Philippine Journal of Science 149 (S1): 1-10, Special Issue on Nuclear S&T ISSN 0031 - 7683 Date Received: 18 Mar 2019

Characterization and Flowering Behavior of Eleven Philippine Native Species and Gamma Irradiation Effects on

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

1Institute of Crop Science, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna 4031 Philippines 2Institute of 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

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 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.

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

INTRODUCTION of cultivation have made Phalaenopsis one of the most widely traded groups of horticultural plants throughout Phalaenopsis or butterfly orchid is a of orchids the world. The center of distribution is in the Philippines consisting of 63 species that occur primarily as epiphytes and Indonesia. Phalaenopsis thrives in low elevations throughout South East Asia and the Pacific Islands up to 1,500 m asl. Normally, they grow as epiphyte near (Padolina 2006). It is also found in India and in the running water (Cootes 2011). With the Philippines as a north of Australia. Their showy, exotic flowers and ease primary center of distribution of the genus, it was believed that 14 species and 17 varieties are endemic or native to *Corresponding author: [email protected] [email protected] the Philippines (Valmayor 1984). Endemic Phalaenopsis

1 Special Issue on Nuclear Science and Technology Magdalita et al.: Phalaenopsis Characterization, Flowering, and γ Irradiation species have a restricted distribution and are confined to schilleriana, sanderiana, and Dendrobium was certain localities. For instance, the purple flower group conducted at the Philippine Nuclear Research Institute P. schilleriana is found in Quezon, Laguna, Cavite, Bicol (PNRI) in Quezon City (Lapade et al. 2001). Immature Region, Marinduque, and Eastern Visayas. Phalaenopsis embryos derived from artificial pollination and cultured sanderiana is localized in the provinces in Mindanao. in an artificial medium (Knudson 1946) were used. flourishes in Surigao, Agusan, Protocorms that developed were cultured on the same Misamis, and Bukidnon. thrives medium added with tomato and used as explants for in high elevation like Mt. Province and Nueva Vizcaya, irradiation studies. Irradiation of immature embryo with while P. lueddemanniana is widely distributed in the gamma rays at doses ranging 5–10 Gy increased the islands (Valmayor 1984). percent germination of P. schilleriana and Dendrobium. The protocorms of V. sanderiana irradiated at 10 Gy and The greatest number of orchid species is found in Luzon – grown in artificial culture medium developed plantlets that followed by Mindanao, Visayas, and Palawan. Apparently, are bigger and more vigorous than those irradiated at 20 Gy, the larger landmasses have a much wider area for catching as well as the control plants. A decrease in seedling height minute wind-borne orchid seeds. Once an orchid becomes was observed with increasing dose of gamma radiation established on a landmass, small-range dispersal becomes (Lapade et al. 2001). This study aimed to i) characterize the quite easy and so does evolution to form new species by flower traits, leaf characters, and growth habit of different the process of mutation, hybridization, and selection by Phalaenopsis species; ii) assess their flowering behavior, environmental differences (Valmayor 1984). self-compatibility, and capsule setting under ambient Outstanding endemic species that contributed to the conditions; and iii) assess their effects of irradiation on the improvement of the genus through hybridization are in vitro regeneration, growth, and flowering of P. aphrodite. 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 MATERIALS AND METHODS P. stuartiana for the red lips of hybrid flowers. These orchids have contributed greatly to the improvement and Characterization of 11 Phalaenopsis Species diversification of Phalaenopsis hybrids (Vergara 1997). Eleven Phalaenopsis species previously established The chromosome number of orchid species ranges from and grown at the Institute of Plant Breeding, College of 2n = 10 to as high as 2n = 200, with Phalaenopsis species Agriculture and Food Science, University of the Philippines having 2n = 38 chromosomes. Pollination in orchids is Los Baños (UPLB), College, Laguna were utilized in the done artificially by transferring pollinium with the aid of phenotypic characterization of the flowers, leaves, and tweezers or toothpick to the stigmatic surface. Shortly growth habit. Identification of the different Phalaenopsis after pollination, lobes of the often close around species was carried out based on the books “Orchidiana the stigma and then the flower wilts. Later, the peduncle Philippiniana Vol. I” of Dr. Helen L. Valmayor (1984), expands and the ovary develops. The union of egg and “Philippine Orchid Species” by Mr. Jim Cootes (2011), sperm nuclei results to the formation of the embryo and, and Co’s Digital Flora of the Philippines (Pelser et al. 2011 depending on the species, this fertilization may occur in onwards). Flower color and labellum including the size several days to months from pollination. This embryo/ were assessed. Ten (10) fully developed flowers from 5 to seed has no endosperm. They need an artificial medium 10 plants were used. First two to three uppermost flowers to grow and germinate; hence, tissue culture is necessary per plant were measured. Leaf length was measured from to continue their growth (PCARRD 1994). 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 The continuous destruction of all various types of forest and was described. Two fully expanded, uppermost leaves other areas for mining probably caused the extinction of from five plants were measured. The growth habit was also narrowly endemic species, which will never be known to the described accordingly. In this study, phytography was used scientific world or nature-lovers in general. Approximately to deal with the descriptive terminology of plants and their 85% of the orchid species found in the Philippines are component parts for the purpose of providing an accurate endemic and found nowhere else in the world, but they and complete vocabulary for description and identification. are being destroyed due to forest degradation, particularly Phytographic studies in this research provide the user with a the lowland rainforests. Hence, collecting and maintaining vocabulary for intelligent communication about orchids and them will save their biodiversity in the country. Further an understanding of the use of relative terms – and help the improvement will enhance their breeding and commercial user observe the plants more critically and describe more values. For instance, mutation breeding of Phalaenopsis precisely (Radford et al. 1974).

