Acta agriculturae Slovenica, 118/2, 1–11, Ljubljana 2022 doi:10.14720/aas.2022.118.2.2416 Original research article / izvirni znanstveni članek Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of pseudostem and anti-lipid peroxida- tion activity of Curcuma mangga Val. Fariz ABRAHAM 1, Lai Keng CHAN 1, 2, 3, Gunawan INDRAYANTO 4, 5, Peng Lim BOEY 6, 7 Received November 10, 2021; accepted June 06, 2022. Delo je prispelo 10. novembra 2021, sprejeto 6. junija 2022 1 Universiti Sains Malaysia, School of Biological Sciences, Penang, Malaysia 2 D’Arboretum, Bukit Rambai, Melaka, Malaysia 3 Corresponding author, e-mail: merrilynchan@gmail.com 4 Airlangga University, Faculty of Pharmacy, Surabaya, Indonesia 5 University of Surabaya, Faculty of Pharmacy, Surabaya, Indonesia 6 Universiti Sains Malaysia, School of Chemical Sciences, Penang, Malaysia 7 Sunrich Biotech Sdn. Bhd., Kuala Ketil, Kedah, Malaysia Enhancement of shoot proliferation and evaluation of biot- ic elicitation effects on anatomical changes of pseudo stem and anti-lipid peroxidation activity of Curcuma mangga Val. Abstract: Mango turmeric (Curcuma mangga Val.) con- tains many bioactive compounds that are used for traditional treatment of various health problems and ailments. Slow propagation nature of C. mangga have resulted in short sup- ply to meet the market demand. The longitudinally incised half shoot explants promote 100 % increased of shoot num- ber compared with non-incised shoots with the formation of average 6.6 shoots/explant when they were cultured either vertically or horizontally on MS medium supplemented with 2.0 mg l-1 BA and 0.5 mg l-1 NAA. Biotic elicitation with 3.5 mg l-1 or 5.0 mg l-1 yeast extract or combination of 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract did not promote shoot proliferation but exhibited anti-lipid peroxidation activ- ity slightly lower than quercetin, a potent plant antioxidant flavonoid and butyl hydroxyl toluene (BHT), a commercial preservative agent which is used as a positive control. While absolute ethanol which served as a negative control did not show any anti-lipid peroxidation activity. Biotic elicitation of C. mangga plantlets using similar elicitors resulted in anatom- ical changes of its pseudostem with reduced number of thin lignified xylem cells and the presence of druse suspected to be oxalate crystals inside the cortex cells with delicate cell wall. Key words: anti-lipid peroxidation activity; chitosan; mango turmeric; pseudostem; shoot proliferation; yeast ex- tract Pospeševanje tvorbe poganjkov in ovrednotenje elicitacij- skih učinkov na anatomske spremembe navideznih stebel in proti maščobne peroksidacijske aktivnosti kurkume (Cur- cuma mangga Val.) Izvleček: Kurkuma (Curcuma mangga Val.) vsebuje šte- vilne bioaktivne snovi, ki se uporabljajo pri tradicionalnem obravnavanju številnih zdravstvenih težav in obolenj. Poča- sen način njenega razmnoževanja povzroča njeno pomanj- kanje glede na veliko povpraševanje na trgu. Do polovice vzdolžno zarezani stebelni izsečki so stoodstotno povečali število poganjkov v primerjavi z nezarezanimi s tvorbo pov- prečno 6,6 poganjkov na izseček, če so bili gojeni navpično ali vodoravno v MS gojišču, obogatenim z 2,0 mg l-1 BA in 0,5 mg l-1 NAA. Biotično vzpodbujanje s 3,5 mg l-1 ali 5,0 mg l-1 izvlečka kvasa v kombinaciji z 150 mg l-1 hitozana in 3,5 mg l-1 izvlečka kvasa ni pospešilo tvorbe poganjkov ampak je pokazalo malo manjšo antiperoksidacijsko aktivnost v pri- merjavi s kvercetinom, močnim rastlinskim flavonoidnim an- tioksidatom in butil hidroksi toluenom (BHT), komercialnim zaščitnim sredstvom, ki sta bila uporabljena kot pozitivna kontrola. Uporaba absolutnega etanola kot negativne kon- trole ni pokazala nobene antiperoksidacijske aktivnosti. Bio- tično vzpodbujanje nastanka rastlinic korkume s podobnimi eliciatorji je povzročilo anatomske spremembe v nastajajočih navideznih steblih z zmanjšanjem števila tankih lignificiranih celic