callus induction and plant regeneration from an ancient and forgotten crop plant vicia ervilia l....

ISSN 1068�3674, Russian Agricultural Sciences, 2011, Vol. 37, No. 2, pp. 120–125. © Allerton Press, Inc., 2011.

120

1 The International Plant Genetic Resources Insti�tute (IPGRI) forage working group has ascribed Viciasp. as a high priority for collection, conservation andforage development because of their current economicvalue and potential for future utilization [5, 11, 17].Recently ICARDA has recommended the cultivationof annual legume of vetch group specially bitter vetch(Vicia ervilia L.) in fallow lands for right exploitationof pastures rectify feed shortage at marginal dry landsareas, adaptability to unfavorable environments withcold and low rainfall areas [1, 3, 12, 17], the Mediter�ranean, West Asia, and North Africa regions [1, 2, 6,17, 18, 22]. But over the last 20 years, the area of bittervetch has been reduced dramatically to just a few thou�sand hectares. Formerly it was used to feed oxen, butmechanization has diminished this issue substantially[13]. This species is grown in fall season, before theplanting of the second crops [8, 23, 24] or it can berotated with cereal grains such as reducing soil nitro�gen depletion and breaking the pest and disease cycles[7, 3, 10, 16]. Bitter vetch has different usages in pas�tures and marginal soils furthermore more it has sig�nificant role on fertility and soil protection as a protein

1 The article is published in the original.

source for poultries and animals. Genetic engineeringof crop plants relies on the development of efficientmethods for regeneration of viable shoots from cul�tured tissues. Shoot regeneration has been establishedfrom immature cotyledons and embryo axes of Hun�garian vetch supplemented various concentration of 6�benzylaminopurine (BA) and α�naphthaleneaceticacid (NAA) [19]. Callus development has beenobserved from Vicia narbonensis L. which was supple�mented with low concentrations of picloram and BAP[4]. Some reports on shoot regeneration from imma�ture cotyledons are available. The highest frequency ofshoot regeneration and the highest number of shootsper explant were determined from various concentra�tions of TDZ [9] This study was conducted to developa protocol for efficient shoot and plant regenerationfrom seedling explants and immature cotyledons ofbitter vetch (Vicia ervilia L.) and compare the regener�ation capacity of the most commonly used explants:cotyledon and hypocotyls in high and low levels ofhormonal concentrations and study the impact of hor�monal shock on improving callus induction and alsoplant regeneration from in vitro culture of Iraniangenotypes.

PLANT INDUSTRY

Callus Induction and Plant Regeneration from an Ancient and Forgotten Crop Plant Vicia ervilia L. (WILLD)1

F. Etedalia, B. Baghban Kohnehrouza, A. Motallebi�Azarb, F. Khazaeic, A. Gholizadehd, and V. Razavi Aharic

aAll�Faculty of Agriculture, University of Tabriz, 51666 Tabriz, IRANbDepartment of Horticultural Sciences, Faculty of Agriculture, University of Tabriz, 51666 Tabriz, IRAN

cAll�Seed and plant certification and registration institute, Karaj, Tehran, IRANdResearch Centre in fundamental Sciences of University of Tabriz, 51666 Tabriz, IRAN

e�mail: [email protected], [email protected]

Abstract—Recently ICARDA has recommended the cultivation of annual legume of vetch group especiallybitter vetch (Vicia ervilia L.) in fallow lands for right exploitation of pastures and rectifies feed shortage atmarginal dry lands areas. To reach this aim a research was carried out in Tabriz university of IRAN. At thisresearch bitter vetch plants and embryo axis were grown and developed under some levels of hormonal con�centrations (high and low) on MS medium containing BAP and NAA (0, 0.1, 1, 20 and 40 mg/l). After sta�tistical comparison, hormonal combination of 5 mg/l of BAP with 20 mg/l NAA and without NAA was con�sidered as the best treatment for callus induction and maintenance. Then some callus and embryonic axiswere transferred on regeneration medium containing (NAA 0.1 mg/l, BAP 0.2 mg/l), (NAA 2 mg/l + BAP0.2 mg/l), (NAA 1 mg/l + BAP 0.1 mg/l) and without hormones. Three different hormonal media did notshow significant differences for callus size and fresh weights but shooting frequency was high (94%) and thehighest number of shoot (6–28 per explant) were obtained and recognized significant difference in compar�ison to previous experiments which carried out without hormonal shocks.

