Abstract: Two field experiments were carried out to evaluate the effects of GA3 on the growth and development of cowpea cultivars Blackeye and Tswana. Exogenous application of GA3, 7 days after emergence at 30, 60 or 90 mg L-1 significantly increased cowpea plant height, first node height, leaf area and leaf number/plant, nodulation, plant dry matter accumulation, pod length, pod number/plant, seed number/pod, 100 seed weight, harvest index and seed yield ha-1. Gibberellic acid had no significant effect on cowpea plant senescence. The results of this study suggests that exogenous application of GA3 can be used to modify growth and development of some cowpea varieties.
INTRODUCTION
Cowpea (Vigna unguiculata (L.) Walp.) is cultivated under diverse soil and climatic conditions in Africa. Traditionally cowpea is intercropped with crops such as millet, sorghum, maize, cotton, yam and cassava. However, cowpea is also planted as a pure stand in some countries such as Botswana, Kenya and Republic of South Africa. Cowpea is adapted to warm weather and requires less rainfall than most crops; therefore, it is cultivated in the semi-arid and arid regions of lowland tropics and subtropics, where soils are poor and rainfall is limited (Mortimore et al., 1997).
Cowpea is a very important legume crop in Eastern, Southern, Central and Western Africa. It is utilized as grain, vegetable (leaves and green immature pods) and fodder for livestock. It is a major cheap source of protein in human diets with the grains containing about 23-25% protein (Bressani, 1985). In Botswana, more than 30,000 ha of land is planted annually with cowpea, yielding over 600 tons of grain (Mo, 1996). The leaves are harvested and used either fresh or preserved by blanching and kneading to a pulp, followed by squeezing to form small balls (size of tennis ball), which are then dried in the sun (deMooy, 1987). The end product is known as morogo wa dinawa (setswana), which is utilized during winter when many leafy vegetables are scarce. Cowpea is also an important fodder crop especially during the drought years.
The average grain yield in Botswana is less than 100 kg ha-1 (Mo, 2001). In Botswana, the constraints to cowpea are drought (rainfall failure), pest attack, diseases, parasitic weeds and soils of low intrinsic fertility, lack of good quality seed and distribution system and inappropriate technologies and management. The poor growth of cowpea observed in the dry season has been reported to be due to lack of elongation of internodes (Ishiyaku and Singh, 2001; Mukhtar and Singh, 2004). For large economic yields, leaf area indices of 1 and 2 are required for as long as possible after flowering but need to be coupled with efficient partitioning of dry matter into fruits (Summerfield et al., 1985). Leaf Area Duration (LAD), which is the integral of Leaf Area Index (LAI) from emergence to physiological maturity significantly determines seed yield of cowpea (Laing et al., 1983). Any technology that can delay leaf senescence, therefore, increasing LAD would increase seed yield of cowpea. Pate and Minchin (1980) reported that cowpea yields seem to be limited by poor ability of genotypes to assimilate carbon and nitrogen during the reproductive period and to partition large amounts of their daily gains of these two elements into fruits. The challenge is to find ways of improving cowpea productivity. Variety improvement, modified cropping systems and use of Plant Growth Regulators (PGRs) may improve cowpea yields.
Plant growth regulators play an important role in high value horticultural crops to increase yield, enhance crop quality and management (Emongor, 1997). Exogenous applications of gibberellins to Arabidopsis induced early flowering and affected flower morphology (Richards et al., 2001). Gianfagna and Merrit (1998) reported that GA4+7 increased flowering in a genotype of Aquilegia species. Yang et al. (1992) reported that GA4+7 increased fruit set and fresh weight in cucumber. Emongor (2002) reported that gibberellins (GA4+7 and GA3) and a combination of cytokinins and gibberellins (benzyladine (BA) plus GA4+7) increased vegetative growth of common beans (Phaseolus vulgaris) but had no significant effect on seed yield. Gibberellins (GA4+7) significantly reduced 100 seed weight and seed yield of common beans. However, a combination of 25 mg L-1 of BA and 2.5 mg L-1 GA4+7 significantly increased the number of pods per plant, seeds per pod, 100 seed weight and seed yield ha-1. Williams and de Mallorca (1984) reported that foliar application of GA3 at 2.89x10-6 M to soybean plants delayed the formation of nodule initials and reduced the numbers mass nodule-1 and specific activity of nodules by 43, 31 and 47%, respectively, without affecting growth. Similar effects on nodulation were produced by foliar application of GA4 (3.01x10-5 M) or GA7 (3.03x10-5 M), or by the addition of GA3 (2.89x10-6 M) to the rooting medium.