ksilema in prisotnostjo kritalnih kopuč, domnevno iz kalcijevega oksalata, v celicah primarne skorje, ki so imele zelo tanko celično steno. Ključne besede: antiperoksidacijska aktivnost za ma- ščobe; hitozan; kurkuma; navidezna stebla; tvorba poganjkov; izvlečki kvasa Acta agriculturae Slovenica, 118/2 – 20222 F. ABRAHAM et al. 1 INTRODUCTION Mango turmeric (Curcuma mangga Val.), a mem- ber of Zingiberaceae family, is commonly used in Ayurvedic, traditional Chinese medicine (TCM) and alternative Malay medicines (Ramadanil et al., 2019). It has been used as food spices, food preservatives and treatment of various health problems and minor ail- ments such as fever, stomach-ache, general debility, and postpartum care (Subositi and Wahyono, 2019; Furmuly and Azemi, 2020). It has been proven to have many health beneficial activities due to some bioactive compounds or secondary metabolites such as the alka- loids, flavonoids, tannins, terpenoid and curcuminoid that are present mainly in the rhizomes (Muchtaromah et al., 2018; Yuandani et al., 2019; Awin et al., 2020; Fi- triastuti et al., 2020; Maryam and Martiningsih, 2021). Conventionally, C. mangga is propagated via its rhizome during the rainy season. However, C. mangga and many other Curcuma species have slow propaga- tion rate and very prone to soil borne diseases and rhizome dormancy (Škorničková, 2007; Pikulthong et al., 2016; Soonthornkalump et al., 2020; Leong-Škornič- ková et al. 2021). This has resulted in insufficient sup- ply of C. mangga to meet the market demand. In vitro culture techniques have been employed as alternative measures for improving the production of plantlets for some Curcuma species such as C. longa (El-Hawaz et al., 2015; Marchant et al., 2021), C. aromatica (Sharmin et al., 2013; Mohanty et al., 2015), C. alismatifolia (Li et al., 2021) and C. zedoaria (Sudipta et al., 2020). Addition of elicitors into culture medium had been reported to enhance the production of useful secondary metabo- lites but high amount of elicitation was found to cause cell or tissue damage of the cultured materials (Espi- nosa-Leal et al., 2018). Our previous study (Abraham et al., 2011) had reported that biotic elicitation using yeast extract and chitosan did not enhance shoot pro- liferation but increased the production of total phenolic compounds which resulted in severe abnormality of the C. mangga in vitro plantlets especially the pseudo stems. Hence, the present study was carried out with three main objectives. Firstly, to determine whether a shoot incision technique could enhance in vitro shoot proliferation and used it as an alternative means for mass production of C. mangga plantlets. Secondly, to determine whether yeast extract and chitosan that have been found to promote production of total phenolic compounds with good free radical scavenging activity could also enhance the anti-lipid peroxidation activity of C. mangga. Thirdly, to study the effect of biotic elici- tation of yeast extract and chitosan on the anatomical structures of C. mangga pseudostem. 2 MATERIALS AND METHODS 2.1 ESTABLISHMENT OF IN VITRO PLANTLETS Young buds of C. mangga of approximately 1.5 cm3 in size, excised from the actively growing rhizomes dur- ing the raining season. They were washed with com- mercial detergent solution (Sunlight®, Unilever, Selan- gor, Malaysia) to remove all soil and organic matters and rinsed under running tap water for 40 min. The cleansed bud explants (1.5 cm3) were then immersed in 70 % ethanol (Chemical Industries (M) Sdn. Bhd., Selangor, Malaysia) for 10 min followed by surface ster- ilization with 20 % Clorox®, a commercial bleach solu- tion containing 5.3 % sodium hypochlorite (The Clorox Company, Oakland, CA), for 20 min. The surface-ster- ilized buds were then rinsed three times with sterile distilled water before being inoculated onto gelled MS (Murashige and Skoog, 1962) medium without any plant growth regulator (PGR). The cultures were incu- bated in a culture room regulated at 25 ± 2 ºC with con- tinuous illumination at average light intensity of 32.5 µmol m-2 s-1 for 6 weeks (Abraham, 2010). 