Histological observations of adventitious root derived from in vitro plantlet and shoot bud of Boesenbergia rotunda (Zingiberaceae)
1Siti Zulaiha Ahamad Azhar, 1Khairunnisa Abd. Ghani, 123Nor Azma Yusuf*
1Centre of Graduate Studies, Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA (UiTM) 40450 Shah Alam, Selangor, Malaysia
2Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA (UiTM) Kampus Jasin, 7730 Merlimau, Melaka, Malaysia
3Agricultural Biotechnology Research Group, Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA (UiTM) 40450 Shah Alam, Selangor Darul Ehsan, Malaysia
*corresponding author: [email protected]
ABSTRACT
Boesenbergia rotunda has been locally known as ‘Temu kunci’ and has renowned to possess some of important bioactive compounds that are promising for pharmacological applications. It is essential to obtain structural information on the inner parts of the roots since growing roots undertake various anatomical and morphological changes that influence their activity and nutrient uptake processes. Although root functions are thought to be very important for the growth of shoot, the morphological and anatomical knowledge of adventitious root for this plant are very limited. This study aims to characterize and determine the presence of three main components in root system; epidermis, cortex, and the vascular bundle of adventitious root derived from in vitro plantlet and shoot bud of B. rotunda. Histological sections using resin were done to study the anatomical of adventitious root of B. rotunda. The root samples were fixed in a Glutaraldehyde-Paraformaldehyde-Caffeine (GPC) fixative, dehydrated, infiltrated, embedded, cut, stained and mounted with Surgipath mounting medium for observations under light microscope. From the histological observations, adventitious roots derived from in vitro plantlet and shoot bud of B. rotunda have showed the presence of all the three main systems and have the same internal structures containing epidermis, exodermis, suberized sclerenchyma cells, cortex and stele. Both adventitious roots derived from in vitro plantlet and shoot bud showed normal growth morphological, have same normal cell structures and arrangements.

Key words: Histological, adventitious root, HYPERLINK “http://www.tandfonline.com/keyword/Metroxylon+Sagu+Rottb.”Boesenbergia rotunda, in vitro
Abbreviations: MS – Murashige and Skoog; BAP – benzylaminopurine; NAA – ?-napththalene acetic acid; KIN – Kinetin
INTRODUCTION
Boesenbergia rotunda which locally known as ‘Temu kunci’ in Malaysia and Indonesia is a herbaceous monocotyledon plant belongs to Zingiberaceae family. This medicinal herb is widely distributed throughout Southeast Asia regions mainly Southern China, Sri Lanka and India. Owed to its aromatic feature, this herb has been regularly used as a condiment in various Asian dishes to stimulate appetite. Traditionally, the rhizomes of B. rotunda have been used to treat ailments such as muscle pain, rheumatism, febrifuge, and gout. It is also capable to treat gastrointestinal diseases including stomach ache, flatulence, peptic ulcer, carminative and dyspepsia (Tan et al., 2012). Boesenbergia rotunda has been recognized by its valuable bioactive compounds derived from the flavonoids and essential oils. These compounds are known to be the most abundant secondary metabolites found in this plant. There are three main groups of flavonoids particularly flavanones, flavones and chalcones that have been found in the rhizome of this plant (Chahyadi et al., 2014) which have been reported to have potentials as antimicrobial (Bhamarapravati et al., 2006; Pattaratanawadee et al., 2006), anti-human immunodeficiency virus 1 (anti-HIV-1) (Cheenpracha et al., 2006) and showed antioxidant activity (Shindo et al., 2006). 4-hydroxypanduratin A and panduratin A in rhizome of B. rotunda were found to display high inhibition towards dengue-2 virus protease (Kiat et al., 2006).

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In recent years, plant biotechnology approaches including organ, tissue and cell cultures have been widely used to produce various secondary metabolites in medicinal plants that are worth for pharmaceuticals (Baque et al., 2012). Although plant cell culture has been acknowledged to be one of the efficient culture techniques to obtain secondary metabolites, the yields are often too small for commercialization level. Roots of plants are known to be the main location for synthesis and amassing of a wide range of highly potential secondary metabolites (Mahdieh et al., 2015). Thus, the differentiated tissues application by organ culture technique particularly root culture has been studied and has been chosen as an alternative method to obtain large scale of secondary metabolites. According to Hahn et al. (2003), the in vitro adventitious roots which were induced using medium with additions of specific plant growth regulators under optimum aseptic culture conditions have displayed higher root biomass and secondary metabolites production.

