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In vitro shoot regeneration of fenugreek (Trigonella foenumgraceum L.).

Introduction

Fenugreek (Trigonella foenum-graecum L.) is a legume crop used as a forage, as a green manure and as a source of medicinally important steroid sapogenins (Provorovl et al. 1996). This annual self-fertilizing species with a short life cycle (usually 80-85 days from emergence to the seed maturity) is spread from the Central and South Europe to the South-East Asia (Tubives 2008, Flora of Pkistan 2008, Flora of China 2008). Fenugreek seeds are a rich source of the polysaccharide galactomannan. They are also a source of saponins such as diosgenin, yamogenin, gitogenin, tigogenin, and neotigogens. Other bioactive constituents of fenugreek include mucilage, volatile oils, and alkaloids such as choline and trigonelline (Pribacl and Ardelean 2008). Fresh leaves of fenugreek are also used as green vegetable and fodder in some parts of the world; while seeds are used for making pickles in South Asia.

It has high forage value and has ability to fix nitrogen. Moreover, it has been observed that fenugreek has more sustained release of Nitrogen in the rumen for cattle feeding and greater mass digestion than alfalfa (Anonymous 1998).

There are no reports on in vitro regeneration of fenugreek. Thus, the object of this research was to obtain a rapid, reliable and simple method to regenerate from different explants of Fenugreek under in vitro conditions

Materials and Methods

2.1. Seed germination, regeneration and rooting

The seeds of cv. Kasuri of fenugreek were obtained from the Directorate of vegetable research, Ayub agricultural research Institute, Faisalabad, Pakistan. Seed variation and contamination was minimized by selecting healthy and uniform seeds free of injuries, skin imperfections or blemishes. They were surface sterilized using 70% commercial bleach (Ace Turkey) for ten minutes and rinsed three times with autoclaved distilled water. The sterilized seeds were germinated on MS medium solidified with 0.22 % gelrite.

Shoot tips and cotyledon explants were isolated from 6-10 days old germinated seedlings and germinated on MS medium (Murashige and Skoog 1962) supplemented with 0.05 0.80 mg/l TDZ with or without 0.1 mg/l IBA (Table 1. When developing shoots were 1 cm long they were removed and rooted on MS medium supplemented with 0.1, 0.5 and 1 mg/l IBA in magenta [GA.sup.7] vessels.

The pH of all media was adjusted to 5.6-5.8 with 1 N NaOH or 1N HCl before autoclaving at 121 oC, 118 kPa for 21 minutes. The germination, regeneration and rooting cultures were maintained at 24 +2 oC at a photosynthetically active radiance of 30 000 lux with 16 h photoperiod.

Statistical analysis

Each treatment had 3 replicates with 10 explants. Significance was determined by analysis of variance (ANOVA) and the differences between the means were compared by Duncan's Multiple Range Test using "SPSS for Windows" computer program. Data given in percentages were subjected to arcsine (Vx) transformation (Snedecor and Cochran 1967) before statistical analysis.

Results and discussion

The study present efficient shoot regeneration of fenugreek using apical meristem, and cotyledon leaf explants on different concentrations of TDZ with or without IBA. It was observed that shoot regeneration frequency of shoot tip and cotyledon explant varied when the regeneration medium contained TDZ with and without 0.1 mg/l IBA in the regeneration medium.

Cotyledon was very recalcitrant, where shoot regeneration was recorded on two culture media only containing 0.2 and 0.4 mg/l TDZ and one culture media containing 0.4 mg/l TDZ with 0.1 mg/l IBA with shoot regeneration frequency of 11.11-22.22 (Table 1). Number of shoots per explants ranged 9.50-16.75. The maximum number of 16.75 shoots per explants were recorded on MS medium containing 0.4 mg/l TDZ with shoot length of 1.20 cm. However, addition of 0.1 mg/l IBA to the same concentration of BAP resulted in decreased number but longer shoots (1.40 cm).

Apical meristem showed higher shoot regeneration potential compared to cotyledon leaf explants. Higher frequency of regeneration was recorded when the regeneration medium was devoid of IBA with range of 88.89-100% (Table 2). Addition of IBA in the regeneration medium was inhibitory and reduced frequency of germination in the range of 55.55 to 88.89% (Table 1).

Shoot regeneration increased with each increase in the concentration of TDZ with maximum number of shoots at 0.40 mg/l TDZ. Thereafter, a sharp decrease in the number of shoots/explants was very evident at 0.80 mg/l TDZ. More number of shoots per explants were recorded when the TDZ was used alone with range of 16.10 to 27.75 shoots per explant.

