Influence of Silver Nitrate on Enhancing in vitro Rooting of Gardenia jasminoides Ellis

Journal of Environmental Science and Technology

Year: 2018 | Volume: 11 | Issue: 5 | Page No.: 238-245
DOI: 10.3923/jest.2018.238.245

**Influence of Silver Nitrate on Enhancing in vitro Rooting of Gardenia jasminoides Ellis**

Amal Abd El-Latif El-Ashry , Ahmed Mohamed Magdy Gabr, Nancy Danial Girgis and Mohamed Kamal El-Bahr

Abstract: Background and Objectives: Silver ion (Ag+), which added in vitro in the form of silver nitrate (AgNO₃) or as silver thiosulfate (STS) complex can act as an effective inhibitor of ethylene action. In addition to inhibiting ethylene action, Ag+promotes indole acetic acid (IAA). The current study was conducted to evaluate the impact of AgNo₃ on improvement root formation during micropropagation of Gardenia jasminoides Ellis. Materials and Methods: For multiplication, stem segments were cultured on MS medium supplemented with 0, 3, 5 or 7 mg L^–¹ benzyl adenine (BA). Elongated shootlets were transferred to different rooting treatments; MS supplemented with naphthalene acetic acid (NAA) or indole butyric acid (IBA) in combination with different concentrations of AgNo₃ 1, 2 and 3 mg L^–¹. The antioxidant activity of extracts from each rooting treatment was determined through 2,2-diphenyl-1-picrylhydrazyl(DPPH) radical scavenging activity. Results: Using high concentration of BA (7 mg L^–¹) led to obtain the highest multiplication rate. Results observed that addition of AgNo₃to medium contained NAA alone doubled the rooting formation percentage. Meanwhile, addition of AgNo₃to MS medium contained IBA alone or both NAA and IBA increased the root formation percentage from 83.33-100%. DPPH radical scavenging activity of different rooting treatments revealed that 2 NAA+2 IBA+2 AgNo₃ recorded the highest DPPH percentage (79. 2%). Conclusion: It could be concluded that, the medium supplemented with2 NAA+2 IBA gave the highest number of roots/plantlet. While, the addition of 3 mg L^–¹ AgNo₃ to MS medium supplemented with 2 NAA+2 IBA gave the highest mean length of root/plantlet. Also, it could be concluded that using AgNo₃with 2 mg L^–¹ NAA+2 mg L^–¹ IBAA improved the antioxidant capacity of gardenia extracts.

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Amal Abd El-Latif El-Ashry, Ahmed Mohamed Magdy Gabr, Nancy Danial Girgis and Mohamed Kamal El-Bahr, 2018. Influence of Silver Nitrate on Enhancing in vitro Rooting of Gardenia jasminoides Ellis. Journal of Environmental Science and Technology, 11: 238-245.

Keywords: DPPH radical scavenging act, Gardenia jasminoides Ellis, rooting, silver nitrate and antioxidant capacity

INTRODUCTION

Gardenia jasminoides Ellis is an evergreen ornamental plant belongs to the genus Gardenia, family Rubiaceae, widely grown in the tropics, subtropics and warm temperate regions. It is used as a cut flower and a garden shrub as screens, hedges or borders. Gardenia has thick, glossy, dark-green leaves and white flowers with sweet fragrance. Gardenia is propagated by terminal shoot cuttings. However, it takes several years to obtain enough plants by cuttings. In vitro propagation of Gardenia plants was a promising tool to overcome this problem^1-3. Micropropagation became a reliable and routine approach for large-scale rapid plant multiplication. Rooting stage may involve not only rooting of shoots derived in vitro, but also conditioning of the in vitro plants to increase their potential for ex vitro acclimatization and survival during transplanting process⁴.

Incubation of plant tissue cultures resulted in ethylene accumulation in the culture vessels, which is unfavorable for culture growth⁵.

Silver ion (Ag+), which added in vitro in the form of silver nitrate (AgNO₃) or as silver thiosulfate (STS) complex can act as an effective inhibitor of ethylene action⁶. As well as, it was found that in addition to inhibiting ethylene action, Ag⁺ promotes⁷ IAA. Thus, silver supplementation to the culture medium has been used to improve plant regeneration in vitro^8,9. In this regard, silver nitrate is widely used in plant tissue culture. Many reports have shown the positive effect of AgNO₃ on shoot multiplication, root formation and flowering in vitro^10-16.

