Piriformospora indica improves micropropagation, growth and phytochemical content of Aloe vera L. plants Priyanka Sharma, Amit C. Kharkwal, M. Z. Abdin & Ajit Varma
Symbiosis ISSN 0334-5114 Volume 64 Number 1 Symbiosis (2014) 64:11-23 DOI 10.1007/s13199-014-0298-7
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Author's personal copy Symbiosis (2014) 64:11–23 DOI 10.1007/s13199-014-0298-7
Piriformospora indica improves micropropagation, growth and phytochemical content of Aloe vera L. plants Priyanka Sharma & Amit C. Kharkwal & M. Z. Abdin & Ajit Varma
Received: 8 August 2014 / Accepted: 7 November 2014 / Published online: 16 November 2014 # Springer Science+Business Media Dordrecht 2014
Abstract The study was undertaken to investigate the effect of symbiotic fungus Piriformospora indica on the phytochemical content of medicinal plant Aloe vera L. The in vitro culture of A. vera was initiated with rhizomatous stem, where MS medium containing 0.5 mg/l BAP and 0.5 mg/l NAA was found to be the best for multiple shoot proliferation. Interaction of A. vera with P. indica resulted in an overall increase in plant biomass and chlorophyll content. Improved growth was observed in terms of higher shoot and root length, number of shoots and roots in P. indica colonised A. vera plantlets, both under in vitro and ex vitro environment. The simple acclimatization procedure ensured 100 % survival rate for the colonised A. vera plantlets. Root colonization by P. indica increased the gel content in A. vera by 16.5 %. The symbiotic interaction resulted in an increase in phenol content and radical scavenging capacity. The colonised plantlets also had higher aloin content, which is used as a laxative and is a potential anticancer agent. The study demonstrated the potential of P. indica as a biopriming agent for achieving better growth, survival of in vitro raised plantlets and enhancement in secondary metabolite content. Key message: Symbiotic root endophyte primed A. vera plants exhibit better growth, quality and can meet ever increasing industrial demand. Keywords Micropropagation . Mycorrhiza . Piriformospora indica . Acclimatization Aloe vera L . Aloin . HPLC . DPPH P. Sharma : A. C. Kharkwal (*) : A. Varma Amity Institute of Microbial Technology, Amity University Uttar Pradesh, E-3 Block, Fourth Floor, Sector 125, Noida, Uttar Pradesh 201303, India e-mail:
[email protected] M. Z. Abdin Jamia Hamdard University, Hamdard Nagar New Delhi 110062, India
Abbreviations MS Murashige and Skoog BAP 6-Benzylaminopurine NAA α-Naphthaleneacetic acid HPLC High performance liquid chromatography DPPH 1,1-diphenyl-2-picryl hydrazyl AMF Arbuscular mycorrhizal fungi BSA Bovine serum albumin TFA Trifluoroacetic acid MQ Milli Q UV Ultraviolet
1 Introduction Aloe vera L. is a perennial succulent herb that belongs to Asphodelaceae family (Bhuvana et al. 2014). The pharmacologically active ingredients of Aloe are concentrated in inner parenchymatous tissue, called Aloe gel and outer pericyclic tissue, called Aloe sap (Sharma et al. 2014; Tarro 1993). Aloe gel from the leaves is an excellent treatment for wounds, burns and other skin disorders (Facciola 1990; Hart et al. 1988; Anshoo et al. 2005; Ahmed et al. 2007). Aloin, the major anthraquinone in Aloe sap is an active yellow brown compound, used as a laxative, anti inflammatory and anti cancer agent (Lee et al. 2014). The A. vera explants secrete phenolics that lead to browning and often death of the explants (Abrie and Staden 2001). A. vera L. is mostly propagated vegetatively in its natural state, but due to male sterility and low propagation rate, its commercial production and high industry demand is not being met (Natali et al. 1990; Abdi et al. 2013). To catch up with the demand, micropropagation approach for rapid production of this plant has gained importance (Meyer and Staden 1991; Abadi and Kaviani 2010; Marfori and Malasa 2005; Pandhair et al. 2011; Natali et al.
