Journal

Journal of Applied Horticulture, 10(1): 40-43, 2008

Appl

Improved plant regeneration in cowpea through

shoot meristem

Muthusamy Manoharan*, Sharmin Khan and James O. Garner

Department of Agriculture, University of Arkansas at Pine Bluff, Pine Bluff, AR 71601, USA.
*E-mail: manoharanm@uapb.edu

Abstract
Cowpea is a highly recalcitrant nutrient-rich leguminous vegetable crop. Efforts to genetically transform cowpea with insect-resistant
genes remains a challenging task due to lack of an efficient regeneration system. We have established an efficient regeneration system
in cowpea through shoot meristem. Shoot meristems were isolated from embryos that were precultured for 3-5 days on Murashige
and Skoog (MS) medium containing 8.9 μM benzylaminopurine (BA). The isolated shoot meristems were cultured on MS medium
containing 0.89 μM BA. After 3-4 weeks, multiple shoots were separated from the explant and cultured on half-strength MS medium
for elongation and rooting. More than 90% of the regenerants formed roots. The rooted plantlets were transferred first to peat pellets
and subsequently to the greenhouse. The plants were allowed to flower and set seed. The efficiency of regeneration in all four cultivars
ranged from 76-87%, demonstrating a significant improvement over the published protocols (1-32%). At least six to seven plantlets
were obtained from each meristem. The protocol using shoot meristems is simple, efficient, rapid and genotype-independent and may
be amenable for transformation through particle bombardment.
Key words: Vigna unguiculata, shoot meristem, regeneration, transformation, legumes

Introduction in these reports is too low (1-32%) to reliably obtain transgenic

plants. Consequently, efforts to transform cowpea were mostly
Cowpea (Vigna unguiculata L. Walp.), an annual vegetable, is unsuccessful or resulted in very few transgenic plants (Garcia et
one of the world’s important legume food crops. Cowpea grain al., 1986, 1987; Penza et al., 1991; Muthukumar et al., 1996;
contains about 25% protein, especially rich in folate, potassium, Ikea et al., 2003). In recent years, regeneration of shoots from
iron, magnesium, and the essential amino acids lysine and cotyledon nodes or from other meristematic explants has emerged
tryptophan. Cowpea is also rich in phytochemicals that may help as a rapid and relatively efficient method of transformation in a
prevent chronic diseases such as cardiovascular disease, cancer number of legumes that are highly recalcitrant in tissue culture
and diabetes. In addition, cowpea is a good source of fiber. A (Oger et al., 1996; Trieu and Harrison, 1996; Larkin et al.,
diet high in fiber can help lower blood cholesterol levels, which 1996; Olhoft et al., 2001). In cowpea, transgenic plants were
can reduce risk of heart disease (www.mayoclinic.com/health/ regenerated using cotyledon nodes containing axillary meristems
legumes/NU00260) (Popelka et al., 2006), although at low frequency (0.05-0.15%),
Cowpea is severely infected by insects such as thrips demonstrating the feasibility of using meristems as an alternate
(Megalurothrips sjostedti), aphids (Aphis craccivora), curculio source for genetic transformation.
(Chalcodermus aeneus), pod borer (Maruca vitrata), weevils In this report, we present a simple, efficient, rapid and genotype-
(Callosobruchus maculatus) etc. that cause significant damage to independent regeneration system for cowpea plants from four
crop production and yield (Singh et al., 1990). However, current cultivars by using shoot meristems. The regeneration method
cultivars do not offer protection against insect damage. Efforts has potential use in transforming cowpea with insect resistant
to develop durable insect resistance did not succeed because the genes.
genome of cowpea may be devoid of major resistance genes to
many insect pests that attack cowpea. Also, attempts to bring Materials and methods
insect resistance into cowpea from wild Vigna species have failed
Four cowpea cultivars (Early Scarlet, Coronet, Quick Pick and
because of high genetic barriers between wild Vigna and cultivated
AR87-435-68) were selected for regeneration. Mature seeds
cowpea (Singh et al., 1997).
(kindly provided by Dr. S. Okiror, University of Arkansas at Pine
Cowpea is an ideal vegetable crop for the application of genetic Bluff, USA) were surface sterilized in 70% alcohol for 5 minutes,
engineering technologies for developing insect resistance. rinsed in sterile water and placed in 0.2% sodium hypochlorite
However, cowpea is highly recalcitrant to tissue culture and solution. After 1 h, the seeds were rinsed thrice with sterile water.
therefore genetic transformation is difficult to achieve. There have Finally, the seeds were allowed to soak in sterile water overnight.
been a few reports of plant regeneration through organogenesis Murashige and Skoog (1962) medium (MS) supplemented with
and somatic embryogenesis (Muthukumar et al., 1995; 3% (w/v) sucrose (Sigma, USA) and various concentrations of
Pellegrineschi, 1997; Brar et al., 1999a, b; Anand et al., 2000, growth regulators were used for tissue culture and regeneration.
2001; Ramakrishnan et al., 2005). The efficiency of regeneration The media were adjusted to pH 5.8 with 1 N NaOH or 1 N HCl,
 Improved plant regeneration in cowpea through shoot meristema 41

