AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: 2151-7517, ISSN Online: 2151-7525, doi:10.5251/abjna.2013.4.3.243.251

© 2013, ScienceHuβ, http://www.scihub.org/ABJNA

In vitro Callus induction of Fenugreek (Trigonellafoenum-graecumL.)Using Different Media with Different Auxins Concentrations

MawahibE.M. ELNour *, 2, LamiaS, Mohammed and Bader EldinA.Saeed

Department of Biology and Environmental sciences, Faculty of Science and Biotechnology, AL-Neelain University

ABSTRACT

A protocol of callus induction in fenugreek (Trigonellafoenum-graecumL.) from 8 - 20 days old in vitro grown cotyledons node explants cultured on Murashige and Skoog (MS) and B5((Gamborget al., 1958) media supplemented with different types and concentrations of growth regulators were tested in order to obtain the best callus formation. Two auxin types, α-naphthalene acetic acid (NAA), and 2,4-dichloro-phenoxyacetic acid (2,4-D) at four concentrations(0.5, 0.1, 1.5, and 2.0 mgL-1) with (0,0) as control in the two media were used in this study .The objective of this study was to determine the effect of different concentration of two types of auxins 2,4-D (2,4Dichlorophenoxy acetic acid) and NAA (Naphthalene acetic acid), on callus induction using cotyledons and hypocotyls explants of T.foenum –graecum. 100 percentage of primary callus induction rate and 2.5 callusing index were achieved on MS medium containing 2mg/l ,2,4-D hormone hypocotyls explants .The maximum value of callusing index (2.8) was obtained from MS medium containing 1.5 mg/l ,2,4- D using hypocotyls and cotyledons explants. The maximum callus formation observed in the MS media containing 2.0mg/l NAA was 3.9±o.o8 in hypocotyls segment. The callus was compact in cotyledons and variable in hypocotyls segments and the colour is creamy.

Key words: Fenugreek ,cotyledons, hypocotyls, callus induction, growth regulators.

INTRODUCTĐON

Fenugreek (Trigonellafoenum-graecumL.) is an important vegetable, spice and medicinal legume plant used as fresh and dried leaves and seeds in many parts of the world (Lewis et al., 2005).Fenugreek seeds have been extracted for Polysaccharide, galactomannan ,different saponins such as diosgenin, yamogenin, mucilage, volatile oil and alkaloids such as choline and trigonelline (Aasim et al., 2010). Trigonelline, coumarin and nicotinic acid have been isolated from fenugreek seeds and shown to be useful in diabetes(Moorthy et al., 2010). Fenugreek seeds mixed with yogurt are used as a conditioner for hair. Seeds are used for making oily pickles in South Asia. Galactagogue in fenugreek seeds are used to increase milk supply in lactating women (Chantry et al., 2004). The T.foenum –graecum) has many traditional uses in Sudan country, like it used for digestive system attractions, and many other extra uses.The young leaves and sprouts are good source of protein, mineral and vitamin C (Khan et al., 2005; Chhibba et al., 2007) and are used as green vegetable in Pakistan and

India alone or with potatoes, spinach and meat. The dried leaves have a bitter taste and a strong characteristic smell. Fenugreek leaves and seeds have been used extensively for preparing extracts and powders in medicinal performance (Basch et al., 2003; Nithya and Ramachandramurty, 2007)The potential benefits of using advanced agricultural biotechnology in Fenugreek (T.foenum-graecumL.) genetic improvement have not yet been realized in Sudan. Establishment of an efficient callus induction protocol is an essential prerequisite in harnessing the advantage of cell and tissue culture for genetic improvement. For the successful application of the tissue culture technique in plant breeding, callus induction and plant regeneration potential of each plant must be determined (wang,2011). Many investigations on tissue culture and plant regeneration in a variety of plants including Fenugreek (T.foenum-graecumL.) have been well documented. fenugreek(T foenum –graecum callus induction was reported by (Aasim et al., 2009)in Fenugreek (T.foenum-graecumL.) hypocotyl explants were most responsive to callus induction and proliferation (Zhang et al., 2003). Thus, first of

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all, for biotechnological research on fenugreek(T.foenum –graecum a reliable callus induction protocol using hypocotyl is essential.Research on the medicinal aspects of fenugreek biproducts has taken great importance in the past few years (Bahram., 2005). However, it lacks the efficient and reliable in vitro micropropagation protocols. The present study was designed to develop reliable in vitro shoot regeneration protocol of fenugreek using different explants and type of plant growth regulator.The present investigation was undertaken with an objective to develop an efficient callus induction protocol which is a major prerequisite for in vitro system involving fenugreek(T.foenum –

graecumin Sudan.

