Seed Germination of Orchid | Research

Seed Germination of Orchid ResearchThere atomic turning 18 a lot of orchidaceous plantaceous plantaceous plant species being listed as endangered species due to all 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 fundamental additives on in vitro ejaculate sprouting of Vanda hastfera, an endemic species of orchid to Borneo. By the end of this investigation, we expect to determine the fair composition and germinating chassiss that atomic number 18 favourable for in vitro seed sprouting of Vanda hastifera. Various organic additives such as stump spud show, peptone, coconut pissing, love apple juice and banana tree pulp will be added into the intermediate to probe their effects on the seed sprouting. The physical conditions such as slatternly colour, temperature, pH and relative humidity will be adjusted to the near suitable condition in compliance with the percent age of seed germination. The cultivation room will be maintained at a 16 light/8 dark photoperiod with environmental temperature of 252C. 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, MS long suit1.0 IntroductionThe family Orchidaceae is considered as one of the largest mensesering plant families which comprised of over 880 genera and approximately 25,000 to 30,000 species worldwide (Paek, Hahn Park, 2011 Bektas, Cce Skmen, 2013). Paek et al. (2011) indicated that the high score 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% argon said to be endemic species. Borneo is whereby denoted a s 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 atomic number 18 generally found at hill-forests or tropic 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 br testify spots. The uniqueness of this species is that it is very hairy at the margin of its mouth as well as the auricles (Beaman et al., 2001).Over-collection of orchids has endangered few of the species which flip high commercialised value in a commixture of indu stries like the medical industry, horticultural, or denotental so on. The survival of orchid in unsubdued 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). establish on the study that is fulfilled by Beaman et al. (2001), poorly preserved herbarium collections and wanting(p) information about the flower species for genus Vanda have made it material 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- pull stringsled. This can prevent the waste of resources and conserve the process of seed germination in making certain(a) of the efficacy of the process. Furthermore, the addition of organic additives in appropriate slow-wittedness under prop er circumstances can also enhance the effectiveness of seed germination carried out in vitro with relatively low expenses.The objectives of the study areTo 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 Review2.1 Vanda hastifera (Orchidaceae)According to Metusala and OByrne (2012), on that point 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 composite 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 OByrne, 2012). Among the species of orchids in genus Vanda, Vanda s candens 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 discolor 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 2-2.5 cm which is dense and linked to a sheathing base.The embryo of Vanda differentiates into three sections in its early victimisation, 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 germinationThe orchid seed is very fine and delicate. The weight of the seed is varied from 0.3 g to 14 g 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 em bryo during germination will bust 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 eldest 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 starting signal 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 chemical element 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 mycorr hizal 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 orchidIn 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 follow up 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 used 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 b ooming 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 market-gardening suitable for more species (Kauth, 2005).Table 1 Media composition of MS, VW and Knudson media (Paek et al., 2011).Component MS (mg/L)VW (mg/L)Knudson (mg/L)MacronutrientsNH4NO3825(NH4)2SO4500500Ca3(PO4)2200Ca(NO3)2. 4H2O1000CaCl2.H2O220MgSO47H2O185250250KNO3950525KH2PO485250250MicronutrientsNa2EDTA18.65FeSO4.7H2O13.925Fe2(C4H4O4).2H2O28H3BO33.1CoCl2.6H2O0.0125CuSO4.5H2O0.0125MnSO4.4H2O11.157.5KI0.415Na2MoO4.2H2O0.125ZnSO4.4H2O4.3OrganicsGlycine2Myo-inositol100100Nicotinic acid0.51Pyridoxine0.5Thiamine HCl0.11Adenine sulphate10The media composition is an important factor that will affect the efficiency of seed germination in vitro. M S 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. tell 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 change Hyponex media composition (Paek et al., 2011).ComponentSeed germinationProtocorm multiplicationFirst transplantingSecond transplantingHyponex (g/L)NPK= 6.56193.01.01.01.0NPK= 2020201.01.01.0Adenine Sulfate (g/L)5.0Peptone (g/L)2.02.03.0Coconut water (%)201010Potato or banana hom ogenate (g/L)30-10030-100Activated charcoal (%)0.050.050.050.05Germination of seed can be affected by seed maturity. By using asymbiotic in vitro seed germination technique, childlike seed has found to be more effective than mature seed to germinate. Claiming that the embryos have developed completely but not yet dedicated to go into 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 germinationNatural 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 process inhibitors. Nevertheless, Paek et al. (2011) suggested that th e 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 involve 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 turn out 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 r oxburgii 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, overawe milk and peptone on the development of Dendrobium discolors 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 maxi mum fresh-cut 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 discolors protocorms were obtained in Hyponex medium containing potato extract with 2.0 g/L peptone (Tharapan et al., 2014).3.0 Materials and Methods3.1 List of MaterialsSeeds of Vanda hastifera, 0.2% (w/v) HgCl2, 70% neutral spirits, 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 seedsImmature 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 orangeness to red colour under the stereoscopic microscope observation (Lauzer, Renaut, St-Arnaud Barab, 2007). This procedure has proven effective for testing the viability of epiphytic tropical orchids seeds (Singh as cited in Vujanovic, St-Arnaud, Barab Thibeault, 2000, p.79).3.3 Media preparationHalf 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 conc entration 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 forward to autoclave at 121 under 15 p.s.i. for 20 minutes. (Jawan, Gansau Abdullah, 2010 Ali, Murdad Latip, 2011)3.4 InoculationThe 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 252. The light intensity is to be at 20-50 molm-2s-1 provided by cool white fl uorescent tubes (Jawan et al., 2010 Ali et al., 2011). The relative humidity is adjusted at 70-80%.3.5 Observation and analytical techniqueSeed 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 Duncans Multiple Range Test (DMRT) at 5% level of probability (Aktar et al., 2008 Ali et al., 2011).4.0 Expected outcomeThe 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 in tensity.It is expected that the 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.