Hindawi Publishing Corporation Advances in Biology Volume 2015, Article ID 639678, 6 pages http://dx.doi.org/10.1155/2015/639678

Research Article Direct Organogenesis from Rhizome Explants in Marsilea quadrifolia L.: A Threatened Fern Species

Mahipal S. Shekhawat and M. Manokari

Biotechnology Laboratory, Department of Plant Science, MGGAC, Mahe, Pondicherry 673 311, India

Correspondence should be addressed to Mahipal S. Shekhawat; [email protected]

Received 17 August 2015; Revised 2 November 2015; Accepted 12 November 2015

Academic Editor: Elisa Rubino

Copyright © 2015 M. S. Shekhawat and M. Manokari. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

An efficient micropropagation protocol has been developed for Marsilea quadrifolia L. through direct organogenesis. The mature rhizomes were used as explants and successfully sterilized using 0.1% HgCl2 for the establishment of cultures. The multiple shoots were differentiated from the explants on Murashige and Skoog (MS) medium augmented with 6-benzylaminopurin (BAP). Full strength MS medium was reported to be effective for the induction of sporophytes from the rhizomes after four weeks of inoculation. Maximum response (96%) with average of 6.2 shoots (2.72 cm length) was achieved on full strength of MS medium augmented with 0.5 mg/L BAP in culture initiation experiments. The cultures were further proliferated in clusters79.0 ( ± 0.37 shoots per explant) with stunted growth on half strength MS medium supplemented with 0.25 mg/L BAP after four weeks. These stunted shoots were elongated (5.30 cm long) on half MS medium devoid of growth hormones. Root induction and proliferation (3.0–4.0 cm long) were observed after 4th subculture of sporophytes on hormone-free half strength MS medium. The rooted plantlets were hardened in the fern house for 4-5 weeks and transferred to the field with 92% survival rate. There were no observable differences in between in vivo grown and in vitro propagated plantlets in the field.