solidified with 3 g L-1 phytagel (Sigma, USA) and autoclaved shoots could be seen from the cut end of the meristem. After 3-4
at 1 kg cm-2 for 20 min. Media (50 mL) were dispensed into weeks, individual shoots were separated from each meristem and
20- by 100-mm sterile Petri dishes (Falcon, Becton Dickinson transferred to half-strength MS medium for elongation and rooting.
Labware, USA). The cultures were maintained at 25 ± 2 0C with Among the different concentrations tried, MS medium containing
a 16-h photoperiod (25-40 μmol cm-2 s-1). All growth regulators
0.89 μM BA produced more shoots (3.2 shoots per meristem) than
were filter sterilized before adding to the media. Embryos were
isolated and precultured either on MS basal medium or MS other concentrations. To improve the number of multiple shoots,
medium containing BA (8.9 μM) (Table 1). After 3-5 days, the embryos were precultured on higher concentration of BA (8.9
shoot meristems (0.5-1 mm) were carefully isolated and cultured μM) for 3-5 days and the isolated shoot meristems were cultured
on MS media with different concentrations of BA (0.4-22.2 μM) on different concentrations of BA (Table 1). Significantly, more
for three week. After three weeks, the regenerated multiple shoots plants were regenerated after transfer to MS medium containing
were transferred to elongation and rooting medium (half-strength 0.89 μM BA from all four cultivars tested, indicating the positive
MS with no growth regulators). Plantlets with well-developed effect of preculture in inducing multiple shoots (Fig. 1A). More
roots were removed from the culture medium, the roots washed
than six shoots per meristem were regenerated from all four
thoroughly with tap water, and transferred to peat pellets [Jiffy-
7, Jiffy Products (N.B.) Ltd., Shippagan, Canada] for initial cultivars with the preculture on BA (8.9 μM) medium. Increasing
acclimatization. The plantlets were covered with plastic wrap the concentration of BA beyond 0.89 μM in the culture media
to maintain high humidity for first few days. Gradually the resulted in callus growth that significantly reduced the number
humidity was reduced by slowly removing the plastic wrap and of multiple shoots. The addition of other cytokinins such as
the hardened plants were transferred to the greenhouse [24-28ºC, zeatin and kinetin in the regeneration media also led to callus
16/8 h (day/night) photoperiod supplemented by sodium halide growth from shoot meristems, thereby significantly reducing the
lights]. Plants were allowed to flower and set seed. The root tips number of shoots (data not shown). Shoots were separated and
of the regenerated plants were collected in cold distilled water,
transferred to half-strength MS medium for elongation (Fig. 1B)
kept at 40C for 24 h and then fixed in Farmer’s fixative (3:1 95%
ethanol: glacial acetic acid). The root tips were squashed with and rooting. More than 90% of the regenerants formed roots in the
carbol fuchsin and observed under a microscope. half-strength MS medium (Fig. 1C). The regenerated plants with
roots were initially transferred to peat pellets for hardening. Only
Results 50% of the plants survived during hardening. After 10-12 days of
Shoot meristems (0.5-1 mm) were carefully isolated from hardening, the surviving plants were transferred to the greenhouse
precultured embryos and cultured on different concentrations (Fig. 1D). No phenotypic and chromosomal abnormalities (2n =
of BA (Table 1). After 3-5 d of culture, newly formed multiple 22) were noticed.