2.2 EFFECT OF SHOOT INCISION ON SHOOT PROLIFERATION The shoot explants of 1.0 cm length were obtained from the 6 weeks old in vitro plantlets. Each shoot ex- plant was vertically cut into half or quarter while the non-incised whole shoot explants were used as control. These shoot explants were then cultured onto shoot proliferation medium, MS supplemented with 2 mg l-1 6-benzylaminopurine (BAP) and 0.5 mg l-1 1-naphtha- lene acetic acid (NAA) (Sigma-Aldrich (M) Sdn. Bhd. Subang Jaya, Malaysia) (Abraham, 2010). Three shoot explants were used for each experimental unit and ten experimental units were used for each explant type. The explants were placed vertically and horizontally on the proliferation medium. The number of shoots produced from each explant with different mode of placement was determined after 6 weeks of culture. 2.3 ANTI-LIPID PEROXIDATION ACTIVITY OF CURCUMA MANGGA 2.3.1 Sample extraction Our previous study (Abraham et al., 2011) have indicated that plantlets cultured in proliferation me- dium added with 3.5 mg l-1 yeast extract, 5.0 mg l-1 yeast Acta agriculturae Slovenica, 118/2 – 2022 3 Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of ... Curcuma mangga Val. extract, and plantlets cultured in proliferation medium supplemented with150 mg l-1 chitosan plus 3.5 mg l-1 yeast extract produced high total phenolic compounds with high free radical scavenging activity (RSA). Hence, these plantlets together with plantlets cultured in shoot proliferation medium (Control) were selected for their anti-lipid peroxidation activity. Two grams (g) of dried sample derived from each treatment condition was grounded into powder form using blender (Philips, Se- langor, Malaysia). Each sample was placed into 250 ml conical flask and soaked with 100 ml methanol (Chemi- cal Industries Sdn. Bhd., Selangor, Malaysia) at 40 ºC for two hours and the soaking process was repeated three times. The methanol extracts from each sample were collected, combined, filtered, and evaporated using ro- tary evaporator machine (EYELA, N-N Series, Japan). 2.3.2 Anti-lipid peroxidation activity Anti-lipid peroxidation activity was determined using modified Ferric thiocyannate (FTC) method (Os- awa and Namiki, 1981). C. mangga sample extract (4 mg) was dissolved in 4 ml absolute ethanol (99.5 %) followed by addition of 8 ml 0.05M phosphate buffer (pH 7.0), 4.1 ml 2.5 % linoleic acid solution (Sigma, Ronkonkoma, NY) and 3.9 ml distilled water. The ex- tract solutions were kept in aluminium foil wrapped vessels and incubated at 40 °C. Butyl hydroxyl toluene (BTH) (Sigma, Ronkonkoma, NY) was used as a posi- tive control and 4 ml absolute ethanol served as a nega- tive control. A volume of 0.1 ml extract solution was added to 9.7 ml 7.5 % ethanol followed by 0.1 ml 30 % ammonium thiocyannate solution and 0.1 ml 0.02 M ferric chloride (Sigma, Ronkonkoma, NY) in 3.5 % HCl (v/v). The reaction was incubated for three minutes un- der dark condition. Three replicates were prepared for each sample and three repetition of spectrophotometer reading were applied for each sample. The absorbance of sample was measured at 500 nm wavelength using UV-Vis spectrophotometer (Mettler Toledo, Colum- bus, Ohio). This procedure was repeated every 24 hours until both the positive and negative controls gave the maximum absorbance. The degree of lipid peroxidation was represented by percentage of oxidized lipid in test- ed samples at the day before the absorbance decreased. 