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MULTIPLE SHOOT REGENERATION VIA INDIRECT ORGANOGENESIS FROM SHOOT TIP AND NODAL MERISTEM EXPLANTS OF CERATOPHYLLUM DEMERSUM L.

Byline: M. Dogan

Keywords: Callus induction, C. demersum, nodal explant, photosynthetic pigment.

INTRODUCTION

Medical and aromatic plants produce a wide variety of hydrocarbons and their analogues such as aldehydes, ketones and esters (Qiming et al, 2006). Industrial and commercial pharmaceuticals and chemicals are usually products of secondary metabolism in plants. Out of the 350, 000 plant species known to date, about 35, 000 are used worldwide for medicinal purposes (Shasany et al., 2007). Approximately 25% of all medicines prescribed in developed countries have about 100 plant species (Comer and Debus, 1996). Ceratophyllum demersum L., a perennial plant of family Ceratophyllaceae (Arber, 2010), is an aquatic medicinal plant and it has been traditionally used in the human treatment such as ulcer, dysentery, wounds, fever (Taranhalli et al., 2011). Karale et al. (2013) reported that the methenolic extracts of C. demersum in albino rats show analgesic, antipyretic and anti-inflammatory effects.

The essential oil of C. demersum has been found to have anti-inflammatory, antifungal, antibacterial, and antineoplastic activity (Kurashov et al., 2016). Moreover, it has been reported that the phytochemical components such as flavonoids, sterols, glycosides, tannins, saponins, alkaloids, terpenes, chlorides of C. demersum have allelopathic effects on algae (El-Sheekh et al., 2017). The demand for medicinal and aromatic plants is rapidly increasing, due to their valuable chemistry. One of the most important production methods to be applied to meet this demand is plant tissue culture techniques. Medical plants have a number of advantages in producing by optimizing culture conditions through cell and tissue cultures.

These can be listed as (i) the environmental factors (climate, geographical difficulties, seasonal constraints) encountered during cultivation of the plant are removed, (ii) less land use is provided, (iii) prevent the plant from being collected from the nature and protect its generation, (iv) it is ensured that economically valuable metabolites, which are present in plants in low quantities, can be produced in sufficient quantity, (v) homogeneity, standard quality and productivity are provided in production, (vi) helps in the understanding of the biosynthesis mechanisms of the metabolites (Erkoyuncu and Yorgancilar, 2015). Thanks to all these advantages, the production of medical and aromatic plants by tissue culture techniques is an important research topic.

The technique is used in the production of many medical plants such as Scorzonera ahmet-duranii (Surucu et al., 2018), Ephedra gerardiana (Rautela et al., 2018), Stahlianthus campanulatus Kuntze (Mongkolsawat et al., 2018) and Artemisia abrotanum (Bolyard, 2018). The production of callus form from various explants (leaf, shoot tip, internode etc.) is carried out primarily to determine the culture conditions required for the survival and growth of the explants, examine cell growth, obtain products from primary and secondary metabolism, and obtain cell suspension. Large amounts of secondary and bioactive compounds can be obtained from the plants via the callus and cell culture (Ogitaet al., 2009; Sen et al., 2014). The present study was designed for in vitro callus formation and plantlet regeneration of C. demersum, a high value medicinal aquatic plant, using shoot tip and different nodal meristem explants. In addition, photosynthetic pigment contents of shoots formed on calli were examined.

In vitro production studies on C. demersum have been carried out previously by Karatas et al. (2014), Dogan et al. (2015) and Emsen and Dogan (2018). However, studies on plant regeneration following callus formation have never been conducted before. In this respect, our current work is potentially a significant contribution to the literature.

MATERIALS AND METHODS

The plant materials were obtained from Karamanoglu Mehmetbey University, Karaman, Turkey. Surface sterilization of the explants was accomplished by the method of Karatas et al. (2014). After sterilization, shoot meristem, 1st and 2nd nodal meristem explants were isolated under sterile conditions and incubated on MS (Murashige and Skoog, 1962) medium fortify with 30 g sucrose (Duchefa) per liter, 0.65% agar and 0.05, 0.10, 0.20, 0.40 and 0.80 mg/L Thidiazuron (TDZ) in Magenta GA7 vessels for in vitro callus formation. Seven weeks after inoculation, the densities and frequencies of callus were recorded (Table 1). After seven weeks, the identical compact callus obtained from shoot tip, 1st and 2nd nodal meristem were cultured on MS nutrient media supplemented with 3% sucrose (Duchefa), 0.65% agar (Duchefa) and 0.10 mg/L 6-Benzylaminopurine (BAP), 0.10 mg/L Gibberellic Acid (GA3) and on MSO (hormone-free) medium to evaluate shoot regeneration (Table 2).