DPPH radical scavenging assay is a good method depend on the antioxidant efficiency of plant crude extracts, based on the conversion of the free radical 2,2-diphenyl-1-picrylhydrazyl to a stable molecule diphenylpicryl hydrazine by reduction using various plant extracts as hydrogen donors, through change the color from purple to yellow in a short time¹⁷. The aim of the current study is to evaluate the impact of silver nitrate on improvement root formation during micro propagation of Gardenia jasminoides Ellis. and its effect on the antioxidant efficiency of plant crude extracts through DPPH radical scavenging assay.

MATERIALS AND METHODS

This study was carried out in Plant Biotechnology Department, National Research Centre, Giza, Egypt, during the period from January-December, 2017.

Plant material: In vitro growing Gardenia plant lets were subjected in this study as a source of plant material.

Multiplication stage: Stem segments (about 1 cm length) were used as explants. For multiplication explants were cultured on MS medium supplemented with 0 (control), 3, 5 or 7 mg L^–¹ BA, for two subculture, each of them was 4 weeks. Mean number of lateral buds/explant and mean numbers of lateral shoots were recorded after the two subcultures.

Elongation stage: Cultures from the previous stage were transferred to MS medium supplemented with 2 mg L^–¹ BA or 0.4 mg L^–¹ IAA as well as interaction between 1 mg L^–¹ BA+0.2 mg L^–¹ IAA as an elongation medium for two subcultures each of them was 4 weeks. Average number of lateral shoots, shoot height and number of leaves per shoot were recorded after the two subcultures.

Rooting stage: The elongated shoots (about 3 cm) were transferred to different rooting medium supplemented with different concentrations of NAA and IBA as well as silver nitrate as follow:

•	Treatment
•	3 mg L^–¹ NAA
•	3 mg L^–¹ NAA+1 mg L^–¹ AgNo₃
•	3 mg L^–¹ NAA+2 mg L^–¹ AgNo₃
•	3 mg L^–¹ NAA+3 mg L^–¹ AgNo₃
•	3 mg L^–¹ IBA
•	3 mg L^–¹ IBA+1 mg L^–¹ AgNo₃
•	3 mg L^–¹ IBA+2 mg L^–¹ AgNo₃
•	3 mg L^–¹ IBA+3 mg L^–¹ AgNo₃
•	2 mg L^–¹ NAA+2 mg L^–¹ IBA
•	2 mg L^–¹ NAA+2 mg L^–¹ IBA+1 mg L^–¹ AgNo₃
•	2 mg L^–¹ NAA+2 mg L^–¹ IBA+2 mg L^–¹ AgNo₃
•	2 mg L^–¹ NAA+2 mg L^–¹ IBA+3 mg L^–¹ AgNo₃

At the end of the second subculture, rooting formation percentage, number of roots/plantlet, length of roots (cm)/plantlet and plantlet height (cm) were recorded.

Biochemical analysis
Sample extraction: According to Gabr et al.¹⁸, 50 mg dry weight (from all rooting treatments which were air dried at room temperature for 3 days) was grounded and extracted with methanol 80% (1 mL) for 48 h on a shaker (120 rpm) at room temperature. The extraction procedure was carried out in an ultrasonic water bath for 20 min. Samples were centrifuged for 5 min at 6000 rpm. Then the supernatants were collected. The extracts were stored at -20°C until further use.

DPPH radical scavenging activity: The DPPH assay according to Gabr et al.¹⁸ was used with some modifications. Methanolic extract of different treatments of rooting (0.1 mL of each) were vortexed for 30 sec with1.9 mL of DPPH solution and left to react for 30 min, after which the absorbance at 515 nm was recorded. A control with no added extract was also analyzed. Scavenging activity was calculated as follow:

where, A is the absorbance at 515 nm.

Statistical analysis: All analysis was in triplicate and data reported as Mean±Standard Deviation (SD). Data were subjected to analysis of variance (One-way ANOVA) (p<0.05). Results were processed by Excel (Microsoft office 2010) and SPSS version 17.0 (SPSS inc, Chicago, IL, USA).

RESULTS AND DISCUSSION

Multiplication stage: The effect of different concentrations of BA on number of lateral buds and lateral shoots was recorded in Table 1.

Data generally showed a remarkable effect of the different BA concentrations on mean number of lateral bud/explants and number of lateral shoots compared with control treatment (without BA).

