Urgent propagation of the endemic and possibly extinct in the wild tree Cassine koordersii (Celastraceae)

Cassine koordersii Kosterm. (Celastraceae) is a narrow endemic tree species known only to Mt. Puger, Jember, Indonesia. The IUCN Red List classifies it as Critically Endangered (CR) possibly extinct in the wild. Currently, there are only two living collections at the Bogor Botanic Gardens. As an urgent measure to anticipate extinction, vegetative propagation through shoot cuttings was conducted. This study aimed to determine the effects of rooting media and Rootone-F hormone on the successfulness of shoot cuttings of C. koordersii. It was carried out at the glass house in April 2022 with a 3-months observations period. The Komatsu-FORDA Fog Cooling (KOFFCO) system was adopted. The research used a completely randomized factorial design with rooting media and Rootone-F hormone as treatments. The media were sand (M1), cocopeat+burnt husk 2:1 (M2), and perlite+vermiculite 1:1 (M3). The Rootone-F hormone treatments included control (R1) and the addition of Rootone-F (R2). Six treatment combinations and four replications by 12 units obtained a total of 288 cuttings. The results indicate that in general, M2 and R2 have the best average yield: percentage of rooted cuttings (14.58%), number of primary roots (1.7), primary root length (1.58 cm), and percentage of survival cuttings (78.12%). The interaction between rooting media and Rootone-F hormone was not significant for all parameters. Despite only few new individuals are generated, they are valuable material for ex-situ collection backup. In addition, new juvenile seedlings are designed as mother trees for clonal orchards. Their high percentage rooting is expected to support the reintroduction program to their natural habitats.


Introduction
Indonesia is an archipelagic country and known worldwide as one of the top most biodiverse countries [1,2,3].Yet it must face the threat of rapid forest destruction due to human 1255 (2023) 012024 IOP Publishing doi:10.1088/1755-1315/1255/1/012024 2 intervention, leading to deforestation and land degradation as well as the extinction of plant species [4].Java is the most densely populated island in Indonesia [5], home to 56.10% or 151.58 million people on only 7% of the total land area of the country [6].In fact, approximately 30% of the population of Java live in villages adjacent to forests.Dense population and high living needs, however, may adversely impact on the utilization and sustainability of natural resources [7].According to [8], in April 2023, the number of rare plant species in Indonesia include: 1 Extinct (EX) and 3 Extinct in the Wild (EW); Critically Endangered (CR), Endangered (EN) and Vulnerable (VU) are 214, 392, and 464 respectively.
Cassine koordersii Kosterm.(Celastraceae) is one of endangered plants endemic to East Java Indonesia.It has been recorded that this species is endemic specially in the Lampesan Valley and Watangan Mountains, Puger, Besuki Regency [9,10], now Jember, East Java Province.It grew well in the limestone sedimentary rock soil of the deciduous teak forest ecosystem on elevation 300 m a.s.l.[9,11].C. koordersii is a plant habitus of large tree with height reaches 15.86 m and diameter of 1.47 cm, dense crown [12], and flower and fruit throughout the year with peaks occurring in August, September, and October [13].The currently conservation status of C. koordersii is Critically Endangered Possibly Extinct in the Wild A2c; B1 ab(iii)+2ab(iii) [11,12,14].A plant is considered Extinct in the Wild when it no longer exists in its natural habitat and can only be found surviving in cultivation, captivity, or as a naturalized population far from its original range, and an exhaustive survey has been carried out in known and/or expected habitats have failed to record an individual [15].
This species is also one of the critically endangered plants that has been successfully conserved at ex-situ area in Bogor Botanical Gardens [12,13], despite the mother tree is already dead.In 2020, field survey was carried out to find this species in its natural habitat, but the effort was unsuccessful [11].The only living collection is two 18-year-old trees in the ex-situ area of the Bogor Botanical Gardens, but they bear no fruits or flowers.Previously, trial propagation of C. koordersii had been attempted using conventional methods such as grafting and shoot cuttings with limited plant materials.However, the rooting ability was low, with only one out of ten grafting successfully rooting and five out of twenty-eight shoot cuttings successfully producing roots [11].Currently, research on vegetative propagation adopting KOFFCO methods is immediately required in order to improve shoot cuttings and produce more viable new individuals in the form of seedlings.This is crucial as a preventive measure against the risk of extinction and to support the reintroduction program aimed at enriching the natural habitat's population.

