Phytochemical Analysis and Antimicrobial Activities of Different Callus Extracts of Pelargonium Sidoides DC . Against Food Borne Pathogenic Bacteria

© 2018 Mona M. Ibrahim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Mona M. Ibrahim, Plant Biotechnology Department, Genetic Engineering & Biotechnology Division, National Research Centre, Dokki, Giza, Egypt. E-mail: monaibrahem27 @ yahoo.com Phytochemical Analysis and Antimicrobial Activities of Different Callus Extracts of Pelargonium Sidoides DC. Against Food Borne Pathogenic Bacteria


INTRODUCTION
Pelargonium species family, Geraniaceae are predestined for their colorful garish flowers and odd leaf shapes (Wojtania et al., 2004).Pelargonium species have contributed decidedly to the annual industry in North America and Europe, which is account an estimated $700 million annually (Mithila et al., 2001).The essential oils of Pelargonium species are in high request in the pharmaceutical, aromatherapy, perfumery and cosmetic industries because of different phytochemical property.The aerial parts of these Pelargonium species are active as wound healing agents (Kolodziej, 2000).
Seeds and cuttings are the main propagation methods for most Pelargonium species (Wojtania et al., 2004).Plant tissue culture techniques can be used to get a large number of plants species for preservation and upgrade target.Studies on plant metabolites obtaining by using callus cultures have been accomplished on the large scale since the end of the 1950's.Such culturing technique is used for secondary metabolites production like hormones, enzymes, proteins, antigens, food additives and natural pesticides, these active compounds are used for pharmaceuticals and cosmetics (Terrier et al., 2007).Duchow et al. (2014) found that the blend of dichlorophenoxy acetic acid (2,4-D) and kinetin (Kin) together gives rise to excellent growth for calli but with browning formation, even though the calli which induced on the medium containing both 6-benzylaminopurine (BAP) and naphthalene acetic acid (NAA) obtained rapid growth with a few browning formation.The combination of NAA and Kin was used for induction calli from only petioles resulted in calli with crumbly consistency (Moyo et al., 2012).Prosperous callus induction from leaves, stems, and seeds of P. sidoides was first achieved on the medium containing 2,4-D and Indole-3-butyric acid (IBA) (Lewu et al., 2007).Therefore, the aim of this work was to study to produce some secondary metabolites (phenolics, flavonoids, and proanthocyanidins) in callus cultures of P. sidoides derived from different explants.The effect of solvent type on the contents of these metabolites was highlighted.The antimicrobial activity of the different extracts obtained from different explants calli were tested against food borne poisoning bacteria and fungi.

Plant material
Seeds of Pelargonium sidoides DC. were kindly supplied by SEKEM company, Egypt.

Sterilization and incubation conditions
Seeds were surface sterilized by thoroughly washed under tap water then rinsed with 70% ethanol for 30 seconds followed by twice washing with sterile water.They were then immersed in 50% sodium hypochlorite solution for 15 min after which they were rinsed with sterile water four times and cultured on MS-medium (Murashige and Skoog, 1962) free hormones containing 3% sucrose and 0.7% agar.Seeds were transferred in a growth chamber at 26 ± 1ºC under light conditions of 16 h photoperiod.After one-month seedlings were used as the starting plant material.

Callus culture initiation
Leaf, root, and shoot tip segments were separated from the produced seedlings and cultured on solidified MS medium with different supplementation: a) 1.0 mg/l Dichlorophenoxy acetic acid (2,4-D) + 0.2 mg/l Kinetin (Kin), b) 1.0 mg/l naphthalene acetic acid (NAA) + 0.2 mg/l Kin, c) 1.0 mg/l NAA + 5.0 mg/l BA.Cultures were kept in the culture room under a temperature of 26 ± 1ºC and light conditions of 16 h photoperiod.Initiated callus was observed after 4 weeks of cultivation then the callus initiation frequency (%) was calculated based on the following equation: Callus initiation frequency % = ((Number of initiated calli)/(Number of inculated explants)) × 100.

Callus production and growth index
The growth characterization of callus culture and their optimal time of sub-culturing were studied.Therefore, 0.3 g of P. sidoides callus which formed in the previous experiment was transferred to the best combination medium (1.0 mg/l NAA + 0.2 mg/l Kin).Average fresh weight (g) was recorded weekly for six weeks, then growth index was calculated according to the following equation of Dung et al. (1981): Growth index = (Final callus fresh weight − Initial callus fresh weight)/(Initial callus fresh weight).

