Increase production of some secondary products in callus ...ijiset.com/vol4/v4s3/IJISET_V4_I03_34.pdf · Figure (1): The chemical structure of ABA [6] Copper sulfate is a chemical

IJISET - International Journal of Innovative Science, Engineering & Technology, Vol. 4 Issue 3, March 2017

ISSN (Online) 2348 – 7968 | Impact Factor (2016) – 5.264

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Increase production of some secondary products in callus of Citrullus colocynthis L. using chemical and

physical elicitors.

Dr. Alla Jabbar Taha and Halla H. Mutasher.

Al-Muustansiriyah University, College of Science, Biology Department.

UAbstract:

The results showed different concentration in some of the secondary metabolites in

methanol extracts of callus (control), compare with callus treated by physical and

chemical elicitors. . Callus were treated with Abscisic Acid (ABA) at 1mg/l gave

highest significantly concentration of quercetin and rutin reached to 223.6 and 229.9

µg/ml respectively, while the compound cucurbitacin C reached to highest value

significantly 149. 4µg/ml at 2.5 mg/l of ABA. CuSOR4R.5HR2RO at 5mg/l concentrations

gave highest significantly value of quercetin, rutin, cucurbitacin A and colocynthinin

reached to 210.1, 242.8, 70.3 and 135.4 µg/ml respectively. UV exposure for 20

minutes, gave highest significantly concentration of citrullol and cucurbitacinB ,

reached to 29.3 and 45.6µg/ml respectively, while cucurbitacin C reached to highest

value significantly 230.8µg/ml for 10 min. The dark treatment lead to increased

significantly concentration of quercetin ,citrullol and cucurbitacinA reched to 225.7,

20.9 and 22 µg/ml respectively, while the light treatment for 24 hrs affected

significantly to rise concentration of cucurbitacinC to 247.1µg/ml . morover the

results showed that all chemical and physical treatments led to an increase in the

concentrations of cucurbitacin C compared with the control treatment and the highest

concentration reached to 549µg/ml when CuSOR4R.5HR2RO was used at 10mg/l

concentration.

Key words: Chemical and physical elicitors, InVitro, Plant secondary products,

Citrullus colocynthis

UIntroduction:

From decays, plants were take an important caring because their complex medicinal

contents called secondary metabolites which used in traditional treatment like

purgative, insecticide, anti-tumor, anti-diabetic, and others [1]. Plant tissue culture

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technique play an important role for increasing some of chemical contents in

medicinal plants by adding abiotic and biotic stimulators [2] Which include in this

study Abscisic Acid (ABA) and copper sulfate CuSOR4R.5HR2RO as chemical abiotic

elicitors and (light and ultra violet UV) as physical abiotic elicitors. ABA is a weak

acid (CR15RHR20ROR4R) show in figure (1) [3].this hormone is growth and development

regulator and inter in other physiology operations like dormancy, germination, cell

division, elongation and flowering [4]. ABA increase in drought and cooling

condition to protect and survive the plant cells [5].

Figure (1): The chemical structure of ABA [6]

Copper sulfate is a chemical compound in powder or blue crystals shape in structure CuSOR4R.5HR2RO [7]. It used for increasing some of cardiac glycosides for Digitalis lanata [8].

Materials and methods: U

Callus induction U

The callus initiated from explant stem, cultured on MS media supported by NAA at 0.5 and BA at 2 mg/l concentration used as maintenance media.

Preparation of chemical elicitors.

The maintenance media is using for stimulating callus to produce secondary metabolites after adding chemicals elicitors in different concentrations table (1).




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Table (1): chemicals preparation with concentrations.

Preparation of physical elicitors. The callus induced from maintenance media, were use after dividing it into two groups one of them were treated with UV-C exposure for 10 min and the other with 20min.The other physical elicitor was light, the callus was exposed for different periods of light (0h, 12h, and 24h). All treatments were in five replicates.

Quantitative and qualitative determination of secondary metabolites: HPLC technique used to determine the quantitative and qualitative of some compound of glycosides and flavonoids from stimulator callus.

Statically analysis. All experiments designated by completely random design (CRD) to study the effect of the different characters and compering the means to the least significant differences (LSD) at 5% probability.

U nd DiscussionResults a

Mg/l chemicals

Full strength MS

0.5 NAA

2 BA

0, 0.5, 1, 2.5 ABA

0, 2.5, 5, 10 CuSOR4R.5HR2RO

30000 Sucrose

7000 Agar




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Effect of ABA concentration on quantitative and qualitative of secondary metabolite in callus by HPLC technique.