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1990; Roy and Sarkar 1991; Abrie and Staden 2001; Corneanu et al. 1994). The micropropagated plantlets performed better than conventionally propagated plantlets in a study conducted by Gantait et al. (2011). An auxin free culture system for high frequency Aloe vera production has also been developed by Haque and Ghosh (2013). Piriformospora indica was isolated from the Thar Desert of western India from roots of several xerophytic plants (Varma et al. 1999; Verma et al. 1998). It is an endophytic root colonizing basidiomycete fungus and is one of the best studied members of the group Sebacinales (Weiß et al. 2011; Verma et al. 1998). Unlike Arbuscular Mycorrhizal Fungi (AMF), it can be cultured axenically. It colonizes the roots, grow inter and intracelluarly and forms pear shaped spores within the cortex. Symbiotic association of P. indica increases the biomass of both monocot and dicot plants (Varma et al. 2012a). The fungus promotes nutrient uptake and allows the plant to survive under stress conditions (Varma et al. 2012a). It also induces resistance against root and shoot pathogens (Waller et al. 2005; Serfling et al. 2007; Varma et al. 2012a). P. indica mobilizes insoluble phosphates and translocates them via high affinity phosphate transporter (Kumar et al. 2011; Pedersen et al. 2013). Its properties have been patented in Germany, India and other countries (European patent office, Muenchen, Germany, patent No. 97121440.8-2105, Nov. 1998) and the culture of strain DSM 11827 has been deposited at the Deutsche Sammlung für Mikroorganismen und Zellkulturen, Braunschweig, Germany (Verma et al. 1998) The symbiotic interaction of plant and fungi provides a promising strategy for the better establishment of tissue culture raised plants and enhancing various phytochemicals present in the plant (Varma et al. 2012b; Bajaj et al. 2014). These phytochemicals protect not only the plant but also humans and animals who feed upon it, from various oxidative damages (Etten et al. 1994; Kispotta et al. 2012). Hence, it offers a system to study plant microbe interaction and its effect on plant growth related parameters. It has great potential for agricultural, industrial and commercial exploitation for secondary metabolites (Bagde et al. 2010; Varma et al. 2012a; 2013; Pham et al. 2004; Singh et al. 2000; Waller et al. 2005; Deshmukh et al. 2006; Oelmüller et al. 2009; Yadav et al. 2010; Peŝkan-Berghofer et al. 2004; Bajaj et al. 2014). We have standardized composition of growth regulators for rapid and efficient micropropagation of A. vera using underground rhizomatous stem pieces as primary explants. A subsequent study has been undertaken to analyse the effect of P. indica on the growth performance of A. vera plantlets under in vitro and ex vitro conditions. The colonised and non colonised micropropagated plantlets have also been evaluated for various biochemical parameters, including chlorophyll, sugar, protein, phenol, flavonoid, flavonol content, radical scavenging activity and secondary metabolite content.
