Seed Germination of Orchid | Research

Seed Germination of Orchid | Research There are a lot of orchid species being listed as endangered species due to over-collection and lack of conservation. The survival rate of orchid in the nature is relatively low. This research is aimed to investigate the effect of organic additives on in vitro seed germination of Vanda hastfera, an endemic species of orchid to Borneo. By the end of this investigation, we expect to determine the medium composition and germinating conditions that are favourable for in vitro seed germination of Vanda hastifera. Various organic additives such as potato extract, peptone, coconut water, tomato juice and banana pulp will be added into the medium to examine their effects on the seed germination. The physical conditions such as light intensity, temperature, pH and relative humidity will be adjusted to the most suitable condition in compliance with the percentage of seed germination. The culture room will be maintained at a 16 light/8 dark photoperiod with environmental temperature of 25 ±2à ‚ °C. Also, the protocol will be designed by taking into account of the cost-effectiveness of the project. Keywords: Vanda hastifera, in vitro seed germination, protocorm,  ½ MS medium 1.0 Introduction The family Orchidaceae is considered as one of the largest flowering plant families which comprised of over 880 genera and approximately 25,000 to 30,000 species worldwide (Paek, Hahn Park, 2011; Bektas, Cà ¼ce Sà ¶kmen, 2013). Paek et al. (2011) indicated that the high degree of compatibility among genera and species allowed the registration of bi- and plurigeneric hybrids to reach over the number of 100,000. Lamb (as cited in Chan, Lamb, Shim Wood, 1994, p. 5) claimed that approximately 10% of orchids in the world were found in Borneo, which is around 2500-3000 species. Among these about 30-40% are said to be endemic species. Borneo is whereby denoted as â€Å"Orchid Island† (Chan et al., 1994). As discussed by Beaman, Wood, Beaman and Beaman (2001), Vanda is a monopodial genus of orchids that produce attractive flowers which are generally found at hill-forests or tropical lowlands. Vanda hastifera Rchb.f. (Orchidaceae) is endemic to Borneo in which this epiphytic species inhabits at hill forest, lowland or coasted area (Chan et al., 1994). Vanda hastifera can be found at Kinabatang district in Sabah, Pontianak area in Kalimantan Barat, and Kuching area in Sarawak. Vanda hastifera is scented flower that can blooms for around 10 days in which it produces reflexed petals and sepals (Chan et al., 1994). The sepals of Vanda hastifera are marked by a few reddish brown spots. The uniqueness of this species is that it is very hairy at the margin of its lip as well as the auricles (Beaman et al., 2001). Over-collection of orchids has endangered some of the species which have high commercial value in a variety of industries like the medical industry, horticultural, ornamental so on. The survival of orchid in wild habitat is very low which also press the need to perform in vitro germination of the seeds. Vanda is one of the commonly cultivated genera that have high commercial value (Uchida, 1994). Based on the study that is fulfilled by Beaman et al. (2001), poorly preserved herbarium collections and insufficient information about the flower species for genus Vanda have made it substantial to practise cultivation upon the species. The significance in performing in vitro seed germination of Vanda hastifera is to optimize the seed germination process under in vitro condition in which the conditions and requirements of seed germination can be well-controlled. This can prevent the waste of resources and conserve the process of seed germination in making sure of the efficacy of the process . Furthermore, the addition of organic additives in appropriate concentration under proper circumstances can also enhance the effectiveness of seed germination carried out in vitro with relatively low expenses. The objectives of the study are: To investigate the effect of organic additives on in vitro seed germination of Vanda hastifera. To optimize the in vitro seed germination process of Vanda hastifera. To develop an appropriate and cost-effective protocol for in vitro seed germination of Vanda hastifera. 