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

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 different 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, chemical 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 germination 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 temperatures 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 storage, 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 followed 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 temperatures 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. The results are shown in Figure 1. The bananas stored at room temperature (30°C) showed that the color of the banana skin turned from green to dark brown. The banana peel and skin were also covered with a fungus (Figure 1b). In the next experiment, the temperature was increased to the range 32 to 40°C. The fruit stored at these temperatures did not show any fungal growth, but the fruit became very dry, shrunk in size and the fruit color turned black. This indicated that the temperature range of 32 to 40°C was not a good temperature for banana fruit storage.

Figure 1. Performance of wild bananas (Musa acuminata Colla var microcarpa) before and after storage: (a) fruit after harvest; (b) stored at room temperature (30°C) for 2 weeks; (c) stored at above room temperature (32 to 40°C); (d) storage at freezing temperatures (-20 to -25°C); (e) storage in cold temperatures (1 to 5°C); and (f) banana seeds at room temperature. 73 Most of the fruit and seeds stored for 30 and 60 days at freezing and cold temperatures showed an alteration in performance. For the fruit stored at freezing temperatures (-20 to -25°C), the fruit size was relatively large, and the skin color changed from green to dark brown; the seeds also swelled. The storage of fruit at freezing temperatures suggests fruit damage occurred due to chilling injury (Figure 1d). According to Mangrich and Saltveit (2000), this is a physiological disorder that occurs in plants sensitive to temperatures below 12°C. Fruit and seeds stored at cold temperatures (1 to 5°C) shrink and turn blackish-brown (Figure 1e). Oxidative browning is a common occurrence and is often a severe problem for plant cells. This brown pigment occurs because of the accumulation of a phenolic compound. It is a chemical process and produces a brown pigment called melanin (Jones & Saxena, 2013). Storage of plant material at temperatures of 0 to -5°C is used for species that are tolerant to cold temperatures, but for plant species that are sensitive to cold temperatures, storage is at temperatures ranging from 0 to 15°C (Engelman, 1991; Kaviani, 2011). This experiment showed that fruits stored at above room temperature, freezing and cold temperatures for 30 days indicated that the banana fruit is sensitive to cold temperatures. Seed storage is a common method for genetic conservation of plants. Banana seeds are naturally orthodox seeds and have various dormancy types (Fortescue & Turner, 2011). Orthodox and sub-orthodox seeds could be stored at temperatures from -15 to -20°C (for those tolerant to cold temperatures) or 0 to -5°C (for subtropical and tropical species) (Kaviani, 2011). Banana seeds could germinate immediately after extraction, but the seeds showed secondary dormancy after storage (Uma et al., 2011). This experiment showed that wild banana seeds stored at above room temperatures, cold temperatures, and freezing temperatures did not show changes in performance of seeds (Figure 1f). According to Nagano et al. (2008) storage of banana seed can promote the germination of dormant seeds and immature seeds

3.2 Effects of the period of fruit and seed storage on in vitro embryo germination of wild bananas The germination of wild bananas was carried out through an in vitro embryo culture technique after storage. At the initiation stage, it showed that for most seeds stored for 60 days at freezing temperatures of -20 to -25°C, their embryos appeared to swell. Seeds stored in cold temperatures of 1 to 5°C) had an embryo size relatively no different from embryos that had been inoculated at 4 to 10 days after harvest. Whereas at temperatures of 32 to 40°C, the embryo shrank. The embryo was then germinated on the medium containing 2.25 mg/l−1 BA and 0.175 mg/l−1 IAA (Indrayanti et al., 2011; 2012). The ability to germinate wild banana embryos after storage was determined by calculat- ing germination potential. Wild banana seed germination is determined by the number of normal germinating embryos characterized by the emergence of a radicle. Germination is a measure of potential viability with the formula for the number of normal germinating embryos per number of embryos planted (Sadjad et al., 1999). The results of this study are shown in Figures 2 and 3 and Table 1. The effect of fruit stored at freezing and above room

Figure 2. Non-germinating embryos: (a) after the seeds were stored at -20 to -25°C for 30 days

(S2T1P1); and (b) after the fruit was stored at 32 to 40°C for 60 days (S1T3P2). 74 Figure 3. Embryo growth to shoot after the seeds are stored at a temperature of 1 to 5°C for 30 days

(S1T2P1): (a) 14 days after initiation; and (b) 60 days after initiation. The growth of the embryo became callused after the fruit is stored at a temperature of 1 to 5°C for 4 to 10 days (S1T2P0) in 30 days (c) and 60 days (d) after initiation.

