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In Vitro Shoot Regeneration of Fenugreek (Trigonella Foenumgraceum L.)
Department of Field Crops, Faculty of Agriculture, Ank ara University, Disk api, 06110, Ank ara, Turk ey
M uhammad A as im, Khalid M ahmood K hawar, Cengiz Sancak, Sebahattin Özcan, In Vitro Shoot
Regeneration of Fenugreek (Trigonella Foenumgraceum L.), Am.-Eurasian J. Sustain. Agri c ., C ( ):
CC-CC, 2008
ABS TRACT
Fenugreek (Trigonella foenumgrac e u m L.) is legume crop us ed as medicinal and s pice crop throughout
t he world. Fres h leaves of fenugreek are als o us ed as green vegetable and fodder in s ome parts of the world ;
while s eeds are us ed for making p ic kle s in South A s ia. The s tudy pres ent efficient s hoot regeneration of
fenugreek us ing apical meris tem, and cotyledon leaf explan t s o n different concentrations of TDZ with or
without IBA . A pical meris tem s howed higher s hoot regeneration potential compared to cotyledon leaf explants .
Irres pective of the concentration of plant growth regulators , s hoot regeneration was obs erved on all culture
mediums from s hoot meris t e m e xp lants . Higher number of s hoots were recorded on M S medium containing
TDZ without IBA compared to the medium containing b o th TDZ and IBA . However, pres ence of IBA had
pos itive effect on s hoot length. Regenerated s hoots could not be rooted on any medium containing IBA .
Introduction
Fenugreek (Trigonella foenum-graecum L.) is a legume crop us ed as a forage, as a green manure and as
a s ource of medicinally important s teroid s apogenins (Provorovl et al. 1996). This annual s elf-fertilizing s pecies
with a s hort life c y c le (us ually 80-85 days from emergence to the s eed maturity) is s pread from the Central
and South Europe to the South-Eas t A s ia (Tubives 2008, Flora of Pkis tan 2008, Flora of Chin a 2008).
Fenugreek s eeds are a rich s ource of the polys accharide galacto ma n n a n . T h ey are als o a s ource of s aponins
s uch as dios genin, yamogenin, gitogenin, tigogenin, and neotigogens . Other bioactive cons tituents of fenugreek
include mucilage, volatile oils , and a lka lo id s s u c h a s c holine and trigonelline (Pribac1 and A rdelean
2008). F res h leaves of fenugreek are als o us ed as green vegetable and fodder in s ome parts of the world;
while s eeds are us ed for making pickles in South A s ia.
It has high forage value and has ability to fix nitrogen. M oreover, it has been obs erved that fenugreek has
more s us tained releas e of Nitrogen in t h e ru me n fo r cattle feeding and greater mas s diges tion than alfalfa
(A nonymous 1998).
There are no reports on in vitro regeneration of fenugreek. Thus , the object of this res earch was to obtain
a rapid, reliable and s imple method to regenerate from different explants of Fenugreek under in vitro conditions
The s eeds of cv. Kas uri of fenug re e k w e re obtained from the Directorate of vegetable res earch, A yub
agricultural res earch Ins titute, Fais alabad, Pakis ta n . Seed variation and contamination was minimized by
Corresponding Author: M uhammad 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
Am.-Eurasian J. Sustain. Agric., 3(2): 135-138, 2009 136
s electing healthy and uniform s e e d s free of injuries , s kin imperfections or blemis hes . They were s urface
s terilized us ing 70% commercial bleach (A ce Turkey) for ten minutes and rins ed three times with autoclaved
dis tilled water. The s terilized s eeds were germinated on M S medium s olidified with 0.22 % gelrite.
Shoot tips and cotyledon explants were is olated from 6-10 days old germinat e d s eedlings and germinated
o n M S me dium (M uras hige and Skoog 1962) s upplemented with 0.05 0.80 mg/l TDZ with or without 0.1 mg / l
IBA (Table 1. W hen developing s hoo t s w e re 1 cm long they were removed and rooted on M S medium
s upplemented with 0.1, 0.5 and 1 mg/l IBA in magenta GA 7 ves s els .
The pH of all media was adjus ted to 5.6-5.8 with 1 N N a O H o r 1N H Cl before autoclaving at 121 o C,
118 kPa for 21 minutes . The germination, re g e n e ra t io n and rooting cultures were maintained at 24 +2 o C at
a photos ynthetically active radiance of 30 000 lux with 16 h photoperiod.