Monocotyledonous crop does not allow much explants diversity for micropropagation. Rhizome and shoot bud was often used as a source of responsive explant. Adventitious roots can be simply understood as roots that emerged from unusual plant parts other than the radicle including leaves, twigs, branches underground stems, aerial stems, from old roots or even branches of secondary roots from the primary root itself (Esau, 1943). The development of a single root has always been illustrated through orthodox route involving division, elongation, and finally maturation phase (Alarcon et al., 2014). Frequently, the formation of the root initials was highly influenced by parenchyma cells. These cells are usually competence to revert themselves to meristematic activity that eventually will started to divide into real root (Tukey, 1979). This work had been done particularly to compare anatomical structures of adventitious root derived from in vitro plantlet and shoot bud of B. rotunda.

MATERIALS & METHODS
Stock plants
The roots used in this study were obtained from the in vitro plantlet and shoot bud of B. rotunda of four subculture cycle. For the in vitro plantlet, micropropagation medium of B. rotunda was established using Yusuf et al. (2011), which is MS medium with addition of 2.0 mg/L 6-Benzylaminopurine (BAP) and 0.5 mg/L 1-Naphthaleneacetic acid (NAA). The medium had been used to culture and continuously maintained the in vitro plantlets of B. rotunda for subsequent uses. The cultures were placed in a control in vitro environment of culture room. This was included constant lighting exposure from white fluorescent cylinder tube with the light intensity of 30 – 35 µE m-2 s-1. The temperature also was controlled at 25 ± 2°C. Adventitious roots that derived from shoot bud of B. rotunda were obtained from direct induction of shoot bud explants, which were cultured onto ½ MS medium added with 0.1 mg/L Kinetin (KIN) and 1-Naphthaleneacetic acid (NAA) (Siti Zulaiha et al., 2018). The adventitious root cultures were maintained in total darkness at temperature of 25 ± 2 ºC.

Microscopy
Histological sections using resin were done to study the anatomical of adventitious root of B. rotunda. Both adventitious root samples were taken and were cut into small pieces. The samples were fixed in a Glutaraldehyde-Paraformaldehyde-Caffeine (GPC) (Sigma Chemical Co., USA) fixative (50 ml 0.2 M, pH 7.2 Phosphate buffer; 20 ml 10% (v/v) Paraformaldehyde; 4 ml 25% (v/v) Glutaraldehyde;1 g Caffeine and distilled water to a total volume of 100 ml) for 24-48 hours at room temperature. The samples were then underwent dehydration process in ethanol (EtOH) dilution series: 30% for 30 min; 50%, 45 min; 70%, 45 min; 80%, 60 min; 90%, 60 min; 95%, 60 min and finally twice for 60 min in pure EtOH. The tissues were ready for infiltration with Technovit 7100 resin for 24 hour at 4 ºC. The specimens were embedded in a mould and left for 24 hours to solidify at room temperature before sectioning. Semi-thin sections of 3.5 µm were cut using a microtome. The slides were soaked in 100% EtOH. The sections were placed in distilled water in a glass container with black paper underneath to facilitate picking up of the sections. The sections were arranged in clean dry slide and each section was stained with 0.5% (w/v) toluidine blue stain (Sigma, USA) to check tissues. Good sections were double-stained with 1% (w/v) periodic acid for 5 min, rinsed four times in distilled water (pH 4.5). Then they were soaked in Schiff’s reaction in the dark for 20 min and rinsed again in distilled water (pH 4.5) four times. Lastly, the slides were stained with napthol blue black (Sigma, USA) at 60 ºC for 5 min and rinsed well with distilled water. The slides were mounted with Surgipath mounting medium and were left dried for 24 hours. Photomicrographs were taken with a Leica camera on a Leica (LEITZ DMRB) light microscope (x20/0.5; x40/0.7; and x100/1.3). Observations were made to determine the presence of three main components in root system; epidermis, cortex, and the vascular bundle in the adventitious root of B. rotunda.