1. Introduction they are young and arise alternately in two rows from the upper surface of the creeping rhizome [5]. The pteridophytes are fragile and vulnerable to anthro- M. quadrifolia is chosen for ecotoxicogenomic studies pogenic disturbances and climate changes due to their micro- because it is sensitive to aquatic environmental toxicant climatic dependence and having a strong affinity on high exposure [6]. It is a high bioaccumulator of heavy metals moisture for sexual reproduction [1]. Marsilea quadrifolia L. with maximum bioconcentration factor for cadmium and (commonly known as water clover, four-leaf clover, or water chromium and could be used for phytosequestration of shamrock) is an aquatic, perennial, and heterosporous fern these metals from contaminated sites [7]. Marsilea was that belongs to the family Marsileaceae (water clover family) studied exhaustively for its valuable biochemical prop- andnativetoEuropeandAsia[2].Itiswidespreadnaturally erties [8–10]. The fern has potential antibacterial, anti- in central and southern Europe, Caucasia, western Siberia, inflammatory, diuretic, depurative, febrifuge, refrigerant, Afghanistan,southwestIndia,China,andJapan[3]. antiepileptic, antioxidant, and cytotoxic activities [11–13]. The plant body of M. quadrifolia is sporophytic with M. quadrifolia is facing major threats due to the climate well branched slender creeping rhizome which is capable aridisation, limited distribution of the species, drainage and of indefinite growth. There are four leaflets of equal size drying up of the wetlands, redirection of the river beds, atthetipofeachpetiole,hencepopularlyknownasfour- overgrowth of marshes, and water pollution [14]. This fern leaf clovers. The aerial or land leaves exhibit circadian and is classified as threatened species and enlisted in the IUCN phototropic movements [4], which have trichomes on adaxial RedDataBook[15,16].Duetotheincreasinghuman and abaxial surfaces. The leaves show circinate venation when activities, all Marsilea species in Europe are considered as 2 Advances in Biology rare, vulnerable, endangered, at extinction, or extinct in the of 0.1 N NaOH/KOH or HCl. The culture tubes (vessels) ∘ wild [17]. As per the assessment of World Conservation were finally incubated at 25 ± 2 C temperature under cool Monitoring Centre [18], M. quadrifolia is threatened in 21 white fluorescent light (Philips, India) with 16 h artificial −2 −1 European countries, vulnerable in eastern Palaearctic region photoperiod (40 𝜇mol m s Spectral Flux Photon Density, (ecozone), and known to be extinct in Germany, Poland, and SFPD) at 55–60% relative humidity (RH). Switzerland [19]. The National Biodiversity Act stressed on the conservation needs to protect M. quadrifolia in its natural 2.4. Aseptic Culture Proliferation. MS basal medium sup- habitats [20]. The fern is also ranked as endangered plant in plemented with different concentrations of cytokinins (BAP eastern India [21]. and Kin) with varying concentrations (ranging from 0.0 Biotechnologicalapproachescanbeusedinpreservation to 1.0 mg/L) was used for multiplication of cultures of M. of genetic material and explored for the reintroduction of quadrifolia.Theculturemediumwaschangedasperthe the species in nature [22, 23]. In vitro culture of endangered morphogenic response of the cultures using different concen- and rare fern species at mass scale can be exploited as an trations of basal salts fortified with different ranges (0.0 to alternative tool for ex situ conservation. Nephrolepis exaltata 1.0 mg/L) of growth hormones. The cultures were transferred bostoniensis was the first fern in which the in vitro propa- to the fresh medium after 4-5-week intervals and the number gation protocol was developed for commercial exploitation and lengths of shoots and roots from rhizomatous sporophyte [24, 25]. Sporocarps and spores were used as explanting were evaluated after every 4 weeks of subculture. materials in the previous attempts on micropropagation of M. quadrifolia [26, 27] which failed to respond under in vitro culture experiments [28]. This is an attempt to develop 2.5.HardeningandAcclimatizationofPlantlets.Invitro an efficient micropropagation protocol of aquatic fern M. raised plantlets were rooted simultaneously with culture quadrifolia using rhizomes as explants. proliferation stage on same medium combination. Hardening aimed at the introduction of micropropagated plantlets of M. quadrifolia into the greenhouse and finally to the field. 2. Materials and Methods The rooted sporophytes were separated from culture medium carefully and washed with distilled water to remove the 2.1. Plant Material and Explants Selection. The plant M. remainsofmediumandtransferredtosterilesoilrite(inpaper quadrifolia was collected from southeast India (swampy area cups) moistened with one-fourth MS salts solution. The setup near Villupuram, Pondicherry State) in the months of April- was maintained in the fern house for 10 days to ensure high November 2013 and successfully grown in fern house. The humidity and open shade (diffused light) for hardening. After rhizomatous nodal explants (stolons) of about 1-2 cm in aweek,theplantswereshiftedtonurserypolybagscontaining length were harvested and brought to the laboratory in order garden soil and clay (1 : 1) to maintain aquatic environment. to initiate the cultures in vitro. Finally the hardened plantlets were transferred to the aquatic, shade field. 2.2. Surface Sterilization of Explants. Since the stolons were used as explants, certain procedures followed to reduce the 2.6. Statistical Analysis. The values presented in each table are contamination in cultures: (1) Rhizomatous explants were means of triplicates with ten replicates each. The results were rinsed with sterile running tap water for 15 min, (2) explants presented as mean ± standard deviation (SD) of triplicates. were rinsed with sterile double distilled water and centrifuged The resulting data were analyzed by analysis of variance with at 100 rpm for 5 min, (3) the explants were treated with 0.1% Duncan’s multiple range tests where the level of significance Bavistin (a systemic fungicide, BASF India Ltd. India) for was 𝑝 < 0.05 using SPSS software (version 16.0). 15 min, and then (4) surface sterilization was carried out with 0.1% HgCl2 for 4-5 min which is followed by washing with sterile distilled water for 5 times under laminar air flow 3. Results and Discussion cabinet. 3.1. Establishment of Cultures. M. quadrifolia cultures were established on MS medium augmented with cytokinins in 2.3. Culture Medium and Inoculation. Murashige and Skoog present study. Cytokinins regulate a considerable number of (MS) medium [29] was used for the establishment of cultures. different developmental and physiological processes in aerial The sterilized explants were inoculated aseptically on the and subterranean organs, like cell divisions in the shoot and half and full strength MS basal medium supplemented with root meristems and chloroplast differentiation [30]. These additives (50 mg/L ascorbic acid, 25 mg/L each of citric also promote axillary buds proliferation by antagonizing the acid, L-arginine, and adenine sulphate). The medium was activity of auxins [31]. The effect of strength of MS medium augmented with 6-benzylaminopurine (BAP) and 6-furfuryl and the different concentration of plant growth regulators amino purine (Kinetin) in different concentrations ranging were tested for multiplication and rooting of the shoots. Full from 0.0 to 3.0 mg/L to induce shoots from the explants. strength MS medium was reported to be effective for the The amendment of carbon source in the medium was 1.5% induction of fronds from the rhizome explants after four to 3% of sucrose as per MS salt concentrations, and the gel weeks of inoculation. Brownish green meristematic areas strength was maintained with the help of 0.8% agar-agar. developed within two weeks around the notch (nodal part) The pH of medium was adjusted to 5.8 ± 0.02 with help of rhizome on full strength MS medium supplemented with Advances in Biology 3

