Endangered Durio spp. conservation and seed germination in Indonesia

Abstract Wild king fruit (Durio spp, Malvaceae) is a threatened native tree species in Kalimantan, Indonesia which needs significant and urgent cultivation. This study characterized the appearance, flavor, and germination rate of four durian species D. zibethinus, D. dulcis, D. kutejensis, and D. oxleyanus. The results showed that D. oxleyanus was one of the superior fruits with a very sweet flavor, smooth texture, and juicy flesh despite its green thorny exterior. Four months after sowing, the germination capacities (p-value 0.18) were as follows: D. kutejensis 7% (± 1.4%), D. dulcis 0%, D. oxleyanus 1% (± 0%), and D. zibethinus 21% (± 26.2%) at Banua. In Bogor, West Java, seed germination was: D. zibethinus 58% (± 2.8%) which developed into seedlings, D. kutejensis 10% (± 0%), and 0% for both D. dulcis and D. oxleyanus). Ex-situ conservation of the four Durio species by seed propagation needs to be conducted carefully to avoid the failure of seedling growth.


Background
In Southeast Asia, durian (Durio spp) is named after the Malayan word 'duri' which means 'spine' due to its spiny appearance. The durian is known as the 'king of fruits' (Wallace 1856) due to its large and expensive fruit and distinctive taste. It is also favored by people in the Southeast Asia, however its distinctive strong aroma makes it forbidden to be carried on planes or in hotel rooms. Indonesia is one of the world's centers of plant genetic diversity, particularly for tropical fruits such as durian (Sastrapradja and Rifai 1989).
The island of Borneo, in particular, is the most important distribution area for relatives of the genus Durio in the family Malvaceae. Durio was initially classified into the Bombacaceae family (Bremer et al. 2009), but is now revised to be included in the Malvaceae. According to an examination of 270 herbarium specimens at Herbarium Bogoriense (Uji 2005), 20 species of durian were identified in Indonesia. Eighteen of the 20 species are found in Kalimantan, 7 species in Sumatra and 1 species in Java, Bali, Sulawesi, and Maluku. There are approximately 27 species of Durians in the world, 18 of which grow in the Borneo islands (Kalimantan, Sabah-Sarawak, and Brunei Darussalam), 11 in Malaya, and 7 in Sumatra (Uji 2005). Fourteen of the 18 Durio species in Borneo are listed as endemic (Kostermans 1958). Four of 9 species are reported by Seibert and Nephelium (1991) as having edible fruit (Table 1), but its population has declined, and seedling numbers have significantly and quickly been reduced.
This study reports on an inventory and descriptions of the fruit of species found on Pulau Laut, Kota Baru District, and South Kalimantan Province, Indonesia, and the cultivation of them in the Banua Botanical Garden (South Kalimantan), and in the Bogor Botanic Garden (West Java).

Study area and botanical identification
The research was conducted in March 2018 in the INHUTANI II forest and surrounding area in Pulau Laut forest (Figure 1). Purposive sampling methods used to King fruits explored. Global Positioning Sites were noted, and fruit and leaves were collected as herbarium vouchers for identification.

Organoleptic test
Organoleptic tests (Lestari and Purwayantie 2018) were carried out by 50 panelists using a scoring method on King fruits species. The purpose of this was to determine the level of acceptance of King fruits. The scoring scale was based on a preference level from 1 to 3, in which 1 was for dislike, 2 was for normal, and 3 was for like (Table 2).

Seed propagations
Seeds were sown and grown to the seedling stages at Banua Botanical Garden, Banjar Baru, South Kalimantan and Bogor Botanical Garden, West Java (hereafter as Banua BG and Bogor BG). The two botanical gardens were not considered treatment but were used as nursery sites and for their living collections. Observations were also made on the seed germination and seedling growth of each species. One hundred seed were germinated at each site for each species. The number of germinated seeds and seedling survival were recorded during the first four months of growth, which is considered a suitable age for developing strong roots and stems.