Keywords: Embryo axis culture, Immature cotyledons, Shoot and Root regeneration

DOI: 10.3103/S1068367411020108

RUSSIAN AGRICULTURAL SCIENCES Vol. 37 No. 2 2011

CALLUS INDUCTION AND PLANT REGENERATION 121

METHOD

Plant Materials

Seeds of Vicia ervilia L. landrace cultivars collectedfrom the fields in the North West of Iran were har�vested individually and sown in a row and also 20 daysold siliques were used to provide cotyledonaryexplants.

Callus Induction and Plant Regeneration

Siliques on 20 days old containing immature seedswere surface sterilized for 20 min in commercialbleach (5% NaOCl) and then 1 min in 70% ethanol,followed by several changes of sterile distilled water.Immature seeds were separated from the siliques andthe cotyledons then carefully dissected. Ten immaturecotyledons and embryo axis that have been isolatedfrom same�sized kernels were placed upward on MSmedium [14] supplemented with low levels of hor�monal concentrations containing combination ofBAP and NAA (0, 0.1 and 1 mg/l) with two replicationand three samples within each experimental unit atfirst experiment. Then second experiment supple�mented with high levels of hormonal concentrationscontaining BAP (0, 5 and 15 mg/l), NAA (0, 20 and40 mg/l) and without hormones (control) for callusand snoot induction by eight replications under 16�hphotoperiod Then, in the third experiment, callusfrom plantlets of embryo axis under high levels of hor�monal concentrations were micropropagated on MSmedium containing (NAA 0.1 mg/l + BAP 0.2 mg/l),(NAA 2 mg/l + BAP 0.2 mg/l), (NAA 1 mg/l + BAP0.1 mg/l) and without hormones by ten replicates andfive explants in each replicate for regeneration. Callussize, fresh weight growth of callus (FWG) and regener�ation rate was measured 20 days after subculturing.Regenerated shoots were rooted in MS medium with0.6% agar.

Statistical Analysis

Data was analyzed in three experiments: 1—As a3 × 3 factorial experiment with completely random�ized basic design (CRD) with two replication for cul�ture in low levels of hormonal concentration as well asthree samples within each experimental unit 2—As a3 × 3 factorial experiment with complete randomizedbasic design (CRD) with eight replications for culturein high levels of hormonal concentrations as well as tensamples within each experimental unit. 3—Incom�pletely randomized basic design (CRD) with ten rep�lications was performed for culture of shocked callusin 3 low levels of hormonal concentrations for regen�eration (plus five samples per experimental unit).Analysis was carried out by using of PROC GLM ofSAS [20] Mean comparisons were conducted usingDuncan in 5% significantly.

RESULTS AND DISCUSSION

First Experiment: Callus Induction and Plant Regeneration in Low Hormonal Concentrations

Analysis of variance for callus diameter in low lev�els of hormonal concentration is present in Table 1 andsummarized in Fig. 1. Significant differences (p < 0.05and p < 0.01) were observed among different low levelsof hormonal concentration combinations of auxin(NAA) and cytokinin (BAP) with control treatmentdata on means callus formation from immature coty�ledons and embryo axis explants. The average diame�ter of the induced callus within the experiment wascalculated to be 0.59 cm. When the induced callusstructures transferred to regeneration medium (hor�mone�free), didn’t start to form green spots for regen�eration in low hormonal concentration media in ourexperiments. The amount of callus developed fromhypocotyls explants increased with increment of BAPconcentrations in the culture medium The highestBAP concentration (1 mg/l) resulted in significantlymore callus production than other BAP (0.1 and0 mg/l) (Fig. 1) levels. However, higher amounts ofcallus were obtained when 1 mg/l NAA was combinedwith a cytokinin, especially with 0.5 or 1 mg/l BAP.

Second Experiment: Callus Induction and Plant Regeneration in High Hormonal Concentrations

In the other experiment, we used high level of hor�monal concentration combinations of auxin (NAA)

Table 1. Analysis of variances (ANOVA) for callus diameterunder low levels of hormonal concentrations in bitter vetch

Source of variation Degree of freedom

Mean squares (MS)

Formed shoots

Callus diameter, cm

BAP 2 0.0 1.66**

NAA 2 0.0 0.19*

BAP × NAA 4 0.0 0.015ns

Experimental error 9 0.0 0.03

Note: * p < 0.05, ** p < 0.01.

