Effect of 6-BA on the Plant Regeneration via Organogenesis from Cotyledonary Node of Cowpea (Vigna unguiculata L. Walp)

The present study compares effects of different concentrations of 6-BA on regeneration from cotyledonary node explants of cowpea (Vigna unguiculata L. Walp). The seeds were inoculated on MSB5 medium [Murashige and Skoog (1962) salts and Gamborg B5 vitamins (1968)] containing different concentrations (0, 1, 2, 3, 4, 5 mg/L) of 6-BA for 4 days. The cotyledonary node explants with one cotyledon excised from 4-day-old seedlings, placed in vitro on MSB5 medium containing 6-BA at different dose (0, 0.5, 1.0, 1.5 mg/L) for shoot induction and elongation. Best response in terms of shoot number and shoot length were obtained with explants derived from seedling preconditioning with 3mg/L 6-BA followed by the induction and elongation stage pretreated with 0.5mg/L 6-BA. The elongated shoots were rooted on MSB5 medium without hormone.


Introduction
Cowpea (Vigna unguiculata L.) is widely grown in Africa, Latin America, Southeast Asia and southwestern regions of North America, and is a major source of high-quality dietary protein and energy for local people.It plays an important role in the lives of millions of people in developing countries of Africa and Asia.In spite of the great importance of this crop, its productivity is low, which is mainly limited by the damage caused by biotic and abiotic stresses (Singh et al., 1997).In addition, limited genetic diversity in cowpea breeding programs is of special concern because cowpea appears to have lower inherent genetic diversity than other cultivated crops as a result of a hypothesized single domestication event (Fang et al., 2007).Although some resistance genes to insect pests and fungi have been identified in some IITA cowpea varieties and other closely related Vigna species (Latunde-Dada et al., 1990), the attempts using conventional breeding methods to introduce the resistance genes into the cultivated cowpea have made little progress for the strong hybrid incompatibility.Hence, genetic engineering approaches stand out as the most effective alternative strategy to overcome the production constraints (Zaidi et al., 2005).An effective and rapid regeneration protocol is essential for genetic transformation.Plant regeneration of cowpea via organogenesis has been achieved from epicotyls, hypocotyls, primary leaves, cotyledons, cotyledonary nodes, shoot tips, plumular apices and shoot meristem.Of these, cotyledonary node explants seemed the most responsive for the induction of multiple shoots, which was appropriate to agrobacterium-mediated transformation (Chaudhury et al., 2007;Raji et al., 2008;Solleti et al., 2008aSolleti et al., , 2008b;;Adesoye et al., 2010).
Previous work has studied the effect of varied hormones used together on the regeneration of cowpea.But the regeneration of cowpea via cotyledonary node uses 6-BA alone has not been explored.The aim of this paper is to explore the effect of 6-BA on different stages of regeneration of cowpea, to provide a theoretical and technical basis for rapid propagation.

Plant Materials and Seeds Preconditioning
Mature seeds of cv.Cheng-jiang VII of cowpea was obtained from the Research Institute of Horticulture, Academy of Chengdu Agriculture and Forestry Science, Chengdu, China.The seeds were soaked with 70% ethanol for 1 min, surface-sterilized with 0.2% (w/v) HgCl 2 for 5 min, followed by rinsed five times with sterile www.ccsen distilled w supplemen Figure 1.

Shoot I
The cotyle with the hy (0, 0.5, 1.0 hypocotyls buds were explants a weeks (Fig  )

Data Collection and Statistical Analysis
The length and the number of adventitious buds were recorded after 2 weeks of culture on the shoot induction and elongation medium.The date of the shoots began to take roots were also recorded.The experiments were arranged to repeat thrice with 20 replicates per treatment.The data were determined by analysis of variance and the significant difference between the means were compared using Duncan's new multiple range method with the help of statistical software DPS.

Effect of 6-BA Preconditioning on Seed Germinating
Cowpea is recalcitrant to regeneration from shoot proliferation and genetic manipulation (Dita et al., 2006).The regenerative competence could be increased via seedling preconditioning using high dose of cytokinin because of its promoting in cell division.
In this study, the seeds on the medium containing 6-BA grew obviously stronger than those cultured on hormone-free medium.The differences are expressed in following aspects made up of dramatic enlarged primary leaves, stubby hypocotyls, hyperplastic region of the cotyledonary node, and thick and short roots (Figure 1a).This conclusion was similar to some other scholars (Bakshi et al., 2012;Tang et al., 2012).The effect was significantly more pronounced when the concentration of 6-BA was up to 3 mg/L.Looking just from morphological terms, the seedlings showed no obvious differences at high dose (3,4,5 mg/L) of 6-BA.

Effect of 6-BA on Shoot Induction, Elongation and Rooting
Table1.Effect of different concentration of 6-BA on shoot regeneration from cotyledonary node explants of cowpea following culture on MSB 5 medium for two weeks At the stage of shoot induction and elongation, more adventitious buds were observed on the media supplement with 6-BA compared with that of control.When the concentration of 6-BA was 0.5 mg/L, 1.0 mg/L and 1.5 mg/L, the number of shoots per explant displayed increasing but the distinction was not very significant (Table 1).This might be because higher concentration of 6-BA precondition at the stage of seedlings made a large impact on the following stage (Brar et al., 1999;Le et al., 2002;Raveendar et al., 2009).The shoot length was decreased with the concentration of 6-BA increasing, which was in agreement with the research of pioneers (Diallo et al., 2008;Aasim et al., 2009;Tang et al., 2012).Besides, it is important to note that when the concentration of 6-BA was 1.5 mg/L, abnormal morphology of shoots would be observed.The stems grew and bend downwards, the leaves were shrunken.And the abnormal shoots were difficult to elongate.
After induction and elongation, the regenerated shoots were removed on MSB 5 medium without hormone for rooting.More than 95% of the regenerative shoots could produce roots.But the time at the beginning of forming roots were different.If the concentration of 6-BA was higher at the stages of induction and elongation, it was difficult to produce roots, which manifested as it would take a longer time to start rooting.The experiment showed that the optimal concentration of 6-BA promoted the propagation of adventitious buds, but inhibit both the shoot elongation and rhizogenesis.
In the present investigation, 3 mg/L 6-BA preconditioning during the seedlings and 0.5 mg/L 6-BA at the induction and elongation stages was the best concentration to induce adventitious buds and to elongate comprehensively considered efficacy and cost.
NaOH or HCl before autoclaving at 121°C for 15 min.All the cultures were maintained at 26±2°C temperature with 16 h light photoperiod.The experiment started from mid-August and finished in mid to end of October in 2012.
Means having the same letters are not significantly different according to Duncan's multiple range test at P = 0.05.