Appearances of fruits
Data were analyzed with the descriptive method shown in Table 3. The results showed that D. oxleyanus fruit had the highest score (10) and were favored by panelists. Four species, D. zibethinus, D. dulcis, D. kutejensis, and D. oxleyanus have the largest potential to be developed as edible fruits and have high economic value., D. oxleyanus has a yellow flesh which was the thickest, a strong sweet taste, and juicy, soft texture.
Observations of four durian species are listed in Table 4. The texture and taste (sweetness) quality of all species were considered the same. From the results in Table 4, D. oxleyanus (Merdaun) seems to be one of the best quality fruits in terms of appearance. Its advantages include thick fleshy arils, with a very sweet taste, and smooth, watery texture, although there are fewer seeds with an unattractive oval shape, and it has green, prickly skin ( Figure 2). In contrast, D. kutejensis (Pampaken), has a more rounded fruit with an attractive yellow skin and orange to yellow flesh which is D. oxleyanus Griff.
Kerantung (Seibert and Nephelium 1991), merdaun (present study) Not evaluated (Malaysia Plant Red List Plants 2010b) a A taxon is not evaluated against IUCN criteria; Vulnerable A1c: the best available evidence indicates that the population has reduced more than 50% in the past (A1) and its endemic area, extent of occurrence, and/or habitat quality has reduced (c).  thin and dry. D. dulcis has a relatively longer fruit, a red spiny skin, and fewer seeds with thick, sweet, but fibrous arils. Although it is attractive, the thickness of the flesh was recorded as medium. D. zybethinus has many varieties in forests and is widely cultivated in agricultural land. D. zybethinus in forests produces the smallest fruit (13 cm) and has a medium thickness of flesh. In summary, the ripe fruit of D. oxleyanus ( Figure 2b) is relatively small and oval in shape with a spiny green skin, and is less attractive in appearance than D. kutejensis ( Figure 2c) with a large, round, bright yellow fruit with short spines. However, the savory, fragrant, juicy, thick flesh aril of D. oxleyanus was considered superior in organoleptic quality compared to D. kutejensis and D. dulcis.

Appearances of fruits
Although the results of the survey in this study indicated that D. oxleyanus was more popular and superior than D. kutejensis, D. dulcis and D. zibethinus in Pulau Laut, South Kalimantan, it sometimes differed from other locations in terms of taste. Another study reported that D. kutejensis has a yellow to orange-gold color due to its beta-carotene level (Charoensiri et al. Bogor BG (100 seeds) 0 ± 0 10 ± 0 0 ± 0 58 ± 2.8 2009) which is considered more attractive to consumers than other species with light yellow coloring. The solid, dry, and fibrous texture of D. kutejensis is caused by lower levels of water content (Sipahutar 2008). Other research shows that D. kutejensis taste, based on organoleptic testing, is sweeter (Belgis et al. 2016). The sweetness of durian increases with ripeness and is caused by the production of sucrose, followed by glucose, fructose, and maltose (Shaffiq et al. 2013;Belgis et al. 2016). reported that D. kutejensis has a higher value of sucrose (13%). The appearance of D. kutejensis is more attractive, although for the sweet taste and soft texture, D. oxleyanus is preferred. In general, durian fruits in Kalimantan has a various taste because it comes from the seed. It's just a offspring of the fruit from parent trees that grows in the forest. Whereas germplasm from parents with sweet fruit can be crossed with parents whose fruit looks attractive. D. oxleyanus had the best quality compared to D. kutejensis and D. dulcis in having delicious, aromatic, juicy, and thick flesh. Durian is considered superior if it meets the following criteria: (1) the overall appearance of the fruit is attractive; (2) the spines on the skin are large and pyramidal in shape; (3) the fruit is elliptical and regular, i.e. faintly or not too star-shaped; (4) it has a relatively short fruit stalk; (5) the flesh is smooth, the texture is fibrous, moist, yellow-honey (copper) in color, thick, and sweet; (6) the shape of the tree is symmetrical, and is in the form of a pyramid or an umbrella; (7) branches appear regularly from the main trunk; and (8) trees have high productivity and resistance to pests and diseases (Setiadi 2003). In addition, an ideal durian fruit should weigh between 1.5 to 2.0 kg, with an edible component as high as 30% to 46% of the total fruit weight. In general, a superior tree must have the following characteristics: (1) high fruit productivity in one season; (2) fruiting at a relatively young age; (3) resistance to pests and diseases; (4) propagation by stem or shoot grafting; (5) fruit with a pleasant taste and aroma, and (6) fruit with uniform shape, size, and color (Mansur 2007). To obtain this superior type of durian from germplasm in Pulau Laut, it is necessary to make careful selections according to these criteria. Furthermore, crossbreeding between the four main species may be necessary to combine these features.