2.4 HISTOLOGY STUDY OF BASAL PSEU- DOSTEM The basal pseudostems of the morphological ab- normal C. mangga plantlets were selected for anatomi- cal study. They were trimmed into approximately 0.5 cm3 in size and were then immersed in FAA solution (40 % formaldehyde: acetic acid glacial: 95 % ethanol = 5: 5: 90) for fixation. During preparation of sections, the fixed tissues were passed through a series of alco- hol solutions, starting with 50 % ethanol/tetra butyl alcohol (TBA) (ethanol 95 %: absolute TBA: water = 4: 1: 5) and finally treated with absolute ethanol/TBA (ethanol 95 %: absolute TBA = 2.5: 7.5). After which the tissues were immersed in mixture solution of TBA and liquid wax with ratio 1:1 at 60-62 ºC for 24 hours. At the embedding stage, the tissues were immersed in liquid wax at 60-62 ºC for 12 hours. After the wax solidified, the wax blocks containing the tissue samples were sliced into thin slices with 15 µm thickness using rotary microtome (Leica, Germany) for slide prepara- tion. Double stained standard technique was used for the preparation of permanent slides. Each sliced section treated with a few drops of safranin after it was placed on the glass slide. This was followed by a few drops of 95 % ethanol to remove the excess safranin. After this, a few drops of Fast Green and 95 % ethanol were added respectively. Finally, one drop of xylene was added on the section. The sliced section was then covered with a glass cover slip and sealed with Shandon Mount (Shan- don, USA) as a mounting agent. The prepared slides were observed under light microscope (Olympus BX- 50, Japan) fitted with coloured video camera (JVC KF- 55B, Japan) and image analysing system (analySIS docu version 3.1, Germany) for determination of cell size and cell morphology. 2.5 STATISTICAL ANALYSIS For the effect of shoot incision and mode of place- ment on shoot proliferation, the experiment was con- ducted in complete randomized block design (CRBD). The data was analysed using two-way ANOVA and the best explant type was determined using Tukey’s HSD test at p ≤ 0.05. The anti-lipid peroxidation activity for all the selected samples was analysed using one-way ANOVA and the comparison of means was determined using to Tukey’s HSD test at p ≤ 0.05 3 RESULTS AND DISCUSSION 3.1 EFFECTS OF SHOOT INCISION ON SHOOT PROLIFERATION Shoot proliferation is an essential step for mass production of in vitro plantlets which is normally car- Acta agriculturae Slovenica, 118/2 – 20224 F. ABRAHAM et al. ried out by inducing multiple shoots formation using plant growth regulators. Many researchers have been using 6-benzylaminopurine (BAP) or benzyl adenine (BA) combination with naphthalene acetic acid (NAA) for multiple shoot induction in Zingiber species (Ab- bas et al., 2011; Zahid et al., 2021) and Curcuma species (Bejoy et al., 2012; Jala, 2012; Ferrari et al., 2016) with the formation of an average of 3 to 5 shoots per ex- plants. Similar result was obtained in the present study whereby an average of 3.3 and 3.7 shoots per shoot ex- plant were formed when the shoot explants were cul- tured horizontally and vertically respectively on MS medium supplemented with 2.0 mg l-1 BA and 0.5 mg l-1 NAA for induction of multiple shoot formation. When the shoot explants were cut longitudinally into half, the number of shoots formed per shoot explant was greatly enhanced with the formation of 6.6 shoots per shoot explant and the number of shots formed were not sig- nificantly different when the explants were placed hori- zontally or vertically. The quarterly cut shoot explants further enhanced the formation of multiple shoots with 7.6 and 8.4 shoots per shoot explant formed when the shoot explants were cultured horizontally and vertically respectively (Table 1). However, the multiple shoots de- rived from the quarterly cut shoot explants were small and became necrotic with copious release of phenolic compounds in the culture medium and eventually re- sulted in death of plantlets after two subculture cycles (6 weeks/cycle). Mode of inoculation either vertically or horizontally was statistically found to have no effect on promoting shoot proliferation in C. mangga. The half shoot explants grow into healthy multiple shoots when cultured on the shoot proliferation