Materials and Methods
This research was conducted in the greenhouse of threatened plant species in Research Center for Plant Conservation, Botanic Gardens and Forestry BRIN in April-July 2022.The shoot cutting materials were collected from the 18-year-old C. koordersii conserved in Bogor Botanic Gardens (Figure 1a).Vegetative propagation was carried out, adopting Komatsu-FORDA Fog Cooling System KOFFCO systems with a slight modification [16,17].
Three types of media were used: sand, cocopeat+burnt husk 2:1, and perlite+vermiculite 1:1.The media were sterilized by steaming for approximately 4 hours at 100°C and subsequently put into the pot tray in the KOFFCO propagation box.
The shoot cutting materials were obtained from branches by cutting off about 12 cm long at a 45° angle and leaving the top 4 leaves.To minimize transpiration, the leaves were cut in 2/3 parts.They were soaked in a tub filled with water and a mixture of fungicides.Rootone-F hormone was prepared by mixing Rootone-F powder with water to form a paste according to the directions for use.Rootone-F paste was applied to the cuttings and stored for 30 minutes (Figure 1b).
Cuttings were planted on the prepared media, namely in KOFFCO propagation boxes based on the treatment (Figure 1c).The propagation boxes were stored in a greenhouse with controlled air temperature <30°C with automatic water fog sprinkle, light intensity reduced using shadenet to about 60 %, and humidity of 95%.Watering was done in the beginning of planting and later when required, about twice a week to ensure the seedlings received enough water during the initial growing stage.Fungicide was applied once a week to protect the cuttings from fungus attacks.The research used a completely randomized factorial design with rooting media and Rootone-F hormone as treatments.The media were sand (M1), cocopeat+burnt husk 2:1 (M2), and perlite+vermiculite 1:1 (M3).The Rootone-F hormone treatments included control (R1) and the addition of Rootone-F (R2).Six treatment combinations and four replications by 12 units obtained a total of 288 cuttings.Observation was done after 12 weeks with the observed parameters including percentage of rooted cuttings, number of primary roots, primary root length, and percentage of survival cuttings.