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Flowering Behavior of Different Phalaenopsis 10, 15, 20, and 25 Gy serving as treatments. They were Species irradiated at the PNRI. Untreated germinated embryos The flowering behavior of the different Phalaenopsis were sub-cultured and incubated in a similar manner serve species was monitored for 2 yr and the peak of flowering as the control. After irradiation, sub-culturing in a fresh was recorded. Self-pollination of the 11 Phalaenopsis artificial culture medium followed and then incubated in species was done to assess self-compatibility and capsule the culture shelf. The cultures were grown for 6–8 mo from setting. A varying number of flowers (5–8) from each initial culture and assessed for different growth parameters species were self-pollinated and the pollinated flower was such as the number of regenerants with complete and bagged and labeled accordingly. The percent success of incomplete (shoot only) regeneration, and length and self-fertilization was assessed 3–4 wk after pollination width of the fully expanded leaf. using the following formula: Seedlings were potted out in 12-cm diameter clay pots % success of self-fertilization = (number of self-pollinated containing chopped tree fern bark and acclimatized inside flowers that developed into capsules / total number of the net house provided with 60% shade using a black net self-pollinated flowers) x 100 with plastic roofing. The seedlings were not watered for 2–3 d, after which the seedlings were misted with a dilute The duration of maturity of capsules was determined by solution of fungicide and fertilized weekly with a water- monitoring the yellowing of the capsules and abscission soluble fertilizer. After 2 yr, growth of seedlings inside the of the remnant at the tip of the capsules. Capsule net house was assessed. Leaf length and leaf width were length was measured using a ruler from the base to the determined, while leaf thickness was measured using a tip of the capsule, while capsule width was taken from micrometer caliper. the middle of the capsule. The number of self-pollinated capsules that reached maturity was counted for each Phalaenopsis species. Statistical Design and Analysis Radiation experiments and growth of the irradiated seedlings of P. aphrodite was conducted in completely Embryo Culture of 11 Phalaenopsis Species randomized design (CRD) with five replications and Mature self-pollinated capsule harvested from each five experimental culture vessels in each replicate. Four Phalaenopsis species was washed with soap and water and gamma irradiation doses (10, 15, 20, and 25 Gy) doses and dried in a Petri dish lined with sterile tissue paper inside an untreated as control served as treatments. In assessing the laminar flow hood. Capsules were sterilized by dipping the regenerated plants of P. aphrodite after 2 yr of growth in 70% (v/v) ethanol followed by flaming two to three in the net house, CRD with 5 replications and 20 sample times for 10 s. The dust-like seeds embedded on cottony- plants in each replicate was used. The data gathered were like structures were scraped off using sterile scalpel and subjected to one-way ANOVA using F-test. Significant were inoculated into an artificial culture medium based differences between treatment means were detected using –1 on Knudson (1946) with coconut water (150 mL L ), the least significant difference (LSD) at 0.05 level of –1 tomato puree (5 g L ), and sucrose (2.0%, w/v) as organic significance. Descriptive statistics such as the percent, additives, and Bacto-agar (0.8%, w/v; Difco Laboratories, frequency, or count data for the different Phalaenopsis Detroit, MI, USA) to solidify the medium. The pH of species were used. 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 RESULTS AND DISCUSSION 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 Characterization of Flowers, Leaves, and Plant fluorescent tube as a light source at photosynthetic photon Habit flux density (PPFD) of 120 µmol m–2s–1 at a prevailing Characterization of different Phalaenopsis orchids temperature of 25 ± 1 °C. The number of regenerants was done, and morphological descriptions of each from each Phalaenopsis species was counted 6–8 mo Phalaenopsis species were described in Table 1 and their from initial inoculation onto the artificial culture medium. flowers were presented in Figure 1.