Statistical analysis
Each treatment had 3 replicates w it h 10 e xp lants. Significance was determined by analys is of variance
(A NOVA ) and the differences between the means were compared by D u n c a n 's M ultiple Range Tes t us ing
" S PSS for W indows " computer program. Data given in percentages were s ubjected to arcs ine (Vx)
trans formation (Snedecor and Cochran 1967) before s tatis tical analys is .
The s tudy pres ent efficient s hoot reg e n e ra t io n of fenugreek us ing apical meris tem, and cotyledon leaf
explants on different conce n t ra t io n s o f T DZ with or without IBA . It was obs erved that s hoot regeneration
frequency of s hoot tip and cotyledon explant varied w h e n the regeneration medium contained TDZ with and
without 0.1 mg/l IBA in the regeneration medium.
Cotyledon was very recalcit ra n t , w h e re s hoot regeneration was recorded on two culture media only
containing 0.2 a n d 0.4 mg / l TDZ and one culture media containing 0.4 mg/l TDZ with 0.1 mg/l IBA with
s hoot regeneration frequency of 11.11-22.22 (Table 1). Number of s hoots per explants ranged 9.50-16.75. The
maximum number of 16.75 s hoots per explants were record e d o n M S medium containing 0.4 mg/l TDZ with
s hoot length of 1.20 cm. However, addition of 0.1 mg / l IBA to the s ame concentration of BA P res ulted in
decreas ed number but longer s hoots (1.40 cm).
Table 1: E ffect s o f v arious concentrations of T DZ-IBA on shoot regeneration from apical meristem and cotyledon node expl an t s o f
fenugreek
T DZ 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 T ukeys b test (p < 0.05)
A pical meris tem s howed h ig h e r s hoot regeneration potential compared to cotyledon leaf explants . Higher
frequency of regeneration was recorded when the regeneration mediu m was devoid of IBA with range of
88.89-100% (Table 2). A ddition of IBA in the regenerat io n me d iu m w as inhibitory and reduced frequency
of germination in the range of 55.55 to 88.89% (Table 1).
Shoot regeneration inc re a s e d with each increas e in the concentration of TDZ with maximum number of
s hoots at 0.40 mg/l TDZ. Thereafter, a s harp decreas e in the nu mb e r o f s h o o t s /explants was very evident at
0.80 mg/l TDZ . M o re number of s hoots per explants were recorded when the TDZ was us ed alone with range
of 16.10 to 27.75 s hoots per explant.
Similarly, an increas e in the number of s hoots per explants was recorded w it h each increas e in the
c o n c e n t ration of TDZ with 0.1 mg/l IBA in as cending order with a range of 11.88 to 20.40 s hoots per explant .
Number of s hoots per explants were les s compared to the number of s hoots per explants on each corres ponding
concentration of TDZ with out IBA .
Am.-Eurasian J. Sustain. Agric., 3(2): 135-138, 2009 137
Table 2: E ffect s o f v ari ous concentrations of T DZ-IBA on shoot regeneration from apical meristem and cotyledon node explant s o f
fenugreek
T DZ 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
T ukeys b test (p < 0.05).
Contrarily, each increas e in the concentration o f T D Z with or with out IBA res ulted in decreas e in the
s hoot length. M oreover, addition of 0.1 mg/l IBA in the regeneration me d iu m h a d promotory effect and
res ulted in longer s hoots per explants compared to th e s hoot length on regeneration medium with out 0.1 mg/l
IBA with range of 0.95 to 1.55 cm. W hereas , increas ed s hoot length was recorded on M S me dium containing
various concentrations of TDZ with 0.1 mg/l IBA with range of 1.14 to 1.95 cm.
No s hoot could be rooted on an y ro o t in g medium. Experiments are in progres s to s olve the problem and
induce roots on the regenerated s hoots .
TDZ is among the mos t active cytokinin – like s ubs tances and it induces greater in vitro s hoot
proliferation than many other cytokinins in many p la n t s p e c ies (Khawar et al. 2004). W e found that apical
meris tem explants were more res pons ive than cotyledon leaf explants o n a ll TDZ concentrations (Tables 1
and 2). Fratini & Ruiz (2002) found that TDZ inhibits rooting. Similar re s u lt s w e re als o obtained in Cercis
canadensis L.var. alba (Rehder) Bean. (Yus nita et al., 1990), H ib is cus ros a-s inens is L. (Preece et al., 1987) and
in mus cadine grape (Gray & Benton, 1991) us ing TDZ. TDZ concentrations did n o t reduced s hoot
regeneration but res ulted in s tunted s hoots , as has been reported for pea (M alik & Saxena, 1992a and Khawar
et al 2004). The highes t s hoot regeneration capacity w a s achieved on a M S medium s upplemented with 0.4
mg/l TDZ without IBA . Thes e res ults underline the importance of TDZ and s ugges t that a us e of TDZ without
IBA is more favorable and induces high frequency of s hoot regene ra t io n fro m apical meris tems . Similarly,
M alik & Saxena (1992b) obtained the highes t s hoot regeneration from nodal and bas al regions of primary
s hoots developed from s eed cultures of lentil on media s upplemented with relatively low concent ra t io n s of
T D Z . Gill & Saxena (1992) des cribed organogenes is and s omatic embryogenes is in intact s eedlings of s eve ra l
Phas eolus L. s p e cies and explants cultures of peanut by us ing TDZ or BA P. They s ugges ted a crucial role of
TDZ in th e in teraction with endogenous hormones in reprogramming the mode of morphogenes is from
organogenes is to s omatic embryogenes is pos s ibly by releas ing, s ynthes is ing, protecting or even inhibiting auxins
in s itu in combination with other s ub-cellular met a b o lic c hanges , particularly in key regulatory enzyme and
related proteins .