MATERIALS AND METHODS

The experiment was carried out in the Tissue Culture Laboratory of the Department of Biology, Faculty of Science and Biotechnology, Al Neelain University, Khartoum, Sudan during June 2012.

Seed Material:Fenugreek ((T. foenum-graecumL.)seeds were prepared by Agricultural

Research.

SeedSurface and Sterilization: Seeds were sterilized in 70% ethanol for 25 sec and 2% hypo chloride sodium solution for 3 minute seeds were then rinsed several times in sterile water.

Seed Germination and Explant: After surface sterilization the seeds were transferred to culture bottle and directly inoculated on the basic media at 25± 1ºC and photoperiod of 16 hrs light and 8 hrs dark. Two media namely, 1- MS (Murshige and Skoog’s, (1962) salts and vitamins 2- B5 (Gamborget al., 1958) salts and vitamins were tested for their effect on seed supplementary with varies plant growth regulator .Hypocotyls segment of 0.5 cm length excised from 7-10 days- old seedling were used as explant in this study. Cultures were maintained at 25°C with continuous light.

Effect of Axuin on Callus Induction :Two auxin types were used to assess their effect on callus induction. Hypocotyls explants were cultured in culture bottles containing B5,M.S media supplemented with (0.0, 0.5, 1.0, 1.5 and 2.0 mgl-1) α-naphthalene acetic acid (NAA) and 2,4-dichloro-phenoxyacetic acid (2,4-D).The media were autoclaving before the agar gelled. All types of media

were sterilized at 121°C for 20 min. The calluses were grown and maintained at 25°C with a 16 h light photoperiod provided by fluorescent tubes (F36W/Gro). Tissues were subculture at 3-week intervals. Three replicates were maintained for each analysis. All reagents, solvents and standards were of analytical reagent grade.

Statistical analysis: Data were analyzed by ANOVA, considering each bottle with ten seeds as one replicate of each treatment. Treatments were grouped by the Scott Knott test (Scott and Knott 1974) using the statistical software (Cruz 1997). The percentage of callus induction (%), texture of callus, callus colour and callus index (Mean±SE) were evaluated.

RESULTS AND DISCUSSION:

In the present study two explants of each plant (cotyledons and hypocotyls segment). Significant differences (p<0.05) were detected among different level of hormones in two medium MS and B5 , The results in table (1) showed that callus index increased significantly(P<0.o5) through the increasing of 2,4-D hormone concentrations to 1.5mg/l, while it is increasing up to 2mg/l decreased the callusing index and percentage of callus. These results agree with that obtained by(Johri and Mitra 2001), (Johri and Mitra, 2001). Also, Fitch and Moore (1993) expressed higher callus formation in meristems attributed to easier differentiation of these areas. Slesak et al. (2005) determined the best hormonal status for callus generation of Brassica napus is 1 mg L

-1 2,4-D in MS culture. Also the

maximum callus generation for Tanacetumbalsamita L. determined 1 and 0.5 mg L

-1 2,4-D hormone for leaf explant (Shoja et al.,

2010). Among the various concentrations of 2,4-D used for callus induction ,1.5 mg/l proved to give the maximum mean of callusing index , ( 2.8) with 100%of callus induction of hypocotyls(fig 1). The minimum mean of callusing index was recorded by hypocotyls segment is 1.5 by concentration 0.5mg/l with 66%.The results also revealed that cotyledons segments gave the same results of mean callusing index ,which was recorded 2.8 by concentration 1.5 mg/l ,of 2,4-D (fig2), and the minimum callusing index was 0.9 in concentration 0.5 mg/l of hormone . Table 3 showed that different 2,4-D hormonal in MS Medium of callus in days 18, 17, 15 and 16 days after culture presented. During the experiments the highest and lowest percentage (%) callus attributed to 1 mg L