Table 2. Analysis of variances (ANOVA) for fresh weightgrowth of callus (FWG) (g) and callus diameter (CD) (cm)under high levels of hormonal concentrations of bitter vetch

Source of variation Degree of freedom

Mean squares (MS)

FWG, g CD, cm

Hormonal concentration, mg/l

7 0.15* 0.74**

Experimental error 56 0.069 0.085

Note: * p < 0.05, ** p < 0.01.

122


ETEDALI et al.

and cytokinin (BAP) containing BAP (0, 5 and15 mg/l), NAA (0, 20 and 40 mg/l) and without hor�mones (control) with eight replications to study theimpact of hormonal shock on improving callus induc�tion and plant regeneration. Callus formation was ini�tiated at the cut surface of the immature cotyledon andembryo axis explants (frequency 100%). Analyses ofvariance for FWG and callus diameter are existing inTable 2. Significant differences (p < 0.05 and p < 0.01)were observed among different high levels of hormonal

concentrations combinations of auxin (NAA) andcytokinin (BAP) without control treatment on meancallus formation from these explants. The effects ofhigh levels of hormonal concentrations on FWG anddiameter of callus that summarized in Figs. 2 and 3shows that 5 mg/l of BAP with 20 mg/l NAA and with�out NAA were considered as the best treatment for cal�lus induction and maintenance. Two types of calluswere usually obtained; white and friable callus or greenand compact callus, referred to as Type I and Type II,respectively. Type I callus mostly consisted of large andtranslucent cells (Fig. 5), while Type II callus con�tained small and green cells (Fig. 6). Type I callus wasusually formed on medium containing (BAP 0 mg/l,NAA 20 mg/l) and (BAP 15 mg/l, NAA mg/l).

Third Experiment: Plant Regeneration from Shocked Callus in Low Hormonal Concentrations

Callus under hormonal shock were transferred onMS supplemented with (NAA 0.1 mg/l + BAP0.2 mg/l), (NAA 2 mg/l + BAP 0.2 mg/l), (NAA1 mg/l + BAP 0.1 mg/l) and hormone free MS medialFresh weight growth of callus (FWG) and regeneration

1.2

1.0

0.8

0.6

0.4

0.2

010.101

0.54

0

1.1730.0830.140.8980.708

0.1Callus diameter, cm

Cal

lus

diam

eter

, cm

NAA BAP

abb

a

bc

a


Fig. 1. Means of callus diameter from immature cotyledon and embryo axis explants affected by different low levels of hormonalconcentration combinations of auxin (NAA) and cytokinin (BAP); a–c: Means followed by same letter are not significantly dif�ferent at p < 0.05.

1.5

1.0

0.5

0

0.0620.0780.0850.10.140.31 0.45

5BAP0NAA

0BAP20NAA

5BAP0NAA

15BAP20NAA

5BAP40NAA

15BAP40NAA

0BAR40NAA

5BAP20NAA

FWG, g

FW

G, g


Fig. 2. Means of fresh weight growth of callus (FWG) from immature cotyledon and embryo axis explants affected by differenthigh levels of hormonal concentration combinations of auxin (NAA) and cytokinin (BAP); a–c: Means followed by same letterare not significantly different at p < 0.05.

Table 3. Shoot regeneration from hormonal shock of callusafter transfer to low levels of hormonal concentrations inbitter vetch


Plantlets regenera�tion frequency

Fresh weight of callus, g

BAP NAA Per explant Mean ± S.E

0.1 0.2 6–22 1.22 ± 0.95

0.2 2 6–28 1.5 ± 0.92

0.1 1 6–26 1.45 ± 1.04

a

bbc

bc c c c c



rate were measured 20 days after subculturing. No sig�nificant differences (p < 0.05 and p < 0.01) in shootregeneration were noticed among these different levelsof hormonal concentration of auxin (NAA) and cyto�kinin (BAP) (Table 3, Fig. 3) but the highest numberof shoot (6–28 per explant) with shooting frequency(94.4%) were obtained Callus growth was high anddisplayed significant difference in compare to previousexperiments which carried out without hormonalshocks (Fig. 4). Regenerated shoots were rooted in MSmedium with 0.6% agar (Fig. 9). Two different modesof adventitious shoot regeneration were obtained fromimmature cotyledon and embryo axes explants. Thefirst was direct whereby shoot originated directly from

the subepidermal tissues (Fig. 7), in the second sys�tem, shoot proliferation occurred through a callusphase which was initiated at the end of the explantswithin 3–4 weeks in high and low levels of hormonalconcentrations (Fig. 8).

