INTRODUCTION
Yam (Dioscorea Spp.) is among the oldest recorded crops eaten in many continents (Dansi et al., Reference Dansi, Mignouna, Zoundjihekpon, Sangare, Asiedu and Quin1998). In West Africa, yam ranks second after cassava (FAO, 1997), providing millions of people with a source of nutritious diet and income. Its cultural value is high, especially in West Africa; e.g. in fertility and marriage ceremonies, and in many cultures, the New Yam Festival is celebrated at the beginning of the first harvesting season (about the months of August). Today, the use of yams is diverse: as raw material in bakeries, pharmaceutical, textile, and energy generating industries.
The yam growing season is long. In West Africa and Nigeria in particular, the main planting season begins in the month of February with the planting of sprouting tubers, and ends with harvesting of new tubers about the month of August (first harvest season) or between November and January of the subsequent year (second/ main harvest season). Harvested tubers serve dual purpose as a source of planting material/ seed tuber and food (Coursey, Reference Coursey1967; Hahn, Reference Hahn1995). Seed tubers refer to whole tubers or pieces of it that are set aside for planting (Orkwor and Ekanayake, Reference Orkwor, Ekanayake, Orkwor, Asadu and Ekanayake1998). Whether tubers are harvested in the month of August (i.e. about 180 days after planting) or November (i.e. about 270 days after planting), most of such tubers do not resume shoot growth/sprouting until about 210 days or 150 days respectively. The long wait for the resumption of sprouting, imposes the need for prolonged storage of seed tubers, restricts planting to once per annum, exposes up to 40% of highly-valued tubers to loss (due to pests and diseases during the compulsory storage period), exposes whole seed tubers to unplanned consumption, and these in turn contribute to high cost of yam production and low availability of tubers especially during the planting season (FAO, 2008; Onayemi, Reference Onayemi1983). The cost of planting material alone constitutes about 40% of the total cost of yam production (Nweke et al., Reference Nweke, Ugwu, Asadu and Ay1991; Ugwu, Reference Ugwu1990).
Tuber dormancy is the major cause of the prolonged inability of ware or seed tubers to sprout. Dormancy is an inherent plant physiological mechanism that regulates the timing of sprouting of affected plant parts (Craufurd et al., Reference Craufurd, Summerfield, Asiedu and Vara Prasad2001). The consequence of dormancy is severe in yam, because the duration of dormancy is very long; as much as 270 days, depending on the time of tuber harvest and the definition of the start of dormancy (Hamadina, Reference Hamadina2011a). According to Ile et al. (Reference Ile, Craufurd, Battey and Asiedu2006), dormancy in yam occurs in phases: long Phase I of dormancy (i.e. the period from some point around tuber initiation to the formation of the tuber germinating meristem-TGM, which is up to 200 days long), moderately long Phase II (i.e. the period from TGM to the initiation of foliar primordium-FP, which is about 40 days long) and a short Phase III i.e. the period from FP to the appearance of shoot bud on the surface of the tuber, which is only about 10 days long). Thus, two key approaches to solving the problem of dormancy in yam and its associated consequences are identified. These are: (1) induction of early sprouting through the prevention/inhibition of the initiation of dormancy in seed yams such that shoot growth/sprouting can resume soon after tuber formation or (2) shortening of the duration of dormancy such that shoot growth/sprouting can resume soon after the first harvesting season (i.e. 180–200 days after vine emergence), which coincides with the months of August–September for an early February planting and a late February vine emergence dates in Nigeria. From the findings of Ile et al. (Reference Ile, Craufurd, Battey and Asiedu2006), it is clear that a promising approach to solving the problem of long tuber dormancy in yam is one that is aimed at preventing the initiation of dormancy rather than to shorten the duration of the long Phase I of dormancy. Unfortunately, there are no known methods for preventing/inhibiting the commencement of Phase I of dormancy in seed yams.