Extracts preparation
Dried powdered samples of in vitro callus cultures derived from leaf, root, and shoot tip explants were extracted using three different solvents.These are water, methanol (85%) and acetone (80%) for 24 hrs at room temperature.The extracts were collected, filtered and evaporated to dryness.Each residue was dissolved in same extract solvent and stored at 4°C until further use.

Determination of total phenolic compounds
The concentration of phenolic compounds was determined by a spectrophotometric method using the Folin-Ciocalteu reagent according to Singleton et al. (1999).A calibration curve of gallic acid (20,40,40,60,80, and 100 μg/ml) was prepared and the absorbance for tests and standard solutions were determined against the reagent blank at 550 nm with an Ultraviolet (UV)/Visible spectrophotometer.The total phenolic content was expressed as mg of gallic acid equivalent (GAE)/g of dry weight (DW).

Determination of total flavonoids
The total flavonoids content was measured using a modified colorimetric method of Vabkova and Neugebauerova (2012).The standard curve was prepared using a different concentration of quercetin.The flavonoids content was expressed as mg quercetin equivalents per g of dry weight (DW).

Determination of Total proanthocyanidins
Total proanthocyanidins were determined by acid/nbutanol method according to the method described by Porter et al. (1986).The absorbance was recorded at 550 nm using an Ultraviolet (UV)/Visible spectrophotometer.The results were expressed as units at 550 nm per mg of extract (A550 nm/mg).

Antimicrobial activity
The antimicrobial activity of the given P. sidiodes extracts was tested against microbial reference cultures, including bacterial strains; two Gram positive (Clostridium perfringenes and Staphylococcus aureus ATCC 6538) and four Gram negative (Shigella flexnerii ATCC 9199, Escherichia coli ATCC 25922, Escherichia coli O157 ATCC 700728, Salmonella typhimurium ATCC 14028) as well as one fungal strain (Candida albicans ATCC 2091).The antimicrobial activates were evaluated using agar well diffusion test (Sgouras et al., 2004).Hundred μl of cell culture suspension matching with 0.5 McFarland of target strains were spread onto the plates.For the investigation of the antimicrobial activity, 100 μl of extracts were added into wells of agar plates directly, oxytetracycline disc (Oxoid) (Oxytetracycline 30 µg/ disc) as control positive for bacterial stains and Fluconazole (10 μg/disc) as control positive for tested mycotic strain.Plates were left for 1 h at 25°C to allow a period of pre-incubation diffusion in order to minimize the effects of variation in time between the applications of different solutions.The plates were re-incubated at 37 and 30°C for bacterial and mycotic strains respectively for 24 h.After incubation, plates were observed for antimicrobial activities by determining the diameters of the zones of inhibition for each strain.For an accurate analysis, tests were run in triplicate for each strain to avoid any error and the means ± SD were tabulated in the given tables.

Statistical analysis
The experiments were carried out in triplicate and the results were expressed as means ± standard errors.Statistical analysis of variance was performed by ANOVA and significant differences (LSD) at P value ≤ 0.05 between the means were determined by Duncan's Multiple Range Test.

Sterilization and incubation conditions
Disinfection of seeds and subsequently germination is crucial to obtain success explants under the establishment stage of callus cultures.Sterilization with 50% sodium hypochlorite for 15 min resulted in highest percentage of contaminated free seedling.Unfortunately, low seed germination percentage (about 30% of seeds) succeeded to germinate under these sterilization conditions (Figure 1).Seedlings were used as a source of explants in the current study.

Callus culture formation
This experiment aims to find any combination of growth regulator which give the highest productivity of callus cultures through two steps protocol including callus initiation and callus production.