Table 1. Percentage of embryo germination of wild bananas after storage at various temperatures and periods after 30 days of culture.

Percentage of Storage embryo germination Storage Temperature period Code of (%) after 30 days organ (°C) (days) treatment of culture

Fruit -20 to -25 4 to 10 S1T1P0 70

Fruit 1 to 5 4 to 10 S1T2P0 74

Fruit 32 to 40 4 to 10 S1T3P0 64

Fruit -20 to -25 30 S1T1P1 0

Fruit 1 to 5 30 S1T2P1 76

Fruit 32 to 40 30 S1T3P1 0

Fruit -20 to -25 60 S1T1P2 0

Fruit 1 to 5 60 S1T2P2 0

Fruit 32 to 40 60 S1T3P2 0

Seed -20 to -25 4 to 10 S2T1P0 70

Seed 1 to 5 4 to 10 S2T2P0 66

Seed 32 to 40 4 to 10 S2T3P0 76

Seed -20 to -25 30 S2T1P1 0

Seed 1 to 5 30 S2T2P1 84

Seed 32 to 40 30 S2T3P1 0

Seed -20 to -25 60 S2T1P2 0

Seed 1 to 5 60 S2T2P2 68

Seed -20 to -25 60 S2T3P2 0

temperature for 30 to 60 days on seed germination was that all the embryos did not germinate (Figure 2a–b). At those two temperatures, the embryo of the wild banana Musa acuminata Colla var. microcarpa germinated only at the storage period of 4 to 10 days and their germination percentages were 64–74% (Table 1 and Figure 3a–d). Germination is a physiological reaction; the ability of the embryo to germinate depends on specific temperatures and is related to starch digestion by amylase (Nagano et al., 2008). 75 Temperature during seed storage affects embryo development (Nagano et al., 2008). This study showed that there was no embryo germination after fruit storage for 60 days at all temperature treatments, after 30 days storage at freezing temperatures, and above room temperature (Table 1). Among these various temperature treatments, embryos germinated mostly at a temperature of 1°C to 5°C for all storage periods with their germination percentages invariably being 68–84% after 30 days of culture on the Murashige Skoog medium. The highest percentage of embryo germination (84%) was produced from seeds stored for 30 days. In the wild banana (Musa acuminata subsp. burmannica), the percentage seed germination ranged from 84.12% to 88.5% under in vitro conditions and 4 weeks after culture (Vineesh et al., 2015). The zygotic embryos of wild bananas from Mexico, Musa ornata Robx, took 21 days to germinate (Burgos-Hernandes et al., 2014). The developed embryo from stored seeds is still able to germinate (68%) until 60 days of expandable storage, although the number of days to germination increased (16 to 17, see Figure 4). According to Nagano et al. (2008), seed storage can promote the germination of dormant and immature seeds. Embryos grown from 30 days of fruit storage need 24 to 25 days to germinate, and their germination percentages were 76%. This occurs because of the activation of inhibitory compounds, at the stage of embryo maturation, which causes seed dormancy (Reed, 2005; Roy et al., 2006; Uma et al., 2011). All germinated seedlings grew and developed normally. The number of days to germination is the average of the time the embryo grows a normal sprout. Most of the embryos stored for 4 to 10 days germinated at 8 to 9 days after culture with the percentage germinating being 6–12% (Figure 4). For the fruit of the bananas stored at 1 to 5°C for 30 days of storage, embryos germinated at 18 to 19 days after culture (13%). For banana seeds stored at 1 to 5°C for 30 days of storage, embryos germinated 16 to 17 days after culture (11%), while for banana seeds stored for 60 days, the embryos germinated 24 to 25 days after culture (11%, see Figure 4). This study shows that Musa acuminata Colla var microcarpa stored for 60 days has a faster germination time than Musa veluntina Wendl. & Drude. Seeds of Musa velutina stored for 60 days took 42 days to germinate, but seeds stored for 4 to 8 months germinated in 14 to 21 days (Nagano et al., 2008). Lee et al. (2018) reported that for Thalictrum uchiyamae (Ranunculaceae), a species of herbaceous perennial, the physiological dormancy of the embryo was broken by a cold temperature of 5 °C for 60 days, but warm temperatures (15°C to 20°C) were required to promote embryo elongation after the physiological dormancy was broken. The percentage of embryos to germinate is at its maximum at the temperature storage range of 1 to 5°C (cold temperature) for 30 days of storage (84%). As the temperature declines to freezing temperatures or advances at above room temperatures, the percentage of embryos to germinate decreases and the number of days to germination increases. Bananas

