Scientia Horticulturae 122 (2009) 468–471

In vitro micropropagation from plumular apices of Turkish cowpea(Vigna unguiculata L.) cultivar Akkiz

Muhammad Aasim, Khalid Mahmood Khawar *, Sebahattin Ozcan

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

A R T I C L E I N F O

Article history:

Received 7 December 2008

Received in revised form 15 April 2009

Accepted 25 May 2009

Keywords:

Cowpea

Micropropagation

Plumular apices

Pulse treatment

A B S T R A C T

Multiple shoot formation from the plumular apices excised from mature embryos of cowpea cv. Akkiz

was obtained after pulse treatment with 10 mg/l BAP for 5 days followed by culture on MS medium

containing 0.25, 0.50, 0.75 and 1.00, 1.25 mg/l BAP – with or without 0.10 mg/l NAA. Callus induction and

shoot regeneration was recorded on all cultures containing BAP with or without NAA. However, inclusion

of 0.1 mg/l NAA had positive effect on callus diameter and shoot length. Maximum mean number of 7.11

shoots per explant were obtained on MS medium containing 1.00 mg/l BAP. Longer shoots were recorded

on MS medium containing various concentration of BAP+ 0.1 mg/l NAA compared to those containing

various concentrations of BAP singly. All shoots cultured on MS medium containing 1 mg/l BAP were

rooted on MS medium containing 0.50 mg/l IBA. Rooted plants were acclimatized at room temprature in

soil contained in pots. All plants flowered and set seeds in the greenhouse after 3 months.

� 2009 Elsevier B.V. All rights reserved.

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1. Introduction

Cowpea (Vigna unguiculata (L) Walp.) is one of the most ancientfood sources and has probably been used as a crop plant sinceNeolithic times (Summerfield et al., 1974). It is a broadly-adaptedand highly-variable crop cultivated around the world primarily asa pulse, especially in tropical and subtropical areas. Cowpea is anutritious source of food, providing proteins, vitamins, mineralsand carbohydrates (Smatt, 1976). It is used as a vegetable legumefor both greens and grain and as a cover crop. Today, cowpea islargely cultivated on tens of millions of small holder farms in thedrier zones of Africa, in a great arc from Senegal eastward to Sudanand Somalia and southward to Zimbabwe, Botswana andMozambique (NRC, 2006). It is grown successfully in extremeenvironments such as high temperature and low rainfall, and doeswell on poor soils with few economic inputs. It is a legume with adeep root system that binds soil particles together preventingerosion. In Africa, more than 200 million people consume cowpeaon daily basis as vegetable in the form of green leaves, green pods,fresh shelled green peas and shelled dried peas (Popelka et al.,2004). It is also used as fodder, cover crop and green manure.

In vitro regeneration in cowpea using different explantsincluding; primary leaves (Muthukumar et al., 1995; Prem Anandet al., 2000; Ramakrishnan et al., 2005), cotyledonary node (Van Le

Abbreviations: NAA, a-naphthalene acetic acid; BAP, 6-benzylaminopurine; IBA,

indole-3-butyric acid; PVP, polyvinylpyrrolidone.

* Corresponding author. Tel.: +90 312 596 1540; fax: +90 312 318 2666.

E-mail address: kmkhawar@gmail.com (K.M. Khawar).

0304-4238/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2009.05.023

et al., 2002; Chaudhury et al., 2007), shoot meristem/apices/tip(Kartha et al., 1981; Brar et al., 1997; Mao et al., 2006; Aasim et al.,2008) mature cotyledon (Muthukumar et al., 1996; Brar et al.,1999), embryonic axis (Popelka et al., 2006) and mature embryo(Odutayo et al., 2005; Popelka et al., 2006) has been reportedpreviously.

The plumule explant has proved one of the most responsiveexplants for inducing shoot regeneration on calli derived fromplumules of pea (Molnar et al., 1999) and pigeon pea (Surekhaet al., 2005). However, no report covers the use of plumule orplumular apices as explant for shoot regeneration in cowpea.

The objective of the present study was to investigate factorsresponsible for plant regeneration from cowpea plumular apicesexcised from mature zygotic embryos with aim to use the results inbreeding and genetic transformation experiments in future.