Seed propagation
So far there has been limited information on the successful growth of D. oxleyenus, D. kutejensis and D. dulcis seedlings except for D. zibethinus which even has many and varied cultivars (Fitmawati and Betty 2012). reported on five local name cultivars of D. zibethinus seeds having a germination rate i.e. Tembaga (96.5%), Terong (94.5%), Kunyit (94.1%), Malukuik (84.9%), and Jantung (79.6%). The germination rate of D. kutejensis is similar to other durian species, about 85% over a ten-day period (Anonymous , 2013). D. zibethinus or Durian seeds are recalcitrant and are able to germinate within 3-20 days after planting, but generally do so within one week (Uji 2005; Shih et al. 2008). reported that unlike orthodox seeds, most recalcitrant seeds could not survive during dehydration or chilling (less than 10 C) and thus were not able to store for long periods. Observations of the emerging durian seedlings in this study indicated that fungi and other types of organisms were the main cause of spoilage. The ideal temperature for durian seed germination was reported by (Uji 2004;Navia and Chikmawati 2018) to be a constant 28-29 C although temperatures lower than 24 C still resulted in adequate germination (Kartikasari 2001). Fusarium solani and the pathogenic fungus Phytophthora palmivora were identified by a pathogenicity test to cause durian to not grow (Yep et al. 2001). Durian seedlings were also found to be susceptible to rat, fungal, and bacterial diseases, particularly Rhizoctonia and Phytophthora (Pongpisutta et al. 2020). Many factors such as watering and humidity levels affected growing of bacteria and caused rotting of seeds. Therefore durian seeds should be treated with fungicide and media sterilized.

Implications for conservation
In the lowlands of West Halmahera Island, the distribution and growth of durian is influenced by local microclimate (Sirajudin 2018). Sunlight factors determine tree height and growth rate, thereby affecting fruiting time. The recalcitrant nature of durian seedlings requires careful and immediate handling. The original durian tree is threatened with extinction in Kalimantan if the conversion of forests and traditional agricultural land to oil palm and rubber monocultures continues at its current rate. At the Katingan Botanical Garden (Katingan, Central Kalimantan Province) an ex-situ conservation effort has been made to collect 7 species from the forest around Central Kalimantan: D. dulcis, D. grandiflorus, D. graveolens, D. kutejensis, D. oxleyanus, D. testudinarius, and D. zibethinus (Aprilianti 2019). Only four of these species were found that have been cultivated, while the rest still grow wild in cultivated forests, namely D. zibethinus, D. kutejensis, D. dulcis, and D. lowianus (Ketsa et al. 2019). Durio is included in the category of rare plants that need to be preserved (Love et al. 2019). Seed propagation is still common in Indonesia and Malaysia but high genetic variability in offspring due to crosspollination (Yumoto 2000) requires intensive care in order to ensure seedling survival and growth to tree maturation. In addition, the ideal fruit should weigh between 1.5 to 2.0 kg, with edible components as high as 30-46% of the total fruit weight (Haryanto and Royananingsih 2003).
The conservation status (Table 1) of the IUCN Red List has D. dulcis and D. kutejensis susceptible to vulnerable (A1c) (World Conservation Monitoring Centre 1998; Rahman 2021). D. oxleyanus and wild D. zibethinus were not evaluated (Malaysia Plant Red List Plants 2010a; 2010b). The Indonesian government has made efforts to protect Kalimantan's forests and have arrangements not only for in-situ protected areas such as national parks, nature reserves, and protected forests, but also for ex-situ conservation of local Kalimantan fruits. The three wild species, D. kutejensis, D. dulcis, and D. oxleyanus, should be considered for conservation, via arboreta, the development of traditional agroforestry (the local name of agroforestries i.e. lembo, munan, simpung, pulong bua 0 , dalung bua 0 , tundang kemurlan, karloka kemurlan, tembawang and dusun) and conservation through cultivation in parks, city parks, campuses, roadsides, experimental gardens, and residential gardens. The research location is in West Kutai District, East Kalimantan Province in the Lembo forest which is a former shifting cultivation area. The most prominent trees have a canopy height of more than 30 meters, occupy only 3.85% of the area and are dominated by D. zibethinus, Octomeles sumatrana, and Macaranga triloba which have a generally large diameter (Siregar 2006). The existence of local fruit trees in Kalimantan is threatened by the presence of external cultivars and other commercial trees such as coconut (Cocos nucifera), oil palm (Elaeis guinensis) and rubber trees (Hevea braziliensis) (Mansur 2007). For this reason, it is necessary to have an ex-situ conservation program specifically for fruit trees endemic to Kalimantan (Thorogood et al., 2022). stated that there needs to be support from the conservation community in Southeast Asia to preserve and develop Durio species.

Conclusions
In this study, the texture and taste (sweetness) quality of all durians were descriptively the same. D. oxleyanus had a yellow flesh which was the thickest with strong sweet taste and juicy, soft texture. Although D. kutejensis had a yellow to orange-gold color which is considered more attractive compared to the other species. Seeds of Durio are recalcitrant and seedlings were difficult to grow beyond the early growing stage and survive. Seed germination was very low and the conservation status under IUCN RedList categories is not yet evaluated. Protection of forests and vegetative propagation are necessary for conservation. This study was limited to report only on D. kutejensis, D. dulcis, and D. oxleyanus seed germination.