Using high concentration of BA (7 mg L^–¹) led to obtain the highest mean number of shoots per explant (2.33). On the other hand, the lowest mean number of shoot per explant (0.66 shoot/explant) was observed with 3 mg L^–¹ BA. The high concentrations of BA (5 or 7 mg L^–¹) produce swelling lateral buds after 4 weeks of culturing which, developed to very short shoots as a cluster shape after 8 weeks of culturing. These results are in agreement with those reported by Chuenboonngarm et al.¹⁹ on Gardenia jasminoides who found that using B5 medium supplemented with BA gave high shoots number compared with those cultured on a medium free from BA. On the same respect, Duhoky and Rasheed²⁰ reported that using a medium supplemented with 2 mg L^–¹ BA gave the highest shoot number/each explant (2.20 shoots/explant), which may be related to the efficiency of BA in cell division. Furthermore, in Gardenia gummifera, multiple shoots were obtained from nodal explants on MS medium fortified²¹ with 2 mg L^–¹ BA+0.5 mg L^–¹ IBA. While, MS medium supplemented with 1 mg L^–¹ BA+0.5 mg L^–¹ IBA gave the highest number of shoots from leaf and internode explants of Gardenia gummifera²². In this connection, Lakshmi and Jaganmohanreddy¹⁴ reported that, using inter-nodal segments explants of Gardenia resinifera Roth, the medium supplemented with 2 mg L^–¹ BA+0.25 mg L^–¹ NAA gave two shoots with an average shoot length 3.75 cm. As well as, using a medium fortified with 2 mg L^–¹ BA+0.1 mg L^–¹ IAA gave two shoots with average shoot length 3.53 cm.

Elongation stage: The new shoots obtained in the previous stage were transferred to culture medium supplemented with BA (2 mg L^–¹) or IAA (0.4 mg L^–¹) or 1 mg L^–¹ BA+0.2 mg L^–¹ IAA aiming to prolongate the shoots. Data presented in Table 2 showed that highest mean number of lateral shoots (4 shoots) was obtained with 2 mg L^–¹ BA whereas, the lowest mean number of lateral shoots (2.5 shoot) was recorded with 0.4 mg L^–¹ IAA. On the other hand, the treatment of 1 mg L^–¹ BA+0.2 mg L^–¹ IAA gave the highest mean number of leaves (8.5 leaves/shoot). However, the highest of the main shoot height (3.33 cm) was recorded with 2 mg L^–¹ BA. These results were partially in agreement with those found with Duhoky and Rasheed²³, who reported that using MS medium supplemented with 3 mg L^–¹/BA+0.9 mg L^–¹/1 IAA with single nodes of gardenia gave the highest number of shoots and leaves.

Table 1:	Effect of different concentrations of BA on number of lateral buds and number of lateral shoots after 8 weeks of culturing

Table 2:	Effect of BA, IAA and their interaction added to MS-medium on number of lateral shoots, number of leaves per shoot and shoot height, after 8 weeks of culturing

Mean±SD

Table 3:	Effect of NAA, IBA and AgNo₃ on root formation, mean number of roots/plant and mean length of root/plantlet

Mean±SD

Also, Salim and Hamza²⁴ reported that using a medium supplemented with 3.0 mg L^–¹ TDZ+0.3 mg L^–¹ IAA with Gardenia jasmonides gave the best results for shoot multiplication as shoot number, shoot length and number of leaves/shoot.

It is well known that auxins (IAA) is involved in root initiation. Apical dominance may occur in which the growth of lateral buds is inhibited by the growth of apical buds and that declared that in case of adding IAA alone to the elongation medium gave the lowest number of lateral shoots. Whereas, cytokinins (BA) is produced in the regions where cell division occurs; i.e., in the roots and shoots. They help in overcoming apical dominance, leaves production and growth of lateral shoots. This may explain why the addition of IAA in combination with BA gave the highest mean number of leaves and also, the highest of the main shoot height was recorded with 2 mg L^–¹ BA.

Rooting stage: One of the major obstacles of the in vitro propagation is the rooting and conditioning of the plantlets. The elongated shoots were transferred to MS-medium contained two concentrations (2 and 3 mg L^–¹) of NAA or IBA added individually or in combination and supplemented with AgNo₃as reported in Table 3. Root formation percentage, number of roots/plantlet, root length and plantlet height was recorded after 8 weeks of culturing. The results obtained indicated that the highest root formation percentage (100%) were recorded with IBA at 3 mg L^–¹ with silver nitrate at any concentration (1, 2, 3 mg L^–¹ ) and with 2 mg L^–¹ NAA+2 mg L^–¹ IBA in the presence of silver nitrate at any concentration.