Fig (2,3,4) and table (2) showed the effect of different concentration of ABA on the concentration of secondary metabolites in colocynthis callus. There are differences in average of compounds depend on ABA concentrations. The table show high value of quercetin and Rutin (223.6 and 299.9) µg/ml at 1mg/l of ABA, also all the ABA concentration show significant increasing in value of CucurbitacinC, which reached to 196.5µg/ml at 0.5mg/l ABA. While Kaempferol and Cucurbitacin B decreased in value when ABA concentration increased. The reason of increasing the flavonoid compounds values with ABA concentrations increased belong to that ABA is causing a high stress to induce the callus to produce these compounds [9]. As well as ABA has a certain role in metabolism operation development like protein and lipid manufacturing [10]. Also,ABA induced phenolic compounds synthesis in 1TVitisrotun difolia1T [11].

Table (2): the effect of different concentration of ABA mg/l on the concentration of secondary metabolites µg/ml from callus of Citrullus colocynthis

LSD 0.05 ABA concentration mg/l Secondary metabolites µg/ml

2.5 1 0.5 Control callus without treatment

7.02 109.4 122.5 187.1 382.5 Kaempferol

11.99 100.1 223.6 178 131.3 Quercetin

49.95 137.8 299.9 193.5 231.1 Rutin

5.65 8.8 7.4 7.4 7.4 Citrullol

9.03 17.6 14.1 13.3 8.8 Cucurbitacin A

6.8 5.3 8.5 12.2 19.2 Cucurbitacin B

19.98 149.4 139.6 196.5 103.2 Cucurbitacin C

9.99 102 53.8 77.7 128.2 Colocynthin

11.53 93.5 68.3 97.5 104.7 Colocynthinin




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Effect of CuSo4.5H2Oconcentration on quantitative and qualitative of secondary metabolite in callus by HPLC technique.

Observed from fig ( 5,6,7) and table (3) the effect of CuSOR4R.5HR2RO in different concentration in glycosides and flavonoids compound of callus. The CuSo4.5H2O play a role to change the value of this compounds, the highest value significantly of Quercetin, cucurbitacinA and colocynthinin , reached to 210.1 , 70.3 and 135.4 µg/ml respectively at 5mg/l from CuSo4.5H2O, which significantly different from other treatments. The value of Cucurbitacin C increased in highly significant from the other reached to 549 µg/ml at 10mg/lCuSo4.5H2O. The increasing of some of secondary metabolites with increasing of concentration of CuSo4.5H2O belong to the activity of sulfate for inducing the compound accumulation, [12] reported that the high levels of copper sulfate in Capsicum annum L. cell culture were active to accumulation capsidiol by destroyed cell membrane of these culture. Also [13] found that sulfate was increasing level of Sanguinarine concentration in Eschscholtzia californica cell suspension. µg/ml

Table (3): the effect of different concentrations of CuSo4.5H2o) mg/l on the concentration of Secondary metabolites µg/ml from callus of Citrullus colocynthis

LSD 0.05 CuSo4.5H2o concentration mg/l Secondary metabolites. µg/ml 10 5 2.5 Control

callus without treatment

55.32 96.3 107.7 135.4 382.5 Kaempferol

23.07 170.9 210.1 122.1 131.3 Quercetin

29.97 224 242.8 162.7 231.1 Rutin

8.16 7.5 7.5 10.4 7.5 Citrullol

18.91 50.1 70.3 25.2 8.8 Cucurbitacin A

16.56 9.1 11.3 16.4 19.2 Cucurbitacin B

28.56 549 85 120 103.2 Cucurbitacin C

29.97 70.2 117.5 93.2 128.2 Colocynthin

19.98 81.3 135.4 107.2 104.7 Colocynthinin




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Effect of the period UV exposure (min) in quantitative and qualitative of secondary metabolites in callus by HPLC technique.

Fig(8,9) and the result of table(4) show the effect of (10 and 20)min of UV exposure on the concentration of secondary metabolites, the 20min exposure led to significantly increasing the concentration of citrullol and Cucurbutacin B values reached to 29.3 and 45.6 µg/ml respectively. While Cucurbitacin C value was increasing significantly reached to 230.8 µg/ml for 10min exposing. Flavonoid were decrease in their values at 10 and 20min exposing. These results agree with [14].

Table (4): the effect of different exposure period of UV (min) on the concentration of Secondary metabolites µg/ml from callus of Citrullus colocynthis.

