In vitro Callus induction of Fenugreek (Trigonellafoenum ... characteristic smell. Fenugreek leaves

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  • 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

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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(Mea n±SE)

    Brawn creamy creamy White 747±747 Creamy Creamy Creamy Crea my

    747± 747

    Callus colour

    Cotyledon

    compact compact compact compact 747±747 compact compact compact com pact

    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±