Foliar application of cowpea plants with 15 mg L-1 naphthaleneacetic acid (NAA) at 15, 30 and 45 days after sowing increased vegetative growth, fruit set and grain yield (Resmi and Gopalakrishnan, 2004). Resmi and Gopalakrishnan (2004) further reported that foliar application of 2-chloroethyl trimethyl ammonium chloride (CCC) at 300, 400 and 500 mg L-1 to cowpea plants increased vegetative growth, fruit set and yield and grain yield. However, 2,4-dichlorophenoxyacetic acid (2,4-D) at 2,4 or 6 mg L-1 and para-chlorophenoxyacetic acid (PCPA) at 25, 50 or 75 mg L-1 depressed vegetative growth and seed yield ha-1 of cowpea. Mukhtar and Singh (2006) reported that GA3 stimulated an increase in growth, flowering, pod maturity and grain yield of cowpea. Okelana and Adedipe (1982) reported that foliar application of GA3, BA and 2-chloroethylphosphonic acid (CEPA) to cowpea cultivars (Adzuki, Ife Brown and New Era) had no significant effects on vegetative growth, however, CEPA suppressed stem elongation and root dry weight and caused defoliation. As efforts are being made towards maximizing the yield of cowpea in the dry season with irrigation, efforts should be made on improving not only reproductive development but vegetative growth as well. Foliar application of GA3 appears promising (Mukhtar and Singh, 2006) but the authors concluded that more research be done to determine the optimum concentration of GA3 as well as explore the possibility of using other hormones or PGRs and/or combination of PGRs. The objective of this study was to evaluate the effects of GA3 on the growth and development of cowpea. The cowpea cultivars used in the study, Blackeye and Tswana are what the farmers grow commercially or traditionally in Botswana.
MATERIALS AND METHODS
Experimental site: Two field experiments were conducted at Botswana College of Agriculture (BCA) Gaborone Botswana from 18th February 2002 to 21st January 2004. Botswana College of Agriculture is located at Sebele Content farm (latitude 24° 34`S and latitude 25° 57` at altitude of 994 m above sea level). The climate in Sebele is semi-arid with an average annual rainfall (30 year mean) of 538 mm (Bekker and de Wilt, 1991). Most rain falls in summer, which generally starts in late October and continues to March/April. Prolonged dry spells during rainy seasons are common and rainfall tends to be localized. The soils are shallow, ferruginous tropical soils, mainly consisting of medium to coarse grain sands and sandy loams with a low water holding capacity and subject to crusting after heavy rains. The soils are deficient in phosphorus, have low levels of mineral nitrogen and low organic matter (Persaud et al., 1992). The cowpea cultivars Blackeye and Tswana were used in the study because of their popularity and availability in Botswana. Before the start of the experiment soil was randomly sampled at 0-30 cm depth within the study field. The soil was analyzed in the laboratory for pH, cation exchange capacity, calcium, sodium, potassium, magnesium and organic carbon. The soil used for the study had the following properties: sodium, potassium, magnesium and calcium contents have 0.09, 0.39, 0.16 and 9.53 meq/100 g, respectively. The organic carbon and CEC contents were 1.04 and 7.53 meq/100 g, respectively and pH of 6.5.
Experimental design: The experiments were laid out in a completely randomized design with three replicates. Each replication was made up of 3 plots of 3x3 m. The cowpea plants were sprayed to run-off with Gibberellic acid (GA3) at 0, 30, 60 and 90 mg L-1 (GA3, a liquid concentrate containing 4% g ai (w/V), Abbott Laboratories, North Chicago, USA) using a pressurized hand knapsack sprayer. The control plants (0 mg L-1 GA3) were sprayed with distilled water.