-1 of 2, 4-D and control plants (0 mg L

-1 2, 4-

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D).Our results revealing that 2, 4-D gave high frequency of callus induction, these results agree with the finding of (Rezaeian 2011). The other auxin used in this study was NAA, the effect of this hormone showed in table (1) ,the callus generation increased through increasing the NAA hormone concentrations in MS medium. The maximum mean callusing index and proliferation from hypocotyls and cotyledons segments were 3.9 and2.o by concentration 2mg/l of NAA (fig3,4) while the minimum callusing index were recorded in hypocotyls and cotyledons segments were 2.7&1.5 by the concentration 0.5mg/l

respectively.(Bahramet al., 2005).found that NAA inhanced callusing from leaf of T.corniculata L. and T.foenum- graecumL.The same results obtained in this study , but explants used are hypocotyls and cotyledons .Callus of T.foenum- graecum showed creamy colour, where callus fromconcentration, 2mg/l of NAA showed brown colour, this may be due to accumulation of secondary metabolite in the callus.The texture was compact in cotyledon segments and variable in hypocotyls segments ( table 1).

Table (1) Effects of different concentrations of 2,4-D & NAA on callus induction of Cotyledons and hypocotyls of Trigonellafoenum- graecum culture on MS Medium.Callus induction in MS medium:

Growth regulators mg/l

parameter Explants

NAA CONCENTRATION 2,4-D CONCENTRATION 2 1.5 1 0.5 0 2 1.5 1 0.5 0

74.0±047

74..±841

7400±840

7480±840

747±747 74.0±040

74..±041

7400±84.

0.9±0.28

747±747

Callus indix(Mean±SE)

Brawn creamy creamy White 747±747 Creamy Creamy Creamy Creamy

747±747

Callus colour

Cotyledon

compact compact compact compact 747±747 compact compact compact compact

747±747

Texture of callus

90 10 00 747±747 83 75 75 60 747±747

% of callusing

7471±.40

74.0±.47

74.8±041

7408±040

747±747 74.1±040

74..±041

74.0±040

74.8±840

747±747

Callus indix(Mean±SE)

Creamy Creamy Creamy Creamy 747±747 Creamy Creamy Creamy Creamy

747±747

Callus colour

Variable Variable Variable Variable 747±747 Variable Variable Variable Variable

747±747

Texture of callus

Hypocotyls

100 877 877 877 747±747 877 00 10 66 747±747

% of callusing

8 8 8 8 0 16 9 9 10 747±747

Day of callus

The results in table (2) showed that callus generation increased significantly (<0.05) through increasing the 2,4-D hormone concentrations in B5 medium .Among the various concentrations of 2,4-D used for callus induction, 2.0 mg/l proved to give the maximum mean of callusing index , ( 3.8) with 100% of callus induction of hypocotyls(fig5). The minimum mean of callusing index was recorded In hypocotyls segment is 2.5 by concentration 0.5mg/l with 92%.The results also revealed that cotyledons segments gave maximum results of mean of callusing index ,which was recorded 1.9 by concentration 2.0 mg/l, and the minimum callusing

index was 1.2 in concentration 0.5 mg/l 2,4-D hormone.The other auxin used in this study was NAA, the effect of this hormone showed in table (2).The callus generation increased through the increasing of NAA hormone concentrations in B5 medium. The maximum mean callusing index and proliferation in hypocotyls and cotyledons segments were 3.8 and 2.9 by concentration 2mg/l (fig 6,7) , while the minimum callusing index were recorded in hypocotyls and cotyledons segments were 3.5 &1.1 by the concentration 0.5mg/l respectively. Callus of Trigonellafoenum- graecum showed creamy color and the texture was compact in cotyledon segments

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and variable in hypocotyls segments (table 2).The results on table (1) and table(2) revealed that there is no significant difference between callus formation culture MS and B5 media , but table (3) and table (4)

showed that the starting day of callus initiation faster on B5 than MS medium, when hypocotyls explants were used.

