Effect of Storage Temperature on Seed Viability and in Vitro Germination of Nobile Dendrobium Hybrids
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Effect of Storage Temperature on Seed Viability and In Vitro Germination of Nobile Dendrobium Hybrids Waraporn Udomdee 1/ Pei-Jung Wen 2/ Shih-Wen Chin 2/ Fure-Chyi Chen 2/ ABSTRACT Nowadays, seed storage plays an important role for long-term conservation for plant genetic resources including orchids. Besides, asymbiotic germination provides an essential tool for commercial propagation and conservation purpose. Nobile-type Dendrobium hybrids are in high demand as decorative pot plants due in part to their showy flowers. In this study the effect of storage temperatures on seed viability and subsequent germination capability of nobile Dendrobium hybrids was evaluated. Mature seeds from ripe capsules were collected from three cross combinations of nobile Dendrobium hybrids; D. Spring Dream ‘Apollon’ x D. Hamana Lake ‘Dream’, D. Golden Tower x D. 012-3, and D. 184-1 x D. 140-1 and air-drieD. The dried seeds were stored at either room temperature (25+2° ํC) or -20 ํ°C for twenty four months. In vitro seed germination and viability test by TTC stain were investigated. The germinability and viability varied on storage temperature as well as hybrid combinations and duration of seed storage. The viability of seed stored at -20° ํC was higher than at room temperature as revealed by their germination percentage. Stainability percentage of seed stored at room temperature was severely low after 9 months of storage, while seed stored at -20°C could maintain viability longer than at room temperature. Seed germination decreased rapidly after 6 months at room temperature, whereas seeds stored at -20° ํC the germination decreased after 20 months. Also, the seeds rarely developed to advance seedling stages (stage 5 and 6) after 20 months of storage at -20 ํ°C. Key-words: nobile Dendrobium, TTC staining, seed storage, low temperature 1/ Tak Provincial Agricultural Research and Development Center, Department of Agriculture, Tak Province, 63000 Thailand 2/ Department of Plant Industry, Graduate Institute of Biotechnology, National Pingtung University of Science & Technology, Pingtung, 921 Taiwan ROC Thai Agricultural Research Journal Vol. 32 No. 2 May - August 2014 201 INTRODUCTION al., 2007; 2008). According to Kamemoto Increased demand for orchids in et al. (1999), the success of Dendrobium the global floriculture trade is creating breeding program has depended on the interest among orchid growers in utilization of amphidiploids which were producing new high-quality hybrids produced from seed propagation method. (Ngampanya and Homla-aor, 2010). Observation and evaluation of progenies Orchid hybridization is a common and are usually carried out for a minimum of essential practice among orchid growers, two years of flower production. Therefore, its main goal is to generate new and seed storage could obtain seeds for the improved material with characteristics of germination of selected amphidiploid commercial interest such as new flower progenies. Seed storage is of great colors, color patterns, and flower size and importance for plant breeding as well as number (Vendrame et al., 2008). an efficient means for conservation of Dendrobium hybrids are considered to be plant genetic resources and germplasm one of the most globally popular orchids preservation of rare or endangered since their high number of flowers per species (Pritchard and Seaton, 1993; inflorescence, longlasting and recurrent Vendrame et al., 2007; Ngampanya and flowering (Martin and Madassery, 2006; Homla-aor, 2010). Kananont et al., 2010). Among them, the Seaton and Pritchard (2011) high heterozygosity of nobile type demonstrated that the time of banking Dendrobium hybrids has attracted the orchid seeds has started to be used as a attention of orchid industry for large scale key component of an effective ex situ clonal propagation of elite genotypes due conservation strategy. Seed banking, to their strong vigor and showy flowers either short-term (seeds lasting for 1-5 (Faria et al., 2004). In vitro germination of years) or long-term (seeds lasting >5 hybrid seeds is a common practice years) persistent (Bakker et al., 1996), has among orchid growers and most orchid been recognized as being the most seeds can either be readily germinated efficient way of storing large numbers of after harvest from the mother plant or living plants in one place (FAO, 1996; stored for later germination (Vendrame et Linington and Pritchard, 2001). There are 202 วารสารวิชาการเกษตร ปีที่ 32 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2557 currently very few genebanks that Although plenty of Dendrobium hybrids preserve orchid germplasm and this have high commercially value in the creates the need to develop procedures global floriculture trade, there is rarely to improve orchid seed storage for information on low temperature seed germplasm conservation (Mweetwa et al., storage of nobile Dendrobium hybrids are 2007). described. There are only some reports Even though, cryopreservation has demonstrated seed cryopreservation in been reported as a tool for long-term Dendrobium hybrids (Vendrame et al., seed conservation in various of orchids 2007), D. crystallinum Rchb.f. and D. such as Spathoglottis plicata (Khor et al., virgineum Rchb.f. (Huehne and Bhinija, 1998), Bratonia spp. (Popova et al., 2003), 2012) and D. candidum PLBs by Bletilla striata (Hirano et al., 2005), Brassia encapsulation (Yin and Hong, 2009) and and Phalaenopsis spp. (Mweetwa et al., air-drying method (Bian et al., 2002). 