2. Material and methods

Seeds of cv. Akkiz of cowpea were obtained from theDepartment of Field Crops, Faculty of Agriculture, Ege University,Izmir, Turkey. Seeds were surface sterilized with 70% commercialbleach (Ace – Turkey containing 5–6% NaOCl) for 5 min. Thereafter,the seeds were rinsed 3� 5 min with bidistilled sterilized water.Mature zygotic embryos were separated aseptically from seedsand pulse treated with 10 mg/l BAP for 5 days (Fig. 1a) on MS basalmedium (Murashige and Skoog, 1962) supplemented with 0.65%agar (Duchefa, Germany) and 3.0% sucrose. A control was alsoplanted without pulse treatment of explants followed by culturingon MS medium.

Fig. 1. Shoot regeneration of Turkish cowpea cultivar Akkiz from plumular apices. (a) Mature zygotic embryos separated aseptically from seeds and pulse treated with 10 mg/l

BAP for 5 days, (b) shoot regeneration on MS medium containing 1 mg/l BAP and (c) 1 mg/l BAP–0.1 mg/l NAA. Bar (a–c) = 0.2 cm.

Table 1Effects of various concentrations of BAP–NAA on shoot regeneration behavior from

plumular apices of Turkish cowpea (V. unguiculata L.) cv. Akkiz.

Medium Callus

diameter

(cm)

Frequency

(%) of shoot

regeneration

Mean number

of Shoots per

explants

Shoot

length

(cm)BAP NAA

0.25 0.00 1.11d 100.00 4.33b 1.67b

0.50 0.00 1.13d 100.00 4.00b 1.24b

0.75 0.00 1.21cd 100.00 4.45b 1.17b

1.00 0.00 1.22cd 100.00 7.11a 1.09b

1.25 0.00 1.14d 100.00 6.42ab 0.91b

0.25 0.10 1.40bc 100.00 1.44c 3.18a

0.50 0.10 1.48b 100.00 4.00b 2.16b

0.75 0.10 1.71a 100.00 4.56b 2.01b

1.00 0.10 1.38bc 100.00 6.11ab 1.92b

1.25 0.10 1.30bc 100.00 4.77b 1.77b

Values with in a column followed by different letters are significantly different at

0.05 level of significance using Duncan’s Multiple Range Test.

M. Aasim et al. / Scientia Horticulturae 122 (2009) 468–471 469

Thereafter 5 days of pulse treatment, plumular apices withplumular leaves were cultured on MS basal medium containing0.25, 0.50, 0.75, 1.00 and 1.25 mg/l BAP – with or without 0.10 mg/lNAA supplemented with 3.0% sucrose and gelled with 0.65% agar(Duchefa, Germany). Culture media was also supplemented with5 g/l activated charcoal, 1.00 mg/l polyvinylpyrrolidone (PVP) as anantioxidant to overcome the problem of chlorosis due to presenceof phenolic compounds on explants. Endogenic latent bacterialcontaminations were eradicated by adding 500 mg/l Augmentin(Smith–Klein–Beecham) in all culture medium.

All culture were incubated in growth chamber at 24 � 2 8C with16 h light photoperiod. All experiments were repeated twice.

Regenerated shoots were excised aseptically and rooted on MSmedium containing 0.5 mg/l IBA. After 2 weeks of culture, agar wascarefully removed from the roots and the plants were keptsubmerged in water for 15 min before transferring to potscontaining clay, sand and organic matter (1:1:1). Pots werecovered with transparent polyethylene bags to maintain theinternal humidity and placed in growth room at room tempera-ture. After 1 week, transparent polyethylene bags were removedgradually and pots containing in vitro regenerated plants were leftin the growth room at room temperature with relative humidity of50%. Thereafter 1 week these were transferred to greenhouse toflower and obtain seeds.

All treatments of regeneration experiments had three replicatescontaining 6 explants and all experiments were repeated twice (3replications � 6 explants � 2 repeats = 36 explants). Data forfrequency (%) of callus induction, frequency (%) of shootregeneration, mean number of shoots per explant, shoot lengthand frequency of rooting were recorded and analyzed using oneway ANOVA. The post hoc tests were performed using DuncansMultiple Range Test with the help of statistical software SPSS 15.00for windows. Data given in percentages were subjected to arcsinetransformation (Snedecor and Cochran, 1967) before statisticalanalysis.