IBA concentrations failed to induce roots in agreement with Huetteman & Preece, (1993), who emphas is e
that rooting of excis ed s hoots may be difficult due to the “carry over” effect of TDZ.
The des cribed protocol meets the objects of s tudy of s hoot multiplication fro m a p ic a l meris tem and
cotyledon leaf explants ; however, it fails to root the regenerated s hoots . M ore experiments are needed to
overcome this problem.
References
Provorovl, N.A ., D. Yury, Y.D. S o s ko v, A . Ludmila, L.A . Lutova, A . Olga, O.A . Sokolova, S.S. Bairamov,
1996. Inves tigation of the fenugreek (Trigonella foenum-graecum L.) genotypes for fres h w eight, s eed
productivity, s ymbiotic activity, callus formation and accumulation of s teroids . Euphytica, 88: 129-138.
Pribac1, C., A . A rdelean, 2008. In vitro c u lt u re of Trigonella foenum-graecum plantules and their anatomic
characterization. EM C 2008 14th European M icros copy Congres s 1-5 Septembe r 2008, Springer Berlin
Heidelberg, A achen, Germany, 3: 181-182.
A nonymous , 1998. Fenugreek. A gri-fax. A lberta, A griculture, Food and Development, A gdex, 147: 20-5.
Tubives , 2008. http.www.tubitak.gov.tr/tubives .
Flora of Pakis tan, 2008. http://www.efloras .org/brows e.as px?flora_id=5&name_s tr=trigonella.
Flora of China, 2008. http://www.efloras .org/flora_page.as px?flora_id=2.
Fratini, R., a n d M .L. Ruiz, 2002. Comparative s tudy of different cytokinins in the induction of morphogenes is
in lentil (Lens culinaris M edik.). In vitro cellular and Developmental Biol Plant, 38: 46-51.
Am.-Eurasian J. Sustain. Agric., 3(2): 135-138, 2009 138
Yus nita, S., R.L. Geneve a n d S.T. Kes ter, 1990. M icropropagation of white flowering eas tern redbud (Cercis
canadens is var. alba L). J. Environ. Hort, 8: 177-179.
Preece, J.E., C.A . Huetteman, C.H. Puello and M .C. Neuma n , 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 es tablis hment of mus ca d in e grape
cultivars (Vitis rotundifolia). Plant Cell Tis s Org Cult., 27: 7-14.
M alik, K.A . and P.K. S a xe n a , 1992a. Thidiazuron induces high frequency of s hoot regeneration in intact
s eedlings of pea (Pis um s ativum) chickpea (Cicer arietinum) and lentil (Lens culinaris M edik). A us t J Plant
Phys iol, 19: 731-740.
M alik, K.A . and P.K. Saxena, 1992b. In vitro regen e ration of plants : a novel approach. Naturwis s ens chaften,
79: 136-137.
Gill, R. and P.K. Saxena, 1992. Direct s omatic embryogenes is and regeneration of plant from s eedling explant
of peanut (Arachis hypogae L): Promotive role of thidiazuron. Can. J. Bot., 70: 1186- 1192.
H u e t t e mane, C.A . and J.E. Preece, 1993. Thidiazuron: a potent cytokinin for woody plant tis s ue culture. P la n t
Cell Tis s Org Cult., 33: 105-119.
M uras hige, T. and F. Skoog, 1962. A revis ed medium for ra p id g ro w t h a nd bioas s ays with tobacco tis s ue
cultures . Phys iol Plant, 15: 473- 497.
Snedecor, G.W . and W .G. Cocharan, 1967. Statis tical methods . The Iowa State Univers ity Pres s . Iowa. USA .