Biotechnological Approaches for the Improvement of Magnolia Genus Grown in Indonesia

Magnolia sp., also known as Michelia, are woody fragrant flowering plants that have been used in traditional medicinal purposes. This review covers past, curent and future potential studies of Magnolia species grown in Indonesia. There are 28 species and a hybrid of Magnolia that have been recognized and distributed in Indonesia. Conventional cultivation of Magnolia becomes very hard due to poor seed germination. It is caused by hard seed coat, short-lived seed, a fleshy red outer layer of seed called aeril. There are a few studies about in vitro culture and volatile compounds of Magnolia genus at the world literture. M. champaca , M. liliifera , and M. alba (hybrid) and others widely grow in Indonesia, yet the output of studies are anadequate about in vitro or ex vitro cultivation in Indonesia. This review compiles the works of Magnolia species carried out in the past and approaches for future breeding and production studies. These new approaches will significantly contribute to the economic production of the Magnolia species grown in Indonesia.


Introduction
The genus Magnolia known as Michelia in Magnoliaceae family is woody flowering plant of about 223 species in the world that are widely distributed in tropical and subtropical regions [1]. Some species of Michelia have been used for use in traditional medicine. It has fragrant attractive flowers [2,3,4,5]. The 25 species of Magnoliaceae are discovered in Indonesia [6]. There is limited report on Magnolia research especially species that is commonly grown and distributed in Indonesia known as a region of Indomalaya ecozone. Indomalaya ecozone spreads most of South (Indian subcontinent) and Southeast Asia to the southern part of East Asia (lowland southern China) through Indonesia, Phillipines, lowland Taiwan and Japan Ryuku's islands. These species are native to Indomalaya ecozone especially in Indonesia is M. champaca and M. liliifera [7,8,9].
There are a few tissue culture studies on M. alba, M. liliifera and Magnolia that grow in Indonesia. Advanced studies are needed to develop genus Magnolia that grows in Indonesia; due to its high economic value and advantages to individuals, industries and state.

Growth of Magnolia Genus and Their Its Distribution in Indonesia
In contrast to Rozak [6], there are 28 recognized species native to Indonesia (Table 1, Figure 1). There is also a species of hybrid origin, known as M. alba. More than half of Magnolia's species are distributed in Borneo with the oldest rainsforest in the world that makes it one of the most biodiverse area in the world. Others species are mostly found in Sumatra, Sulawesi and Jawa. Several species are spread in Maluku, New Guinea and Lesser Sunda Islands. Rozak [6] reported that there is inadequate information based on IUCN's listing criteria to evaluate the current conservation that lead to the distribution and evaluation of the species distributed in Indonesia yet. Therefore, there is danger that some of these rare species might become rare and threatened as a consequence of restricted distribution, threats of deforestation, illegal logging or natural disasters.  [21,22].
Some ex situ studies of saprobic fungi on M. liliifera leaves in Thailand has been reported by Promputtha et al. [23,24]. It had been concluced that its large and thick leaves are a good source for saprobic fungi. Another study has mentioned that saprobic fungi could be a potential resource of bioactive compounds for medicinal utilization [25].   [26,27].