This study supports that the onset of tuber dormancy in yam is linked to early tuber formation stages (Claassens and Vreugdenhil, Reference Claassens and Vreugdenhil2000; Ile et al., Reference Ile, Craufurd, Battey and Asiedu2006). It thus, hypothesizes that the onset of tuber dormancy, which is linked to early yam tuber formation stages, is regulated by endogenous plant growth regulators such as ABA and the application of an ABA inhibitor, before or just after seed tuber formation shortens drastically, the duration from vine emergence/sprouting of the older tuber to sprouting of the new tuber (seed tuber). ABA is an endogenous growth regulator that is reported to maintain and induce dormancy in potato (Claassens and Vreugdenhil, Reference Claassens and Vreugdenhil2000; Suttle and Hulstrand, Reference Suttle and Hultstrand1994). In an in vitro study reported by Hamadina et al. (Reference Hamadina, Craufurd, Battery and Asiedu2010), dormancy in yam micro tubers was prevented using 10 µM and 30 µM fluridone, but there are no reports on the effects of fluridone on seed yams in vivo. Fluridone is an agrochemical that putatively inhibits the biosynthesis of ABA, therefore, capable of controlling dormancy of affected plant tissues (Suttle and Hulstrand, Reference Suttle and Hultstrand1994).
The hydroponics system (water culture or medium culture) is widely used for small scale and commercial premium food crops production. In this study, the hydroponics system was used as a medium for manipulating tuber dormancy of seed yams. The advantage of this system in this study lies in the fact that it provides adequate nutrients for plant growth while allowing for easy inclusion of test chemicals into the nutrient solution. Furthermore, the probability that growing plants will absorb the test chemical alongside the nutrient solution is higher in a hydroponics system than in the use of other methods such as: injecting of test chemical(s) into plant tissue or the spraying of same on leaf surfaces. The choice of coco coir medium culture hydroponics system, in this study, is due to its inert nature in nutrient solution and its capacity to provide support for plant root system. Therefore, this study was conducted to determine the effects of fluridone in the induction of sprout/shoots on developing seed tubers of yams, and to evaluate the effect of fluridone on early shoot growth.
MATERIALS AND METHODS
Study environment
This study was conducted in a screen-house at the Faculty of Agriculture, University of Port Harcourt, Rivers State, Nigeria. The temperature and relative humidity of the screen-house were monitored daily using a cable free weather station (model WMR968) equipped with a digital sensor (model BTHR968) to provide some information on macro-environmental conditions during the study. Temperature in the screen house ranged from 24 °C–38 °C with an average temperature of 32 °C while relative humidity ranged from 45–77%. This study was conducted during the raining season, beginning in the month of May.
Planting materials
Two species of yam were used in this study Dioscorea rotundata var. TDr 131 and Dioscorea alata var. TDa 98/01166. Non-dormant tubers of these species were obtained from the International Institute of Tropical Agriculture (IITA) Ibadan, Nigeria. These varieties were chosen because they both exhibit long tuber dormancy, dormancy in TDr 131 had been prevented, in vitro using fluridone (Hamadina et al., Reference Hamadina, Craufurd, Battery and Asiedu2010), while TDa 98/01166 has potential commercial value as source of flour for confectionary. Therefore, eliminating dormancy period in TDa98/01166 could lead to more plantings per year, hence reduce the pressure on D. rotundata for uses other than food.
Preparation of minisetts and pre-sprouting of minisetts
The non-dormant whole tubers collected for this study weighted between 50 and 200 g. The tuber-head; described by Hamadina (Reference Hamadina2011a, Reference Hamadinab) as the corm-like structure attached to the proximal/head region of the tuber, was detached from the tuber (where present) to inhibit apical dominance and increase the propensity of shoot emergence from other regions of the tuber. Each tuber (i.e. tubers whose tuber-head was detached) was cut longitudinally into six setts. Each sett was cut continuously at the distal-middle region end until it weighed 25 g. Because the head region of tubers tend to sprout faster than the distal or middle regions (Orkwor and Ekanayake, Reference Orkwor, Ekanayake, Orkwor, Asadu and Ekanayake1998), all the minisetts (25 g setts) used in this study had a head region but not necessarily a tail region. The cut areas of the minisetts were treated with wood ash to minimize rotting (Orkwor and Asadu, Reference Orkwor, Asadu, Orkwor, Asadu and Ekanayake1998). To pre-sprout the minisetts, they were sown in baskets containing moist sawdust in the month of May. The minisetts were observed daily, and the dates of vine emergence were noted. When most minisetts had sprouted, (after 9 days), the sprouted minisetts were planted in black polypots (containing 500 g surface soil). Watering was done when necessary. The sprouted minisetts were grown in polypots for 69 days to enable them root before transferring of the plants to the hydroponics system.