2.0 Literature Review 2.1 Vanda hastifera (Orchidaceae) According to Metusala and O’Byrne (2012), there are around ten taxa comprised in the genus Vanda section Hastifera which are distributed in the area of Borneo, Philippines, Lesser Sunda Island, Maluku, Sulawesi and New Guinea. The typical characteristics of section Hastifera are like complex lip with its apex divided into two buttock-like lobules and two dagger-like lobules bulging sideways from the base of the thickened mid-lobe (Metusala O’Byrne, 2012). Among the species of orchids in genus Vanda, Vanda scandens and Vanda hastifera have exhibiting all of the characteristics of section Hastifera. The name of Vanda hastifera is derived from Latin hastifer. Vanda hastifera is a fascinating species of orchid that has outstanding appearance which the fragrant flower for about 4.5 cm to 5 cm is fleshy and marked unevenly with brownish red blotches (Chan et al., 1994). Beautiful pale cream or pale yellow sepals and petals are reflexed. As revealed by Chan et al. (1994), the both the dorsal sepals and lateral sepals are spathulate with undulant margin. The white side lobes of the lip has shiny mid-lode at the top and side of the apex which joined to a short column-foot. The lower surface of the apex is purple to brownish purple with two reddish lines near the base. There is found to be a very hairy auricle at the base of the lip (Chan et al., 1994). Likewise, the edge of the mid-lobe is also hairy. The ligulate leaves of Vanda hastifera is about 15-20 Ãâ€" 2-2.5 cm which is dense and linked to a sheathing base. The embryo of Vanda differentiates into three sections in its early development, which are parenchymatous, meristematic and suspensor (Arditti, 1967). Based on the research done by Alvarez (as cited in Arditti, 1967, p. 4), the parenchymatous tissue is acting on supplying nutrition to meristem after the early degeneration of suspensor. 2.2 Orchid seed germination The orchid seed is very fine and delicate. The weight of the seed is varied from 0.3  µg to 14  µg; the length is to be at a range of 0.25 mm to 1.2 mm; and the width is around that of 0.09 mm to 0.27 mm (Arditti, 1967). The seed of orchid is normally produced in a large quantity in which Arditti (1967) indicated that a capsule may contains about 1,300 to 4,000,000 seeds. The orchid embryo usually maintains its globular or spherical shape in contrast to the great variety of the shape of the seed coat which may be in globular, elliptical, rounded, butterfly shaped or fusiform (Arditti, 1967). The swelling of embryo during germination will rupture the seed coat. This will lead to the formation of spherical or cone-shaped seedling which is the protocorm stage for orchid seed germination (Arditti, 1967). The protocorm is an undifferentiated mass of cells (McKendrick, 2000). Subsequently the first leaf primordium will project out of the upper flat surface. The protocorm then starts growing and the absorbing hairs starts to emerge at the periphery of the lower surface. Next, the first minute leaf is produced (Arditti, 1967). Soon after this, the first root will be formed. The development continues until a small plant takes it shape. In relation to orchid seed germination and development in nature, fungus infection is seemed to be a substantial factor for certain tropical epiphytic orchids (Arditti, 1967). This is because as the seeds are insufficient with the carbohydrate reserves, the young plant requires the supply of nutrients, sugar and organic material from the mycorrhizal fungus until the plant is capable of producing its own food (McKendrick, 2000). Once the fungus is penetrated into the seed, it is to be as an exogenous carbohydrate for the growing embryo upon the digestion of the fungal hyphae (Kauth, 2005). Moreover, fungi may be treated as a water supply as germination is started by imbibition (Yoder et al. as cited in Kauth, 2005, p. 2). 2.3 Research history of In vitro seed germination of orchid In vitro methods are used to improve and assist the development of plants that are vulnerable to grow in the nature (Fay, 1992). The use of symbiotic and asymbiotic in vitro germination techniques have been used for the seed germination of some orchid species (Fay, 1992). For the seeds that are to be germinated symbiotically, sowing is performed with a piece of mycorrhizal fungus. Symbiotic relationship is established when the fungus propagates and colonized the seed germination media (Mckendrick, 2000). Before the plant capable of making its own food, the fungus is thought to be sustaining the protocorm. Nonetheless, the proper strain of mycorrhizal fungus is required or else it might lead to seedling death as the fungus strain becomes parasitic. Proliferation of temperate terrestrial orchids is suitable to apply with this technique. On the other hand, tropical orchids are easier to grow as compared to temperate terrestrial orchids. Thus, asymbiotic germination method is normally us ed for the in vitro germination of tropical orchids. The media used appeared to be more intricate than that used in symbiotic germination (McKendrick, 2000). Without the mycorrhizal fungus, the nutrients required for proper germination have to be supplied fully. There are examples of simple