RESULTS AND DISCUSSION
Based on the morphological observations, we found that both adventitious roots of B. rotunda derived from in vitro plantlet and shoot bud were mutually showed almost similar growth pattern physically when cultured on their specific media formulation; MS medium with addition of 2.0 mg/L BAP + 0.5 mg/L NAA and ½ MS medium with addition of 0.1 mg/L Kinetin + 0.5 mg/L NAA respectively for four weeks. Thus, in order to determine any differences in cell structures and arrangements, the histological cross sections were used by observing and comparing them to clarify this situation.
Generally, the development of a single primary root can be studied by observing and defining the arrangement of its tissues through longitudinal and transverse sections. The arrangement of tissues commonly made up of three main systems in plant roots which include the epidermis, cortex and vascular cylinder (Alarcon et al., 2014). During staining procedure, it is very crucial to select the good quality of targeted tissues. Consequently, for any recalcitrant tissues, an extensive dehydration method could be carried out using 100% (v/v) butanol for three times with minimum 24 hours for each treatment to soften the tissue (Schwendiman et al., 1988). Periodic acid Schiff particularly stains polyssacharides (starch reserves and walls) while blue black naphtol particularly stains reserve protein blue-black or soluble (Fisher, 1968).
From the histological examinations, adventitious roots derived from in vitro plantlet and shoot bud of B.rotunda have showed the presence of all three main root systems which particularly similar in roots from monocot plants. The arrangement of tissues that form the body of the primary root can be studied through the longitudinal sections.
Based on the histological examinations in Figure 1, both single segment of the adventitious root displayed all the main structures in a whole primary root development consists of root meristem, root cap, distal elongation zone and elongation zone. Root meristem and root cap can be seen clearly at the end of the distal elongation zone. This findings was supported by Jones and Dolan (2012), which had stated that a root cap was derived to protect the root apical meristem(RAM).

For monocots, comprise to their root systems; primary root which was often temporary, adventitious roots and seminal roots can produce lateral roots (Bell ; Bryan, 2008). Based on Figure 2, there are generally no differences in anatomy of adventitious root derived from in vitro plantlet and shoot bud of B. rotunda. It has been hypothesized by Seago and Fernando (2013) that the differences in the anatomy of primary and adventitious roots lie in the types of groups of plants and their environments, not from the origin of the roots.

-339090-423545Fig. 1: Longitudinal cross section of adventitious roots segments of B. rotunda derived from (1a) shoot bud. (1b) in vitro plantlet. The root sections of both (1a) and (1b) displayed the cell structures and development that include RM root meristem, EZ elongation zone, DEZ distal elongation zone, and RC root cap, Magnification : x800.

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Fig. 2: Longitudinal cross section of column pericycle of the B. rotunda adventitious roots derived from (2a) in vitro plantlet. (2b) shoot bud. Stained cross section showed rh root hair, co cortex, ep epidermis, en endodermis, xy xylem. Magnification : x300.

Like any other organs in plant, root also comprises of the three main specific tissues arrangements that include cortex, vascular bundle and epidermis. According to Figure 3, both of the transverse sections of adventitious root of B. rotunda shows the present of epidermis, endodermis, pericycle, phloem, cortex, late metaxylem and protoxylem pole. As confirmed by Seago and Fernando (2013), all these specifications were very important as the endodermis ensures a one-way mode of water transport into the plant, whereas pericycle was usually where the lateral roots initiated.

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Fig. 3: Transverse section of adventitious roots of B. rotunda segments for (3a) in vitro plantlet. (3b) shoot bud. Stained cross section showed, co cortex, ep epidermis, en endodermis, ph phloem, lmx late metaxylem, pe pericycle, px protoxylem pole. Magnification : x200.

CONCLUSIONS
In vitro cultures offer an economical importance in plant development of B. rotunda. As an organ culture, in vitro adventitious roots are known to have high potential to produce important secondary metabolites. However, the lack of growth studies on adventitious roots leads to questionable uncertainties on their capabilities. Although the growth and morphological characteristics of adventitious roots were seen as proficient as the roots from in vitro plantlets, histological analysis had to be done to further identify and clarify the root growth at cellular level. This gave confirmation that the adventitious roots are competent with the adventitious roots from in vitro plantlets in their morphological growth and they have same normal cell structures and arrangements. This study gave a validation that the adventitious roots of
B. rotunda offer many interesting perspectives which can be useful for the future studies.

ACKNOWLEDGEMENTS
We thanked Universiti Teknologi MARA (UiTM) for financially supporting this particular study through a research grant by the No. 600-IRMI/Dana KCM 5/3/GIP (090/2017) UiTM Excellence Fund.

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