Abstract
Couroupita guianensis is a medicinally important tree (family: Lecythidaceae) distributed in tropics and widely used in traditional medicine. An efficient micropropagation protocol has been established for C. guianensis using cotyledonary nodes from one wk old-in vitro germinated seedlings. Murashige and Skoog’s (MS) medium supplemented with 3.0 mg/l kinetin (KIN) and 1.0 mg/l ”-naphthaleneacetic acid (NAA) exhibited highest shoot regeneration frequency (96.65%) with an average of 5.66”0.08 shoots per explant and 3.50”0.04 cm shoot length. Micropropagated shoots (6 wk old) were rooted on MS medium amended with indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). Rooting medium supplemented with 3.0 mg/l IBA and 6.0 mg/l IAA produced more number of roots (2.66”0.11; 2.40”0.13) with an average root length of 2.96”0.06 cm and 2.90”0.06 cm, respectively. An increase in the number of roots (5.33”0.16) and root length (5.73”0.35 cm) was observed with the addition of activated charcoal (1.0 g/l) to the rooting medium. Rooted shoots transferred to quarter-strength liquid MS basal salts produced secondary roots. After 7 d of secondary roots initiation, healthy shoots were transferred to the soil mixture containing sand, red soil and organic manure (1:1:1, v/v/v). About seventy percent of the in vitro grown plantlets survived the transfer to field conditions after 3 months. Monomorphic DNA fingerprinting pattern obtained using RAPD and ISSR markers confirmed the clonal fidelity of micropropagated plantlets. This study promotes the large-scale clonal propagation of C. guianensis for use in traditional medicine.
Keywords
Couroupita guianensis; micropropagation; cotyledonary node; plant growth regulators; activated charcoal
Key message
Conservation of medicinal plants through in vitro propagation is a viable option for their sustainable use in pharmaceuticals. The protocol developed herein would facilitate mass production of Couroupita guianensis.
Introduction
Couroupita guianensis, an important medicinal tree of Lecythidaceae family is widely used in folk medicine. It is commonly called as cannonball tree because of large, ball-shaped fruits hanging down from the trunk (Manimegalai et al. 2012). In tamil, it is referred as nagalingam as the flowers contain snake-shaped pollen at the centre (Shah et al. 2013). In traditional medicine system, whole tree parts of C. guianensis are used to treat tumors, inflammation, malaria, skin infections and toothache (Sivakumar et al. 2012; Lanvanya and John 2014). The leaves possess anti-inflammatory (Pinheiro et al. 2013), antibacterial, antimicrobial, antimycobacterial, antibiofilm (Umachigi et al. 2007; Al-Dhabi et al. 2012), antiulcer, antiarthritic, antidiarrheal (Elumalai et al. 2012 a,b; 2013), anticancer (Ramalakshmi et al. 2014), anti-depressant (Gupta et al. 2012), ovicidal (Baskar and Ignacimuthu 2013; Baskar et al. 2014), and antinociceptive properties (Pinheiro et al. 2010). The pharmaceutical industry uses the tree parts (leaf, flower and root) to prepare a syrup ‘Betalupe’ for treating female infertility (Tamilselvi et al. 2012).
C. guianensis is found mostly in sacred groves of temples. However, its distribution in natural habitats is threatened by human settlements (Rai 2014). Propagation of this plant species through seeds is not feasible under natural conditions, as the seeds are consumed along with fruit pulp by peccaries, pigs and monkeys when the fruits split open. Rarely, seeds that escape the degradation of their digestive enzymes germinate under suitable conditions. However, the seedling emergence and growth is greatly affected by abiotic stress like heat and drought. An efficient micropropagation system is therefore necessary for mass multiplication, germplasm conservation and genetic improvement of this tree to meet the demands of the pharmaceutical industry. Micropropagation has been successfully employed for the large scale clonal propagation of medicinally important tree species like, Stevia rebaudiana (Lata et al. 2013) and Morus alba (Saha et al. 2015).
Micropropagated plantlets, in general, are genetically identical as multiple shoots are formed from meristematic cells (Swarna and Ravindhran 2012). Genetic stability of in vitro regenerated plantlets is commonly analyzed using molecular markers such as randomly amplified polymorphic DNA [RAPD], inter simple sequence repeat [ISSR], restriction fragment length polymorphism [RFLP] and amplified fragment length polymorphism [AFLP] (Akdemir et al. 2014). Of these, RAPD and ISSR are rapid, cost effective, and highly discriminating markers (Vinoth and Ravindhran 2015). Both these markers were useful in analyzing the genetic integrity of micropropagated medicinal plants like Lecythis pisonis Cambess (Borges et al. 2016) and Harpagophytum procumbens (Muzila et al. 2014).