Also, it can be noticed that addition of AgNo₃ at one of the three concentrations (1, 2 or 3 mg L^–¹) to 3 mg L^–¹ NAA doubled the rooting formation percentage (from 33-66%). Meanwhile, addition of AgNo₃to MS medium contained IBA alone or both NAA and IBA increased the root formation percentage from 83.33-100%. Also, it was observed that the highest mean number of roots/plantlet was 20.66 roots/plantlet on medium supplemented with 2 mg L^–¹ NAA+2 mg L^–¹ IBA (Fig. 1i) followed by 16.3 roots/plantlet with medium supplemented with 3 mg L^–1 IBA+1 mg L^–¹ AgNo₃(Fig. 1f).

On the other hand, it was found that addition of 3 mg L^–¹ AgNo₃to both NAA and IBA at 2 mg L^–¹ gave the highest mean of length of roots/plantlet (2.66 cm) (Fig. 1l).

Regarding plantlet height, results presented in Table 3 revealed that, the highest plantlet height (6.66 cm) was obtained on medium supplemented with 3 IBA+3 AgNo₃(Fig. 1h).

It was noticed that thickness of roots was affected by the type of the auxins as generally roots were thick with NAA alone or with AgNo₃whereas it were thin with IBA alone or with AgNo₃.

Incubation of plant tissue cultures in vitro resulted in ethylene accumulation in the culture vessels, which is unfavorable for culture growth⁵. Silver ion (Ag+), which added in vitro in the form of silver nitrate (AgNO₃) or as silver thiosulfate (STS) complex can act as an effective inhibitor of ethylene action^6,25. In this regard, Strader et al.⁷ reported that in addition to inhibiting ethylene action, Ag⁺ promotes IAA. Thus, silver supplementation to the medium has been used to improve plant regeneration in vitro^8,9.

The results assured that silver nitrate could be the solution of slow multiplication and difficult in vitro root formation problems. These results were in harmony with Harsh et al.²⁶, who reported that in case of adding 0.5 mg L^–¹ IAA to the rooting medium of Decalepis hamiltonii poor rooting was observed. The resultant roots were stunted. While, addition of 40 μM AgNO₃ improved rooting as root initiation and root elongation. Similar results were conducted in Vanilla planifolia as AgNo₃ not only induced shoot multiplication but also influenced rooting¹¹.

Fig. 1(a-l):

Gardenia plantlet after 8 weeks on rooting medium supplemented with, (a) 3 mg L^–¹ NAA, (b) 3 mg L^–¹ NAA+ 1 mg L^–¹ AgNo₃, (c) 3 mg L^–¹ NAA+2 mg L^–¹ AgNo₃, (d) 3 mg L^–¹ NAA+3 mg L^–¹ AgNo₃, (e) 3 mg L^–¹ IBA, (f) 3 mg L^–¹ IBA+1 mg L^–¹ AgNo₃, (g) 3 mg L^–¹ IBA+2 mg L^–¹ AgNo₃, (h) 3 mg L^–¹ IBA+3 mg L^–¹ AgNo₃, (i) 2 mg L^–¹ NAA+2 mg L^–¹ IBA, (j) 2 mg L^–¹ NAA+2 mg L^–¹ IBA+1 mg L^–¹ AgNo₃, (k) mg L^–¹ NAA+2 mg L^–¹ IBA+2 mg L^–¹ AgNo₃and (l) 2 mg L^–¹ NAA+2 mg L^–¹ IBA+3 mg L^–¹ AgNo₃

Also, Chithra et al.²⁷ reported that silver nitrate induce in vitro rooting on Rotula aquatica L. They added that the addition of 11.7 μM silver nitrate to half strength MS liquid medium containing 2.69 μM NAA increased the number of roots to reach 16.8/explant comparing with 7.3 roots/shoot without the addition of silver nitrate. Also, Petrova et al.¹³ reported that using half strength MS supplemented with 1 mg L^–¹ IBA+1 mg L^–¹ AgNo₃with Gentiana lutea gave 90% rooting with 4.5 roots/plant with 1.5 cm mean length. Mahmoud and Kosar²⁸ reported that the highest elongation and number of roots per strawberry explant were obtained on MS medium supplemented with 0.5 mg L^–¹ BAP+0.2 mg L^–¹ KIN+4 mg L^–¹ Ag(NO ).