Table 1. Effects of benzylaminopurine (BA, μM) on shoot regeneration from cultured meristems in cowpea
Genotype Preculture medium Culture medium Explants forming shoots (%) Number of shoots per explant†
Early Scarlet MS MS 56.25 1.16 j
MS MS + 0.4 BA 59.37 2.62 f,g,h,i,j
MS MS + 0.9 BA 60.94 3.24 c,d,e,f,g
MS MS + 2.2 BA 59.37 2.83 e,f,g,h,i
MS MS + 4.4 BA 51.28 2.37 f,g,h,i,j
MS MS + 8.9 BA 69.58 2.16 g,h,i,j
MS MS + 13.3 BA 60.35 1.89 h,i,j
MS MS + 22.2 BA 68.71 1.22 j
MS + 8.9 BA MS + 0.4 BA 70.89 4.87 b,c
MS + 8.9 BA MS + 0.9 BA 72.33 6.73 a
MS + 8.9 BA MS + 2.2 BA 76.82 5.45 b
MS + 8.9 BA MS + 4.4 BA 74.02 4.21 c,d,e
Coronet MS MS 72.66 1.99 g,h,i,j
MS MS + 0.9 BA 73.08 3.34 d,e,f,g,h
MS + 8.9 BA MS + 0.9 BA 87.65 6.89 a
MS + 8.9 BA MS + 2.2 BA 86.23 5.32 b
Quick Pick MS MS 81.23 1.89 h,i,j
MS MS + 0.9 BA 83.78 3.87 c,d,e,f
MS + 8.9 BA MS + 0.9 BA 86.66 6.46 a
MS + 8.9 BA MS + 2.2 BA 85.62 5.12 b
AR87-43568 MS MS 75.83 1.71 i,j
MS MS + 0.9 BA 79.21 3.43 c,d
MS + 8.9 BA MS + 0.9 BA 84.02 6.29 a
MS + 8.9 BA MS + 2.2 BA 82.17 5.18 b
†
means followed by the same letters are not significantly different based on Duncan’s multiple range test (P<0.05).
42 Improved plant regeneration in cowpea through shoot meristema

explants on media containing cytokinins like BA on shoot

regeneration has been reported in other legumes such as grain
legume (Vigna mungo), Phaseolus sp. and Cajanus cajan (Shiv
Prakash et al., 1994; Santalla et al., 1998; Saini and Jaiwal,
2005). Once the regeneration system for the cultivar Early Scarlet
was optimized, the system was applied to the cultivars Coronet,
Quick Pick and AR87-43568. The shoot meristems from these
cultivars were isolated from the embryos precultured on 8.9 μM
BA and cultured on 0.89 μM BA. All three cultivars produced
six to seven shoots per meristem, demonstrating the applicability
of the shoot meristem-based protocol to different genotypes. The
regeneration efficiency of shoot meristems that produced multiple
shoots ranged from 72-88% in the four cultivars compared to 1-
32% obtained through organogenesis or somatic embryogenesis.
In conclusion, we have successfully established a simple,
rapid and efficient regeneration system in cowpea using shoot
meristems. The regenerated plants exhibited no phenotypic and
Fig.1. In vitro regeneration of cowpea plants cv Early Scarlet from shoot genotypic abnormalities. Moreover, the regeneration response of
meristem. A. Development of multiple shoots from precultured shoot
meristems on BA medium (0.89 μM); B. Multiple shoots, separated and four cultivars to the shoot meristem based system was similar,
transferred to rooting medium; C. Rooted plants in half-strength MS demonstrating the genotype-independent nature and applicability
medium; and D. Fully established plants in the greenhouse. of this protocol. The regeneration procedure developed from this
study may be used to transform cowpea with insecticidal genes
Discussion
through biolistics transformation.
Cowpea is one of the most recalcitrant leguminous vegetable
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