2007) and Phaius tankervilleae (Hirano et Therefore, establishment of an efficient al., 2009) but it faces significant obstacles method for nobile type Dendrobium because of methodological problems hybrids seeds storage has been needed involved with determination of seed for breeding programs and conservation germination rate in vitro and selection of purpose. In this study, we investigated the optimum freezing conditions (Pritchard, effect of storage temperature and 1984). Moreover, seeds of some species duration on viability and germination do not tolerate desiccation or cold ability of mature seeds of nobile storage so lower temperature storage Dendrobium hybrids. may be more suitable for long-term storage (Machado-Neto and Custodio, MATERIALS AND METHODS 2005; Seaton and Pritchard, 2008). Plant materials According to Seaton and Pritchard (2003), Three cross combinations of orchid seeds can be stored successfully nobile type Dendrobium hybrids; D. at either 5º ํC, the temperature of a Spring Dream ‘Apollon’ x D. Hamana Lake domestic refrigerator, or -20º ํC, the ‘Dream’, D. Golden Tower x D. 012-3, and temperature of a domestic deep freezer. D. 184-1 x D. 140-1 were hand-pollinated Thai Agricultural Research Journal Vol. 32 No. 2 May - August 2014 203 under greenhouse in National Pingtung 30 min. Petri plates were sealed with University of Science and Technology. clear plastic tape and maintained at Mature seeds from ripening capsules 25+2 ํC under a 12/12-h (day/night) were collected and seeds were stored in photoperiod provided by cool white 1.5 ml centrifuge tubes provided by fluorescent lamps (StarcoatTM F28W/T5/ wrapping in heavy-duty aluminum foil and 840,170 MA, Hungary) at photosynthetic stored at two different conditions; 25+2° ํC photon flux (PPF) of 40+10 µmol/m2/s. (room temperature) and -20° ํC (low Seed germination was observed weekly temperature) over silica gel desiccant until under stereomicroscope (Stemi SV6 experimentation. Zeiss, Germany) and germination Seed sterilization, germination and percentage was calculated by dividing the culture conditions number of germinating seeds by total Seeds from 0, 1, 2, 3, 6, 9, 16, 20 number of seeds in the sample 6 weeks and 24 months after storage were after sowing. Four replicates (each surface-sterilized with 0.6% sodium replicate includes 300-400 seeds hypochlorite for 10 min, and then washed approximately) were performed for all with sterile water for 3 times. Sterilized hybrids. seeds were resuspended in a small Seed viability test amount of sterile water and the seeds Seed viability was assessed using were extracted with a sterile glass the 2,3,5-triphenyltetrazolium chloride Pasteur pipette spreading thinly as (TTC) staining method. A small amount of possible over the surface of medium in seeds were placed in 1.5 ml centrifuge 90x15 mm Petri plates (Alpha Plus tubes and surface-fertilized as above Scientific Corp., Taiwan). Basal medium methods before being resuspended in consisted of half-strength Murashige and 0.5% TTC aqueous solution and Skoog (1962) supplemented with 20 g/L incubated for 24 h at 25+2° ํC in darkness. sucrose and solidified with 8 g/L Sigma The 0.5% TTC aqueous solution was agar (Sigma-Aldrich, St Louis). The pH of prepared by dissolving 0.5 g TTC in the media was adjusted to 5.6-5.8 prior to solution with 0.2 M NaH2PO4 30.5 ml, 2 autoclaving at 121° ํC and 1.21 kg/cm for 0.2M NaH2PO4 19.5 ml and total volume 204 วารสารวิชาการเกษตร ปีที่ 32 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2557 to 100 ml by water. Then seeds were genotype (Table 1). Before storage, seeds examined under a fluorescence of D. Spring Dream ‘Apollon’ x D. microscope (Olympus CX40, USA); seeds Hamana Lake ‘Dream’ and D. Golden containing embryos with any degree of Tower x D. 012-3 showed higher pink to red staining throughout the germination percentage than D. 184-1 x embryos were considered viable while D. 140-1. After one and two months wholly unstained embryos were storage, there is no significant different on considered non-viable. Three replicates germinability between 25° ํC and -20 ํ°C but (each replicate includes 150-200 seeds significant different among species. The approximately) were performed for all germination percentage of D. Golden genotypes and percent viability was Tower x D. 012-3 and D. 184-1 x D. 140-1 calculated by dividing the number of higher than D. Spring Dream ‘Apollon’ x seeds with viable embryos by the total D. Hamana Lake ‘Dream’ after one month number of seeds with embryos.