Figure 4. Days of emergence and percentage of embryo germination of wild bananas Musa acuminata

Colla var. microcarpa (Becc.) at 8 to 30 days after culture. S1 = fruit storage, S2 = seed storage, T1 = temperature -20 to -25°C, T2 = temperature 1 to 5°C, S3 = temperature 32 to 40°C, P0 = 4 to 10 days of storage, P1 = 30 days of storage, and P2 = 60 days of storage. 76 are sensitive to low temperatures. The fruit will suffer damage after a storage period below 10 to 15°C (Mangrich & Salveit, 2000), 11 to 20°C (Hardaningsih & Alfi, 2010) or 11.5 to 13°C (Wang, 1991), with a critical temperature of 13°C. This damage occurs due to physiological disturbances where the color of the fruit will become decolorized. Therefore storage at freezing temperatures (-20 to -25°C) causes chilling injuries. Seeds stored in freezing conditions for 30 to 60 days showed damage to the embryo and caused dormancy. According to Fortescue and Turner (2011), banana seeds show secondary dormancy after storage. In this experiment, the fruit and seeds were stored at freezing temperatures caus- ing the seeds’ water content to become high. The duration of storage for 60 days can cause a decrease in the moisture content of the seeds which is closely related to the evaporation process of seeds during the storage period. Germination of ginger seeds (Zingiber officinale Rosc.) showed similar results in that the viability of the embryos decreased after being stored for 60 to 90 days (Sukarman et al., 2007). This study showed that improperly stored banana fruit and seeds at freezing temperatures (-20 to -25°C) and above room temperature (32 to 40°C) was, in contrast, not the right method for banana storage. The number of days needed to germinate and the total percentage of wild banana germination showed that temperature, and prolonged duration of storage organs, can critically affect the ability of the embryo to germinate in a culture medium. To prolong the duration of wild bananas, their seeds should be stored at a temperature of 1 to 5°C for 30 to 60 days.

3.3 Effect of various storage conditions to in vitro shoots and calli growth of wild bananas The embryos that were germinated in the MS medium, partially grew into shoots or calli (Figure 3b–d). The banana shoots and calli were subcultured at regular intervals every month in a fresh media. The number of shoots and calli were recorded 1 to 4 months after culture. At this stage, leaf plumula will grow to shoots, and the radicle will develop into roots. The effect of various storages on the number of in vitro shoots at 120 days after culture showed that embryos developed from fruit stored for 4 to 10 days at a cold temperature (1 to 5°C) had the highest number of shoots (5.00 ± 0.5). There was no significant difference in the number of shoots from seeds stored at cold temperatures for 4 to 10 days (4.60 ± 0.83) and 30 days of storage (4.60 ± 0.34, see Table 2). The lowest number of shoots produced from fruits stored at above room temperature (32 to 40°C, 1.90 ± 0.41, and see Table 2). This study shows that cold temperatures (1 to 5°C) are an optimum temperature for seed storage. According to Burgos-Hernandez (2014), the differences in the width of the water channel in banana seeds may result in differences in the ability of cells to form shoots. In Musa acuminata it has been reported that fresh seeds have a narrower water channel than dry seeds, so the uptake of water is higher for dry seeds (Puteh et al., 2011). The high percentage of germination (84%) and the high number of shoots (5.00 ± 0.78) produced from seeds stored at cold temperatures could be a good indicator for the success of plants in the acclimatization phases.

Table 2. Effect of various storage conditions on the average number of in vitro wild banana shoots 30 to 120 days after culture.