Callus initiation
Leaf, root, and shoot tip explants derived from 30 days old seedling were transferred to MS solid media containing different growth regulators.After one month the callus initiation frequency (%) on the three different combinations was calculated and shown in Figure 2. Data reveals that callus initiation frequency (%) of root and shoot tip was the same in all different combinations.These values were 100% at the a and b combinations and the lowest in the c combination (70%).While leaf explant showed minimum callus initiation frequency (%) on all different combinations (72%, 85%, and 33%) on a, b, and c combinations respectively.To investigate the effect of these different combinations for getting healthy calli, the brown deposits formation was followed in all different combinations.We observed that, the two combinations (1.0 mg/l 2,4-D and 0.2 mg/l Kin and 1.0 mg/l NAA + 5.0 mg/l BA) obtained calli with brown color abounding, while the NAA and Kin combination was the best to get friable calli with little brown deposits as shown in Figure 3.
The present combination (NAA and Kin) was first used by Moyo et al. (2012) to initiate calli from petioles explant of P. sidoides and obtained calli with crumbly consistence.Our results agree with Duchow et al. (2014) in that the 2,4-D and Kin combination obtained calli with many brown deposits and does not agree with them in that the combination of NAA and BAP resulted in rapid growth of calli with little brown color abounding.In this respect, successfully achieved of callus culture induction from leaves, stems, and seeds of P. sidoides using auxin alone like 2,4-D and indole butyric acid (IBA) was reported by Lewu et al. (2007).Other pelargonium species induced calli with optimal growth by using balancing amount of auxins and cytokinins (Brown and Charlwood, 1986).
Leaves formation were observed with callus induction by using single shoot tip explant (Figure 4A) on different combinations.These shoots were taken after calli isolation and transferred to the same media to examine shoot tip development.Obtained results after one month showed that no development occurred in these shoots (Figure 4B).In the present study, 1.0 mg/l of BA was also used to observe shoot tip development.After one month the new leaves were formed on this medium, but no root formation was observed (Figure 5).These shoots were sub-cultured on the same medium (1.0 mg/l BA) for three months further to obtain new shoots.In this further experiment, we observed that shoots failed in multiplication and new shoots formation resulted after three months.

Callus production and growth index
As previously mentioned the combination of NAA and Kin was the best for obtaining healthy callus cultures, so it was applied for getting the highest productivity of callus cultures.Initiated calli derived from leaf, root, and shoot tip explants were examined for callus production depending on their fresh weights and growth index as shown in Table 1.
Callus fresh weights and growth index which obtained for six weeks showed that the maximum increasing value of fresh weight was observed slightly at the fifth week in all explants then rapidly decreased at the sixth week.
Concerning the influence of explant type on the productivity of calli, the maximum values of fresh weight and growth index were recorded with root explant (4.01 g and 12.37) followed by leaf explant (3.98 g and 12.27) respectively.On the other hand, the calli from shoot tip explant recorded the minimum values (2.72 g and 8.07) in the fifth week respectively.Obtained calli was sub-cultured on the best combination (NAA and Kin), after three months rapid mass production of calli was obtained (Figure 5) then it used in phytochemical analysis and antimicrobial activity tests.

Extraction method and determination analysis
Extraction process of the bioactive components from medicinal plants is a substantial step for retrieval of these components and obtained it in a pure form.Using different solvents for extraction and determination of phenolic and flavonoids compounds were studied and suggested in various researches (Tatiya et al., 2011;Addai et al., 2013).In the current experiment, we discuss the effect of two variables (explant type and extraction solvents) on the yield percentage and total bioactive components content (phenolics, flavonoids, and proanthocyanidins).

Extraction yield percent evaluation
Estimation of percentage yield extracts (w/w) of P. sidoides calli obtained with different solvents is displayed in Table 2. Results show that the extraction calli of shoot tip explant with aqueous acetone solvent gave maximum extraction yield percent (77.8%) followed by water solvent extraction of the same calli explant (44.4%).Generally, calli from shoot tip explant extracted by various solvents (aqueous methanol, aqueous acetone, and water) were the highest extraction yield percent from calli of leaf and root explants extracted with the same solvent.Contrary the extraction with aqueous methanol solvent gave the minimum extraction yield percent of different calli explants compared to their counterpart in other extracts (water and aqueous acetone), where minimum extraction yield percent was recorded with calli from root explant extracted by aqueous methanol solvent (12.8%).Our results are in contrast with Chatha et al. (2006) who achieved maximum extraction yield by using aqueous methanol from rice bran.Similarly, Vaghasiya and Chanda (2007) found that the extraction yield of fourteen Indian medicinal plants was more in methanol than acetone.These differences may be attributed to the diversity of the existing components in different plant materials.In the present work, the differences of extraction yield possibly could be attributed to the variations of solvents or quantity of components in different calli explants.