Table 1: Effect of different concentrations of cytokinins (BAP and Table 2: Effect of growth hormones (BAP and Kin) on multiplica- Kin) on induction of shoots from rhizome explants of M. quadrifolia. tion of sporophytes on agar gelled MS medium.

Conc. of Conc. of BAP Conc. of Number of shoots Shoot length (cm) Response Number of shoots Shoot length (cm) cytokinins (mg/L) Kin (mg/L) (Mean ± SD) (Mean ± SD) (%) (Mean ± SD) (Mean ± SD) (mg/L) 0.00 0.00 79.0 ± 0.37k 5.30 ± 0.51g ± a ± a Control (0.0) 0.00 0.0 0.00 0.00 0.00 0.10 — 54.3 ± 0.46fg 4.13 ± 0.30e BAP 0.25 — 50.9 ± 0.23de 4.08 ± 0.38e ± h ± f 0.5 96 6.2 1.39 2.72 0.40 0.50 — 43.5 ± 0.51c 3.20 ± 0.29b ± f ± g 1.0 69 3.7 0.51 1.60 1.25 1.00 — 40.1 ± 0.26b 3.06 ± 0.57b ± g ± e 1.5 73 4.8 1.04 1.33 0.18 — 0.10 55.0 ± 0.71g 3.74 ± 0.20cd ± de ± b 2.0 61 3.0 0.73 1.10 0.12 — 0.25 49.5 ± 0.37d 3.51 ± 0.35c ± g ± e 2.5 48 4.8 1.04 1.33 0.18 — 0.50 44.2 ± 0.29c 3.00 ± 0.62ab ± cd ± c 3.0 22 2.5 0.30 0.44 0.31 — 1.00 36.6 ± 0.18a 2.79 ± 0.48a Kin 0.10 0.10 68.2 ± 0.49j 4.78 ± 0.42f ± ef ± e 0.5 57 3.3 0.26 1.31 0.22 0.25 0.25 61.9 ± 0.35i 4.22 ± 0.15e ± de ± d 1.0 49 3.0 0.14 1.08 0.16 0.50 0.50 57.3 ± 0.26h 3.80 ± 0.30d ± de ± f 1.5 42 2.8 0.40 0.85 0.38 1.00 1.00 52.8 ± 0.40ef 3.53 ± 0.41c ± d ± h 2.0 40 2.6 0.39 0.93 0.45 Note: mean separation was analyzed by ANOVA using SPSS software (var. 2.5 35 2.0 ± 0.81c 0.49 ± 0.10d 16.0) and the values represented in corresponding column followed by same letters are not significantly different according to DMRT at 𝑃 < 0.05. 3.0 19 1.2 ± 0.22b 0.32 ± 0.28c Note: mean separation was analyzed by ANOVA using SPSS software (var. 16.0) and the values represented in corresponding column followed by same letters are not significantly different according to DMRT at 𝑃 < 0.05. 3.2. Multiplication of Shoots In Vitro. The enhancement of shoots multiplication rate on various concentrations of cytokinins on culture medium was tested. The fresh shoots 0.5 mg/L BAP. The maximum response in culture initiation with mother explants were subcultured to the fresh medium was achieved in presence of low concentrations of BAP after four weeks of incubation. Cultures were proliferated in (0.5 mg/L), and 96% of explants responded well with average clusters on half strength MS medium augmented with addi- tivesand0.25mg/LBAPwithstuntedgrowth(Figure1(b)). of 6.2 shoots with 2.72 cm length on this combination Highestmeannumberofjuvenileshoots(79.0 ± 0.37 shoots (Figure 1(a), Table 1). Delayed response of the explants with per explant) was regenerated on this concentration (Table 2). less number of shoots was observed on half strength MS The further growth and elongation of shoots with large num- medium on same hormone concentrations. The shoots were ber of identical individuals from small parts of inoculums was induced after eight weeks of inoculation of explants on the achieved on half strength MS medium without any growth medium. The response of explants with Kin was reported hormone in present study (Figure 1(c)). poor as compared to the BAP, and only 57% explants The leaflets with long petiole arise solitarily from the responded on full strength MS medium supplemented with nodes on the rhizomes. The response of the cultures under Kin with 3.3 shoots per explant. The number of shoots was in vitro conditions was very slow, but frequent subculturing reported stagnant on MS medium supplemented with Kin on fresh medium was found the only feasible option to (0.5 mg/L), even after four months of incubation. multiply this plant species. It was observed that the shoots Generally, ferns micropropagated using spores in most with mother explants failed to respond better if they were of the studies like Salvinia natans [32], Dryopteris affinis directly transferred to the hormone-free medium. There- [33], Equisetum arvense [34], Marsilea minuta [35], and Pteris fore, the cultures were first stabilized on 0.25 mg/L BAP tripartita [36], but the shoots were induced from the nodal and later transferred to the hormone-free medium. Higher partoftherhizomesinpresentstudy.Ithasbeenreportedthat concentrations of cytokinins (both BAP and Kin) showed the supplementation of abscisic acid in the medium induced stunted growth of sporophytic shoots and decreased rate of shoots, roots, and elongation of petioles of M. quadrifolia shoots multiplication. The elongated shoots (5.30 cm long) [37, 38], whereas the supplementation of auxins has no with good roots (3.0–4.0 cm length) were developed on MS ameliorative effects on the regenerative response in this plant medium without any growth hormones. In contrast to the species [28]. The shoots were proliferated from the mother present results the effects of cytokinins were found inhibitory explants even in absence of abscisic acid and auxins in present on multiplication of cultures of M. quadrifolia by Rolli et study. Rolli et al. [27, 39] also studied the effect of plant al. [39]. Spore germination and conversion of gametophytic hormones on in vitro propagation of M. quadrifolia using stage (haploid) into a sporophytic stage (diploid) could rhizomes as explants but less number of shoots was induced be exempted through micropropagation technique in M. per explant. quadrifolia. M. quadrifolia cultures were also established on 4 Advances in Biology