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2 Material and methods 2.1 Explant preparation and in vitro establishment of A. vera Aloe vera L. in actively growing condition were collected (along with their moist rhizospheric soil) during April 2011 from Amity farm, Issapur (Najafgarh), Delhi, India and maintained in green house facility, until explants preparation. The rhizomatous stems of 35–40 mm diameter were used as primary explants. The explants were thoroughly washed under running tap water and were cleaned using detergent (0.02 % tween 20) for 10 min with vigorous shaking. They were surface disinfected using 0.2 % bavistin and 0.25 % streptocycline treatment for 45 min, followed by a quick rinse with 70 % ethanol. They were kept in 0.1 % HgCl2 for 10 min, with continuous stirring, followed by five rinses with sterile distilled water to remove last traces of chemical (Liao et al. 2004). The rhizome was cut into small pieces having 1–2 buds and further used for cultivation. Disinfected explants were aseptically transferred to MS (Murashige and Skoog 1962) mineral salts and vitamins containing 3 % (w/v) sucrose (carbon source), solidified with 0.8 % (w/v) agar, at a pH of 5.8. Autoclaving was done at 121 °C and 1.034×105 Pa for 20 min. Cultures were incubated in a temperature controlled room at 25±2 °C with 16 h light (2500 lux) provided by cool white fluorescent tubes and a relative humidity of 70 %. 2.2 Shoot and root proliferation The established A. vera plantlets (4 weeks old) having a height of approximately 1.5 cm, were transferred to media containing 0–3 mg/l BAP or the same concentrations of BAP in combination with NAA. They were allowed to grow for 5 weeks, after which the data was recorded. Five replicates of each concentration were taken and the experiment was repeated thrice. The best concentration was then used for the proliferation of plantlets. They were grown on MS basal medium for rooting. 2.3 In vitro co-cultivation of symbiotic fungus Piriformospora indica and A. vera One month (4 weeks) old rooted plantlets of A. vera were cocultivated with P. indica in jam bottles containing 50 ml solidified MS medium. P. indica was grown on solidified potato dextrose medium at 28±2 °C in the dark. An 8 mm disc having spores and fully grown hyphae, extracted from 1 week old culture plate acted as co- inoculum and were placed at a distance of 1 cm from the explant. The noninoculated plantlets acted as control. Cultures were incubated in Plant Tissue Culture Laboratory at 25±2 °C with 16 h light (2500 lux) provided by cool white fluorescent tubes and a relative humidity of 70 %. After 6 weeks of co-cultivation
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(10 weeks old plantlets), 14 samples were randomly selected from each set and various parameters, including the shoot height, root height, number of shoots, number of roots and fresh weight were recorded. The experiment was repeated twice. 2.4 Root colonization Roots of 10 weeks old A. vera plantlets were observed for colonization. Fungal colonization was determined by Phillips
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and Hayman method (1970). Root samples were cleaned by heating in 10 % KOH solution for 15 min then rinsed and acidified with 1 M HCl for 10 min and finally stained by simmering in 0.02 % trypan blue overnight and were destained with 50 % lactophenol for 1–2 h prior to observation. 20 root segments of 1 cm length were randomly chosen from each set and examined under light microscope. The distribution of chlamydospores within the root cortex was taken as an index of colonization. Colonization percentage was determined by using following formula:
Percentage o f root colonization ¼ ½ðnumber o f colonised segments=total number o f segments examinedÞ 100:
2.5 Acclimatization After 6 weeks of co-cultivation, the control and treated A. vera plantlets were transferred to plastic pots (14 cm dia.) containing autoclaved soil, vermiculite and sterilized compost (1:1:1 v/v) for biological hardening. A relative humidity of 75 % and temperature of 24±1 °C was maintained with light intensity of 12000 lux provided by cool white fluorescent tubes and 16 h light photoperiod. Each set consisted of 35 pots (1 plant per pot). The plantlets were covered with transparent plastic poly bags in order to retain moisture and avoid transient shock. To allow proper aeration, small pores were made on plastic bag at regular intervals. They were watered daily with RO water (45 % of maximum water-holding capacity). After 8 and 16 weeks of acclimatization, when the plantlets were 18 and 26 weeks old respectively, the length of shoots, roots and number of shoots, roots and fresh weight of 14 randomly selected samples from each set were recorded. Root segments of 26 weeks old control and treated samples were harvested from randomly selected plants (5 from each set) to examine the colonization by P. indica
Homogenized tissue was centrifuged at 10,000 rpm for 15 min at 4 °C and the supernatant was used to measure the absorbance at 645 and 663 nm. Chlorophyll a, chlorophyll b and total chlorophyll (mg/g fresh weight) were calculated using the equations described by Arnon (1949).