Determination of total phenolic compounds
Determination of total phenolic compounds under the influence of explant type and extraction solvent have been clear in Figure 6.Data indicate that callus from root explant extracted with aqueous acetone exhibited the manifest increase in total phenolic compounds value (12.34 mg/g DW) compared with the others, followed by callus from shoot tip extracted with aqueous acetone also (4.78 mg/g DW).This observation quite agrees with Tatiya et al. (2011) who mentioned that the maximum phenolic content in Bridelia retusa bark extract (14% W/W) was detected using aqueous acetone compared with other extracts (water, aqueous ethanol, and aqueous methanol).In contrast, Addai et al. (2013) recorded high total phenolic content (7.05 mg/g DW) from two papaya cultivars in aqueous methanol extract compared with water, aqueous ethanol, and aqueous acetone.This difference probably due to the variation of existent phenolic compounds in various plant materials, as phenolic compounds varied from simple (phenolic acids, anthocyanins) to highly polymerized substances (e.g., tannins), also phenolic compounds may be linked with other substances like carbohydrates and proteins.So, there is no common solvent appropriate for phenolic extraction because it depends on the type of plant material.
In the present study, using of aqueous methanol for phenolic extraction from different callus explants showed minimum phenolic content values, while the minimum value of phenolic was observed with calli from root explant extracted with aqueous methanol (0.41 mg/g DW).This finding is contradictory with several studies utilized the aqueous methanol for phenolic compounds extraction (Bonoli et al., 2004;Chatha et al., 2006;Sultana et al., 2009;Shabbir et al., 2011).Several studies revealed that phenolic compounds content differed with solvents polarities.For example, pure methanol was used for the extraction of tea polyphenols (Yao et al., 2006) and 50% acetone for extraction of wheat total phenolics (Zhou and Yu, 2004) which were found to be more effective than water.Statistical analysis shows that there are significant differences in total phenolic contents between different extracts whether it is due to the explant type which consists of calli or type of the solvent.This means that these two aforementioned variations have a clear impact on phenolic compounds accumulation and it must be taken into account when phenolic compounds were extracted.

Determination of total flavonoids
Determination of total flavonoids in calli extracts from different explants and diverse solvents was determined and showed in Figure 7. Data elucidate that, total flavonoids concentrations ranged from 1.69 to 20.82 mg/g DW and going in the same trend of total phenolic compounds determination.The maximum total flavonoids concentration was observed with calli from root explant extracted with aqueous acetone (20.82 mg/g DW), followed by calli from shoot tip explant extracted with aqueous acetone (10.72 mg/g DW).This observation agrees with Stankovic et al. (2012) who mentioned that the acetone extract of Teucrium polium L. leaves showed higher amounts of flavonoids content than the other examined extracts (water, methanol, ethyl acetate and petroleum ether).Also, Adaramola and Onigbinde (2016) found that the highest flavonoids of clove bud were obtained for 80% acetone followed by the 80% methanol and the least were obtained for the water extract.In the opposite manner, Mohammadi et al. (2016) detected highest total flavonoids content of Anabasis setifera and Salsola tomentosa plants by using methanol as the solvent for extraction compared with acetone solvent.Also, Butsat and Siriamornpun (2016) found that the 80% methanol extract of Amomum Chinese leaves gave the greatest yield of total flavonoids compared with other extracts (water and aqueous acetone).As well as the differences of phenolic compounds varied depending on type of plant materials, the same explanation is stood for flavonoids.
Total flavonoids contents of calli from different explants extracted with water were higher than aqueous methanol extracts for calli from all explants.Minimum flavonoids content (1.69 mg/g DW) was observed with calli from leaf explant extract by aqueous acetone.
Statistical analysis shows that there are significant differences in total flavonoids contents between calli from different explants in aqueous acetone extract, in contrast with aqueous methanol extracts there are no significant differences between different explants, but in water extract, there are no differences between root and shoot tip explants.These results demonstrated that the solvent type is more important as an effective factor for total flavonoids content.