Propagation
Various authors have classified different storage behaviour of M. champaca as closer to ortodhox [28] and intermediate or semi-recalsitrant [18]. Their storage behaviour has been classified as recalcitrant seeds with its characteristic fast loss of viability by Bahuguna et al. [29], Robbins [30], Bisht and Ahlawat [31], Yuniarti and Nurhasybi [32] and Pudjiono [27]. Seed of M. champaca has aeril lead to reduction of seed viability and small structure called elaiosomes that can enchant animal dispersors due to its colour and lipids and protein compounds [33]. It is difficult to cultivate this plant by seeds due to these mentioned factors like recalcitrant seed which means short-lived seeds, aeril layer, elaiosomes and hard seed coat. The physical, physiological and morphological characteristics as type of dormancy also have been noted by many authors. Yuniarti and Nurhasybi [32] mentioned that appropriate drying time and storage methods could be applicated to maintain seed viability. The room (ambient) temperature is better than DCS (4-8 o C, RH 40-60%) and refrigator (0-5 o C, RH 40-50%) storage for M. champaca storage to keep their viability.
In order to solve these problems of conventional propagation some studies have been established by many authors. Candiani et al. [34] noted that aeril on dropped seeds can be removed by ants and rodents with higher removal in the old seeds compared to the new eucalypt and related seeds parts to improve seed germination of M. champaca. Fernando et al. [18] notified scarcity of natural regeneration and trouble of seed propagation due to its physiological dormancy; which has been solved by using GA3. The study also showed morphological dormancy of M. champaca identified by elongation of embryos inside seeds at warm temperatures following radicle emergence.
The cutting propagation using softwood and semihard wood of M. champaca IBA treatment has been carried by Tan et al. [35]. Their result showed that M. champaca could be propagated by cutting and IBA application to support rooting with the best rooting percentage using 12000 mg/L IBA and softwood cutting. There is also a conventional propagation study of M. champaca in Indonesia. The effecetiveness of combination of media with or without plant growth regulator on M. champaca cutting had been reported by Danu and Putri [26]. They noted three of best combination for M. champaca cutting: sand media only, combination of coconut husk, rice husk and 100 ppm IBA and combination of coconut husk, rice husk, charcoal of rice husk and 50 ppm IBA.
Pudjiono [27] studied effectiveness of coir dust and roasted rice hull as growth medium on survival percentage and root growth of M. champaca shoot cuttings.

Constituent of M. champaca
The volatile compounds of M. Champaca's concrete, absolute and essential oilisolated and extracted from various part of plant contain esters, alcohols, fatty acid, terpenes (monoterpenes and sesquiterpenes). Phenol and oxydes group are also found from the extracts. Dwicandra et al. [36] noted that stem barks of M. champaca extracted by 80% ethanol contain essential oils, triterpenoids, polyphenols, and flavonoids. The obtained total component with different concentration (percentage) is influenced by source, type of oil and isolation or extraction methods ( Table 2). Rout et al. [37] reported that the lower sesquiterpene hydrocarbons which compose more than 60% of headspace were found at absolute and concrete compared to essential oil components of M. champaca flower. The absolute and concrete contents contained higher amounts of esters while they were no or had decreasing in concentrations in the essential oils. The essential oil contained several monoterpenes such as linalool which were not isolated at the absolute, concrete and headspace. Linalool is also identified at the concrete and absolute level through distilation method [38,39,40]. Indoles which were found at the absolute, concrete and headspace dissapeared at essential oil due to loss through distillation. The more components are identified by simultaneous distilation extraction method than other methods [39,40].

Magnolia alba
M. alba syn. M. longifolia Blume is known as white champaca or white jade orchid tree or "Cempaka Putih" in Indonesia and its flowers are used as flower garland with Jasmine at traditional wedding ceremonies. Although it is a typical plant from the province of Central Java, M. alba is spread throughoutIndonesia. Similar to M. champaca, its essential oil can be used as key topnote of perfumes due to various compounds from flower or leaf.

Propagation
Propagation by seed is rarely used for M. alba due to its hybrid origin, infrequent seed availability, erratic germination and long germination period [55]. Vegetative methods such as cutting, grafting and air-layering is an alternative to propagate M. alba. The ethepon and IBA application on softwood cutting of M. alba had been also conducted by Tan et al. [35]. It differs from M. champaca, the ethepon and IBA application showed no significant effect on rooting of softwood cutting due to short period application of ethepon and lower concentration of IBA.
While petals on medium containing BA showed caulogenesis after 20 days culture. Previous studies conducted by Evachristy [55] had also reported that the concentration of 2 mg/l (ppm) NAA and 3 ppm BAP is optimum to induce calli of M. alba young leaves compare to other combinations of NAA and BAP with 1-5 mg/l (ppm) concentration range. Similarly, the appropiate combination of NAA and BAP stimulated calli growth [60]. The auxin and cytokinin roles on callus initiation and somatic embryogenesis and their responses are also influenced by explant sources.  [19] and essential oil production) -Solid MS media + 1,0 mg/L benzyladenine (BA) and 4,0 mg/L 2,4dichlorophenoxyacetic acid (2,4-D) (

