to resist radiation-induced damage is very much evolutionary in nature, and it is manifested in the DNA repair mechanisms of different species. As expected, DNA damage resistance of plants imposes dose-dependent lethality brought by gamma irradiation. As irradiation dosage is increased, an elevated amount of irradiation would be tantamount to greater areas of damage in the plant, whether it is single- or double-stranded mutations that may occur (Mitchel 2006).

CONCLUSIONEleven Phalaenopsis species were characterized for flower and leaf characters, flowering ability under ambient conditions, self-compatibility, capsule setting and characters, embryo germinability, and growth on an

artificial culture medium and irradiation effects of gamma irradiation of P. aphrodite. All of the 11 Phalaenopsis species flowered under ambient conditions consistently for two years. Self-compatibility and capsule setting varied among species. However, P. pulchra is self-incompatible and did not set capsules. Capsule maturity also varied. Phalaenopsis schilleriana produced the longest capsule, while P. lindenii had the shortest capsule. P. hieroglyphica had the widest capsule, while P. equestris had the narrowest capsule. P. aphrodite produced the greatest number of capsules, while P. amabilis produced the fewest. Mature dust-like seeds embedded in cottony-like structures germinated within 1–2 mo at varying degrees in the artificial culture medium and developed into plants 9–10 mo after germination. Regenerated plants were obtained from P. hieroglyphica, P. x intermedia, P. schilleriana, P. stuartiana, P. amabilis, P. equestris, and P. aphrodite. However, P. lueddemanniana, P. lindenii, and P. fasciata embryos did not germinate.

Germinated P. aphrodite seeds subjected to different levels of gamma radiation at 10, 15, 20, and 25 Gy responded differently to treatments. The number of complete regenerants and the number of regenerants with shoot only also differed significantly among treatments. Leaf length, width, and thickness were significantly different among treatments after two years of growth.

In P. aphrodite, early flowering was induced in two plants irradiated with 15 Gy. One flowered 1 yr and 8 mo and the other 2 yr after potting out. Normally, tissue culture-derived P. aphrodite flowers three years after potting out. Overall, irradiation significantly affected the regeneration and growth of P. aphrodite and further induced early flowering compared to the control.

ACKNOWLEDGMENTSThe UPLB Basic Research provided funds to the project “Embryo Culture and Irradiation Studies to Improve Some Native Phalaenopsis Species” from where this article was based. The authors would like to thank Mr. Jonard C.

Table 6. Leaf measurements of Phalaenopsis aphrodite 2 yr after irradiation with different levels of gamma rays.

Irradiation treatments (Gy) Leaf length (cm) Leaf width (cm) Leaf thickness (cm)

Untreated 13.56 ± 2.28 a 5.28 ± 0.63 a 1.57 ± 0.22 a

10 11.14 ± 2.09 a 4.78 ± 0.82 a 1.47 ± 0.15 a

15 10.60 ± 1.92 ab 4.60 ± 0.58 ab 0.43 ± 0.17 b

20 9.78 ± 0.93 bc 4.42 ± 0.31 ab 0.41 ± 0.11 b

25 7.78 ± 1.26 c 3.60 ± 0.34 b 0.23 ± 0.23 b

*Values with the same letter are not significantly different at 0.05 level LSD.


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Valdoz, Mr. Marcelino T. Gregorio, Ms. Besseluz DLC. Abayon, Ms. Mercedes A. Dreje, and Ms. Maria Fe H. Cayaban for their assistance rendered to this project.

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Characterization and Flowering Behavior of Eleven Philippine ......Phalaenopsis species; ii) assess their flowering behavior, self-compatibility, and capsule setting under ambient