3. Results

Pulse treatment resulted in swelling of mature zygoticembryos followed by induction of callus on all pulse treatedexplants 7–8 days after transfer of the explants to the regenera-tion media. No callusing or swelling was recorded on non-pulsetreated explants (control). Callus induction was recorded on allpulse treated explants; however, significantly larger calli wererecorded on adding 0.1 mg/l NAA in the culture media. Callusdiameter ranged 1.11–1.71 cm. Maximum callus diameter of1.71 cm was recorded on MS medium containing 0.75 mg/lBAP–0.10 mg/l NAA (Table 1).

Shoot regeneration began 4–5 days after callus induction withinitiation of shoot primordia. Thereafter, shoots began to emergefrom shoot meristems after 1 week followed by the development oflateral shoots.

Mean number of shoots per explant ranged 1.44–7.11 after 4weeks of culture. MS medium containing 0.25–0.75 mg/l BAPwithout 0.1 mg/l NAA had statistically similar effect on number ofshoots per explant. However, significantly higher number of 7.11shoots per explant were recorded on MS medium containing 1 mg/l BAP (Fig. 1b) and 1 mg/l BAP–0.1 mg/l NAA (Fig. 1c). Acombination of 0.25 mg/l BAP–0.1 mg/l NAA was inhibitorycompared to 0.25 mg/l BAP used singly. However, a combinationof 0.5–0.75 mg/l BAP – with or without 0.1 mg/l NAA in theregeneration media produced statistically similar effects on shootregeneration. Likewise, 1.0 mg/l BAP with or without 0.1 mg/l NAAhad promotory effect and resulted in sharp increase in the numberof shoots per explant in both cases. Higher concentration of1.25 mg/l BAP with or without NAA was inhibitory and resulted insharp decline in the number of shoots per explant in both cases.Inhibition was more severe on MS medium containing 1.25 mg/lBAP–0.1 mg/l NAA.

Data on mean shoot length showed positive effect of thepresence of NAA in the regeneration media. However, all shootswere not uniform in length. First regenerating shoots were longerand later developing shoots were diminished. Shoot lengthdecreased with increase in BAP concentration in all treatmentssupplemented with or without NAA (Table 1). Maximum meanshoot length of 3.18 cm was recorded on MS medium containing0.25 mg/l BAP–0.10 mg/l NAA followed by shoot length of 2.16 cmon MS medium containing 0.5 mg/l BAP–0.10 mg/l NAA.

4. Rooting and acclimatization

No difficulty was recorded in rooting of regenerated shoots onMS medium containing 0.5 mg/l IBA. Rooting in IBA also promoteddevelopment of multiple (2–3) secondary shoots arising from thebase of rooted explants (Fig. 2a). These secondary shoots could alsobe rooted on MS medium containing 0.5 mg/l IBA. Rooted plants

Fig. 2. Rooting of regenerated shoots: (a) rooting and regeneration of multiple secondary shoots arising from the base of rooted explants and (b) flowering and seed setting of

acclimatized plants in the greenhouse. Bar (a) = 1.25 cm and (b) = 1.65 cm.

M. Aasim et al. / Scientia Horticulturae 122 (2009) 468–471470

were not difficult to acclimatize and set flowers, beans and seeds ingreenhouse (Fig. 2b).

5. Discussion

In vitro micropropagation techniques have aided plant breederssince long time and identifying culture conditions that favormicropropagation may aid beneficially towards this end. Multi-plication of cowpea using plumule offers a direct approach formicropropagation. Shoot proliferation on calli derived from plumewas favored in presence of BAP with or without NAA withmaximum number of 7.11 shoots per explant. Presence of NAAincreased the callus diameter which had variable effects on shootregeneration. A comparison of 0.25 mg/l BAP with and withoutNAA, showed sharp inhibition in shoot regeneration due to NAA.Whereas, 0.5 and 0.75 mg/l BAP with or without NAA regeneratedstatistically similar number of shoots per explant. Optimum shootregeneration was achieved on MS medium containing 1 mg/l BAPwith or without NAA. However, numerically more shoots perexplant were recorded on MS medium containing BAP withoutNAA. The results are in partial agreement with Aasim et al. (2008),who found that addition of NAA to the MS medium containing BAPreduced mean number of shoots per shoot meristem explant in cv.Akkiz and increased callusing from shoot tips. Higher number ofshoots per explant were produced on plumular apices compared toshoot meristem (Aasim et al., 2008) of cv. Akkiz (this study). Thisshows advantage of using plumular apices over shoot meristems indifficult to propagate cowpea.