Irradiation Studies on Phalaenopsis aphrodite Flowering Behavior of Different Phalaenopsis Six-month-old germinating embryos of P. aphrodite Species were exposed to four doses of gamma radiation – namely Eleven Phalaenopsis species flowered under ambient conditions at different times of the year for two years

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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 Green, Leathery, 35 cm long, 10 Pendent and repent white with yellow and brown markings cm wide P. aphrodite Rchb.f. Milky-white, 6 cm diameter, labellum is Leathery, 30 cm long, 7 cm Pendent and repent white with yellow and red markings wide, Green in the adaxial and green with purplish tinge on abaxial side of leaves P. equestris (Schauer) Rchb.f. Light violet to pinkish, star-shaped, 2.5 cm Green, Very leathery, 20 cm Pendent and repent diameter long, 4 cm wide P. fasciata Rchb.f. Flower has creamy yellow background and Light green, Leathery, Bright Upright, monopodial cinnamon brown barring, star-shaped, 4 cm green, 20 cm long, 4 cm wide diameter P. hieroglyphica (Rchb.f.) Sweet Creamy white flower with hieroglyphic-like Green, Leathery, Plain green Upright, monopodial maroon markings, 4 cm diameter and shiny, 25 cm long, 5 cm wide P. x intermedia Lindl. White flower with reddish-pink labellum, 3 Green above and purplish Upright, monopodial cm diameter underneath, Leathery, Dark green with purple coloration on the underside P. lindenii Loher White flower with pale pink suffusion, Dull green to mottled silvery Upright, monopodial labellum is darker pink at apex with a number green, Leathery, Dark green of radiating darker rose-colored lines at base, with silvery-white mottling 3 cm diameter P..lueddemanniana Rchb.f. Ivory white flower traversed with magenta Pale green, Leathery, Green Upright, monopodial bars, labellum is carmine and yellow, 4 cm leaves, 25 cm long, 6 cm wide diameter P. pulchra (Rchb.f.) Sweet Solid purple, star-shaped, labellum is purple Green, Leathery, 26 cm long, 7 Upright, monopodial with bright yellow lateral lobes, 3 cm diameter cm wide P. schilleriana Rchb.f. Violet to purplish pink, labellum lateral Dark green and marbled with Pendent and repent lobes upright but flaring outside, mid-lobe is silvery gray upper surface and obovate with two hair-like appendages on its purplish-green underneath, tip, 5 cm diameter Very leathery, Very attractively mottled or has tiger-like patterns, 40 cm long, 8 cm wide P. stuartiana Rchb.f. Creamy white, inner half of the lateral Green, Leathery, Mottled or has Upright, monopodial are beautifully spotted with dark cinnamon tiger-like patterns, 25 cm long, blotches over a base color of sulfur yellow, 7 cm wide 4 cm diameter