The hydroponics system
A coco coir medium culture hydroponics system was used for this study. The coco coir was gotten by dehusking and shredding mature (12 months old) coconuts. The coco coir was then soaked in water for two consecutive 24-hr periods to leach off any inhibitory substances. Thereafter, the coco coir was disinfected by soaking in 5% sodium hypochlorite for 24 hours and then rinsed in clean water. The disinfected coco coir was air-dried and then placed into perforated black pots (∅12.5 cm). The bottom 2.5 cm of the pot was not perforated to enable the pots hold up to 600 ml of liquid.
After 69 days of growth in soil, the plants were transplanted. Soil was washed away from the roots then, the soil-free roots of each plant was placed in the 200 ml of water contained in each pot with the plant held at such height that new tubers would not make contact with the water and then, disinfected coco coir was placed in the pot to help hold the plant in position. The vines were trained on a twine suspended from the roof of the screen house. The experimental treatment commenced a day after transplanting, when the water in each pot was removed and replaced with 100 ml of respective treatment solution.
EXPERIMENTAL TREATMENT AND DESIGN
Two species of yam that exhibit long tuber dormancy were tested at three concentrations of fluridone plus control (nutrient solution without fluridone). Thus, there were eight treatment combinations: (1) TDr 131 + nutrient solution (Control), (2) TDr 131 + nutrient solution with 30 µM fluridone, (3) TDr 131 + nutrient solution with 50 µM fluridone, (4) TDr 131 + nutrient solution with 100 µM fluridone, (5) TDa 98/01166 + nutrient solution (Control), (6) TDa 98/01166 + nutrient solution with 30 µM fluridone, (7) TDa 98/01166 + nutrient solution with 50 µM fluridone, and (8) TDa 98/01166 + nutrient solution with 100 µM fluridone. Hoagland's nutrient solution was used in this experiment, which was prepared following the procedures of Hoagland and Arnon (Reference Hoagland and Arnon1950).
The experiment was a 2×4 factorial arranged in a CRD with three replications and six plants per replicate. The factors were two yam species and four fluridone (0 µM, 30 µM, 50 µM, 100 µM) resulting in eight treatment combinations.
DATA COLLECTION
At the start of this experiment, i.e. when plants were transferred into the hydroponics system, the plants were observed for presence or absence of tubers. Where tuber formation was observed, the size(s) of the bulge/tuber mass was noted, and they were observed for any signs of sprouting e.g. the presence of sprouting loci or shoot bud/sprout. Fifteen (15) leaves were randomly taken per variety for determination of leaf chlorophyll content following the method of Comar and Zscheile (Reference Comar and Zscheile1942).
Weekly, after the start of the experiment, data on the number of leaves, leaf length, and width were recorded. At the first clear evidence of a fluridone effect (that is bleaching of vegetative parts), one plant/treatment/replicate was sampled. All tubers produced by the sampled plants were detached from the main vine and examined for any signs of sprouting. If such young and small tubers are found to show any of the external signs of sprouting, then such tubers are said to have unusually lost the capacity to be dormant (Hamadina et al., Reference Hamadina, Craufurd, Battery and Asiedu2010). The fresh weight of the sampled tubers were taken, and then the plants were further partitioned into their component parts, their fresh weights noted and the oven-dried (at 60 °C) to constant weight. At the end of the experiment (i.e. at five weeks after fluridone treatment), 15 leaves were randomly sampled per treatment for the analysis of chlorophyll content. Also, two plants/treatment/replicate were sampled for the assessment of tuber weight, and for signs of sprouting.
DATA ANALYSIS
Data collected were analysed using the two-way analysis of variance tool run on Genstat software (Release 10.3DE, Edition 4). Count data was transformed using the square root transformation, while treatment means were separated using Standard Error of Difference (SED).
