In Vitro Regeneration of Pakistani Peanut (Arachis Hypogea L.) Varieties Using de-Embryonated Coteledonary Explants

Topics: Peanut, Seed, Arachis Pages: 9 (2503 words) Published: April 9, 2013
African Journal of Biotechnology Vol. 10(43), pp. 8599-8604, 10 August, 2011 Available online at DOI: 10.5897/AJB11.381 ISSN 1684–5315 © 2011 Academic Journals

Full Length Research Paper

In vitro regeneration of Pakistani peanut (Arachis hypogea L.) varieties using de-embryonated coteledonary explants Farhat Nazir1, Mahmood-ul-Hassan2, Zahid Akram2, Muhammad Matloob Javed2, Shoukat Ali1, Ghulam Muhammad Ali1 and Yusuf Zafar1* 1

National Institute of Genomics and Advanced Biotechnology, NARC, Islamabad, Pakistan. 2 Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan. Accepted 20 May, 2011

Peanut (Arachis hypogaea L.) belongs to the family leguminosae and is one of the world’s largest oilseed crops. This study is the first report on peanut regeneration from Pakistan using four commercially released peanut varieties, that is, Golden, BARI-2000, BARD-479 and BARD-92. Longitudinally, halved cotyledons with removed embryos were employed as explants. Among various tested combinations of BAP and NAA, the best combination was 4 and 0.1 mg/L respectively. BARI-2000 proved to be the best responsive variety for in vitro regeneration in terms of number of shoots/explant (133.3%) and number of rooted plants/explant (124.99%). The optimized protocol, which is the first one in the country, would be used to incorporate important traits in future breeding programmes of peanut in Pakistan. Key words: Arachis hypogea, de-embryonated cotyledons, BAP, NAA, in vitro regeneration, Pakistan. INTRODUCTION The oil seed crop Arachis hypogea L. (groundnut/peanut) belongs to family the leguminosae and sub family Papillionacea. The seed containing 50% oil and 25 to 30% proteins can be used as meal for food and feed (Ahmad and Rahim, 2007). In Pakistan, about 85% of total groundnut is grown in Potowar tract of Punjab, 13% in NWFP and 2% in Sindh (Government of Pakistian, 2008). Weeds are one of the major problems causing reduction in yield (Anderson, 1983). Groundnut yield can be reduced up to 80% due to the weed competition with the major loss at early stages of crop development (Gill et al., 1986; Reddy, 1984). Most of the weeds have broad leaves as the groundnut plants so the application of broad leave herbicides becomes impossible. Therefore, it is necessary to incorporate herbicide resistance genes in groundnut so that herbicide can be applied safely. There is also lack of resistant varieties against early leaf spot disease commonly called as Tikka disease caused by Fusarium spp. These are two important constraints (leaf spot disease and weeds) to increase yield of groundnut in the Potohar region of northern Punjab, Pakistan. The introduction of foreign genes from broad resistant sources is possible through genetic transformation. Extensive research has been done in recent years to develop the methods for in vitro regeneration and somatic embryogenesis of peanut crop using various explants (cotyledonary nodes, de-embryonated cotyledons) and media combinations in different countries (Little et al., 2000; Radhakrishnan et al., 2001; Tiwari and Tuli, 2007). Recently, Tiwari and Tulli (2009) developed a protocol using leaflet explants and obtained a high shoot regeneration efficiency (80%). However, in Pakistan the peanut crop remained neglected and currently there are no reports on its genetic improvement through innovative approaches. In this study, we report for the first time, an

*Corresponding author. E-mail: Abbreviations: BAP, NAA, etc

efficient protocol for regeneration of Pakistani peanut cultivars from de-embryonated cotyledonary explants and role of different growth regulator combinations resulting in high frequency of shoot regeneration. These findings will be utilized for genetic manipulation of groundnut for


Afr. J. Biotechnol.

herbicide and disease tolerance.
MATERIALS AND METHODS Plant material Mature seeds of four different peanut...

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Government of Pakistan (2008). Statistical Tables2008/Table-30. Little LI, Magbanua ZV, Parrott WA (2000). A protocol for repetitive somatic embryogenesis from mature peanut epicotyls. Plant Cell Rep., 19: 351-357. McKently AH, Moore GA, Doostdar H, Niedz RP (1995). Agrobacteriummediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants. Plant Cell Rep., 14: 699-703. Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 15(3): 473497. Radhakrishnan T, Murthy TGK, Chandran K, Bandyopadhyay A (2001). Somatic embryogenesis in Arachis hypogaea: revisited. Aust. J. Bot., 49: 753–759. Reddy PS (1984). Groundnut in India present status and strategy. Proc. Meeting of Asian Reg. Res. on grain legumes ICRISAT., pp. 32-35. Swathi TA, Jami SK, Dalta RS, Kirti PB (2006). Genetic transformation of peanut (Arachis hypogea L.) using cotyledonary node as explant and a promoterless gus::nptII fusion gene based vector. J. Biosci., 31(2): 235-246. Tiwari S, Rakesh T (2009). Multiple shoot regeneration in seed-derived immature leaflet explants of peanut (Arachis hypogaea L.). Sci. Hortic., 121: 223-227. Tiwari S, Rakesh T (2008). Factors promoting efficient in vitro regeneration from de-embryonated cotyledon explants of Arachis hypogaea L. Plant Cell Tissue Organ. Cult., 92: 15-24. Venkatachalam P, Geetha N, Abha K, Shaila MS, Sita GL (2000). Agrobacterium-mediated genetic transformation and regeneration of transgenic plants from cotyledon explants of groundnut via somatic embryogenesis. Curr. Sci., 78: 1130-1136.
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