(Figure 2). Year-round flowering was observed on P. in the Philippines. Under natural conditions, P. equestris is equestris. This characteristic makes P. equestris a good self-compatible since it produced capsules throughout the candidate parent in developing a variety or hybrid that year under ambient conditions. Successfully self-fertilized flowers year-round. The success of self-compatibility on Phalaenopsis flower had its ovary enlarged and became the different Phalaenopsis species ranged 0–50% (Table yellowish green for the first two weeks after successful 2). Ten different Phalaenopsis species successfully self- self-pollination. This ovary started to enlarge and turned fertilized, indicating that they were self-compatible (Table green, while and sepals started to become brown 2). The highest percent success of self-fertilization was (Figure 2). obtained with P. equestris and P. hieroglyphica at 50% – followed by P. schilleriana, P. lindenii, and P. stuartiana Characterization of Capsule at 30%. However, the lowest percent success of self- A mature Phalaenopsis capsule is yellowish and plump fertilization was obtained with P. amabilis at 3.8%. This with its petal remaining, which eventually turns brown and is the first report on the assessment of self-compatibility later starts to disappear. However, P. pulchra did not set by hand pollination among different Phalaenopsis species

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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. any capsule. This may suggest self-incompatibility or may after self-fertilization. This result jibed with the previous require lower temperatures in higher elevation or may need report that capsules of P. amabilis matured in 2–4 mo natural pollinating agents for capsule setting. Except for (Lapade et al. 1996). It has been reported that the maturity naturally self-fertilized orchids, all others are pollinated of an orchid capsule may take several months (PCARRD by animals including bees, butterflies and moth, birds, 1994). In contrast, capsules of P. hieroglyphica matured and ants – each of which is attracted in different ways in 8–9 mo, while capsules of P. lindenii matured in 9–10 (Pridgeon 1992). Ten out of the 11 (90.91%) Phalaenopsis mo – indicating that P. hieroglyphica and P. lindenii species that flowered under ambient conditions produced had the longest capsule maturity period among the 10 self-fertilized capsules after assisted pollination. 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 Capsule Maturity disappeared gradually. Capsules of 10 Phalaenopsis species matured 3–5 mo after self-fertilization (Table 3). These species included P. schilleriana, P. equestris, P. aphrodite, P. Length and Width of Capsule lueddemanniana, P. amabilis, P. x intermedia, and P. The 10 different Phalaenopsis species produced capsules stuartiana. The capsules of P. fasciata matured in 4–5 mo after assisted pollination with varying length and width

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(Table 3). The longest self-pollinated capsules among the is by using tissue culture via embryo culture of seeds 10 Phalaenopsis species were shown by P. schilleriana, (Valmayor 1984, PCARRD 1994, Nayak et al. 2006). P. amabilis, and P. aphrodite. On the other hand, P. lindenii had the shortest self-pollinated capsule. Eight The 10 Phalaenopsis species developed capsules in Phalaenopsis species had an average capsule width (5.0– varying numbers via hand pollination (Table 4). P. 5.6 mm) – including P. lindenii, P. fasciata, P. stuartiana, aphrodite developed the highest number of self-pollinated P. schilleriana, P. leuddemanniana, P. x intermedia, P. capsules (Figure 2), followed by P. hieroglyphica. This aphrodite, and P. amabilis. P. hieroglyphica produced the result jibed with the previous observations of orchid widest self-pollinated capsule, while P. equestris had the hunters from Marinduque, Philippines that P. aphrodite is narrowest capsule among the 10 Phalaenopsis species a prolific bearer of capsules even under natural conditions evaluated. These basic data on characteristics and maturity in the forests (Magdalita 2016). This further explains why of self-pollinated capsules of different Phalaenopsis P. aphrodite is widely available naturally in forested areas. species provide useful information for orchid scientists Other Phalaenopsis species including P. x intermedia, P. and growers as this may serve as a guide in the harvesting stuartiana, P. fasciata, P. lindenii, P. lueddemanniana, of self-pollinated capsules for embryo culture. It has been P. equestris, and P. schilleriana produced self-fertilized reported that the most efficient way of multiplying orchids capsules; however, P. amabilis produced the lowest number of self-fertilized capsules. The mature capsule contains dust-like seeds, which are Table 2. Phalaenopsis species collected, the number of self-pollinated flowers, and the percent success of self-fertilization of the 11 embryos devoid of endosperm that are embedded in Phalaenopsis species grown under greenhouse conditions cottony-like structures and are dirty white to creamy white in Los Baños, Laguna, Philippines. (Figure 3b). Capsules were characterized for their length Phalaenopsis No. of self- Percent success of and width (Table 3). Different collections of Phalaenopsis species pollinated flowers self-fertilization species grown in the net house (Figure 3a) were used in P. schilleriana 50 30 pollination. Flowers with successful self-fertilization produced mature capsules (Figure 3b). The embryos P. equestris 20 50 obtained from the mature capsules germinated 1–2 mo P. aphrodite 130 4 after inoculation on the artificial culture medium. Initially, P. lueddemanniana 20 15 they germinate for 3–5 mo after explanting (Figure 3c). P. amabilis 30 3.8 These seeds germinated and developed into plants 9–10 P. lindenii 10 30 mo after inoculation on the culture medium. The highest number of plants was obtained from P. hieroglyphica, P. fasciata 6 17 followed by P. aphrodite (Table 4). P. equestris was P. hieroglyphica 8 50 followed by P. amabilis. No plants were produced from P. x intermedia 40 13 cultures of P. lueddemanniana, P. lindenii, and P. fasciata P. stuartiana 20 30 because embryos did not germinate on the artificial culture P. pulchra 20 0 medium used – suggesting that other media formulations may be needed to germinate these species.