medium. Hence, half shoot explants were used for the subsequent stud- ies. Results obtained in the present study clearly dem- onstrated that shoot incision longitudinally could en- hance 100 % increment of shoot production. The en- hancement of shoot proliferation in longitudinally incised shoots was due to increased cut surface area that exposed to the culture medium for better absorp- tion of nutrients and inhibit apical dominance and hence induce more lateral shoot formation (Mok and Ho, 2019). Longitudinally dissecting of shoot explants to promote high multiple shoot formation has become a common practice in propagation of banana (Ahmed Hasan et al., 2020). Hence, shoot incision can be used as an alternative means to promote in vitro shoot multi- plication of C. mangga that resulted in more and faster production of plantlets. 3.2 ANTI-LIPID PEROXIDATION ACTIVITY OF BIOTIC ELICITED CURCUMA MANGGA Our previous study (Abraham et al., 2011) had shown that C. mangga plantlets, cultured in prolifera- tion medium supplemented with 3.5 mg l-1 yeast extract; 5.0 mg l-1 yeast extract or combination of 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract, exhibited high free radical scavenging activity (RSA). These crude extracts together with the positive and negative controls were tested for their anti-lipid peroxidation activity using the ferric thiocyannate (FTC) assay. This method was used to measure the antioxidant activity of the stud- ied samples toward auto peroxidation of the linoleic acid. BHT (Butyl Hydroxyl Toluene) (Sigma, USA) was used as a positive control and 99.5 % ethanol served as a negative control. Positive control is essential for comparing the biotic elicited C. mangga extracts with BHT, a most used antioxidant as preservative in foods containing fats, pharmaceuticals, petroleum products and it inhibits autoxidation of unsaturated organic compounds. The C. mannga extracts used in this study were dissolved in 99.5 % ethanol, hence 99.5 % ethanol was used as the negative control. The negative control is used to show that any positive effects of the tested sam- ples are not due to the ethanol effect. The selected C. mangga extracts together with BHT (positive control) and 99.5 % ethanol (negative control) and quercetin, a potent plant antioxidants flavonoid, exhibited similar pattern of lipid peroxidation activity from day 0 to day Explants Vertical Horizontal No. of shoots/explant ± Se No. of shoots/shoot No. of shoots/explant ± Se No. of shoots/shoot Whole 3.7 ± 0.2 3.7c c 3.3 ± 0.3 3.3 c c Half 3.3 ± 0.3 6.6b b 3.3 ± 0.3 6.6 b b Quarter 1.9 ± 0.2 7.6a a 2.1 ± 0.2 8.4 a a Table 1: Effect of shoot explant incision and inoculation mode on enhancing mult iple shoot formation of C. mangga Mean values within the row followed by same superscript letter indicate not significantly different when the different explant types were vertically or horizontally placed on the culture medium. Mean values within the same column (for parameter No. of shoots/shoot explant) followed by different subscript alphabet indicate significantly different of shoot numbers for different explant types (Tukey, HSD, p < 0.05) Acta agriculturae Slovenica, 118/2 – 2022 5 Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of ... Curcuma mangga Val. six. However, the oxidation of lipid (linoleic acid) of 99.5 % ethanol (negative control) drastically increased started from day seven until day ten while others re- main constant until day 10 (Figure 1). Quercetin is a potent antioxidant flavonoid found in many plant species such as onions, grapes, berries, broccoli, and citrus (David et al., 2016). Even though the three extracts obtained from biotic elicited plantlets of C. mangga showed slightly lower anti-lipid peroxida- tion activity when compared with quercetin, it could be assumed that C. mangga could also a potent antioxidant. Their anti-lipid peroxidation activity was also found to be lower when compared to BHT, a commercial