The average number of shoot growth in 30 to 120 days

Storage conditions 30 ± SE 60 ± SE 90 ± SE 120 ± SE

Fruit, freezing temp, 4–10 days of storage 2.20 0.39 2.50 0.37 2.70 0.40 3.20 0.33 Fruit, cold temp, 4–10 days of storage 2.88 0.40 2.88 0.40 3.63 0.32 5.00 0.78 Fruit, above room temp, 4–10 days of storage 1.90 0.41 1.90 0.41 1.90 0.41 1.89 0.45 Fruit, cold temp, 30 days of storage 3.20 0.13 3.20 0.13 3.20 0.13 3.40 0.22 Seed, freezing temp, 4–10 days of storage 2.71 0.29 2.71 0.29 2.63 0.26 2.86 0.51 Seed, cold temp, 4–10 days of storage 2.60 0.34 2.70 0.37 3.70 0.54 4.60 0.83 Seed, above room temp, 4–10 days of storage 1.60 0.22 2.10 0.23 2.10 0.23 2.30 0.21 Seed, cold temp, 30 days of storage 3.60 0.22 3.50 0.27 4.50 0.34 4.60 0.34

77 Table 3. Effect of various storage conditions on the average number of in vitro calli of wild bananas in 30 to 120 days after culture.

The average number of callus growth in 30 to 120 days

Storage condition 30 ± SE 60 ± SE 90 ± SE 120 ± SE

Fruit, freezing temp, 4–10 days of storage 1.83 0.40 1.70 0.26 2.00 0.26 2.11 0.26 Fruit, cold temp, 4–10 days of storage 1.33 0.21 1.80 0.29 2.20 0.25 2.29 0.29 Fruit, above room temp, 4–10 days of storage 1.00 0.00 1.67 0.67 1.67 0.67 1.67 0.67 Fruit, cold temp, 30 days of storage 2.30 0.30 2.30 0.30 2.50 0.27 2.80 0.29 Seed, freezing temp, 4–10 days of storage 1.83 0.48 2.11 0.42 2.89 0.42 2.56 0.41 Seed, cold temp, 4–10 days of storage 1.33 0.21 2.10 0.28 3.50 0.40 3.50 0.40 Seed, above room temp, 4–10 days of storage 1.25 0.25 2.43 0.30 3.00 0.38 3.00 0.38 Seed, cold temp, 30 days of storage 2.00 0.30 2.10 0.28 2.10 0.28 2.10 0.28

In this study, some of the embryos of wild bananas that had undergone germination were producing calli. Calli are parenchymal cells that divide abnormally during mitosis under tissue culture processing. There are two types of calli: friable, lignified/hard. Friable calli have a good genetic potential to differentiate and redifferentiate to form a plantlet after passing physiological and biochemical change (Akinyosoye et al., 2015), and through indirect orga- nogenesis or embryogenesis. In this study, the embryo which germinated after storage produced lignified and hard calli (Figure 3c–d). Lignin is a polymer of aromatic subunits derived from phenylalanine that strengthens and waterproofs specialized plant cells. Lignification is part of the normal differentiation and functioning of specific cell types. It also triggers a response to various abiotic and biotic stresses in cells that would not alternatively be lignify- ing (Barros et al., 2015). The effect of various storage methods on calli formation shows that embryos developed from seeds stored at cold temperatures and seeds stored at above room temperatures for 4 to 10 days, have the same number of calli (3.50 ± 0.40 and 3.00 ± 0.38, see Table 3). The lowest number of calli were produced from embryos developed from fruit stored at above room temperature (1.67 ± 0.67). The differences in embryo germination of the Musa species may be a reflection of different degrees of embryo dormancy after storage, and possibly because of the lack of gibberellic acid, and there are lots of plant inhibitors in the seed embryos.

4 CONCLUSIONS

Banana fruit and seeds stored at freezing temperatures and above room temperatures for 60 days were not the right method for banana storage. The most effective temperature for seed storage is in cold temperatures. At these temperatures, the wild banana Musa acuminata Colla var. microcarpa (Becc.) can be stored for two months. 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. In this study, the embryo which germinated after storage and developed shoots, also produced lignified and hard calli.

