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In Vitro Germination of Wild Banana Musa Acuminata Colla Var. Microcarpa (Becc) After Storage Periods at Different Temperatures

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In Vitro Germination of Wild Banana Musa Acuminata Colla Var. Microcarpa (Becc) After Storage Periods at Different Temperatures Empowering Science and Mathematics for Global Competitiveness – Rahmawati & Taylor (Eds) © 2019 Taylor & Francis Group, London, ISBN 978-1-138-61666-0 In vitro germination of wild banana Musa acuminata Colla var. microcarpa (Becc) after storage periods at different temperatures R. Indrayanti, A.R. Putri & A. Adisyahputra Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Jakarta, Indonesia A. Sutanto Indonesian Tropical Fruits Research Institute, Solok Aripan, West Sumatra, Indonesia ABSTRACT: Musa acuminata Colla var. microcarpa (Becc), is one of fifteen types of wild bananas in Indonesia. The storage of bananas is difficult because banana seeds are inside and are a fast decaying fleshy part of the fruit. Germination of intact seeds either fails to germinate or has a low percentage of germination because there is an incompatibility with seed formation. This study aimed to find the ability for wild banana germination after storage through embryo culture. The banana embryos were cultured on a Murashige and Skoog (MS) medium with 2.25 mg/l−1 of 6-benzyladenine and 0.175 mg/l−1 of indole-3-acetic acid. The total number of embryos inoculated was 1,800. This study showed that storage of bananas at three different temperatures for a 60 days period, made the embryo unable to germinate. Most of the embryos germinated in a storage period of 4 to 10 days, whereas 76% germinated when stored at cold temperatures for 30 days. For banana seeds stored at dif- ferent temperatures for 4 to 10 days, 70–76% of the embryos germinated. For banana seeds stored at cold temperatures for a 30 days period, 84% of the embryos germinated, and for a 60 days period, 68%. This research shows that the best short-term storage of wild banana seeds was in a cold temperature (1 to 5°C) for a 30 days period. The germinated embryos of wild banana were then multiplied in MS medium for four months to produce shoots and calli before acclimatization in a greenhouse. 1 INTRODUCTION Banana and plantain (Musa spp) are a giant monocot plant belonging to the family Musaceae. Bananas grow in tropical and subtropical countries at a temperature of 20°C, above and below the equatorial line (Pua, 2007). Bananas consist of many cultivars and there are currently around 1,000 banana cultivars and 50 local races (landraces) (Heslop- Harrison & Schwarzacher, 2007). In Indonesia, around 200 local banana cultivars and wild varieties have been planted in almost all areas at different heights and in various types of soil (Nasution, 1991). Cultivated bananas, plantains and fiber-producing banana species (Musa textilis Nee) are derived from the intraspecific and or interspecific hybridization of the wild banana Musa acuminata Colla and Musa balbisiana Colla (Ploetz et al., 2007; Sipen et al., 2011; Valmayor et al., 2000). The center of diversity and the hybrids of Musa acuminata are in Malaysia and Indonesia (Asif et al., 2001), while hybrids of Musa balbisiana have been predicted to have originated from India. Musa acuminata Colla var. microcarpa (Becc), is one of fifteen types of wild bananas in Indonesia. This banana has a local Indonesian (vernacular) name: forest banana or monkey banana (Nasution, 1991). Wild bananas are a diploid plant (2n = 2x), have fertile pollen which can be cross-pollinated with other diploid bananas, and are relatively free of disease (Heslop-Harrison & Schwarzacher 2007; Ploetz et al., 2007; Uma et al. 2012. Great diversity in wild and cultivated bananas (Musa spp) can be found from Aceh (Sumatera) to Papua 71 (International Network for the Improvement of Banana and Plantain & Central Research Institute for Horticulture, 2002). Musa germplasm can also be found in East Kalimantan, Maluku and Lesser Sunda Islands (Hermanto et al., 2014, and new varieties of wild banana have reportedly been found in Sulawesi (Sulistyaningsih et al., 2014). The variety of wild bananas in Indonesia shows that the genetic diversity in these species is very high, making wild bananas useful as a source of germplasm to assemble superior varieties. The existence of wild bananas needs to be conserved so that a source of plant genetic diversity can be maintained. Germplasm storage methods depend on the nature of germplasm material (seeds or non- seeds) (Imarhiagbe et al., 2016). Banana seeds are naturally orthodox (storable) seeds. They can be stored for a long time under low humidity and low temperatures (Imarhiagbe et al., 2016). Although banana seeds can be germinated immediately after extraction, the seeds show secondary dormancy after storage (Fortescue & Turner, 2011). Previous studies on seed germination of wild bananas at the time of harvest and post-harvest at optimal conditions, have been carried out on Musa velutina Wendl. & Drude (Nagano et al., 2008), Musa ornata