pre- servative agent and a synthetic antioxidant. BHT and quercetin showed similar lipid-peroxidation activity with percentage of oxidized lipid as 29.5 ± 0.1 % and 30.0 ± 0.1 % respectively. Crude extract derived from C. mangga in vitro plantlets cultured in proliferation medium supplemented with 3.5 or 5.0 mg l-1 yeast ex- tract exhibited no difference in anti-lipid peroxidation activity. Plantlets cultured in proliferation medium sup- plemented with 3.5 mg l-1 yeast extract showed better anti-lipid peroxidation activity as compared to extract derived from C. mangga plantlets cultured in prolifera- tion medium supplemented with 150 mg l-1 chitosan plus 3.5 mg l-1 yeast extract. Plantlets cultured in pro- liferation medium without elicitation exhibit the least inhibition of lipid peroxidation (Table 2). It opened an interesting possibility to use C. mangga extracts as food preservative agents or topical medication for treatment of cold sore such as BHT. Besides using as food pre- servative agent, BHT has been used as medicine for the treatment of cold sore (Freeman et al., 1985). 3.3 EFFECT ON ANATOMY OF PSEUDOSTEM Our previous study (Abraham et al., 2011) had shown that the morphology of the plantlets cultured in the proliferation medium without elicitation (Control) were normal. While those cultured in proliferation me- dium with the addition of yeast extracts (3.5 mg l-1 or 5.0 mg l-1 were slightly abnormal with retarded growth. Those plantlets cultured in proliferation medium with combination of 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract were grossly abnormal with chlorosis and glob- ular shaped shoots, fragile leaf petiole and experience severe growth retardation. Results obtained from the histological study of the pseudostem in the present study (Figure 2, Figure 3 & Table 3) clearly showed that these elicited plantlets did affect the anatomical struc- tures of the basal pseudostem of C. mangga and could be linked directly with the morphological character- istics of C. mangga plantlets as reported in Abraham et al. (2011). The plantlets cultured in the shoot pro- liferation medium without elicitation (Control) were consisted of smooth spherical to oval shape cortex cells Sample Oxidized lipid in sample (%) ± se Absolute ethanol (Negative Control) 100 a Quercetine 30.0 ± 0.1 e BHT (Butyl Hydroxyl Toluene) (positive control) 29.5 ± 0.1 e Extract from plantlets cultured without biotic elicitors 33.0 ± 0.2 b Extract of 3.5 mg l-1 yeast-extract treated plantlets 31.6 ± 0.2 d Extract of 5.0 mg l-1 yeast-extract treated plantlets 32.1 ± 0.1 cd Extract of 150 mg l-1 chitosan and 3.5 mg l-1 yeast-extract treated plantlets 32.5 ± 0.1 c Table 2: Percentage of oxidized lipid in tested samples at day 10 using ferric thiocyanate (FTC) assay Mean values within the column followed by same alphabets represent non-significantly different mean values based on Tukey, HSD at p ≤ 0.05 Figure 1: The kinetic of anti-lipid peroxidation of biotic elic- ited C. mangga extracts and controls [BHT (positive control); 95.5 % ethanol (negative control)] Acta agriculturae Slovenica, 118/2 – 20226 F. ABRAHAM et al. and normal xylem cells. The cortex cells did not con- tain any druse, a crystal substance present in plant such as calcium oxalate crystal. The well distributed cortex cells without druse and well-formed lignified xylems (Figure 2 a & b) were able to supply sufficient nutrients to support healthy and normal plantlets. For the plant- lets cultured in medium supplemented with 3.5 mg l-1 yeast extract, the xylems were also well-formed except the lignified layer of these xylem cells were not as thick as the ones observed in the control. Their cortex cells were found to contain druse, which could be seen as black dots inside the cells (Figure 2 c & d). The accu- mulation of druses was more obvious in plantlets that were cultured in proliferation medium supplemented with 5.0 mg l-1 yeast extract (Figure 2 e & f). The