(a) (b)

(c) (d) (e)

(f) (g)

Figure 1: (a) Regeneration of leaflets from the rhizome explants on MS medium. (b) Early stage of multiplication (stunted growth of fronds). (c) Development of multiple sporophytes on half strength MS medium without growth hormones. (d and e) In vitro propagated sporophytes with fronds and roots. (f) Hardening of sporophytes of M. quadrifolia in soilrite. (g) Hardened sporophytes growing in the fern house.

Knop’s nutrient solution and half strength MS medium by et al. [39] and reported development of complete sporophytes various researchers [28, 40]. on hormone-free medium. Banciu et al. [28] reported rooted sporophytes in Knop’s liquid medium. Generally, auxins 3.3. In Vitro Rhizogenesis. Rootsinductionwasobserved areusedinrootingoftheshootsinplanttissueculture after 4th subculture of sporophytic shoots on hormone-free experiments [41, 42] but in case of M. quadrifolia good half strength MS medium. The shoots were separated and rooting was reported without augmentation of any growth transferred to the half strength MS medium, where number regulator with the MS medium. Breznovits and Mohay [43] of shoots with thin and long creeping rhizomes was increased also maintained Marsilea sporophytes on hormone-free half quantitatively in clusters. Best rooting frequency (100%) was strength MS medium. Ravi et al. [36] grown spores derived achieved on hormone-free half strength MS medium (Figures gametophytes on half strength MS medium also support our 1(d)and1(e)).ThepresentresultswerealsosupportedbyRolli results. Advances in Biology 5

3.4.HardeningandAcclimatizationofInVitroGrownSporo- of the Botanical Research Institute of Texas,vol.2,no.1,pp.637– phytes. The in vitro regenerated plantlets were acclimatized 641, 2008. for conservative expression in different environmental con- [3] F. Bonafede, D. Dallai, C. Del Prete, and L. Maffettone, “Marsilea ditions. The plantlets were taken out from the culture vessels quadrifolia L. in Emilia Romagna province, Italy: geographical and the traces of agar were removed using soft brush to avoid distribution, ecology and integrated in and ex situ conservation damage of the roots. Care has been taken while removing the issues,” Atti della Societa dei Naturalisti e Matematici di Modena, agar, since the sporophytic roots were very soft and delicate. vol. 133, pp. 183–211, 2002. The plantlets were initially shifted to sterile soilrite containing [4] W.-Y. Kao and B.-L. 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