from Mexico (Burgos-Hernandez et al., 2014), and on M. acuminata subsp. burmannica Sim- monds from India (Vineesh et al., 2015). Wild bananas are difficult to germinate (Nagano et al., 2008; Roy et al., 2006; Uma et al. 2012. At least 3 to 6 weeks is required to initiate seed germination in soil. Germination occurs over a 3 to 15 week period and often provides a low percentage of growth (Uma et al., 2012. This low growth percentage occurs because the embryo may be in an abnormal condition that does not allow it to grow. Germination of the banana depends on the ripening of the fruit at harvest, and the physiological age of the seed (Harry et al., 2010; Rashid et al., 2013; Reed, 2005 Germination is also inhibited by physical barriers, such as impermeability of seeds, chemi- cal barriers by dormancy induced plant growth, incompatibility of normal seed formation, damaged endosperm, storage period of the seeds (Nagano et al., 2008; Uma et al., 2012, and difficulty with cross-pollinating seeds (Harry et al., 2010; Rashid et al., 2013). Germination of wild bananas through embryo culture is one of the techniques used to increase seed ger- mination so that the potential resource loss of banana genetics can be prevented. This study aimed to find the ability for wild banana seed germination through embryo culture after storage in different treatments. 2 METHOD This study used the Musa acuminata Colla var. microcarpa (Becc.) Nasution, a seeded wild banana from the Aripan area, Solok, West Sumatra, Indonesia. This study was comprised of three factors: the plant storage organ (fruit and seeds); storage temperature (−20 to −25°C, 1 to 5°C, and 32 to 40°C); and storage period (4 to 10, 30, and 60 days). The experimental design used a factorial completely randomized design, and the number of treatments was 18 with 20 replications. The total number of embryos planted was 1,800. 2.1 Storage of wild bananas at different temperatures and storage periods In this previous study, the explanted banana fruit seeds were stored at a freezing temperature, a cold temperature, and at room temperature. This previous study showed that banana fruit stored at room temperature (30°C) is not a suitable temperature for banana storage. The objective of the research was to find the effectiveness of the fruit and seed of the banana as a storage organ, and to achieve a good sterilization technique for fruit and seeds of bananas for an in vitro embryo culture. Fruits and seeds of wild bananas were stored at freezing tempera- tures of −20 to 25°C, cold temperatures of 1 to 5°C, and above room temperatures of 32 to 40°C). They were also stored for 3 different time periods: 4 to 10 days; 30 days; and 60 days. The parameters observed were conducted qualitatively, including fruit phenotype after stor- age, and the best sterilization techniques to obtain aseptic embryo cultures. 72 2.2 Sterilization technique and germination of wild bananas after storage Banana fruit and fertile seeds that were stored in different temperatures and storage periods were grown in vitro through the embryo culture technique. Seeds of the bananas were separated from their pulp by washing in rice husk ash (carbonized rice husk), and then in tap water. Washed seeds were transferred to a beaker glass containing water for 30 minutes. Only sunken seeds were used because most of the floating seeds were generally devoid of the embryo or endosperm. Sterilization of the treated seeds was undertaken in sterile conditions in a transfer box. Banana seeds that were stored for 30 days were soaked with 70% alcohol for 10 minutes fol- lowed by 100% sodium hypochlorite for 10 minutes. The banana seeds were then rinsed with sterile distilled water 4 to 5 times (the first sterilization technique). Banana seeds that were stored for 60 days were soaked with 1–2% mercuric chloride for 10 to 15 minutes followed by 70% alcohol for 10 minutes, then 100% sodium hypochlorite for 10 minutes. The sterile seeds were then soaked in sterile distilled water for 60 to 120 minutes, then dried using sterile filter paper (second sterilization technique). The banana seeds were placed in a sterile petri dish before the embryo was isolated. These two novel techniques sufficiently reduced the embryo contamination by 90–100%. Embryos were extracted under a transfer box and were cultured in Murashige and Skoog salts supplemented with 2.25 mg/l−1 6-benzyl adenine (BA) and 0.175 mg/l−1 indole-3-acetic acid (IAA). The embryos, after initiation, were cultured in a dark culture room at tempera- tures of 16 ± 1°C for 1 to 2 weeks and then transferred to a lighter culture room. The embryos were observed for percentage germination of shoot and calli formation. The experiments were repeated for twenty replications and analyzed statistically by descriptive statistics. 3 RESULTS AND DISCUSSION 3.1 Wild banana fruit and seed performance after a storage period with different temperatures The storage of banana fruit and seeds at various temperatures and periods was conducted to determine the viability of banana seeds after the storage period.
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