size of xylem cells of the control plantlets and that cultured in proliferation added with 3.5 mg l-1 yeast extract was not significantly different, with an average diameter of 32.9 ± 4.0 µm and 40.8 ± 5.1 µm respectively. They were double the size of the xylem cells present in the basal pseudostem of plantlets cultured in proliferation me- dium supplemented with 5.0 mg l-1 yeast extract (15.4 ± 0.8 µm). However, the number of xylem cells found in plantlets cultured with the presence of 3.5 mg l-1 yeast extract was only 22 when compared to that of control with 41 xylem cells. The plantlets cultured in medium supplemented with 5.0 mg l-1 yeast extract produced even much lesser number of not well-lignified xylems, with an average of only 9 xylem cells in each 4x optical magnification field (Table 3). The control plantlets de- veloped bigger cortex cells (43.0 ± 3.0 µm) compared to plantlets cultured with the proliferation medium sup- plemented with 3.5 mg l-1 and 5.0 mg l-1 yeast extract with diameter of cortex cells as 29.9 ± 1.8 µm and 30.8 ± 1.8 µm respectively, which were not significantly dif- ferent in size (Table 3). The smaller size of cortex cells with the presence of druse, and a smaller number of poor lignified xylem cells might not be able to provide sufficient nutrients derived from the culture medium to all parts of plantlets. Hence, it explained the retarded growth with abnormal characteristic of the plantlets that were cultured in proliferation medium supple- mented with 3.5 mg l-1 and 5.0 mg l-1 yeast extract as reported in our previous study. Most of the tissues of the plantlets cultured in proliferation medium supplemented with 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract were fragile, and parts of the section were damaged after dehydration process (Figure 3 a & b). The size of some of the cortex could be measured and determined but not that of xy- lem cells because of cell damage. The number of the xy- lem cells (7 in each 4x optical magnification field) was roughly estimated from the location of the ruptured xylem cells. The size of the cortex cells found in the ba- sal pseudostem of C. mangga plantlets cultured in the shoot proliferation medium supplemented with 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract was found to be not significantly different with those plantlets cultured in proliferation medium added with yeast extract (Ta- ble 3). The small number and fragile xylem cells could greatly reduce the absorption of nutrients, and this was linked directly with the severe growth retardation and gross morphological abnormality of the plantlets cultured in proliferation medium supplemented with yeast extract and chitosan as reported in Abraham et al. (2011). Yeast extract was reported to induce a com- plex stress response resulted in activation of secondary metabolites production (Farjaminezhad and Garoo- si, 2021; Kochan et al., 2017) but resulted in slow cell growth (Hedayati et al., 2021) and cell damage at high concentration (Sánchez-Sampedro et al., 2005). In the present study, the formation of druse (crys- tal inclusion inside cortex cells) was detected in C. mangga plantlets cultured in proliferation medium elic- ited with yeast extract. The druse inside the cells of C. mangga plantlets were suspected to be oxalate crystal because oxalate crystals are the most common crys- tal inclusion of higher plants (Franceschi and Horner, Elicitor treated peudostems Diameter ± s.e. (µm) Estimated No. of xylems/ 4x optical magnification fieldCortex Xylem Control 43.0 ± 3.0 a 32.9 ± 4.0 a 41 5.0 mg l-1 yeast extract 30.8 ± 1.8 b 15.4 ± 0.8 b 9 3.5 mg l-1 yeast extract 29.9 ± 1.8 b 40.8 ± 5.1 a 22 150 mg l-1 chitosan + 3.5 mg l-1 yeast extract 35.2 ± 3.9 b ND 7 Table 3: Summary of cortex and xylem diameter and estimated number of xylem cells in Curcuma mangga plantlets cul- tured in proliferation medium supplemented with biotic elicitors Mean values within the same