3.2 Total soluble sugars Soluble sugar content of A. vera was determined spectrophotometrically (UV- 1800 Shimadzu) as described by Ashwell (1957). 0.1 g leaf sample was homogenized in 5 ml distilled water, followed by centrifugation at 5000 rpm. The supernatant was re suspended in 5 ml distilled water and was pooled with initial extract. 1 ml of extract was thoroughly mixed with 4 ml of Anthrone reagent (0.2 % in concentrated H2SO4) and was kept in boiling water bath for 10 min, followed by cooling at room temperature. The Absorbance was read at 620 nm against the blank of distilled water replacing the extract. Standard curve was prepared by using glucose (10– 100 μg/ml) as standard. Results are expressed in terms of glucose equivalent (mg/g dry weight).
3 Biochemical estimations
3.3 Total phenol content
After 16 weeks of acclimatization, 26 weeks old A. vera plantlets were taken for performing biochemical estimations. Powdered oven dried leaves (50 °C for 48 h) were used for the estimation of total soluble sugars, phenol, protein, flavonoid and flavonol content. Each estimation was repeated twice with eight replicates in both sets.
The total phenol content of A. vera was determined spectrophotometrically (UV- 1800 Shimadzu) using Folin Ciocalteu reagent at 765 nm (McDonald et al. 2001). The dilute methanolic extract (0.5 ml of 1:10 g/ml) or gallic acid (standard phenolic compound) was mixed with Folin Ciocalteu reagent (5 ml, 1:10 diluted with distilled water) and aqueous sodium carbonate (4 ml, 1 M). The mixture was allowed to stand for 15 min and the total phenols were determined by spectrophotometer at 765 nm. The standard curve was prepared using (10–100 mg/l) solutions of gallic acid in methanol: water (50:50, v/v). Total phenol values are expressed in terms of gallic acid equivalent (mg/g dry weight).
3.1 Chlorophyll content Fresh leaves were harvested from A. vera plantlets and were washed and blot dried. 0.1 g leaf sample was homogenized in 10 ml ice-cold 80 % acetone with chilled pestles and mortars.
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3.4 Protein content
3.8 Sample extraction for aloin
Total protein content in the leaves of A. vera was estimated by the method of Lowry et al. (1951). The extract was prepared in 0.1 N NaOH (1:10 g/ml). The standard curve was prepared by using BSA (10–100 μg/ml) as standard and absorbance was taken at 660 nm. The protein values are expressed in terms of BSA equivalent (mg/g dry weight).
Sample extraction protocol of Kispotta et al. (2012) was followed. Fresh leaves of 26 weeks old A. vera plantlets were harvested and cuts were made on the leaf. The brown exudate from the leaf was collected. It was freeze dried and extracted with ethanol for 1 h at 50 °C using sonicator and centrifuged at 3000 g for 10 min. The supernatant was further filtered using 22 μm filter. The filtrate was subjected to HPLC analysis (injection volume - 20 μl). Calibration curve was obtained by plotting the peak area against concentrations of standard solution and they showed linear relationship. Phenolic compound was identified by comparing the retention time with that of standard compound. Quantity of aloin produced was calculated based on area of sample peak, concentration and peak area of standard, with six replicates.