Determination of total proanthocyanidins
Proanthocyanidins are condensed tannin compounds found in some food or medicinal plants, it is a powerful antioxidant agent, so it is a health-promoting factor, some literature suggested that it has antimicrobial properties (Chung et al., 1993;Katiyar and Mukhtar, 1997;Amarowicz et al., 2000b).A colorimetric method of n-butanol/HCl hydrolysis is performed to detect these components.Data in Figure 8 indicate that the A 550 nm/mg extract recorded the maximum value for only callus from root extracted with aqueous acetone (0.290).Other extracts noted low values (from 0.007 to 0.019) and there are no significant differences between them.
Some investigations subjected to proanthocyanidins evaluation in several plants such as legume seeds (Wilska-Jeszka and Stasiak, 1984), the high content of these compounds were detected in faba bean (Amarowicz et al., 2000a).Also, the ethanolic and acetonic extracts of hazelnuts (Alasalvar et al., 2006) and in almonds extract as reported by Amarowicz et al. (2005).The existence of principal plant metabolite predominated in root extract of P. sidoides.Accordingly, high molecular weight proanthocyanidins compound showed the same trend (Kolodziej and Kayser, 1998).Moreover, the most researches on P. sidiodes that were performed on root extract in order to the curative properties of different compounds occurred in such plants (Kolodziej et al., 2003;Mativandlela et al., 2007).

Antimicrobial analysis
Crude methanol and acetone extracts from calli of all explants were tested against two Gram-positive and four Gramnegative bacteria strains as well as they were tested against one fungal strain.The inhibition zone diameters (mm) were recorded to reflect the activity of their extracts.

Antimicrobial activity of methanol extracts
Data in Table 3 clarify that all methanol extracts showed an inhibitory effect against all tested strains.Leaf culture of P. sidiodes extracted with methanol recorded maximum inhibition zone diameters against E. coli ATCC 25922 (20 mm), this value is higher than root and shoot tip cultures extracted with methanol against the same strain.The inhibition zone diameters of the leaf extract against S. aureus ATCC 6538 and Shigella flexneri ATCC 9199 (19 mm) were the same of those in root culture extract and higher than shoot tip culture extract.While leaf extract showed minimum zone diameters against Salmonella typhimurium ATCC 14028 (11.3 mm) and Candida albicans ATCC 2091 (12 mm), these values were lower than inhibition zone diameters resulted from the root and shoot tip cultures.Inhibition zone diameters of root culture extracted with methanol ranged from 10 mm against Clostridium perfringens ATCC and 24 mm against E. coli O157 ATCC 700728 and showed moderate activity against other strains.
The methanol extract of shoot tip culture recorded maximum inhibition (26 mm) against Salmonella typhimurium ATCC 14028 and against E. coli O157 ATCC 700728 (24 mm), while it recorded minimum inhibition (8 mm) against Clostridium perfringens ATCC and showed moderate activity against other tested microbial strains.Inhibition zone diameters of Oxytetracycline or Fluconazole were the highest against all tested microbial strains except against Salmonella typhimurium ATCC 14028 (20 mm) whereas shoot tip extract recorded higher zone diameter (26 mm).