media which are used for the seed germination of orchid which include Vacin and Went (VW), Hyponex and Knudson C medium (Paek et al., 2011). Without using the mycorrhizal fungus as a symbiotic element, Knudson (as cited in Kauth, 200, p. 3) has achieved successful seed germination for several epiphytic orchid genera which lead to the development of Knudson Solution B. After that, Knudson substituted ferric phosphate with ferrous sulphate and supplemented manganese into the medium in order to develop a more complex Knudson C medium that enable the in vitro seed germination and plant tissue culture suitable for more species (Kauth, 2005). Table 1: Media composition of  ½ MS, VW and Knudson media (Paek et al., 2011). Component  ½ MS (mg/L) VW (mg/L) Knudson (mg/L) Macronutrients NH4NO3 825 (NH4)2SO4 500 500 Ca3(PO4)2 200 Ca(NO3)2. 4H2O 1000 CaCl2.H2O 220 MgSO47H2O 185 250 250 KNO3 950 525 KH2PO4 85 250 250 Micronutrients Na2EDTA 18.65 FeSO4.7H2O 13.9 25 Fe2(C4H4O4).2H2O 28 H3BO3 3.1 CoCl2.6H2O 0.0125 CuSO4.5H2O 0.0125 MnSO4.4H2O 11.15 7.5 KI 0.415 Na2MoO4.2H2O 0.125 ZnSO4.4H2O 4.3 Organics Glycine 2 Myo-inositol 100 100 Nicotinic acid 0.5 1 Pyridoxine 0.5 Thiamine HCl 0.1 1 Adenine sulphate 10 The media composition is an important factor that will affect the efficiency of seed germination in vitro. MS medium added with 2,4-Dichlorophenoxyacetic acid (2,4-D) was reported to be favourable for the formation of protocorm-like-bodies (PLBs) and regeneration of plantlet for Dendrobium orchid (Nasiruddin et al. as cited in Aktar, Nasiruddin Hossain, 2008, p. 69). According to Mishra, Rawat, Nema and Shirin (2013), who have done an investigation on the effect of medium strength on in vitro germination of Pterocarpus marsupium Roxb. stated that by using different strength of MS basal medium, there is no significant difference on the rate of germination. Therefore, the concentration of MS medium used can be diluted to half so as to reduce the cost without affecting the efficacy of seed germination. Table 2: Modified Hyponex media composition (Paek et al., 2011). Component Seed germination Protocorm multiplication First transplanting Second transplanting Hyponex (g/L) N:P:K= 6.5:6:19 3.0 1.0 1.0 1.0 N:P:K= 20:20:20 1.0 1.0 1.0 Adenine Sulfate (g/L) 5.0 Peptone (g/L) 2.0 2.0 3.0 Coconut water (%) 20 10 10 Potato or banana homogenate (g/L) 30-100 30-100 Activated charcoal (%) 0.05 0.05 0.05 0.05 Germination of seed can be affected by seed maturity. By using asymbiotic in vitro seed germination technique, immature seed has found to be more effective than mature seed to germinate. Claiming that the embryos have developed completely but not yet dedicated to enter the dormant stage, Light and MacConaill (as cited in Fay, 1992, p. 2) suggested that seeds harvested at 43 to 58 days after pollination has found to be ideal for in vitro seed germination of orchid. 2.4 Effects of organic additives on in vitro seed germination Natural complex additives are added into the simple media lack amino acids or vitamins. Examples of organic additives are like potato extracts, coconut water, banana pulp, peptone, and tomato juice. Activated charcoal is believed to have ameliorated the aeration as well as absorbs ethylene and phenolic inhibitors which are the growth inhibitors. Nevertheless, Paek et al. (2011) suggested that the use of charcoal has to be careful as it also absorbs vitamins and plant growth regulators in the culture medium. Occasionally, low concentration of auxin and cytokinin are needed and supplemented into the media in the early stage of protocorm proliferation for certain species of orchids (Paek et al., 2011). Besides, sucrose is also being added into the media in the early stage of tissue culture. However, Paek et al. (2011) claimed that the plantlet differentiation of some genera of orchids may be improved in a medium which has low concentration of sugar. A research done by Islam, Akter and Prodhan (2011) which used Vanda roxburgii orchid as model has proven that by adding potato extract into the medium of seed germination in vitro, the percentage of seed germination and seedling growth can be increased substantially. From their study, they have found that by supplementing potato extract at the concentration of 200 ml/L showed the best percentage of seed germination for Vanda roxburgii which is 78.24% as compared to the 17.2% of control (Islam et al., 2011). Therefore, it is proposed by Islam et al. (2011) that seed germination of Vanda orchids can be boosted by adding appropriate concentration of potato extract into the medium. It is testified that in 100 g of raw materials of potato extract, there is 1.0 mg of niacin which is believed to be accountable for the orchids’ development. Recent study accomplished by