Table (2) Effects of different concentrations of 2,4-D & NAA on callus induction of Cotyledon and hypocotyls of Trigonellafoenum- graecum culture on B5 Medium.

Growth regulators mg/l

parameter Explants

NAA CONCENTRATION 2,4-D CONCENTRATION 2 1.5 1 0.5 0 2 1.5 1 0.5 0

74.0±040

74.0±047

7408±840

74.7±848

747±747 74.0±840

74.6±840

7401±84.

7408±840

747±747

Callus indix(Mean±SE)

Brawn creamy creamy White 747±747 Creamy Creamy Creamy Creamy 747±747

Callus colour

Cotyledon

compact compact compact compact 747±747 compact compact compact compact 747±747

Texture of callus

100 92 58 58 0747±747

85 80 70 66 747±747

% of callusing

7488±.41

±0.14.46

7401±.40

7400±.40

747±747 7486±.41

74.7±.4.

74.0±.4.

74.8±040

747±747

Callus indix(Mean±SE)

Creamy Creamy Creamy Creamy 747±747 Creamy Creamy Creamy Creamy 747±747

Callus colour

Variable Variable Variable Variable 747±747 Variable Variable Variable Variable 747±747

Texture of callus

Hypocotyls

100 877 877 877 747±747 877 100 100 92 747±747

% of callusing

15 16 18 19 19 19 20 21 Day of callus

Table (1&2) showed the day of callus initiation of different hormones concentrations and explants culture in different media .The result showed that

callus initiation inB5 medium fasterest than MS medium, especially for explants hypocotyls with different concentration of NAA hormone.

Fig(1) Callus induction from hypocotyls segments by 2,4-D( concentration 1.5+control) in MS Media

CONTROL MS+1.5mg/l

2,4-D

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Fig (2) Callus induction from cotyledons segments by 2,4-D (concentration 1.5 , with control) in MS Media

Fig(3), Callus induction from cotyledon segments byNAA( 0.0 as control, 0.5,1.o,1.5 and 2.o) in MS Media

MS+1.5mg/l2.4-D

CONTROL

0mg/l

.5MG

1.0mg

/l 1.5mg

/l 2.0mg/l

CONTROL

MS+NAA

Control

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Fig (4) Callus induction from cotyledons segments by NAA( concentration 2.o+control) in MS Media

Fig (5)Callus induction from hypocotyls segments by 2,4-D( concentration 2.0 +control) in B5 Media

CONTROL

CONTROL

MS+2.0mg/l NAA

B5+2.0 mg/l 2,4-D

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Fig (6)Callus induction from cotyledons segments by NAA(0.5,1.0,1.5 and 2.o concentrations) in B5 Media

Fig (7)Callus induction from cotyledons segments by NAA(concentration 2.o , with control) in B5 Media.

B5+NAA

CONTROL

0.5 mg/l 1.0 mg/l

2.0 mg/l 1.5mg/l

B5+2.0 NAA

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CONCLUSION

The results showed that There is no significant different between MS medium and B5 medium in the percentage of callus rate and mean of callusing index, but it was observed that callusing day initiation was faster in B5 medium than that culture on MS medium. . We concluded that the level of callus induction increase was caused by the increased concentration of 2,4-D hormone. The highest callus induction was obtained in on 1.5 mg L

-1 2, 4-D

hormone in .7 days was significant (p<0.05)

compared with other treatments. It was found that growth regulator type and concentration had a significant effect on the callus induction, the increment of callus index and callus physical appearance. The highest frequencies of callus growth index (8.13 and 8.0) were observed on hypostyles explants cultured on B5 basal medium supplemented with 0.5, 0.1, 1.5, and 2.0 mgL- NAA and 2,4-D.Statistically significant differences were observed between explants plants (cotyledons and hypocotyls), 2,4-D concentrations and days investigated in callus index and callus induction measured. The best concentration for explant for was 2.0 mg/l.

ACKNOWLEDGMENT

The authors would like to acknowledge Department of Biology, Faculty of Science& Technology.

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