Since C. demersum is rootless in its natural environment, in vitro rooting trials have not been conducted. The elongated shoots were transferred to aquariums containing water at pH 7.0 and 23+-1AdegC 16 h light photoperiod for 30 days for acclimatization. The trials were repeated three times. The pH of all culture media was adjusted to 5.7+-1 before autoclaving (1.2 atmospheric pressure, 120AdegC for 20 min). All explants photoperiod using white LED (Light Emitting Diodes) lights (1500 lux). The data for both callus and shoot regeneration in the MS mediums were recorded after 8 weeks of culture. For photosynthetic pigment content analysis, the washed fresh plant samples (100 mg) were extracted with 80% acetone. Photosynthetic pigments (chlorophylls and carotenoids) were measured in a spectrophotometer (Thermo Fisher Scientific, Multiskan Go) at 663 nm, 646 nm and 470 nm. All content values were calculated according to Lichtenthaler and Wellburn (1983).

Total chlorophyll content was given as chlorophyll-a + chlorophyll-b. Data from tissue culture studies and photosynthetic pigment contents were analyzed statistically with IBM SPSS 21 for Windows and post hoc tests were performed using Duncan. Data given in percentages were subjected to arcsine transformation before statistical analysis (Snedecor and Cochran, 1997).

Table 1. Effects of different concentrations of TDZ on callus induction from shoot tip and nodal meristem explants of C. demersum on MS medium.

###Shoot tip###1st nodal meristem###2nd nodal meristem

TDZ###Callus###Callus###First callus###Callus###Callus###First callus###Callus###Callus###First callus

(mg/L)###frequency###density###formation###frequency###density###formation###frequency###density###formation

###(%)###(days)###(%)###(days)###(%)###(days)

0.05###83.33###++###16###100.00###++###14###94.44###+###17

0.25###100.00###++###16###100.00###+++###15###100.00###++###17

0.50###100.00###+++###14###100.00###+++###12###100.00###+++###16

0.75###100.00###+++###15###100.00###+++###15###100.00###+++###15

1.00###94.44###+###18###100.00###++###17###66.66###+###19

Table 2. Effects of free-hormone MS (MSO), 0.10 mg/L BAP and 0.10 mg/L GA3 on regenerative response of callus of C. demersum.

First

Culture###Subculture###Shoot regeneration frequency (%)###Mean number of shoots per callus###Mean shoot length (cm)

Medium

Growth regulators (mg/L)###Shoot tip###1st nodal###2nd nodal###Shoot tip###1st nodal###2nd nodal###Shoot tip###1st nodal###2nd nodal

###meristem###meristem###meristem###meristem###meristem###meristem

###0.10 BAP###100.00a###91.67a###66.67bc###90.75def###86.28bc###82.22b###1.22b###1.35b###1.12c

0.05 TDZ###0.10 GA3###100.00a###100.00a###100.00a###101.25b###91.17ab###85.75a###0.97e###1.10e###0.91e

###MSO###100.00a###91.67a###83.33ab###61.83h###66.44fg###53.00g###0.71h###0.69i###0.74g

###0.10 BAP###100.00a###100.00a###100.00a###96.42bcde###92.92ab###78.75c###1.25a###1.15d###0.95d

0.25 TDZ###0.10 GA3###100.00a###100.00a###100.00a###108.83a###96.25a###87.08a###1.17c###1.39a###1.43a

###MSO###100.00a###100.00a###100.00a###55.50hi###64.33g###61.08f###0.65i###0.62j###0.68h

###0.10 BAP###100.00a###100.00a###100.00a###92.92cde###83.08cd###79.58bc###1.12d###0.97g###0.92e

0.50 TDZ###0.10 GA3###100.00a###100.00a###100.00a###98.50bc###85.92bc###81.75bc###1.13d###1.18c###1.21b

###MSO###100.00a###100.00a###91.67a###51.92i###55.28h###52.05g###0.59j###0.54k###0.57i

###0.10 BAP###100.00a###100.00a###100.00a###89.16efg###74.08ef###72.00d###0.83f###0.96g###0.90e

0.75 TDZ###0.10 GA3###100.00a###100.00a###100.00a###97.17bcd###77.58de###73.16d###0.97e###1.04f###1.14c

###MSO###83.33b###83.33ab###75.00abc###42.22j###45.55i###36.11h###0.51k###0.48l###0.53j

###0.10 BAP###100.00a###100.00a###100.00a###83.41g###66.17fg###62.91f###0.77g###0.91h###0.86f

1.00 TDZ###0.10 GA3###100.00a###100.00a###100.00a###84.92fg###70.75efg###67.83e###0.77g###1.03f###0.91e

###MSO###75.00b###66.67b###58.33c###39.61j###42.08i###33.38h###0.49l###0.47l###0.48k