Crop husbandry: All trials in the experiment used whole cowpea plants, with GA3 sprayed to run-off using a pressurized hand knapsack sprayer. The spacing was 30 cm between rows and 15 cm between plants and one seed was planted per hole. The 1st and 2nd trials were planted on 19th January 2007 and 31st January 2007, respectively. In the first trial, the cowpea cultivar Blackeye was used in the study. While in the second trial, the cowpea cultivar Tswana was used. No fertilizer was applied. Gibberellic acid was sprayed seven days after emergence. Weeds were controlled by hand cultivation (hoeing between and within rows) throughout the growing period.
Dependent variables determined: The dependent variables that were determined included: plant dry mass, shoot and root dry mass, shoot-to-root ratio, plant height, number of leaves/plant, leaf area, plants with 50% or more yellowing 66 days after emergence, height of first node, number of nodules/plant, number of pods per plant, pod length, number of seeds per pod, 100 seed weight, harvest index and seed yield. Plant height and 1st node height were determined by measuring the plant and node heights of 10 randomly selected plants per replicate using a 1 m steel ruler. The leaf number was determined by counting the number of leaves on 10 randomly chosen plants per replication. Leaf area was determined from 3 plants per replicate at the onset of flowering. The leaf area was determined using a leaf area meter (C1-202 CID, Scanman Logitech, Inc., USA). The fresh and dry weights of cowpea plants were determined by destructive harvesting of five plants per replication. The plants were watered 24 h earlier before they were carefully dug to ensure at least 99% of the roots were included. The shoots and roots of five plants per replication were separated and put in already weighed brown paper bags. The fresh shoot and root samples were weighed using Mettler PM 400 digital balance. The fresh mass was determined by subtracting the weight of paper bag from the weight of paper bag plus fresh sample. The plant samples (shoots and roots) were oven dried at 66°C to constant weight. The plant water content was determined by subtracting dry weights from their corresponding fresh weights. The total plant dry mass and water content was determined by adding the shoot and root dry mass and water contents, respectively. The shoot-to-root ratio was determined by dividing the corresponding shoot-dry mass with root-dry mass.
Pod length, number and number of seeds per pod were determined from 10-tagged plants per replication. Pod length was determined by measuring the length of 50 pods per replication using a 30 cm ruler. Pod number was determined by counting all the pods of the 10-tagged plants per replication. The number of seeds per pod was determined by counting the number of seeds per pod using 50 pods were replication. Hundred seed weight was determined by weighing 100 representative seeds per treatment per replication. The seed yield ha-1 was estimated from the seed yield of an area of 2 m2. The harvest index was determined by harvesting (uprooting) 10 whole plants at physiological maturity (roots, shoot, pods) per replication. The whole plants were oven dried at 66°C for 72 h. The whole dried plants were weighed to determine the whole plant mass. Then the pods were removed from the dried plants and threshed to separate the seeds. The seed mass of 10 plants was determined. The Harvest Index (HI) was estimated by diving the seed mass with the corresponding whole plant dry mass of 10 plants (seed mass/whole dry plant mass).
All plants in 9 m2 plots were observed for chlorophyll degradation in order to determine the number of plants with ≥50% leaf yellowing as a sign of senescence. The change in leaf colour from green to yellow was observed.
Data analysis: Analysis of variance was performed on the data collected using the general linear models (Proc GLM) procedure of Statistical Analysis System (SAS) program package. Where a significant F-test was observed, multiple comparisons among treatments was carried out using the Least Significant Difference (LSD) at p≤0.05. Linear, quadratic and cubic orthogonal polynomials were tested using appropriate regression models to examine the nature of the response of cowpea plants to increasing GA3 concentration (Snedecor and Cochran, 1989). Proc univariate procedure was carried out on residuals to support the assumptions of normality made by the researcher.
RESULTS
Vegetative growth: Application of GA3 to cowpea plants at
7 DAE significantly (p≤0.001) increased vegetative growth compared to control
plants (Fig. 1-4). Cowpea plant height and
first node height were significantly (p≤0.001) increased by GA3 (Fig.