3.5 Total flavonoids content Aluminium chloride colorimetric technique was used for flavonoids estimation (Chang et al. 2002). Methanolic extract of sample (0.5 ml of 1:10 g/ml) was sequentially mixed with 1.5 ml of methanol, 0.1 ml of 10 % aluminium chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. It was left at room temperature for 30 min after which the absorbance of the reaction mixture was measured at 415 nm. The standard curve was plotted by preparing the quercetin solutions (12.5 to 100 μg/ml) in methanol. Total flavonoids content is expressed in terms of mg quercetin equivalent/g dry weight. 3.6 Total flavonols Total flavonol content was determined by method given by Kumaran and Karunakaran (2007). The reaction mixture consisted of 2.0 ml of the sample, 2.0 ml of AlCl3 prepared in ethanol and 3.0 ml of (50 g/l) sodium acetate solution. The absorbance at 440 nm was measured after 2.5 h at 20 °C. Total flavonol content is expressed in terms of mg quercetin equivalent/g dry weight. 3.7 DPPH radical scavenging activity The stable 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) was used to evaluate the free radical scavenging activity of the Aloe leaf extract at 517 nm using spectrophotometer (UV1800 Shimadzu) (Williams et al. 1995). Different concentrations of 1 ml methanolic plant extract (0.25, 0.50, 0.75, 1.0, 1.25 mg/ml) were mixed with 2 ml of freshly prepared DPPH solution (0.01 mM) and were kept in dark for 30 min, at room temperature. DPPH dissolves in methanol and give characteristic absorption at 517 nm. The experiment was repeated twice with eight replicates each. Ascorbic acid (0.25–1.25 mg/ml) served as standard control. The antioxidant activity percentage was calculated as described by Williams et al. (1995) Antioxidant activity ð%Þ ¼ ½ðAC−AEÞ=AC 100 Where AC is the absorbance of DPPH solution without extract and AE is the absorbance of the tested extract.
3.8.1 HPLC analysis The aloin content was determined by HPLC analysis. It was Waters system, which consisted of dual wavelength UV absorbance detector (Waters 2487), binary HPLC pump (Waters 1525) and Waters temperature control system. The separation was carried out on a C18 column. Two different mobile phase systems consisting of TFA in MQ water and HPLC grade methanol were used and gradient system was followed (Kispotta et al. 2012). The monitoring was done at wavelength of 293 nm. 3.9 Gel content Aloe vera gel is the mucilaginous jelly obtained from its parenchyma cells (Ramachandra and Rao 2008). Fresh and fully expanded leaves (4 leaves per plant having an approximate weight of 12 g) of 26 weeks old control and treated A. vera plantlets were taken and washed with distilled water. They were then mechanically processed to separate the gel matrix from the outer cortex. The gel obtained was weighed and compared. The experiment was repeated twice with eight replicates each.
4 Statistical analysis One-way ANOVA with Dunnett’s post test was performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. Where applicable, the results were expressed as Mean±Standard deviation or Standard error (SD/SE).
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Fig. 1 a rhizomatous stem of 2 years old A. vera plant used as a primary explants, harvested in April 2011 in actively growing condition. The arrow indicates the emerging bud. Upon establishment of culture, 4 weeks old A. vera plantlets were grown at different concentrations (mg/l) of growth hormones (BAP and NAA) in MS medium. MS
medium was supplemented with 3 % (w/v) sucrose and 0.8 % (w/v) agar at a pH of 5.8. Cultures were incubated in a temperature controlled room at 25±2 °C with 16 h light (2500 lux), provided by cool white fluorescent tubes. These photographs were taken at the end of 5 weeks. b 0.5 BAP +0.5 NAA c 0.5 BAP d 1.5 BAP e 2.5 BAP
5 Results