REFERENCES

Akinyosoye, S.T., Adetumbi, J.A., Amusa, O.D., Olowolafe, M.O. & Olasoji, J.O. (2014). Effect of seed size on in vitro seed germination, seedling growth, embryogenic callus induction and plantlet regeneration from embryo of maize (Zea mays L.) seed. Nigerian Journal of General Practice, 28(2), 1–7. Asif, M.J., Chai, M. & Othman, R.Y. (2001). In vitro zygotic embryo culture of wild Musa acuminata spp. malaccensis and factors affecting germination and seedling growth. Plant Cell, Tissue and Organ Culture, 67, 267–270. Barros. J., Serk, H., Granlund, I. & Pesquet, E. (2015). The cell biology of lignification in higher plants. Annals of Botany, 115(7), 1053–1074. doi:10.1093/aob/mcv046. 78 Burgos-Hernandez, M., Castillo-Campos, G., Mata-Rosas, M., González, D., Vovides, A.P. & Murguía- González, J. (2014). Seed germination of the wild banana Musa ornata (Musaceae). Seed Science and Technology, 42, 16–27. Retrieved from http://doi.org/10.15258/sst.2014.42.1.02. Engelmann, F. (1991). In vitro conservation of tropical plant germplasm—a review. Euphytica, 57, 227–243, Fortescue J.A. & Turner, D.W. (2011). Association between low temperatures and anatomical changes in preanthetic ovules of Musa (Musaceae). Scientia Horticulturae, 104, 433–444. Hardaningsih, W. & Alfi, H. (2010). In vitro medium-term storage of several banana genotypes (Musa spp L.). Journal of Jerami, 3(1), 1–6. Harry, G.I., Ebong, U.U. & Vuylsteke, D.R. (2010). In vitro rescue of zygotic embryos for the enhance- ment of hybrid plantain (Musa, AAB Group) production. Nigerian Journal of Agriculture, Food and Environment, 6(3&4), 8–13. Hermanto, C., Sutanto, A., Edison, H.S., Alfons, R., Hosang, E., Daniells, J. & Hilman, Y. (2013). Triangle banana exploration report, Central Maluku and Lesser Sunda Islands, Indonesia, 16 February–6 March 2013. Montpellier, France: Bioversity International. Retrieved from http://www.musalit.org/. Heslop-Harrison J.S. & Schwarzacher, T. (2007). Domestication, genomics and the future for banana. Annals of Botany, 100, 1073–1084. Imarhiagbe, O., Osazee, J.O., Aiwansoba, R.O. & Shittu, O.H. (2016). In vitro germplasm collection and storage: A review. International Journal of Biology Research, 1(1), 9–14. Retrieved from www.biotechjournals.com. Indrayanti, R., Mattjik, N.A., Setiawan, A. & S. Sudarsono. (2011). Radiosensitivity of Banana cv. Ampyang and Potential Application of Gamma Irradiation for Variant Induction. Journal of Agronomy Indonesia, 39(2), 104–112. Indrayanti, R., Mattjik, N.A., Setiawan, A. & S. Sudarsono. (2012). Evaluation of Phenotypic Vari- ability among Banana cv. Ampyang Regenerated From Gamma Irradiation Induced-Mutation in a Greenhouse. Journal of Horticulture Indonesia 3(1):24–34. International Network for the Improvement of Banana and Plantain & Central Research Institute for Horticulture. (2002). The exploration of Musaceae in Irian Jaya (Papua) (p. 58). Research Institute for Fruits Central Research Institute for Horticulture. http://www.musalit.org. Jones, A.M.P. & Saxena, P.K. (2013). Inhibition of phenylpropanoid biosynthesis in Artemisia annua L.: A novel approach to reduce oxidative browning in plant tissue culture. PLOS One, 8(10), e76802. doi:10.1371/journal.pone.0076802 Kaviani, B. (2011). Conservation of plant genetic resources by cryopreservation. Australian Journal of Crop Science, 5(6), 778–900. Lee, S.Y., Rhiea, Y.H. & Kima, K.S. (2018). Dormancy breaking and germination quirements of seeds of Thalictrum uchiyamae (Ranunculaceae) with underdeveloped