1a, b). The response to increasing GA3 concentration
was linear and quadratic for plant height and first node height, respectively
(Fig. 1a, b). The effect of GA3
on increasing plant height was evident fours days after application (11DAE)
in Tswana cowpea (Table 1). The cultivar Tswana significantly
responded to GA3 application compared to Blackeye with respect to
increase in plant height (Fig. 1). Cowpea plants treated with
GA3 had significantly (p≤0.0001) higher leaf number and leaf area
per plant compared to control plants. The increase in leaf number and leaf area
per plant was linear with increasing GA3 concentration (Fig.
2, 3). At 20 mg L-1 GA3, the response
of both cowpea cultivars was relatively the same with respect to leaf number/plant,
but at higher GA3 concentration Blackeye significantly increased
leaf number/plant than Tswana (Fig. 2).
| Fig. 1a: | Effect of gibberellic acid on cowpea plant height |
| Fig. 1b: | Effect of gibberellic acid on first node height of cowpeas |
| Fig. 2: | Effect of gibberellic acid on number of cowpeas |
Dry matter partitioning and nodulation: Cowpea plants applied with GA3
had significantly higher dry matter contents in whole plant, shoot and root
than control plants. The cultivar Blackeye had significantly higher dry matter
than Tswana (Fig. 4, Table 1). The response
of Blackeye cowpea to increasing GA3 concentration was quadratic
with respect to dry matter accumulation (Fig. 4).
| Fig. 3: | Effect of gibberellic acid on leaf area of Blackeye cowpea |
| Fig. 4: | Effect of gibberellic acid on plant dry matter content of cowpea |
In the cultivar Tswana there were no GA3 concentration differences in respect to shoot and root dry matter accumulation. However, GA3 had relatively more dry matter accumulation in the shoots than roots. Gibberellic acid had no significant effect on Tswana cowpea tap root length (Table 1).
Application of GA3 at 30,60 or 90 mg L-1 on Blackeye cowpea plants significantly (p≤0.01) increased the number of nodules/plant compared to control plants. The response of Blackeye cowpea plants to increasing GA3 concentration with respect to nodulation was quadratic. However, GA3 had no significant effect on number of nodules/plant in Tswana cowpea plants (Fig. 5).
Yield components and yield: Gibberellic acid application to cowpea plants
significantly (p≤0.01) increased pod length compared to control plants. The
response to increasing GA3 concentration was quadratic in both Blackeye
and Tswana cowpea cultivars (Fig. 6). There were no GA3
concentration differences with respect to increase in cowpea pod length. Gibberellic
acid treated cowpea plants significantly (p≤0.01) produced more pod numbers/plant
than control plants (Fig. 7). The response to increasing GA3
concentration was linear and quadratic
| Table 1: | Effect of gibberellic acid on plant height, dry matter partitioning and senescence of cowpeas |
| *, **, ***, NS significant at p = 0.05, 0.01, 0.001, or non-significant, respectively. Means separated using the Least Significant Difference (LSD) at p = 0.05; Means within column(s) followed by the same letter(s) are not significantly different. DAE: Days After Emergence | |
| Fig. 5: | Effect of gibberellic acid on nodulation of cowpea |
| Fig. 6: | Effect of gibberellic acid on cowpea pod length |
in Blackeye and Tswana cowpea plants, respectively, in their ability to increase
pod number/plant. Cowpea plants applied with GA3 had significantly
(p≤0.05) higher seed number/pod than control plants. There were no GA3
concentration differences with respect to increasing seed number/pod (Fig.
8). The lower GA3 concentrations (30 and 60 mg L-1)
had no significant effect on 100 seed weight of Blackeye cowpeas compared to
control plants (Fig. 9). However, 90 mg L-1 GA3
significantly increased 100 seed weight of Blackeye cowpea plants compared to
control plants (Fig. 10). In Tswana cowpea plants, GA3
application significantly (p≤0.05) increased 100 seed weight compared to
control plants (Fig. 9).
| Fig. 7: | Effect of gibberellic acid on pod number of cowpea |
| Fig. 8: | Effect of gibberellic acid on pod seed number |
Cowpea plants sprayed with GA3 at 30, 60, or 90 mg L-1
yielded significantly (p≤0.01) more seed yield ha-1 than control
plants. The increase in seed yield induced by GA3 application was
linear and quadratic in Blackeye and Tswana cowpeas, respectively, with increasing
GA3 concentration (Fig. 10). Gibberellic acid
treated Tswana cowpea plants had a higher harvest index than control plants.