Improvement Study of Magnolia in Indonesia
Many studies under in vitro conditions mentioned above were conducted with the same source explant grown outside Indonesia. There are large chances and opportunities to establish studies under in vitro conditions about both conservation and development of natural resourses like Magnolia that grow in Indonesia.

Propagation
Germination study to solve problem on seeds can be carried out by different pre-treatments. Scarification and stratification are an alternative to solve seed dormancy in seeds of several types of legume plants to improve their germination. The cheap and easy application of methods to optimize germination of seeds can be conducted on Magnolia genus in Indonesia and will be recommended to germinate them both under in vitro and and ex situ conditions to establish their nurseries and orchards.

Tissue culture
Tissue culture which cover plant breeding and plant propagation could be applied to solve problems of Magnolia seed dormancy. Micropopagation in vitro condition is also an alternative when the plant is difficult to propagate by conventional methods. Seeds or others parts of a plant could be used as explant source. Different and combination of plant growth regulator and medium could be used to develop a protocol for callus induction, shoot initiation or root regeneration. The successful acclimatization of appropriate explants and methods can be selected to provide nursery transplant stocks for supporting cultivation of Magnolia in Indonesia. It can also be used for production of synthetic seeds from somatic embryos obtained by tissue culture techniques.
Tissue culture or cell culture methods can be used for production of seconder metabolites from callus or under stress conditions providing large opportunities to study and screen them against antimicrobial, antifungal, antibacterial and antioxidant and antiviral activities in other Magnolia's species of Indonesia which has not been conducted yet. The antimicrobial, antibacterial and antioxidant activities of of M. champaca grown in Bangladesh and India have been carried out by using direct extracts from flowers [66] and leaves and stems [67]. It also contains essential oil from flower [68], fresh leaves [69] and bark [70]. Iyer and Panda [71] reported the potential of callus extract of M. champaca as renewable bio-resource that could be used to control biocompatible gold and silver nanoparticles synthesis due to great size and shape diversity and stability compared to flower extractgenerated particles. However, there is limited information about biological activities of Magnolia' species in Indonesia especially from calli obtained from various explants source under normal or stress conditions that are stimulated for production of more phytochemical components.
Protoplast culture and hybridization of plants by developing cybrids could serve as an alternative to improve colour and aroma of flowers from different varieties and M. liliifera or species in genus Magnolia for use in ornamental plants industry or making garlands. Genetic transformation of species in genus Magnolia could open new chances and avenues to make these tolerant against prevalent biotic or abiotic stresses.

Volatile component
The phytochemical screening and volatile component analysis of M. champaca and M. alba have been reported by many authors in many countries, especially in Indomalaya ecozone. Volatile compounds at the concrete, absolute and essential oil which have different percentage depending on extraction methods and extract sources. These can be developed for medicinal and industrial purposes including their use in perfume, splash cologne, air freshener and aromatherapy industries. M. liliifera and others Magnolia species native to Indonesia could be reviewed on their volatile compound of concrete, absolute and essential oil etc. can be used to extract these directly or from callus under in vitro tissue culture conditions.

Conclusions
This study describes tissue cultures, micropropagation and comparison of compounds obtained from these trees. The results describe variations among different species of genus magnolia. The results indicated more work on the plant in reference to Indonesia and suggest a substantial improvement in vegetative growth and multiplication through these techniques. Differences in species among genus may reflect possible physical or hormone related factors that could prevent growth and release of metablites under in vitro tissue culture conditions of thee plants.

Statement of Conflict of Interest
Authors have declared no conflict of interest.

Author's Contributions
The contribution of the authors is equal.