Variable response of NAA to shoot regeneration could be due todifferent explants of the same cultivar used in two experiments.Whereas, Brar et al. (1997) and Diallo et al. (2008) report negativeeffects of NAA in the regeneration medium on shoot regenerationpotential of explants. Contrarily, Kartha et al. (1981) and Sebastian(1983) did not report any callusing during shoot regeneration fromshoot meristem, shoot apices or shoot tip explants in cowpea

The increase in BAP concentration with or without NAA hadinhibitory effect on mean shoot length as well. However, acomparison of the two (BAP with or without 0.1 mg/l NAA) showsthat presence of NAA in the culture media resulted in longer shootsin agreement with Brar et al. (1997) and Aasim et al. (2008).

Regenerated shoots were easily rooted on MS mediumsupplemented with 0.5 mg/l IBA. IBA had positive effects; notonly on root induction but also on secondary shoot regeneration inthe rooting medium. Similarly, multiple secondary shoots onrooting media containing IBA were observed by Aasim et al. (2008)in cv. Akkiz of cowpea, Contrarily, Mao et al. (2006) reported norooting on cowpea with indole-3-butyric acid in the rootingmedium.

On the basis of the experiment, we conclude that:

� MS medium containing 1.00 mg/l BAP was more potent for shootregeneration compared to other concentrations.� Shoot length reduced with each increase in the concentration of

BAP with or without NAA. However longer shoots were recordedin the presence of NAA.� It was not difficult to root the regenerated shoots on MS medium

containing 0.5 mg/l IBA.� Additional research is needed to know the exact causes and

mechanism of multiple secondary shoot regeneration on rootingmedia containing IBA. Knowledge of this would be beneficial todescribe the behavior of plant more appropriately.

In conclusion, present work opens a new window of opportunity tomicropropagate cowpea using plumular apices of Turkish cowpea cv.Akkiz. This study is a step forward towards the micropropagation andregeneration. It also meets objective of study to develop a reliablemicropropagation system for future use in the mutiplication of in vitro

genetically transformed cowpea plants.

Acknowledgement

Monetary help in the form of fellowship for foreign countrycitizens to the first author by the Scientific and TechnologicalResearch Council of Turkey (Tubitak) through Directorate forFunding Scientists (BIDEB) is acknowledged.

References

Aasim, M., Khawar, K.M., Ozcan, S., 2008. In vitro micropropagation from shootmeristems of turkish cowpea (Vigna unguiculata L.) cultivar Akkiz. Bangladesh J.Bot. 37 (2), 149–154.

Brar, M.S., Al-Khayri, J.M., Shamblin, C.E., McNew, R.W., Morelock, T.E., Anderson,E.J., 1997. In vitro shoot tip multiplication of cowpea Vigna unguiculata (L.)Walp. In Vitro Cell. Dev. Biol. Plant 33, 114–118.

Brar, M.S., Al-Khayri, J.M., Morelock, T.E., Anderson, E.J., 1999. Genotypic response ofcowpea Vigna unguiculata (L.) to in vitro regeneration from cotyledon explants.In Vitro Cell. Dev. Biol. Plant 35, 8–12.

Chaudhury, D., Madanpotra, S., Jaiwal, R., Saini, R., Kumar, P.A., Jaiwal, P.K., 2007.Agrobacterium tumefaciens-mediated high frequency genetic transformation ofan Indian cowpea (Vigna unguiculata L. Walp.) cultivar and transmission oftransgenes into progeny. Plant Sci. 172, 692–700.

Diallo, M.S., Ndiaye, A., Sagna, M., Gassama-Dia, Y.K., 2008. Plants regeneration fromAfrican cowpea variety (Vigna unguiculata L. Walp.). Afr. J. Biotechnol. 7 (16),2828–2833.