Application of GA3 on cowpea plants had no significant effect on
plant senescence (Table 1).
| Fig. 9: | Effect of gibberellic acid on 100 seed weight of cowpea |
| Fig. 10: | Effect of gibberellic acid cowpea seed yield |
DISCUSSION
The cowpea cultivars used in this study Blackeye and Tswana both have a determinate growth habit. Gibberellic acid application to cowpea plants increased plant and first node height. The GA3-induced increase in plant and first node (internode) height was attributed to the role of gibberellins in increasing cell elongation and division and internodal elongation (Taiz and Zeiger, 2002). Sachs (1965) and Sauter and Kende (1992) reported that gibberellins increased both cell elongation and division, as evidenced by increase in cell length and number in peas and rice, respectively. It has also been shown that mitosis increases markedly in the subapical region of the meristem of rosette long-day plants after treatment with gibberellins. Gibberellins might have increased cowpea plant height by increasing cell wall extensibility. Behringer et al. (1990) reported that in peas, gibberellins decreased the minimum force that will cause cell wall extension. Cowpea plants applied with GA3 had significantly higher leaf number/plant and leaf area. The increase in leaf number/plant induced by GA3 could possibly be explained by GA3 reducing the plastochron. The increase in leaf area of cowpea plants due to GA3 application could be due to the role of GA3 in promoting cell division and elongation (Taiz and Zeiger, 2002; Sauter and Kende, 1992). The differences in response to GA3 application by the two cowpea cultivars Blackeye and Tswana in all variables determined was due to differences in the genotypes.
In general, the rate at which a crop increases in dry weight depends on leaf area index and net assimilation rate. However, changes in leaf area index depend on growth in leaf area and plant senescence. In the current study, GA3 had no effect on cowpea plant senescence. The increase in plant dry matter in GA3-treated plants was attributed to the increase in leaf area and leaf number/plant. Large amounts of dry matter are produced when cowpea plants of determinate cultivars maintain relatively large and healthy leaf areas for prolonged periods (Summerfield et al., 1985; Littleton et al., 1979). Emongor (2002) reported that BA+GA4+7 increased dry matter accumulation in common beans (Phaseolus vulgaris), however, GA3 and GA4+7 had no effect on dry matter accumulation.
Legume plants develop a symbiotic interaction with rhizobia by forming root nodules in which the bacteria fix atmospheric nitrogen. Nodule formation integrates several developmental processes, such as induction of cortical and pericycle cell division and rhizobial invasion, which are coordinated in time and space (Lievens et al., 2005). The onset of symbiosis is marked by a complex exchange of signals, involving plant flavonoids and bacterial nodulation (Nod) factors. Recognition of specific nod factors will switch on the nodulation program in the legume host. Plant hormones control all developmental plant processes, including nodulation that is presumably initiated by a change in the cytokinin-to-auxin ratio within the root. Mathesius et al. (1998) reported that nod factor-induced inhibition of auxin transport led to the local accumulation of auxins needed to trigger nodule primordial. Cytokinins have also been reported to be involved in Nod factor induction because non-nodulating bacteria over producing isopentenyl transferase gene (Cooper and Long, 1994) as well as the exogenous application of cytokinin (Libbenga et al., 1973; Reli et al., 1994; Bauer at al., 1996) can provoke nodule-like structures on legume plants. In the current study, application of GA3 on Blackeye cowpea plants increased the number of nodules/plant compared to control plants. However, GA3 had no significant effect on number of nodules/plant in Tswana cowpea plants. This conflicting result in nodulation response by the cowpea cultivars to GA3 application in nodulation response was due to genotype differences between Blackeye and Tswana. This suggests that not all legumes react in the same way regarding to nodulation. Elevated gibberellin levels have been measured in nodules of lima bean (Phaseolus lunatus) and cowpea (Dobert et al., 1992). However, analysis of effects of exogenous gibberellins (GAs) on root nodulation yielded conflicting results (Bishnoi and Krishnamoorthy, 1990; Zhang