RESULTS AND DISCUSSION

The explants of C. demersum for callus induction were cultured in MS nutrient medium with various doses of TDZ (0.05-1.00 mg/L) (Table 1) and a yellowish and light green color were formed on the callus at the end of eight weeks (Fig. 1 a, b, c). Browning at a low rate has been observed on some callus. The first callus formation was observed in the MS nutrient medium containing 0.50 mg/L TDZ for the shoot (on the 14th day) and 1stnodal explant (on the 12th day), while it was obtained in the MS nutrient medium with 0.75 mg/L TDZ for the 2nd nodal explant (on the 12th day). The results showed that callus formation varied according to the explant type and hormone concentration. Irvani et al. (2010) cultured root, hypocotyl and cotyledon explants of Dorema ammoniacum D. on MS medium containing different concentrations of 2, 4-D and NAA alone and in combination with BAP or KIN for callus induction and obtained the 100% effect from root explants on MS nutrient medium with 2 mg/L BAP + 1 mg/L NAA.

Further, they observed that callus development began on the 8th day in root segments and on the 20th day in hypoctyl explants. And also the first callus formation in cotyledon explants was observed at 4 weeks. Khalafalla et al (2010) reported that the earliest calli formation from Solanum tuberosum L. was obtained in MS medium containing 4.0 and 5.0 mg/L 2, 4-D on day 7. Callus regeneration frequencies were determined as 100% in all culture media for 1st nodal explant, as 83.33-100% for shoot tip and as 66.66-100% for 2nd nodal explant. Generally, the calluses are obtained at high frequencies. Similarly, callus formation was reported in other plants cultured on MS medium with TDZ such as Oriental hybrid 'Siberia' (Wu et al., 2017), Brassica oleracea L. var. botrytis (Srivastava et al., 2017), Atropa acuminata (Khan et al., 2017) and Dimorphorchis lowii (Jainol and Gansau, 2017).

Whereas, Dogan et al. (2015) cultured the shoot tip and nodal explants of C. demersum in liquid MS medium supplemented with TDZ at different doses, and reported that callus did not form in the culture medium. This difference in results may be due to the fact the used liquid culture medium. Because the liquid culture medium may have prevented callus formation. In line with these, Karatas et al. (2014) who reported the absence of callus formation of shoot tip and nodal explants of C. demersum cultured in the liquid MS medium. When compared to callus densities for all three explant types, the best hormone application was recorded as MS medium containing 0.50 and 0.75 mg/L TDZ (Fig. 1 a, b, c). The explant that gave the best results in callus experiments was determined as 1st nodal explant compared to other explants.

Jainol and Gansau (2017) cultured leaf tip explants D. lowii on MS medium containing 0.22-3.0 mg/L TDZ and reported that a maximum percentage of callus formation (28.0+-17%) and maximum callus density were determined in MS medium supplemented with 3.0 mg/L TDZ. In vitro shoot regeneration from plant cells and explants is influenced by plant growth regulators that are added exogenously and by hormone present in the plant hormones (Trigiano and Gray, 2000). Different plant tissues can contain endogenous hormones at different levels and therefore the explant varieties have a critical effect on a successful shoot regeneration (Yucesan et al. 2007).

In the present study, the calluses obtained from shoot tip, 1st and 2nd nodal explants in the MS medium fortify with TDZ (0.05-1.00 mg/L) were selected and transferred to the MS nutrient medium without plant growth regulator (MSO) and to MS medium containing 0.10 mg/L BAP and 0.10 mg/L GA3 (Table 2), which is to achieve multiple shoot regeneration by sub-culturing callus. At the end of the first week, shoot tips began to spread on the callus masses, and multiple shoots were clearly observed after three weeks. At the end of eight weeks, shoots were obtained which prolonged and developed on calluses (Fig. 1 d, e, f). Data regarding shoot regeneration frequency of (%), mean number of shoots per callus and mean shoot length were recorded for three explants after eight weeks of culturing (Table 2).

Shoot regeneration frequency was found statistically significant (pBAP>MSO. The longest shoots were determined in the MS medium including 0.10 mg/L GA3 for the 1st and 2nd nodal explants and in the MS medium fortify with 0.10 mg/L BAP for the shoot tip explants. Additionally, explant sources and culture conditions were found to be effective on the photosynthetic pigment contents of the regenerated shoots. This work is a new protocol for efficient and rapid production of C. demersum via callus induction. It may contribute significantly to the literature in this regard.

Author contributions: The design of the work, the experimental applications and the writing of the article were done by Muhammet Dogan.

Conflict of interest disclosure: The author declare no conflict of interest.

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