embryos. Scientia Horticulturae, 231, 82–88. Mangrich, M.E., & Saltveit, M.E. (2000). Effect of chilling, heat shock, and vigor on the growth of cucumber (Cucumis sativus) radicles. Physiologia Plantarum, 109, 137–142. Nagano, S., Mori, G. & Oda, M. (2008). Effects of temperature and moisture content of the substrate during storage on embryo development and germination in seeds of Musa velutina Wendl. & Drude. Journal of Horticultural Science & Biotechnology, 83(10), 33–36. Nasution, R.E. (1991). A taxonomic strudy of species Musa acuminata Colla with its intraspecific taxa in Indonesia (Vol. 32, pp. 1–122). Tokyo, Japan: Memoirs of the Tokyo University of Agriculture. Ploetz, R.C., Kepler, A.K., Daniells, J. & Nelson, S.S. (2007). Banana and plantain and overview with emphasis on Pasific islands cultivars. Specific Profiles for Pacific Island Agroforestry, (26 pp) http:// www.traditionaltree.org. Pua, E.C. (2007). Banana. In E.C. Pua & M.R. Davey (Eds), Biotechnology in agriculture and forestry, Transgenic Crops V (Vol. 60, pp. 3–31). Berlin, Germany: Springer-Verlag. Puteh, A.B., Aris, E.M., Sinniah, U.R., Rahman, M., Rahman, M.M., Mohamad, R.B. & Abdullah, N.A.P. (2011). Seed anatomy, moisture content and scarification influence on imbibition in wild banana (Musa acuminata Colla) ecotypes. African Journal of Biotechnology, 65, 14373–14379. Rashid, K., Mamat, M., Daran, A.B.M., Nezhadahmadi, A., Ruslan, F. & Kayat, F. (2013). Seed prog- eny population of wild banana Musa acuminata ssp.malaccensis for Fusarium screening. Life Science Journal, 10(3), 671–680. Retrieved from http://www.lifesciencesite.com. Reed, S.M. (2005). Embryo rescue. In D.J. Gray (ed.), Plant development and biotechnology (pp. 235– 229). New York, New York: CRC Press. Roy, S., Asaduzzaman, M., Pramanik, M.H.R., & Prodhan, A.K.M.A. (2006). Effect of banana plant extracts on germination and seedling growth of some vegetable crops prodhan. Bangladesh Journal of Crop Science, 17(1), 235–242. 79 Sadjad, S., Muniarti, E. & Ilyas, S. (1999). Seed vigor testing parameters from comparative to simulative. PT Widiasarana Indonesia.Jakarta, Indonesia. Sipen, P., Chubo, J.K., King, P.J.H., Huat, O.K. & Davey, M.R. (2011). Genetic improvement of banana using conventional and in vitro technologies. Journal of Crop Improvement, 25(6), 697–727. Sukarman, D. & Rusmin, M. (2007). Viability of ginger seeds (Zingiber officinale Rosc.) In different ways of cultivation and storage time. Indonesian Spice and Medicinal Crops Research Institute (ISMCRI), 18(1), 1–12. Sulistyaningsih, L.D., Megia, R. & Widjaja, E.A. (2014). Two new records of wild bananas (Musa balbisiana and Musa itinerans) from Sulawesi. Makara Journal of Science, 18(1), 1–6. Uma, S., Lakshmi, S., Saraswathi, M.S., Akbar, A. & Mustaff, M.M. (2011). Plant regeneration through somatic embryogenesis from immature and mature zygotic embryos of Musa acuminata ssp. burmannica. In Vitro Cellular & Development Biology—Plant, 48, 539–545. Valmayor, R.V, Jamaluddin, S.H., Silayoi, B., Kusumo, S., Danh, L.D., Pascua, O.C. & Espiro, R.R.C. (2000). Banana cultivar names dan synonyms in Southeast Asia. (26 pp), INIBAP France. ISBN 971-91751-2-5. Vineesh, P.S., Skaria, R., Mukunthakumar, S., Padmesh, P. & Decruse, S. (2015). Seed germination and cryostorage of Musa acuminata subsp. burmannica from Western Ghats. South African Journal of Botany, 100, 158–163. Wang, C.Y. (1991). Chilling and freezing injury. Retrieved from http://globalripening.com/ne-posthar- vest.com/hb66/017chilling.pdf.