Table 4:	Effect of NAA, IBA and AgNo₃ on free radical scavenging capacity of DPPH (%) for gardenia extracts

Mean of DPPH percentage±SD

Tamimi¹⁵ on his study on banana declared that the rooting media fortified with10 mg L^–¹ AgNO₃ gave the highest number of roots per shoot (21.7) and the longest roots (12.68 cm). Geetha et al.¹⁶ in their study on Solanum nigrum reported that, 95% of rooting, highest number of roots (24.6±0.26) were obtained with MS medium fortified with 2.0 mg L^–¹ IBA and 0.4 mg L^–¹ AgNO₃ and highest mean root length (6.5±0.36 cm) was observed on the same medium. As for Gardenia, Duhoky and Rasheed²⁰ reported that the medium supplemented with 8 mg L^–¹ NAA gave the highest percentage of rooting (90%) and the medium fortified by 12 mg L^–¹ IAA gave (100%) rooting of Gardenia jasmonides. However, Gajakosh et al.²⁹mentioned that using 5.0 mg L^–¹ IBA and 5.0 mg L^–¹ IAA was suitable for induction of roots in Gardenia gummifera.

On the other hand, Lakshmi and Jaganmohanreddy¹⁴ stated that regenerated shoots of Gardenia resinifera roth were rooted on 1/2 MS supplemented with IAA (0.5 mg L^–¹).

It could be concluded that, the medium supplemented with2 NAA+2 IBA gave the highest number of roots/plantlet (Fig. 1i). While, the addition of 3 mg L^–1 AgNo₃to MS medium supplemented with 2 NAA+2 IBA gave the highest mean length of root/plantlet (Fig. 1l). While, the medium supplemented with 3 mg L^–1 IBA+3 mg L^–¹ AgNo₃ gave the highest mean height of plantlet (Fig. 1h).

DPPH radical scavenging assay for gardenia extracts: The DPPH percentage was recorded Table 4. Using 2 NAA+2 IBA+2 AgNo₃ recorded the highest DPPH percentage (79. 2%) following by using 3 NAA+3 AgNo₃ (77.2%). While using 3 IBA+2AgNo₃ recorded the lowest DPPH percentage (49.6%). When using 3 mg L^–¹ NAA or 2 mg L^–¹ NAA+2 mg L^–¹ IBA the DPPH percentage increased with adding different concentrations of AgNo₃. While, using 3 mg L^–¹ IBA with different concentrations of AgNo₃ decreased the DPPH percentage.

From these results it could be concluded that using AgNo₃with 3 mg L^–¹ NAA or 2 mg L^–¹ NAA+2 mg L^–¹ IBAA increased the efficiency of the extracts for donating hydrogen atom to improve their antioxidant capacity. While, using AgNo₃with 3 mg L^–¹ IBA decreased this efficiency.

The current results were in line with those found by Sahandi et al.³⁰, where the content of polyphenols in vegetal tissues of borage showed an increase proportional to the concentration of silver nitrate in the range of 100-300 mg L^–¹. Juarez-Maldonado et al.³¹ stated that silver nitrate had a positive effect on total antioxidant capacity of onion bulbs when it was applied as a nutritive solution. But it differs from those found by De la Fuentei et al.³², who stated that watermelon plants obtained 30 mg L^–¹ silver nitrate showed the greatest total amount of antioxidants in fruits comparing with those obtained higher concentration of silver nitrate.

CONCLUSION

It could be concluded that, the medium supplemented with2 NAA+2 IBA gave the highest number of roots/plantlet. While, the addition of 3 mg L^–¹ AgNo₃ to MS medium supplemented with 2 NAA+2 IBA gave the highest mean length of root/plantlet. Also, it could be concluded that using AgNo₃with 2 mg L^–¹ NAA+2 mg L^–¹ IBAA increased the efficiency of the extracts for donating hydrogen atom to improve their antioxidant capacity.

SIGNIFICANCE STATEMENT

Actually, the main aim of this paper is to study the effect of silver nitrate, as an antioxidant compound as well as promoting rooting of in vitro culture, on gardenia and its effect on the internal antioxidant activity of Gardenia. The results of this study indicate that silver nitrate could be the solution difficult in vitro root formation problems and enhancing the antioxidant activity of Gardenia.

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