LSD 0.05 UV period exposure (min) Secondary metabolites µg/ml 20 10 Control

callus without

treatment

26.18 88.9 141.4 382.5 Kaempferol

25.94 40.1 122.9 131.3 Quercetin

19.97 98 231.9 231.1 Rutin

13.28 29.3 17.8 7.5 Citrullol

NS 8.8 8.8 8.8 Cucurbitacin A

24.04 45.6 5.3 19.2 Cucurbitacin B

26.18 50 230.8 103.2 Cucurbitacin C

22.82 32 13.2 128.2 Colocynthin

15.71 29.8 13.5 104.7 Colocynthinin

Effect of light period (hrs) on the quantitative and qualitative of

Secondary metabolites in callus of Citrullus colocynthis by HPLC technique.

Fig(10,11,12) and table (5) show values of concentration on some compounds, which increased in concentration significantly at 0hr light (darkness) which are Quercetin, citrullol and cucurbitacin A reached to 225.7, 20.9 and 22 µg/ml respectively .While the concentration of cucurbitacin C significantly increased to 247.1 µg/ml at 24hr




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light. The reason of this case was that light has an important role for inducing accumulation for some of chemical compound [14]. While other researcher reported that light has an effect to increase the activity of cell for growth and producing the chemical compounds (15).

Table (5): the effect of different exposure period of light (hr) on the concentration of Secondary metabolites µg/ml from callus of Citrullus colocynthis.

LSD 0.05 Light period exposure (hours) Secondary metabolites µg/ml 24 12 Darkness Control

callus without treatment

19.44 64.1 46.7 234.5 382.5 Kaempferol

19.98 82.3 133.1 225.7 131.3 Quercetin

23.07 189.4 125.5 202.2 231.1 Rutin

11.42 13.2 12.4 20.9 7.5 Citrullol

5.88 8.8 8.8 22 8.8 Cucurbitacin A

14.35 14.2 10.1 15.7 19.2 Cucurbitacin B

25.11 247.1 100 157.7 103.2 Cucurbitacin C

20.96 16.1 33.8 37.9 128.2 Colocynthin

23.07 62.8 23.6 46.9 104.7 Colocynthinin

Conclusion: ABA at 1mg/l and CuSo4.H2o at 5 and 10 mg/l concentration lead to increase the

concentration of some secondary products for callus and moral comparison with control. The physical

elicitors had significant effect to increase some of secondary products when callus exposed to the

darkness, while the secondary products suffered increase or decrease in their concentration when

exposed for 10 and 20 minutes to UV light.




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(A) (B)

Fig (2): Curves of secondary products in callus induced from stem of Citrullus colocynthis after adding 0.5 mg/l of ABA.

)A( Glycoside compound )B:( Flavonoid compound




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)A( )B(

Fig (3): Curves of secondary products in callus induced from stem of Citrullus colocynthis after adding 1 mg/l of ABA.

Glycoside compound: (A) )B:( Flavonoid compound




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)A( )B(

.

Fig (4): Curves of secondary products in callus induced from stem of Citrullus colocynthis after adding 2.5 mg/l of ABA.





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)A( )B(

Fig (5): Curve of secondary products in callus induced from stem of Citrullus colocynthis after adding 2.5 mg/l of CuSo4.5H2o.





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)A( )B(

Fig (6): Curves of secondary products in callus induced from stem of Citrullus colocynthis after adding 5 mg/l of CuSo4.5H2o.





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)A( )B(

Fig (7): Curves of secondary products in callus induced from stem of Citrullus colocynthis after adding 10 mg/l of CuSo4.5H2o.





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)A( )B(

Fig (8): Curves of secondary products in callus induced from stem of Citrullus colocynthis after exposure to UV light for 10 min.





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)A( )B(

Fig (9): Curves of secondary products in callus induced from stem of Citrullus colocynthis after exposure to UV light for 20 min.





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)A( )B(

Fig (10): Curves of secondary products in callus induced from stem of Citrullus colocynthis after exposure to light for12 hours. Flavonoid compound (B) Glycoside compound: (A)




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)A( )B(

Fig (11): Curves of secondary products in callus induced from stem of Citrullus colocynthis after exposure to light for12 hours.





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)B(

.

Fig (12): Curves of secondary products in callus induced from stem of Citrullus colocynthis after exposure to light for 24 hours.





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[12] Ellialtıoğlu, Ş., Üstün, A.S. and Mehmetoğlu, Ü. (2001) Using AgNO3

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