Antimicrobial activity of acetone extract
Data in Table 4 elucidate that all crude acetone extracts have activity against all microbial strains; this activity is different depending on the type of microbes either sensitive or resistant.It is noted that some microbes are sensitive to one extract and resistant in the others like Shigella flexneri ATCC 9199 as it was more sensitive to shoot tip extract as gave inhibition zone diameter 35 mm while it was less sensitive to leaf extract (inhibition zone diameter 16 mm) whereas root extract had moderate activity between them (inhibition zone diameter 22 mm), also Salmonella typhimurium ATCC 14028 going the same line while maximum activity recorded with shoot tip extract.On the contrary we found that the maximum activity of leaf extract (32 mm) recorded against E. coli ATCC 25922, this was asymptotic of the activity of root and shoot tip extracts, furthermore leaf extract had distinguished activity against E. coli O157 ATCC 700728 (inhibition zone diameter was 30 mm) this value is less than the activity of root and shoot tip extracts.
Inhibition zone diameter of root extract ranged from 11.3 mm against Candida albicans ATCC 2091 to 30 mm against E. coli ATCC 25922 and showed middling activity against other microbes.While shoot tip extract had maximum inhibition zone diameters (35 mm) against Shigella flexneri ATCC 9199 and Clostridium perf.ATCC and showed minimum inhibition zone diameter (18 mm) against E. coli O157 ATCC 700728 and has made good activity against other microbes.
Oxytetracycline  In the previous studies of Collins and Franzblau (1997), they discovered that the crude extract of P. sidoides root showed proclaimed growth inhibition of Mycobacterium tuberculosis by 96%.Furthermore, Mativandela et al. (2007) found that butanol root extract of P. sidoides has inhibitory properties against Mycobacterium tuberculosis and Mycobacterium smegmatis.Epigallocatechin and scopoletin showed restrained action against M. smegmatis with minimum inhibitory concentration (MIC) of 7.8 mg/ml.Catechin and umckalin showed MIC values of 31.25 and 62.5 mg/ml respectively.Kolodziej et al. (2003) proved that P. sidoides extracts may have own antibacterial activity as supposed in conventional uses and they also observed antibacterial activity against multi-resistant Staphylococcus aureus strains.Lewu et al. (2006) tested acetone and methanol extracts of P. sidoides from shoot and root as antimicrobial activity against 10 bacterial and 5 fungal strains and reported that two extracts showed considerable activity against all Gram-positive bacteria and two strains of Gramnegative bacteria, as well as showed perceivable activity against all fungal strains with inhibition percent ranged from 52.5% to 82.5%.These results have been interpreted that P. sidiodes extract has antimicrobial activity with possibility wide range effect.This supports the idea of using of P. sidiodes for various infections treatment.

CONCLUSION
Plant cell cultures can certainly contribute to the development of various industries, especially the pharmaceutical industry.For this reason, this study was conducted to clarify the best growth regulator combination which gives the best results for callus initiation and callus production from different explants.The recovery of secondary metabolites relies on the extraction solvent.So, in this work, we used three different solvents (aqueous methanol, water, and aqueous acetone), and results clearly showed that aqueous acetone was the best solvent for phenolic, flavonoids and proanthocyanidins contents.Also, the results show that calli from root explant was the best than the others.This is because the extract of the roots of Pelargonium sidoides DC. has a distinctive therapeutic property and previously used in traditional medicine.Also, this work confirmed that both aqueous methanol and aqueous acetone extracts have a clear impact as an antimicrobial agent with wide range effect.In conclusion, the application of bioactive compounds obtained from different parts of P. sidoides could be applied in both pharmaceuticals and food industries.

Fig. 1 :
Fig. 1: Seedlings of P. sidoides grown on MS-medium after four weeks of cultivation.
Duchow et al. (2014) mentioned that there are little researches about micro-propagation of P. sidoides.On the opposite Moyo et al. (2012) succeed in shoot induction and multiplication with rooting formation by using different types and concentrations of cytokinins alone.

Fig. 5 :
Fig. 5: New leaves formation after one month on the medium containing 1.0 mg/1 BA.

Fig. 9 :
Fig.9: Proanthocyanidins content expressed as Absorbance at 550 nm/mg extract of P. sidiodes calli from different explants extracted with aqueous methanol, water and aqueous acetone.Small letters express LSD significant differences (LSD 0.050 value = 0.0543).

Table 1 :
Fresh weights and growth indexes of callus cultures from leaf, root, and shoot tip explants of P. sidoides for six weeks.

Table 2 :
Extraction yield percent (W/W) of P. sidoides calli derived from leaf, root and shoot tip explants extracted with different solvents.

Table 3 :
Inhibition zone diameters of methanolic P. sidiodes calli crude extracts produced from leaf, root, and shoot tip explants against some microbial species.
or Fluconazole achieved the highest activity from three extracts against some microbes such S. aureus ATCC 6538, E. coli O157 ATCC 700728 and Candida albicans ATCC 2091, but unlike with other microbes such as Clostridium perf.ATCC, Shigella flexneri ATCC 9199 and Salmonella typhimurium ATCC 14028 where higher inhibition zone diameters recorded with shoot tip extract (35 mm, 35 mm and 30 mm) respectively.Also along the same line, E. coli ATCC 25922 recorded higher activity with leaf extract (inhibition zone diameter 32 mm)

Table 4 :
Inhibition zone diameters of acetonic P. sidiodes calli crude extract produced from leaf, root, and shoot tip explants against some microbial species.