Therefore the current investigation was focused to bring out an effective protocol for in vitro shoot multiplication of C. guianensis, using cotyledonary nodes and to authenticate the genetic homogeneity using RAPD and ISSR markers. This is the first report on the assessment of genetic integrity of C. guianensis.
Materials and methods
Collection of plant materials and surface sterilization
Couroupita guianensis fruits were collected from the Loyola College hostel garden, Chennai, India (13”03'41.7"N 80”13'57.5"E). Mature and healthy seeds were extracted from the ripened fruits after removing the outer shell mechanically. The pulp debris was removed from the seeds by washing under running tap water for 15 min and seeds were left for drying overnight at room temperature (28”C). The outer layer of the seed coat with exotestal hairs was removed manually and seeds were surface sterilized for 20 min in disinfecting solution containing 1 ml of Tween-20 per 100 ml in 1% (v/v) sodium hypochlorite (available Chlorine 4% w/v approx, Qualigens, Mumbai, India). The seeds were then subjected to 6 to 8 washes using autoclaved double distilled water. After surface sterilization, seed coat was removed with a sterile blade, embryos were blotted under sterile conditions on a filter paper and germinated on MS medium amended with 1.0 mg/l KIN and 0.1 mg/l IBA. Culture tubes were maintained in dark for 3 days and then transferred to light [16/8-h (day/night) photoperiod] with a photosynthetic photon flux density (PPFD) of 50 ”mol m-2 s-1 supplied with cool-white fluorescent lamps (Philips, Chennai, India). Explants were derived from one wk old-in vitro seedlings. Cotyledonary nodes were separated from the hypocotyl and the cotyledonary leaves were trimmed to expose the shoot apex.
In vitro shoot regeneration
Cotyledonary nodes were cultured in MS (Murashige and Skoog, 1962) medium comprising of 30 g/l sucrose, 8 g/l agar (HiMedia, Mumbai, India), 1% (w/v) polyvinylpyrrolidone (PVP) and supplemented with plant growth regulators (PGRs), such as 6-benzylaminopurine (BAP) and kinetin (KIN) (1.0-5.0 mg/l) individually, or combined with indole-3-acetic acid (IAA), ”-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) (0.5, 1.0 mg/l) (HiMedia, Mumbai, India) to determine the optimum conditions for multiple shoot regeneration. The pH of the medium was adjusted to 5.8 before gelling with agar (HiMedia, Mumbai, India). Ten ml of culture medium was dispensed into tubes (25 mm diameter ”150 mm height) (Borosil, Chennai, India), and autoclaved at 121 ”C for 20 min. The cultures were maintained at 25”2 ”C in light [16/8-h (day/night) photoperiod] supplied by cool white fluorescent lamps (Phillips, Chennai, India) at 50 ”molm-2s-1 PPFD. Newly emerging shoots were sub-cultured at every 2 wk intervals onto the same initial medium.
Rooting and Acclimatization
The proliferated shoots (2-3 cm length) were individually separated and transferred to the rooting medium with (1.0 g/l) or without activated charcoal. Full-strength MS salts and vitamins supplemented with various concentrations (1.0-6.0 mg/l) of IAA or IBA was used for rooting. Rooted plantlets (3-4 cm height) after 4 wk of culture were removed from the culture tubes, and washed with sterile water to remove the agar media, blot dried on filter paper and placed in quarter-strength liquid MS basal salts for the induction of secondary roots. After 2 wk, plantlets (15-18 cm height) with well-established root system were transferred to greenhouse conditions in HDPE grow bags (10 cm diameter) filled with a soil mixture containing sterilized sand, red soil, and organic manure (1:1:1, v/v/v). Hardened plants with six to ten fresh leaves were transferred to clay pots (30 cm height ” 28 cm diameter) and plants grown to about 30 cm in height were transferred to soil.
Statistical analysis
All the experiments were repeated six times with 30 replicates per treatment. Data is presented as mean ” standard error and the analysis of variance (ANOVA) was performed. For shoot proliferation, the regeneration frequency (%), mean number of shoots per explant and shoot length were recorded after 6 wk of culture. For root induction, mean number of roots and root length were recorded after 4 wk of culture on rooting medium. Duncan’s multiple range tests at 5% probability level was performed to detect the significant differences among the treatment means using IBM SPSS statistics version 19.0.
Assessment of genetic fidelity
Genomic DNA was extracted from the leaves of ex vitro micropropagated plants and the mother plant of C. guianensis as described by Michiels et al. (2003). A total of 20 RAPD and 10 ISSR primers were used for genetic fidelity analysis, out of which 10 RAPD and 6 ISSR primers were selected based on the amplification of distinct and scorable bands. PCR reaction setup and cycling conditions were maintained as described by Vinoth and Ravindhran (2015), except for the primer annealing temperature (36 ”C for 30 s – RAPD; 53 to 55 ”C for 30 s – ISSR). PCR products resolved on 1.5 % (w/v) agarose were visualized under UV transilluminator and documented using the Gel documentation system (Gelstan 4X, Chennai, India). DNA fingerprinting profiles were compared using Labimage 1D software version 3.3.0 (Kapelan Bio-Imaging Solutions, Leipzig, Germany) to evaluate the clonal fidelity.