Tharapan, Thepsithar and Obsuwan (2014) has observed the effect of potato extract (PE), soy milk, cow milk and peptone on the development of Dendrobium discolor’s protocorms and seedlings growth of Dendrobium Judy Rutz by using Hyponex medium. After two months, protocorms were found developed in all culture vessels with different combinations of organic supplements in Hyponex medium. For the seedling growth of Dendrobium Judy Rutz, Hyponex medium with supplementation of 100 mL/L of potato extract and 2.0 g/L of peptone has achieved with the maximum fresh and dry weight (Tharapan et al., 2014). Conversely, the dry weights obtained from Hyponex medium supplemented with peptone, organic soy powder and milk powder for Dendrobium Judy Rutz’ seedling growth have no significant difference in comparison to the control. On the other hand, the maximum fresh and dried weight as well as shoot height of Dendrobium discolor’s protocorms were obtain ed in Hyponex medium containing potato extract with 2.0 g/L peptone (Tharapan et al., 2014). 3.0 Materials and Methods 3.1 List of Materials Seeds of Vanda hastifera, 0.2% (w/v) HgCl2, 70% ethanol, 1% 2,3,5-tripheny tetrazolium chloride (TTC), MS medium, KC medium, Hyponex medium, coconut water, potato extract, banana homogenate, peptone, yeast extract, tomato juice, 0.9% (w/v) agar, sucrose, activated charcoal, NaOH and HCl. 3.2 Sterilization of the seeds Immature seeds of Vanda hastifera is surface sterilize by submerging in 0.2% (w/v) HgCl2 for 10 minutes. After this, the seeds will be dipping in 70% ethanol for 15 seconds for further sterilization. Sterilized seeds are then washed with sterile distilled water for 5 to 6 times. These procedures are operated in a laminar flow hood. The viability of the seeds is examined by staining the seed with 1% 2,3,5-triphenyl tetrazolium chloride (TTC) solution (pH 7). The staining is carried out in darkness. Observation is performed by using stereoscopic microscope (Yamazaki Miyoshi, 2006). Viable embryos are those shows orange to red colour under the stereoscopic microscope observation (Lauzer, Renaut, St-Arnaud Barabà ©, 2007). This procedure has proven effective for testing the viability of epiphytic tropical orchid’s seeds (Singh as cited in Vujanovic, St-Arnaud, Barabà © Thibeault, 2000, p.79). 3.3 Media preparation Half and full strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1967), KC medium (Knudson, 1946) and Hyponex medium (Kano, 1965) are supplemented with various organic additives in different concentration to examine their effects on in vitro seed germination of Vanda hastifera. Coconut water (5, 10, 15, 20% v/v), peptone (0.2% w/v), yeast extract (0.2% w/v), potato extract (1, 2, 4, 6 g/L) (w/v), banana homogenate (2.5-12.5% w/v), tomato juice (10-20% v/v) are added into the medium. 30 g/L of sucrose and 2 g/L or activated charcoal are also supplemented into the medium. The medium is solidified with 0.9% (w/v) agar. The pH of the medium is adjusted by using NaOH or HCl to between 5.4 to 5.8 prior to autoclave at 121 under 15 p.s.i. for 20 minutes. (Jawan, Gansau Abdullah, 2010; Ali, Murdad Latip, 2011) 3.4 Inoculation The sterilized seeds are then inoculated on to the medium prepared. The spreading of the seeds is to be even over the entire surface of the medium. The Petri dishes with media inoculated with seeds are kept in the culture room provided with 16 light /8 dark hours of photoperiod at 25 ±2. The light intensity is to be at 20-50  µmolm-2s-1 provided by cool white fluorescent tubes (Jawan et al., 2010; Ali et al., 2011). The relative humidity is adjusted at 70-80%. 3.5 Observation and analytical technique Seed germination is indicated by the development of protocorms. According to Ali et al. (2011), it can be seen by the appearance of protocorm from the testa. Observation is carried out at a 20 days interval for up to 60-70 days by using a dissecting light microscope. The number of germinating seed is recorded and tabulated in a table as the percentage of the total number of seeds inoculated (Ali et al., 2011). Analysis of variance (ANOVA) is used to analyse the data. The significant difference or treatment means is subjected to Duncan’s Multiple Range Test (DMRT) at 5% level of probability (Aktar et al., 2008; Ali et al., 2011). 4.0 Expected outcome The seed germination of Vanda hastifera that is conducted in vitro will be very effective. It is greatly affected by the medium composition, concentration of organic additives supplemented and the light intensity. It is expected that the  ½ MS will be the best medium for in vitro germination of Vanda hastifera. Organic additives like potato extract, peptone and coconut water are expected to give better results in the experiment as compare to other organic additives.