Similarly, an increase in the number of shoots per explants was recorded with each increase in the concentration of TDZ with 0.1 mg/l IBA in ascending order with a range of 11.88 to 20.40 shoots per explant. Number of shoots per explants were less compared to the number of shoots per explants on each corresponding concentration of TDZ with out IBA.

Contrarily, each increase in the concentration of TDZ with or with out IBA resulted in decrease in the shoot length. Moreover, addition of 0.1 mg/l IBA in the regeneration medium had promotory effect and resulted in longer shoots per explants compared to the shoot length on regeneration medium with out 0.1 mg/l IBA with range of 0.95 to 1.55 cm. Whereas, increased shoot length was recorded on MS medium containing various concentrations of TDZ with 0.1 mg/l IBA with range of 1.14 to 1.95 cm.

No shoot could be rooted on any rooting medium. Experiments are in progress to solve the problem and induce roots on the regenerated shoots.

TDZ is among the most active cytokinin--like substances and it induces greater in vitro shoot proliferation than many other cytokinins in many plant species (Khawar et al. 2004). We found that apical meristem explants were more responsive than cotyledon leaf explants on all TDZ concentrations (Tables 1 and 2). Fratini & Ruiz (2002) found that TDZ inhibits rooting. Similar res ults were also obtained in Cercis canadensis L.var. alba (Rehder) Bean. (Yusnita et al., 1990), Hibiscusrosa-sinensis L. (Preece et al., 1987) and in muscadine grape (Gray & Benton, 1991) using TDZ. TDZ concentrations did not reduced shoot regeneration but resulted in stunted shoots, as has been reported for pea (Malik & Saxena, 1992a and Khawar et al 2004). The highest shoot regeneration capacity was achieved on a MS medium supplemented with 0.4 mg/l TDZ without IBA. These results underline the importance of TDZ and suggest that a use of TDZ without IBA is more favorable and induces high frequency of shoot regeneration from apical meristems. Similarly, Malik & Saxena (1992b) obtained the highest shoot regeneration from nodal and basal regions of primary shoots developed from seed cultures of lentil on media supplemented with relatively low concentrations of TDZ. Gill & Saxena (1992) described organogenesis and somatic embryogenesis in intact seedlings of several Phaseolus L. species and explants cultures of peanut by using TDZ or BAP. They suggested a crucial role of TDZ in the interaction with endogenous hormones in reprogramming the mode of morphogenesis from organogenesis to somatic embryogenesis possibly by releasing, synthesising, protecting or even inhibiting auxins in situ in combination with other sub-cellular metabolic changes, particularly in key regulatory enzyme and related proteins.

IBA concentrations failed to induce roots in agreement with Huetteman & Preece, (1993), who emphasise that rooting of excised shoots may be difficult due to the "carry over" effect of TDZ.

The described protocol meets the objects of study of shoot multiplication from apical meristem and cotyledon leaf explants; however, it fails to root the regenerated shoots. More experiments are needed to overcome this problem.

References

Provorovl, N.A., D. Yury, Y.D. Soskov, A. Ludmila, L.A. Lutova, A. Olga, O.A. Sokolova, S.S. Bairamov, 1996. Investigation of the fenugreek (Trigonella foenum-graecum L.) genotypes for fresh weight, seed productivity, symbiotic activity, callus formation and accumulation of steroids. Euphytica, 88: 129-138.

Pribac1, C., A. Ardelean, 2008. In vitro culture of Trigonella foenum-graecum plantules and their anatomic characterization. EMC 2008 14th European Microscopy Congress 1-5 September 2008, Springer Berlin Heidelberg, Aachen, Germany, 3: 181-182.

Anonymous, 1998. Fenugreek. Agri-fax. Alberta, Agriculture, Food and Development, Agdex, 147: 20-5.

Tubives, 2008. http.www.tubitak.gov.tr/tubives.

Flora of Pakistan, 2008. http://www.efloras.org/browse.aspx?flora_id=5&name_str=trigonella.

Flora of China, 2008. http://www.efloras.org/flora_page.aspx?flora_id=2.

Fratini, R., and M.L. Ruiz, 2002. Comparative study of different cytokinins in the induction of morphogenesis in lentil (Lens culinaris Medik.). In vitro cellular and Developmental Biol Plant, 38: 46-51.

Yusnita, S., R.L. Geneve and S.T. Kester, 1990. Micropropagation of white flowering eastern redbud (Cercis canadensis var. alba L). J. Environ. Hort, 8: 177-179.