Results and discussion
In vitro shoot regeneration
Multiple shoots were successfully induced from 7-d-old cotyledonary nodal explants of C. guianensis. The morphogenetic responses to cytokinins alone (BAP and KIN) and in combination with auxins (NAA, IBA and IBA) are summarized in Table 1. No multiple shoots were induced in MS medium devoid of PGRs. The addition of PGRs to shoot regeneration medium (SRM) positively influenced the shoot multiplication. Among the two cytokinins tested, KIN (3.0 mg/l) effectively induced 1.97”0.12 shoots per explant with 3.28”0.14 cm shoot length. BAP (2.5 mg/l), on the other hand, produced a maximum of 1.72”0.08 shoots per explant with 2.64”0.13 cm shoot length. A further increase in the cytokinins greater than 3.0 mg/l resulted in decline of multiple shoot buds. The superiority of KIN for the induction of multiple shoots was previously reported in medicinal plants such as Artemisia pallens (Nathar and Yatoo 2014), Matthiola incana and Eustoma grandiflorum (Kaviani 2014). The synergistic effect of cytokinins with auxins in multiple shoot regeneration was also studied. SRM containing 3.0 mg/l KIN and 1.0 mg /l NAA produced the maximum number of shoots per explant (5.66”0.08) (Fig 1b,c). The positive influence of NAA supplementation in multiple shoot induction was reported in Khaya grandifoliola (Okere and Adegeye 2011), Holarrhena antidysenterica (Kanungo et al. 2012) and Barringtonia racemosa (Behbahani et al. 2007).
Rooting and acclimatization
Rooting of in vitro shoots was carried out on MS medium supplied with various concentrations of IBA or IAA (Fig. 2). IBA was significantly effective compared to IAA for root induction. IBA (3.0 mg/l) induced the highest number of roots per shoot (2.66”0.11) followed by 6.0 mg/l IAA (2.40”0.13 roots per shoot) with an average root length of 2.96”0.06 cm. Similarly, IBA (2.0 mg/l) profusely induced adventitious roots from in vitro leaf explants of C. guianensis Aubl. (Manokari et al. 2016) and ex vitro mini cuttings of tree species like Azadirachta indica (Gehlot et al. 2014) and Spondias pinnata (Tomar 2016). Regenerated shoots showed two folds (5.33”0.16 roots per shoot) increase in the root induction when activated charcoal (1.0 g/l) was added to the rooting medium containing 3.0 mg/l IBA (Fig. 1d, Fig. 3). The use of activated charcoal to improve the rooting performance was reported previously in Acacia leucophloea (Sharma et al. 2012). Acclimatization of rooted shoots in quarter-strength liquid MS basal salts resulted in prominent elongation of the shoots and induction of secondary roots after 2 wk (Fig. 1e). Plantlets with secondary roots survived (70.21%) transferred to the soil and reached a height of 60 cm after 3 months (Fig. 1f). Healthy plantlets were morphologically similar and showed uniform growth characteristics (Fig. 1g,h).
Assessment of genetic fidelity
C. guianensis plantlets propagated under in vitro conditions were subjected to clonal fidelity analysis using ISSR and RAPD markers. A total of 74 monomorphic bands was generated by ten RAPD primers. The number of bands ranged from 5 (OPA2 & OPA4) to 10 (OPA1 & OPA3) with 7.4 bands per primer on average (Table 2). The amplicons ranged in size from 300 to 2400 bp. Likewise, (AG)8 and (GA)8 dinucleotide ISSRs anchored on the 3”-end produced 41 monomorphic bands. Six ISSR primers produced 6.8 bands per primer with a molecular size range of 330 to 1980 bp (Table 2). DNA fingerprinting pattern obtained using RAPD and ISSR primers confirmed the genetic homogeneity of micropropagated plants. (Fig. 4). In concurrence with our study, reports indicate the efficiency of PCR markers in determining the genetic fidelity of tissue cultured plants of Gerbera jamesonii Bolus (Bhatia et al. 2010), Phoenix dactylifera L. (Kumar et al. 2010), Rauvolfia serpentina (Faisal et al. 2012) and Psidium guajava (Rai et al. 2012).
Conclusion
In this paper, we herein report a rapid, reproducible and large-scale micropropagation protocol from cotyledonary nodes of C. guianensis. This protocol facilitates sustainable utilization of C. guianensis in traditional medicine and reintroduction into its natural habitats.