Results and discussion
Root formation is a critical phase that determines the success of rooted cuttings.Roots play an important role in the growth and development of plant.The success of rooted cuttings depends on the plant species, including for woody trees in which some are easy while others are difficult to propagate.The analysis results of various media and Rootone-F treatment on C. koordersii cuttings after 12 weeks are presented in Table 1.Until the end of observation, the cuttings were fresh and in health condition as indicated by green leaves and new shoots.High survival rate of shoot cuttings of C. koordersii is indicated by the survival percentages: 50-78% (Table 1).The survival rate of cuttings is the number of cutting materials undergoing the rooting process in propagation boxes with fresh appearance that denotes their potential to survive prior root formation.The media of Cocopeat+burnt husk 2:1 (M2) was able to provide suitable growing medium as indicated by the highest survival percentage of 78%.According to [18,19], the combination of cocopeat and burnt husk is effective for root growth since the pores effectively bind and store water and nutrients.In addition, this medium contains primary nutrient, phosphorus (P), which able to stimulate root growth and development as well as plant resistance to disease [20].
Based on the observation for 12 weeks, despite the small number, new roots were identified from several cuttings.Cocopeat+burnt husk 2:1 (M2) media had the highest percentage of rooted cuttings of 11.44 %.Meanwhile, the percentage of rooted cutting from the combination of cocopeat+burnt husk 2:1 and Rootone-F (M2R2) was 14.58 %.The relatively low percentage was allegedly caused by the cutting materials, which were obtained directly from 18-year-old mother tress without prior rejuvenation of the branches.Meanwhile, [21,22,23] suggest that the juvenile level of cuttings is significantly influenced by the age of mother trees, and it determines the rooting of cuttings.Materials obtained from old mother tree tend to have low rooting ability [22,24].In addition, the low rooting ability of cuttings is allegedly caused by the addition of Rootone-F hormone, which is unfavorable for callus differentiation [25].Despite the statistically insignificant difference of this treatment to control (Table 2), It indicated positive response toward rooting ability with percentage of rooted cutting of 9.03% while control of 4.85 % (Table 1) (Figure 2).The number of primary roots is major parameter to determine the number of roots formed on the cuttings that will determine the accelerated growth of the cuttings after transplanting.Cocopeat+burnt husk 2:1 (M2) media generated average number of 1.70 primary roots, sand (M1) media of 1.39 roots, while none was identified from Perlite+Vermiculite 1:1 (M3) media (Table 1).The absence of roots on M3 media is allegedly caused by large and porous perlite grains hence not strong enough to store water and to retain moisture that is essential for the rooting process.Nevertheless, the condition of most of the cuttings were still fresh (Figure 2c).We estimated that the observation of shoot cuttings in this medium would require more than 12 weeks with the expectation that roots would emerge.Although statistically the Rootone-F application is not significantly different.Subsequently, the treatment of Rootone-F and control showed statistically insignificant difference (Table 2), yet the addition of Rootone-F positively affected the formation of primary roots (1.15%) while control of 0.91 (Figure 2ab).
The parameter of root length was also examined, showing cocopeat+burnt husk 2:1 (M2) media had the best yield with average length of 1.58 cm, and the longest root of 10.5 cm (Figure 3a).Statistically, the root lengths of cuttings with the addition of Rootone-F and control were insignificantly different, 1.01 and 0.9, respectively.Meanwhile, root formation could not be identified from the cuttings on M3, possibly caused by the coarse and hard texture of perlite and vermiculite that have high porosity and absorb only small amount of water [26].Such texture hinders the roots to penetrate the media so that the growth and development of cutting roots is also hampered.Rooting ability is a key parameter in vegetative propagation, particularly shoot cuttings.During the observation, leaf loss can be used as an indicator of survival and fresh cuttings.Leaves are plant organs where they synthesize food through photosynthesis for plant needs and as food reserves [27,28].Leaf loss occurred when the cuttings were 2 weeks.The highest number was found on perlite+vermiculite 1:1 (M3) with an average of 2.49 strands, while the lowest number was on cocopeat+burnt husk 2:1 (M2) of 2.21 strands.Leaf loss may lead to unsuccessful cutting propagation [29].In addition, [30] argues that high temperatures cause cuttings to lack water, dry out, and drop their leaves.
The treatment of media and Rootone-F had no significant effect on the parameters of leaf loss, the number of shoots and new leaves of C. koordersii cuttings (Table 2).The shoots emerged after 3 weeks and new leaves appeared after 4 weeks.The growth of new shoots and leaves is an indicator of living cuttings and root formation, but some merely grow but then fall off.It possibly occurs because the new shoots and leaves are supported by food reserves in the form of carbohydrates in the stems.Consequently, when the food reserves run out while the roots have not formed yet, the new shoots and leaves will wilt and fall off [31,32].The effort of vegetative propagation by shoot cuttings of C. koorderrsii is the first step in a conservation strategy for critically endangered, endemic and extinct in the wild species.The new individuals yielded from this effort might be small in number (Figure 3) but they are valuable as the materials for the subsequent activity to attain a success cutting propagation.It is a treasure to support treasure ex-situ conservation program, particularly as a backup for the living collection at the Bogor Botanic Gardens.Meanwhile, at the same time, it also supports in-situ conservation program through reintroduction programs and population enrichment in the natural habitat [33].

Conclusion
The present study is the firstly important vegetative propagation for critically endangered tree species, narrowly endemic and possibly extinct in the wild.Eighteen old C. koordersii can be propagated by using shoot cuttings technique adopted KOFFCO systems and resulted in low rooting ability (11.4%).The cocopeat+burnt husk (2:1 v/v) (M2) media with Rootone-F addition produced the highest rooting ability (14.58 %).Despite only a few shoot cuttings are able to form roots due to the old mother trees, these new seedlings are expected to serve as the clonal seed orchard of C. koordersii for further propagation.Finally, it is expected that the percentage of success will be greater in the future.

Figure 2 .
Figure 2. Rooting ability of C. koordersii: (a) rooted cuttings with Rootone-F application, (b) rooted cuttings in control, (c) Cuttings that appear healthy but have not yet developed roots

Figure 3 .
Figure 3. New individual seedlings of C. koordersii shoot cuttings: (a) rooting type the cuttings, (b) transplanting to the soil media, (c) growing new shoots and leaves

Table 1 .
The average value of main parameters of C. koordersii cutting (12 weeks after cutting)

Table 2 .
The summary of variance analysis of shoot cuttings parameters for rooting ability