RESULTS
Effect of fluridone on plant morphology
In the first two weeks after treatment (WAT), no clear visible effect of fluridone (FLU) was observed on any vegetative part of both species of yam. By 3 WAT, however, all concentrations of fluridone tested (30 µM, 50 µM, and 100 µM) resulted in the bleaching of leaves in both species (Figure 1). The bleaching was more pronounced in Dioscorea alata (TDa 98/01166) as >50% of the surface of most leaves appeared whitish, particularly along the leaf veins. In both species, the petioles and portions of the stem appeared purplish, with more intense purple coloration in TDr 131 than in TDa 98/01166. Due to the observed bleaching effect of fluridone, an intuitive analysis of leaf chlorophyll content was conducted. The result showed that percentage chlorophyll in the Controls ranged from 0.010–0.034 in both species. Percentage chlorophyll in the leaves of plants grown in 30 µM and 50 µM fluridone treatments ranged from 0.067–0.125 while the values ranged from 0.001–0.017 in leaves of plants grown in 100 µM fluridone.
Figure 1. Bleaching effect of fluridone on TDa 98/01166 (a) and TDr 131 (b) plants.
In the control, the newly produced seed tubers had adventitious roots around their surfaces and they were attached to a small corm-like structure at their proximal ends (Figure 2). The main shoot/vine was attached to a small corm-like structure but there were no new shoot(s)/vines. The absence of new shoot(s)/sprout on the small corm-like structure and/or from the surface of the tuber close to the point of attachment of the corm-like structure to the tuber is an external indication that the intact tubers were dormant. In contrast, many of the seed tubers that developed in fluridone treatments showed signs of renewed vegetative growth (Figure 3). In these treatments, the new shoot(s) developed from the small corm-like structure and/or from the surface of the tuber close to the point of attachment of the corm to the tuber. The number of new shoots varied from 1–7 shoots per plant or per tuber. These features are external characteristics of non-dormant intact tubers.
Figure 2. External characteristies of an intact dormant seed tuber of D. rotundata attached to the main vine. No new shoots are present.
Figure 3. External characteristics of intact non-dormant seed tubers produced five week after treatment (i.e. at 104 days after vine emergence) in nutrient solution containing fluridone. New shoots present on the seed tuber.
Effect of fluridone and species on number and earliness of new shoots
By 3 WAT (i.e. 90 days after vine emergence) when bleaching was observed in all fluridone treatments, some of the newly produced tubers under some fluridone treatments had already commenced sprouting, while those in the control had not (Table 1). Also, a greater number of new shoots were observed in TDa 98/01166 than TDr 131. However, the observed differences in number of new shoots produced did not vary significantly (p < 0.05) with species and with fluridone concentration.
Table 1. Number of shoots produced as influenced by species, fluridone, and their interaction at 3 and 5 weeks after treatment.
At 5 WAT (i.e. 104 days after vine emergence), most of the tubers sampled from the fluridone treatments had new shoots while those from the Controls had none. The interaction effects of species and fluridone was significant (Table 1). In both TDa 98/01166 and TDr 131, only the tubers that developed in fluridone had new shoots. The effect of fluridone was more pronounced in TDa 98/01166, with up to 7 new shoots per tuber, than TDr 131. New shoots were observed on all fluridone treatments in TDa 98/01166, while in TDr 131, no new shoots were observed at 50 µM fluridone. Also, the number of new shoots on TDa 98/01166 seed tubers appeared to increase with fluridone concentration, but fluridone concentrations greater than 30 µM appeared to suppress the formation of new shoots on TDr 131.
Effect of fluridone on vegetative growth
Leaf width and length
At 1 WAT, leaf width of TDr 131 was significantly wider (by 0.55 cm) than that of TDa 98/01166 (Table 2). However, fluridone concentrations had no significant effects on leaf width in the two species of yam. Leaf length was significantly (p < 0.01) longer in TDa 98/01166 than TDr 131 at 1 WAT, (Table 2) but fluridone concentrations had no significant effects on leaf length in the two species of yam.
Table 2. Species effect on mean leaf width at 1 week after treatment.