column followed by different alphabet indicate significantly different values (Tukey, HSD, p ≤ 0.05). ND = Not determine Acta agriculturae Slovenica, 118/2 – 2022 7 Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of ... Curcuma mangga Val. Figure 2: Histology images of basal pseudostem of C. mangga plantlets cultured in proliferation medium (Control) at 4 x opti- cal magnification (a) and 10 x optical magnification (b); Proliferation medium supplemented with 3.5 mgL-1 YE at 4 x optical magnification (c) and 10x optical magnification (d); Proliferation medium supplemented with 5.0 mg l-1 YE at 4 x optical magnification (e) and 10x optical magnification (f) Acta agriculturae Slovenica, 118/2 – 20228 F. ABRAHAM et al. Figure 3: Histology images of basal pseudostem of C. mangga plantlets cultured in proliferation medium supplemented with 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract at 4x optical magnification (a) and 10x optical magnification (b) Acta agriculturae Slovenica, 118/2 – 2022 9 Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of ... Curcuma mangga Val. 1980; Webb, 1999; Nakata, 2012). Even though oxalate crystals were often found in higher plant, the formation and function of oxalate crystals in plants were still un- clear (Webb, 1999). It was proposed that oxalate crystal might be involved in ion balance, plant defence, tissue rigidity and support, detoxification, light accumula- tor, and reflector (Franceschi and Homer, 1980; Doege, 2003). The possible function of oxalate crystal as part of plant defence might be relevant in C. mangga study. The oxalate crystal could not be detected in plantlets cultured in medium without elicitor (control) (Figure 2 a & b). Most part of the tissue of C. mangga plantlets cultured in medium supplemented with 150 mg l-1 chi- tosan and 3.5 mg l-1 yeast extract were damaged dur- ing dehydration process of histology preparation. The damaged tissue indirectly indicate that the cell walls of those plantlets were more delicate than the cell walls of plantlets cultured in medium supplemented with yeast extract or the control. This finding was also supported the observation on the visual morphological character- istic of those plantlets which exhibited brittleness on the abnormal formed shoots cultured in proliferation medium supplemented with 150 mg l-1 chitosan plus 3.5 mg l-1 yeast extract. Chitosan has been used to study the defence mech- anism of plants towards their fungal pathogens. Unlike yeast extract, chitosan is a compound with defined mo- lecular structure (polycationic b-1,4-linked-d-glucosa- mine polymers) which resemble to cell wall component of fungi (Walker–Simmons et al., 1983). The interaction between oligosaccharins and receptors located in the plant membranes results in the production of many plants defence secondary metabolites and many phe- nolic and terpenoid compounds (Kim and Lee 2011; Pang et al 2021). It was found to induce the synthesis of pathogenesis-related (PR) proteins and several de- fence enzymes (such as phenylalanine ammonia lyase and peroxidase) (Riaz et al., 2014). The plant defence compounds, synthesized after chitosan elicitation, have relevant physiological activity, mainly as antioxidants (the polyphenols), might have resulted in cell damage as well. 4 CONCLUSIONS Longitudinally half incised shoot explants and cultured vertically on shoot proliferation medium, MS supplemented with 2.0 mg l-1 benzyl adenine (BA) and 0.5 mg l-1 NAA, promote shoot proliferation by 100 %. This method can be used as an alternative technique for multiplication of the in vitro plantlets of C. mangga which normally propagate at a slow rate. Supplementa- tion of 3.5 or 5.0 mg l-1 yeast extract, or combination of 3.5 mg l-1 yeast extract and 150 mg l-1 chitosan, into the shoot proliferation medium could be used as an al- ternative mode for enhancing anti-lipid peroxidation activity. 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