Table 3. Capsule characteristics of different Phalaenopsis species. Duration of pollination to Ave. length of capsules Ave. width of capsules Phalaenopsis species capsule maturity in Los (mm) (mm) Baños, Laguna (mo) 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

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Table 4. The number of mature capsules that developed on different Phalaenopsis species. No. of mature No. of plants Phalaenopsis capsules produced by species harvested 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

Irradiation Study on Phalaenopsis aphrodite Germinating 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 Figure 3. Different Phalaenopsis species grow inside the net house at 5 and 10 Gy (Lapade et al. 1993). and used for pollination (A); the self-fertilized capsules of Phalaenopsis aphrodite and the mature capsule However, in the present study, 25 Gy had the lowest containing the seeds that are dust-like and embedded in number of regenerated plantlets – suggesting that higher cottony-like structures (B); and the embryo cultures being doses of radiation could be inhibitory to growth. Similarly, kept in the tissue culture laboratory (C). the number of complete regenerants and incomplete regenerants (shoot only) decreases significantly with increasing amount of irradiation (Table 5). In terms of direct or indirect action – break chromosomes leading to leaf growth of irradiated plants, leaf length and leaf width structural rearrangements and even loss of a part or whole also decrease significantly with increasing amount of chromosome that damage the cells and could bring cell irradiation. death, hence prohibiting growth. The present result is like the previous finding in Vanda sanderiana where there In general, the number of regenerants and their leaf growth was a decrease in seedling height as the dose of gamma decreases with increasing irradiation dose (Table 5). The radiation is increased (Lapade et al. 2001). present result indicates that as the dose of irradiation is increased, the development of regenerants in P. aphrodite An important observation in the gamma-irradiated P. decreases. This could be due to the lethal effects of aphrodite is the induction of early flowering in a plant irradiation on the developing seedling. Medina et al. by 1 yr and 8 mo from the initial culture of germinating (2005) reported that ionizing radiation can – either by 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 No. of plantlets No. of complete No. of incomplete Length of fully Width of fully treatments (Gy) regenerated regenerants produced regenerants (shoot only) expanded leaf (mm) 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.

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 Figure 4. Flowering plants of Phalaenopsis aphrodite whose suggest that increasing irradiation dose is inhibitory to germinated embryos were irradiated with 15 vGy Gamma the growth of P. aphrodite. This result corroborated with radiation. The first plant flowered 1 yr and 8 mo after the previous finding that gamma irradiation at higher protocorms were irradiated (A), while the other plant doses inhibited the growth of cashew (Anacardium flowered 2 yr after germinated embryos were irradiated (B). 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 the control and in the other treatments (Figure 4B). Since and narrowest expanded leaves were observed in 25 Gy, orchids derived from seeds normally flower in three years’ but significant differences between treatments for these time, the induction of early flowering in these two plants characters were not detected. may suggest that irradiation using 15 Gy could shorten juvenility of P. aphrodite. Possibly, mutagenic radiation Further observations on the second year of growth of had some stimulating effects in plants, and this effect irradiated P. aphrodite, indicated that lower doses of could be due to the destruction of inhibitors and release gamma radiation enhanced the growth of P. aphrodite of activators that induces early flowering. While it is a as suggested by the growth of the leaves (Table 6). preliminary finding, to our knowledge, this is the first Consistently, the untreated and the plants treated with 10 report on the induction of early flowering of P. aphrodite Gy radiation dose had plants with significantly longer irradiated with gamma rays at 15 Gy. In a similar and wider leaves than those treated with higher doses of experiment, induction of early flowering by 15 months radiation. This finding corroborated with the previous was observed in gamma-irradiated Dendrocalamus report that gamma radiation at lower doses promoted strictus; however, this species normally flowers 20–40 growth by promoting seedling height and earliness to years after planting without irradiation treatments (Kapoor capsule formation in Cattleya alliances (Thammasiri and Sharma 1992). Also, induction of early maturation 1996) and in soybeans (Glycine max L.) (Barrida and