Between 2–5 WAT, the leaf width of each species was dependent on the interaction of species and fluridone concentration (Table 3). At 2 WAT, leaf width of TDr 131 was significantly increased by growing the plants in 30 and 100 µM fluridone but not in TDa 98/01166. At 3 WAT, the leaf width of TDa 98/01166 plants grown in 100 µM fluridone was significantly wider than that of the Control. In contrast, the leaf width of TDr 131 plants grown in 100 µM fluridone was significantly narrower than the control, and the widest leaf width was observed at 30 µM fluridone. At 4 and 5 WAT, leaf width of TDa 98/01166 was significantly (p ≤ 0.001) narrower in fluridone treatments than in the Control, particularly at 30 and 50 µM fluridone, while leaf width of TDr 131 was significantly (p ≤ 0.001) higher in fluridone treatments than the Control, particularly at 30 µM. In the control, TDa 98/01166 had wider leaf width than TDr 131.
Table 3. Leaf width and length of yam species as influenced by fluridone concentration at 2–5 WAT.
The interaction effect of species and fluridone concentration on leaf length was significant (p < 0.05) at 3, 4, and 5 WAT (Table 3). At 3 WAT, TDa 98/01166 had the longest leaf length in 100 µM fluridone while in TDr 131, the longest leaf length was observed at 30 µM. At 4 and 5 WAT, TDa 98/01166 had longer (p < 0.01) leaf length than TDr 131 in the controls (Table 3). Leaf length of TDa 98/01166 plants was significantly shorter in 30 and 50 µM fluridone compared with the control, while in TDr 131, leaf length was longer in 30 and 50 µM fluridone compared with the control.
In general, TDr 131 had wider leaves than TDa 98/01166 while TDa 98/01166 had longer leaves than TDr 131. In TDa 98/01166, leaf width and length declined mostly at 30 and 50 µM fluridone while in TDr 131, leaf width and length increased mostly at 30 µM fluridone.
Number of leaves
There were no significant differences in the number of leaves across species or fluridone concentrations at 1 WAT. At 2 WAT, however, the mean number of leaves in TDa 98/01166 was significantly (p < 0.01) fewer (4.14; square root transformed) than TDr 131 (4.82); SED 0.26. Nevertheless, fluridone treatments did not differ significantly at 3, 4, and 5 WAT in terms of number of leaves.
Root and stem dry matter content at 3 WAT
Root dry weight in the Control was higher in TDa 98/011166 than TDr 131 (Table 4). Although fresh root mass appeared thicker in fluridone treated plants than the Control, root dry weight of TDa 98/01166 was lower in all concentrations of fluridone compared to the Control. In contrast, in TDr 131 root dry weight was higher in fluridone (particularly at 30 µM fluridone) than the Control. Fluridone did not significantly affect stem dry weight of either TDa 98/01166 or TDr 131.
Table 4. Effect of species and fluridone on root dry weight at 3 WAT.
Effect of fluridone on tuber weight at 5 WAT
The mean fresh tuber weight per plant of TDr 131 was almost two times heavier (p = 0.001) than that of TDa 98/01166 (Table 5). Although tuber weight of both species was lower in fluridone compared to the Control, this difference in fresh tuber weight was not significant and there were no significant interactions between species and fluridone concentrations.
Table 5. Species effect on fresh tuber weight at 5 WAT.
DISCUSSION
In this study, 69 days old D. alata and D. rotundata plants (that were in the process of tuber initiation or had just produced small seed tubers weighing <2.0 g) were grown in a hydroponics system containing nutrient medium, with or without fluridone for five weeks to induce early sprouting by inhibiting the development of dormancy. In both D. alata and D. rotundata, as many as eight unusually early shoots per tuber were observed on tubers that developed in fluridone treated nutrient solution at 90 and 104 days after vine emergence. This unusually early appearance of new shoots on young developing tubers suggests that tubers intended for use as seed tubers can be made to avoid the expression of dormancy by allowing the plants to absorb fluridone during tuber initiation, or early bulking stages. This implies that seed yam tubers can be induced to commence vegetative growth within the first 30 days of tuber formation, perhaps before the induction of endo-dormancy described by Ile et al. (Reference Ile, Craufurd, Battey and Asiedu2006).