Preece, J.E., C.A. Huetteman, C.H. Puello and M.C. Neuman, 1987. The influence of Thidiazuron on in vitro culture of woody plants. Hort. Science, 22: 1071.

Gray, D.J. and C.M. Benton, 1991. In vitro micropropagation and plant establishment of muscadine grape cultivars (Vitis rotundifolia). Plant Cell Tiss Org Cult., 27: 7-14.

Malik, K.A. and P.K. Saxena, 1992a. Thidiazuron induces high frequency of shoot regeneration in intact seedlings of pea (Pisum sativum) chickpea (Cicer arietinum) and lentil (Lens culinaris Medik). Aust J Plant Physiol, 19: 731-740.

Malik, K.A. and P.K. Saxena, 1992b. In vitro regeneration of plants: a novel approach. Naturwissenschaften, 79: 136-137.

Gill, R. and P.K. Saxena, 1992. Direct somatic embryogenesis and regeneration of plant from seedling explant of peanut (Arachis hypogae L): Promotive role of thidiazuron. Can. J. Bot., 70: 1186-1192.

Huettemane, C.A. and J.E. Preece, 1993. Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tiss Org Cult., 33: 105-119.

Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant, 15: 473-497.

Snedecor, G.W. and W.G. Cocharan, 1967. Statistical methods. The Iowa State University Press. Iowa. USA.

Muhammad Aasim, Khalid Mahmood Khawar, Cengiz Sancak, Sebahattin Ozcan

Department of Field Crops, Faculty of Agriculture, Ankara University, Diskapi, 06110, Ankara, Turkey

Muhammad Aasim, Khalid Mahmood Khawar, Cengiz Sancak, Sebahattin Ozcan, In Vitro Shoot Regeneration of Fenugreek (Trigonella Foenumgraceum L.), Am.-Eurasian J. Sustain. Agric., C(): CC-CC, 2008

Corresponding Author: Muhammad Aasim, Department of Field Crops, Faculty of Agriculture, Ankara University, Diskapi, 06110, Ankara, Turkey Tel. No. +903125961540, Fax No. +903123182666) E-mail:mshazim@gmail.com
Table 1: Effects of various concentrations of TDZ-IBA on shoot
regeneration from apical meristem and cotyledon node explants of
fenugreek

TDZ      IBA      Frequency (%)     Number of shoots   Shoot length
(mg/l)   (mg/l)   of regeneration   per explants       (cm)

0.05     -        0.00 c            0.00 c             0.00 c
0.10     -        0.00 c            0.00 c             0.00 c
0.20     -        11.11 b           9.50 b             1.30 ab
0.40     -        22.22 a           16.75 a            1.20 b
0.80     -        0.00 c            0.00 c             0.00 c
0.05     0.1      0.00 c            0.00 c             0.00 c
0.10     0.1      0.00 c            0.00 c             0.00 c
0.20     0.1      0.00 c            0.00 c             0.00 c
0.40     0.1      11.11 b           13.50 ab           1.40 a
0.80     0.1      0.00 c            0.00 c             0.00 c

1: Each valueis the mean of 3 replications with 6 explants each.

2: Values within a column followed by different letters are
significantly different at the 0.05 probability level using
Tukeys b test (p < 0.05).

Table 2: Effects of various concentrations of TDZ-IBA on shoot
regeneration from apical meristem and cotyledon node explants of
fenugreek

TDZ      IBA      Frequency (%) of   Number of shoots   Shoot length
(mg/l)   (mg/l)   regeneration       per explants       (cm)

0.05     -        100.00 a           16.10 c            1.55 b
0.10     -        100.00 a           18.1b c            1.42 b
0.20     -        100.00 a           18.89 bc           1.29 c
0.40     -        88.89 b            27.75 a            1.04 d
0.80     -        100.00 a           22.10 b            0.95 d
0.05     0.1      88.89 b            11.88 d            1.95 a
0.10     0.1      77.78 c            15.72 c            1.74 ab
0.20     0.1      77.78 c            16.43 c            1.33 bc
0.40     0.1      55.55 d            20.40 b            1.20 c
0.80     0.1      55.55 d            18.80 bc           1.14 c

1: Each value is the mean of 3 replications with 6 explants each.

2: Values within a column followed by different letters are
significantly different at the 0.05 probability level using
Tukeys b test (p < 0.05).
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Title Annotation:Original Articles
Author:Aasim, Muhammad; Khawar, Khalid Mahmood; Sancak, Cengiz; Ozcan, Sebahattin
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Geographic Code:7TURK
Date:May 1, 2009
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