et al., 1997). Exogenous application of GA3 has been reported to induce the formation of nodule-like structures on the roots of Lotus japonicus and the response was nitrogen sensitive (Kawaguchi et al., 1996). Some free-living rhizobia have the capacity to produce GAs (Atzorn et al., 1988; Tully et al., 1998), but they probably do not contribute significantly to the amount of GA within the nodule (Atzorn et al., 1988). Nodulation in some pea (Pisum sativum) lines mutated in GA synthesis was hampered, resulting in fewer, nonfunctional nodules, a phenotype that could be completed by exogenous application of GAs (Ferguson et al., 2005). Lievens et al. (2005) reported that GAs were involved in infection (Azorhizobium caulinodans) pocket and infection thread formation, two Nod factor-dependent events that initiate lateral root base nodulation in Sesbania rostrata and that GAs were also needed for nodule primordium development. They further reported that, GAs inhibited the root hair curling process. The results of Ferguson et al. (2005) showed that GAs, are Nod factor downstream signals for nodulation in hydroponic growth of Sesbania rostrata. The results of Dobert et al. (1992) support the hypothesis that the rhizobial strain modifies the endogenous GA status of the symbiotic system. This alteration in GA balance within the plant, presumably, underlies the observed growth response. Fletcher et al. (1959) reported that 25 ppm of GA3 had no effect on nodule numbers of Trifolium repens and up to 1000 ppm GA3 had no effect on the growth of Rhizobium trifolii. Williams and de Mallorca (1984) reported that foliar application of GA3 to soybean plants delayed the formation of nodule initials and reduced the numbers mass per nodule and specific gravity of the nodules, without affecting plant growth.
The increase in seed yield due to GA3 application observed in the current study was attributed to the enhanced vegetative growth (leaf area and leaf number per plant), dry matter accumulation and increase in yield components (pod length, number of pods/plant, seed number/pod and 100 seed weight). Emongor (2002) reported that GA3 and GA4+7 significantly reduced the seed yield and had no effect on seed number/pod and number of pods/plant of common beans (Phaseolus vulgaris). The variability in response by cowpea plants to GA3 application compared to common beans in respect to yield and yield components suggests that not all legumes respond similarly to plant growth regulators. Resmi and Gopalakrishnan (2004) reported that NAA (15, 30 or 45 ppm), 2,4-D (2 ppm) and CCC (300, 400 or 500 ppm) increased the seed yield, pod length, pod weight, pod number per unit area and pod number per plant of cowpea plants. Kannan et al. (2003) reported that application of NAA (30 ppm), mepiquat chloride (120 ppm) and a mixture of NAA (30 ppm) and mepiquat chloride (120 ppm) to blackgram (Vigna mungo) increased leaf area index, leaf chlorophyll content, photosynthetic rate and seed yield. The environment in which cowpeas are grown has been reported to be of great importance to cowpea seed yield (Smithson et al., 1980; Summerfield et al., 1983). This implies that breeding and selection may be more usefully directed to overcoming the constraints imposed by adverse environments than aiming for yield improvements in situations conducive to large yields. Since cowpea seed yield is influenced by leaf area index, net assimilation rate and yield components (number of pods/plant, seed number/pod, pod length and 100 seed weight), variables all enhanced by exogenous GA3 application, breeding and selection should be directed towards varieties probably high in Gas, auxins, cytokinins and abscisic acid (for semi-arid and arid regions).
CONCLUSION
The results of this study showed that exogenous application of GA3 can be used to increase vegetative growth, nodulation, yield components and seed yield of some cowpea varieties. However, more research needs to be undertaken in order to determine the optimum GA3 concentration on several cowpea varieties, timing of application, economics of using GA3 as a cowpea production input, as well as breeding and selection of cowpea varieties high in GAs and other phytohormones such as auxins, cytokinins and abscisic acid.
ACKNOWLEDGMENTS
The author is grateful to the Botswana College of Agriculture for the financial support and providing experimental materials, as well as Abbott Laboratories, North Chicago, USA, for supplying gibberellic acid.