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

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.

Medina 2003). It was purported that in gamma-irradiated artificial culture medium and irradiation effects of gamma plants, nutrient uptake was enhanced while the gas irradiation of P. aphrodite. All of the 11 Phalaenopsis exchange was increased but not in non-radiated plants; species flowered under ambient conditions consistently moreover, there were increased activities on the key for two years. Self-compatibility and capsule setting photosynthetic enzyme RuBisCO and on nitrate reductase, varied among species. However, P. pulchra is self- which is essential in nitrogen assimilation (Babu et al. incompatible and did not set capsules. Capsule maturity 2012). In contrast, higher doses of radiation (15, 20, and also varied. Phalaenopsis schilleriana produced the 25 Gy) had plants with significantly shorter and narrower longest capsule, while P. lindenii had the shortest capsule. leaves, indicating growth was decreased (Table 6). One P. hieroglyphica had the widest capsule, while P. equestris reason for the decreased performance of plants exposed to had the narrowest capsule. P. aphrodite produced the high doses of gamma radiation is the decrease in carbon greatest number of capsules, while P. amabilis produced and nutrient assimilation efficiency, which later translates the fewest. Mature dust-like seeds embedded in cottony- into decreased plant health and growth (Babu et al. 2012). like structures germinated within 1–2 mo at varying degrees in the artificial culture medium and developed Significant differences between different treatments into plants 9–10 mo after germination. Regenerated plants existed for leaf thickness (Table 6). Untreated plants had were obtained from P. hieroglyphica, P. x intermedia, P. significantly thicker leaves followed by those treated with schilleriana, P. stuartiana, P. amabilis, P. equestris, and 10 Gy radiation dose. However, higher doses of gamma P. aphrodite. However, P. lueddemanniana, P. lindenii, irradiation significantly decreased leaf thickness in two- and P. fasciata embryos did not germinate. year-old P. aphrodite. The results indicated that higher radiation doses of 15–25 Gy could have some negative Germinated P. aphrodite seeds subjected to different levels effects on plant growth – suggesting that as the radiation of gamma radiation at 10, 15, 20, and 25 Gy responded dose is increased, lethality is manifested. Since gamma differently to treatments. The number of complete radiation is a type of linear energy transfer radiation, it regenerants and the number of regenerants with shoot is dose-dependent where the effect of exposure to high only also differed significantly among treatments. Leaf dosage could cause an increased lethality to the organism. length, width, and thickness were significantly different According to Mitchel (2006), the ability to resist radiation- among treatments after two years of growth. induced damage is very much evolutionary in nature, and it is manifested in the DNA repair mechanisms of In P. aphrodite, early flowering was induced in two plants different species. As expected, DNA damage resistance irradiated with 15 Gy. One flowered 1 yr and 8 mo and of plants imposes dose-dependent lethality brought by the other 2 yr after potting out. Normally, tissue culture- gamma irradiation. As irradiation dosage is increased, an derived P. aphrodite flowers three years after potting out. elevated amount of irradiation would be tantamount to Overall, irradiation significantly affected the regeneration greater areas of damage in the plant, whether it is single- or and growth of P. aphrodite and further induced early double-stranded mutations that may occur (Mitchel 2006). flowering compared to the control.

CONCLUSION ACKNOWLEDGMENTS Eleven Phalaenopsis species were characterized for The UPLB Basic Research provided funds to the project flower and leaf characters, flowering ability under “Embryo Culture and Irradiation Studies to Improve Some ambient conditions, self-compatibility, capsule setting Native Phalaenopsis Species” from where this article was and characters, embryo germinability, and growth on an based. The authors would like to thank Mr. Jonard C.

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

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