In the present study, immature embryo axis of bittervetch showed higher regeneration capacity thanimmature cotyledons in most media which is in linewith previous reports by Sancak et al. [19] in Hungar�ian vetch and Ozyigit et al. [15] in sunflower.

In conclusion, we have developed an efficientmethod for viable shoot regeneration from immatureembryo and embryo axis of bitter vetch (Vicia ervilia L.).Immature cotyledons in Hungarian vetch appeared to

1.5

1.0

0.5

0

0.320.330.390.50.530.680.71 0.43Callus diameter, cm

Cal

lus

diam

eter

, cm

5BAP20NAA

5BAP0NAA

15BAP20NAA

15BAP40NAA

0BAP20NAA

15BAP0NAA

0BAP40NAA

5BAP40NAA

a ab ab bcc

ab abc


Fig. 3. Means of fresh callus diameter (cm) from immature cotyledons and embryo axis explants affected by different high levelsof hormonal concentration combinations of auxin (NAA) and cytokinin (BAP), a–c: Means followed by same letter are not sig�nificantly different at p < 0.05.

3

0

2

1

4

Low hormonconcentraition, mg/l

2NAA, 0.2BAP

1NAA, 0.1BAP

0.1NAA, 2BAP

5BAR20NAA 0NAA 40NAA

0BAP5BAR 5BAR 0BAP15BAP 15BAP 15BAP20NAA 0NAA 20NAA 40NAA 40NAA

FW

G,

g

Induced high hormon concentreation, mg/l

Fig. 4. Means of fresh weight growth of callus (FWG) from immature cotyledon and embryo axis explants in tow levels of hor�monal concentration affected by different high levels of hormonal concentrations combinations of auxin (NAA) and cytokinin(BAP) to study the impact of hormonal shock on improving callus induction and regeneration.

Fig. 5. Callus induction (Typ I) from immature cotyledon of bitter vetch under high level of hormonal concentration.

124


ETEDALI et al.

produce the highest frequency of shoot regenerationon 20 μM 6�benzylaminopurine (BA) and 2.5 μM α�naphthaleneacetic acid (NAA)�containing medium;whereas, shoot regeneration on immature embryoaxes was the best achieved on a medium supplementedwith 5 μM each of BA and NAA. The average numberof shoots from low hormonal concentrations after hor�monal shock was different from 25.33 [19]. Two stepsprotocol for regeneration of somatic embryos fromprotoplasts in Vicia narbonensis, initially on mediumwith a high auxin concentration followed by a culturephase with lowered auxin amount Regeneration of

plants was achieved via two morphologically distin�guishable pathways [21]. Erdogan et al. [9] used thidi�azuron (TDZ) for regeneration from immatureembryo of six bitter vetches with a shoot regenerationcapacity of 90% and 22 numbers per explant. Thehighest number of shoot (7.33 per explant) wasreported from micropropagation of the best regener�ated tine on MS medium containing 2 mg/l BAP and0.2 mg/l NAA. Our target from this research was eval�uation of this plant response to callus induction andregeneration. We concluded that cotyledons andhypocotyls were the successful explants for regenera�

Fig. 6. Callus induction (Typ II) from immature cotyledon of bitter vetch under high level of hormonal concentration.

Fig. 7. Direct regeneration from subepidermal tissues of the cotyledonary petiole in bitter vetch.

Fig. 8. In direct regeneration from callus in bitter vetch.

Fig. 9. Root induction in MS medium with 0.6% agar.



tion and genetic engineering experiment in future. Werecommend that, this plant can be considered as amodel plant biotechnology researches because of itsshort life cycle and good regeneration.

ACKNOWLEDGMENTS

This research was supported by Talented StudentOffice and in part by a Grant�in�Aid from the TabrizUniversity. We thank Dr. Jalil Shafagh (Faculty ofAgriculture University of Tabriz) for providing seeds ofbitter vetch.

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callus induction and plant regeneration from an ancient and forgotten crop plant vicia ervilia l....

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