The ability of fluridone to successfully induce such early sprouting observed in this study may relate to its ability to inhibit the biosynthesis of ABA, a prime plant growth hormone associated with the induction and maintenance of dormancy in tubers (Suttle and Hulstrand, Reference Suttle and Hultstrand1994). Fluridone is shown to act by inhibiting ABA biosynthesis through the inhibition of the activity of the enzyme phytoene desaturase (PDS), which is responsible for converting phytoene to carotenoids-the precursor of ABA (Mulwa and Nwanza, Reference Mulwa and Nwanza2006; Sanderman and Boger, Reference Sanderman, Boger, Boger and Sanderman1989; Sandmann and Mitchell, Reference Sandmann and Mitchell2001). Many studies have also shown that ABA plays an important role in dormancy of several species/ dormant organs (Kim et al., Reference Kim, Davelaar and De Klerk1994; Suttle, Reference Suttle2004; Yamazaki et al., Reference Yamazaki, Nishijima, Yamato, Hamano and Miura1999). In addition, we propose that its ability to bring about almost instantaneous sprouting in this study relates to its absorption during early tuber initiation and development. In potato and yam, the commencement of tuber dormancy has also been linked to early tuber initiation and development (Claassens and Vreugdenhil, Reference Claassens and Vreugdenhil2000; Hamadina, Reference Hamadina2011a; Ile et al., Reference Ile, Craufurd, Battey and Asiedu2006; Swanell et al., Reference Swanell, Wheeler, Asiedu and Craufurd2003). Furthermore, Ile et al. (Reference Ile, Craufurd, Battey and Asiedu2006) and Hamadina (Reference Hamadina2011b) have argued that dormancy can be prevented or its duration can be drastically shortened, inducing very early sprouting if ABA biosynthesis is inhibited during tuber initiation and development rather than later. Moreover, spontaneous sprouting was reported on micro tubers (in vitro yam tubers) that developed in fluridone (Hamadina et al., Reference Hamadina, Craufurd, Battery and Asiedu2010) while only marginal success have been obtained when sprout promoting chemicals were applied during later stages of yam tuber development (Craufurd et al., Reference Craufurd, Summerfield, Asiedu and Vara Prasad2001; Hamadina and Craufurd, Reference Hamadina and Craufurd2015).
The bleached leaves and purplish stems observed in this study is a characteristic effect of fluridone, which is associated with the accumulation of the whitish hydrophobic substance phytoene rather than the inhibition of chlorophyll (Mulwa and Nwanza, Reference Mulwa and Nwanza2006). This study does not provide conclusive explanation on how fluridone might act on yam. However, intuitive analysis of leaf chlorophyll content of fluridone treated and untreated plants suggest that fluridone at concentrations below 50 µM may not inhibit chlorophyll biosynthesis. Further studies are however required to systematically evaluate the effect of fluridone on leaf chlorophyll content.
The slight reduction in tuber weight observed in the fluridone treated seed tubers is likely related to the utilization of stored reserves for sprouting. Moreover, tuber weight of stored yams is known to reduce sharply with the onset of sprouting (Mijinyawa and Alaba, Reference Mijinyawa and Alaba2013). Also, the presence of larger tubers in TDr 131, which was less affected by fluridone, than TDa 98/01166 might be explained by the presence of fewer new shoots, higher root activity (represented as high root dry matter content) and higher leaf chlorophyll content. Although both species of yam can be induced to commence shoot growth soon after tuber formation using fluridone, seed tubers of TDa 98/01166 will sprout in 30–100 µM fluridone while TDr will sprout more in 30 µM fluridone and perhaps at lower concentrations of fluridone. Further studies to determine the relationship between fluridone concentration and ABA concentration in plant parts is recommended. Also, methods order than the use of fluridone to inhibit dormancy and induce sprouting soon after tuber initiation may be sought.
In conclusion, this study represents a novel protocol for the successful induction of shoot production within 30 days of tuber formation. It has shown that seed yam tubers can commence sprouting soon after tuber initiation by allowing them to absorb fluridone prior to or during early tuber formation. This method shortens the duration from vine emergence to sprouting by more than half. Furthermore, because fluridone inhibits ABA biosynthesis, implies that ABA plays a role in the induction and maintenance of yam tuber dormancy. The practical implication of this study is the potential to lead to rapid production of yam seedlings and seed tubers any time of the year.
Acknowledgement
The authors wish to thank IITA Ibadan, Nigeria, for providing the tubers, and Biogeochem Associates Ltd, Nigeria for providing some of the research equipment used for this study.