Kartha, K.K., Pahl, K., Leung, N.L., Mroginski, L.A., 1981. Plant regeneration frommeristems of grain legumes: soybean, cowpea, peanut, chickpea, and bean. Can.J. Bot. 59, I671–1679.

Mao, J.Q., Zaidi, M.A., Aranson, J.T., Altosaar, I., 2006. In vitro regeneration of Vignaunguiculata (L.) Walp. Cv. Black eye cowpea via shoot organogenesis. Plant CellTissue Organ Cult. 87, 121–125.

Molnar, Z., Jenes, B., Ordog, V., 1999. Genetic transformation of pea (Pisum sativumL.) via particle bombardment. Recent Advances in Plant Biotechnology, StaraLesna, Slovak Republic, September 4–11.

M. Aasim et al. / Scientia Horticulturae 122 (2009) 468–471 471

Murashige, T., Skoog, E.A., 1962. Revised medium for rapid growth and bioassayswith tobacco tissue cultures. Physiol. Plant. 15, 473–497.

Muthukumar, B., Mariamma, M., Gnanam, A., 1995. Regeneration of plantsfrom primary leaves of cowpea. Plant Cell Tissue Organ Cult. 42, 153–155.

Muthukumar, B., Mariamma, M., Valuthambi, K., Gnanam, A., 1996. Genetictransformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp.)using Agrobacterium tumefaciens. Plant Cell Rep. 15, 980–985.

N.R.C. (National Research Council), 2006. Lost crops of Africa. Vegetables. Devel-opment, Security and Cooperation Policy and Global Affairs, vol. II. The NationalAcademics Press, Washington, D.C. , http://www.books.nap.edu/openbook.php?record_id=11763&page=105.

Odutayo, O.I., Akinrimisi, F.B., Ogunbosoye, I., Oso, R.T., 2005. Multiple shoot induc-tion from embryo derived callus cultures of cowpea (Vigna unguiculata l.) Walp.Afr. J. Biotechnol. 4, 1214–1216.

Popelka, J.C., Terryn, N., Higgins, T.J.V., 2004. Gene technology for grain legumes: canit contribute to the food challenge in developing countries? Plant Sci. 167 (2),195–206.

Popelka, J.C., Gollasch, S., Moore, A., Molvig, L., Huggins, T.J.V., 2006. Genetictransformation of cowpea and stable transmission of the transgenes to progeny.Plant Cell Rep. 25, 304–312.

Prem Anand, R., Ganapathi, A., Ramesh, A., Vengadesan, G., Selvaraj, N., 2000. Highfrequency plant regeneration via somatic embryogenesis in cell suspensioncultures of cowpea (Vigna unguiculata L. Walp). In Vitro Cell. Dev. Biol. Plant 36,475–480.

Ramakrishnan, K., Gnanam, R., Sivakumar, P., Manickam, A., 2005. In vitro somaticembryogenesis from cell suspension cultures of cowpea [Vigna unguiculata (L)Walp]. Plant Cell Rep. 24, 449–461.

Sebastian, K.T., 1983. Shoot-tip culture and subsequent regeneration in cowpea. Sci.Hortic. 20, 315–317.

Smatt, J., 1976. Tropical Pulses. Longman, London.Snedecor, G.W., Cochran, W.G., 1967. Statistical Methods. The Iowa State Univ.

Press, Iowa, USA, pp. 327–329.Summerfield, R.J., Huxley, P.A., Steelle, W., 1974. Cowpea (Vigna unguiculata L.

Walp). Field Crops Abstr. 27, 301–312.Surekha, C., Beena, M.R., Arundhati, A., Singh, P.K., Tuli, R., Dutta-Gupta, A., Kirti, P.B.,

2005. Agrobacterium-mediated genetic transformation of pigeon pea (Cajanuscajan (L.) Millsp.) using embryonal segments and development of transgenicplants for resistance against Spodoptera. Plant Sci. 169, 1074–1080.

Van Le, B.U.I., De Carvalho, M.H.C., Zuily-Fodil, Y., Thi, A.T.P., Van, K.T.T., 2002. Directwhole plant regeneration of cowpea [Vigna unguiculata (L.) Walp] from coty-ledonary node thin cell layer explants. J. Plant Physiol. 159, 1255–1258.