0.5 mg/l NAA containing MS medium (9±1 shoots per explant), where shoots were healthy and green and shoot proliferation started very early and reached the maximum height (7±0.5 cm) (Mean±SD) (Table 1 and Fig. 1b). Aloe showed 59 % rooting at this concentration. Multiplication of shoots was maximum on MS medium having 0.5 mg/l BAP (10.67±1.15 shoots per explant) (Table 1 and Fig. 1c). MS medium containing 1.5 mg/l BAP gave 5.67 ± 0.57 shoots per explant (Table 1 and Fig. 1d). Callusing was observed in around 90 % cases in medium containing 2.5 mg/l BAP (Table 1 and Fig. 1e). MS basal medium proved to be best for rooting (99.97 %). Rooting initiated in 40 % cases on the 5th day. Latest by 7th day, rooting was
5.1 In vitro micropropagation and proliferation The culture of A. vera was established using rhizomatous stem as primary explants (Fig. 1a). The disinfection protocol for rhizome was 71 % efficient. Germination was observed 10–15 days post inoculation. The problem of browning was avoided by frequent transfers (once a week) and trimming of the darkened tissues until the cultures were established. A. vera plantlets were cultured on MS media having different concentrations of growth hormones (Table 1). Best response in terms of shoot regeneration (85 %) was seen in 0.5 mg/l BAP + Table 1
Four weeks old A. vera plantlets were grown at different concentrations (mg/l) of growth hormones (BAP and NAA) in MS medium
Concentration (mg/l)
No. of shoots produced after 1st subculture (Mean±SD)
Height of plants (cm) (Mean±SD)
% of plantlets with roots
Comments
MS basal MS +0.5 BAP +0.5 NAA
1.33±0.57 9±1
3.5±0.5 7±0.5
99.97 59
MS +0.5 BAP MS +1 BAP
10.67±1.15 3.67±1.15
3.39±0.34 2.43±0.40
68 42
Best for rooting and growth of the plant Proliferation started at the earliest and max. height achieved Best multiple shoot proliferation
MS +1.5 BAP MS +2 BAP MS +2.5 BAP MS +3 BAP
5.67±0.57 4.33±0.57 3.33±1.15 2±1
1.81±0.68 2.05±0.34 1.38±0.98 1.27±0.25
39 0.5 0 0
Callusing in 90 % explants
MS medium was supplemented with 3 % (w/v) sucrose and 0.8 % (w/v) agar at a pH of 5.8. Cultures were incubated in a temperature controlled room at 25±2 °C with 16 h light (2500 lux), provided by cool white fluorescent tubes. The data was recorded after 5 weeks. n=5 with 3 repetitions
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Fig. 2 Co-cultivation of symbiotic fungus Piriformospora indica with A. vera plantlets under in vitro environment. a – Four weeks old rooted A. vera plantlets having an approximate root length of 2.5 cm, shoot length of 1.5 cm and root density of 2–3 on 1st day of co-cultivation b ten weeks old A. vera plantlets after 6 weeks of co-cultivation in in vitro environment. The arrow indicates the disc of 1 week old culture of
P. indica having spores and actively growing hyphae. –Pi represents the A. vera plantlets without inoculation of P. indica, whereas + Pi represents the A. vera plantlets with inoculation of P. indica. c The plantlets were harvested at the end of 6 weeks and were compared for various growth parameters. The colonised plantlets of A. vera have more number of leaves and roots, as compared to control plantlets. (See also Fig. 3)
obtained in around 80 % cases and almost 100 % after 10th day.
co-cultivated plantlets were grown further for a period of 6 weeks under in vitro environment (Fig. 2b). After 6 weeks of co cultivation, when the plantlets were 10 weeks old, they were harvested (Fig. 2c) and 14 samples were randomly selected from each set and compared for various growth parameters, including shoot height, root height, number of shoots, number of roots and fresh weight (Fig. 3). P. indica colonised A. vera plantlets had enhanced growth characteristics as compared to the non colonised plantlets. Shoot and root length of P. indica colonised plantlets increased by 80.83 and
5.2 Co- cultivation with P. indica Four weeks old rooted A. vera plantlets having an approximate root length of 2.5 cm, shoot length of 1.5 cm and root density of 2–3 were partitioned into two sets- one without P. indica served as control, while the treated set was inoculated with disc of 1 week old culture of P. indica (Fig. 2a). The Fig. 3 Effect of P. indica on growth parameters of 10 weeks old A. vera plantlets after 6 weeks of co-cultivation in in vitro environment. The bar represents the SE. Each data set represents an average of 14 replicates. The (*) over the bar represents the level of significance at p
