In vitro effects of gibberellic acid and sucrose concentration on micropropagation of two elite sweet potato cultivars in Rwanda

Faculty of Agriculture, National University of Rwanda (NUR), P. O. Box117, Huye, Rwanda. Rwanda Agriculture Board (RAB), P. O. Box 5016. Kigali, Rwanda. World Agroforestry Centre (ICRAF) Cote d’ Ivoire Country Program Cocody Mermoz, Abidjan, Côte d’Ivoire | 08 BP 2823 ABIDJAN 08. International Potato Center (CIP), Sub-Saharan Africa Region, P. O. Box 25171, Nairobi, Kenya. Jomo Kenyatta University of Agriculture and Technology, Box 62000, Nairobi, Kenya. Centre National de Recherche Agronomique (CNRA), Laboratoire Central de Biotechnologies (LCB), 01 BP 1740 Abidjan 01, Côte d’Ivoire.


INTRODUCTION
Sweet potato (Ipomoea batatas) is a high yielding crop which is ranked second in the world after potatoes (Deng et al., 2012).It belongs to the family Convolvulacea (Xiansong, 2010) and it originated in America (Burden, *Corresponding author.E-mail: janekahia@yahoo.co.uk. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 2005).Sweet potato is a major food; feed and industrial raw material in China whose total output in the world is estimated to be more than 80% (Farmer et al., 2007;Islam, 2006;Liu, 2011).Studies by Islam (2006) have revealed that sweet potato leaf extracts contains, antimutagenic, anticancer and antibacterial properties.Sweet potato is propagated by stem cuttings in Rwanda.This method of propagation is associated with increase in viral load over time on the planting materials and bacterial diseases that affect sweet potato in the major production zones of the country (ISAR, 2008).To eradicate these constraints, there is a need to develop and transfer new techniques for producing pathogen-free clonal planting materials which can help to significantly increase the potential yield of sweet potato (Zhang, 1995).
Multiplication by tissue culture techniques provides a viable alternative to the traditional methods of sweet potato propagation (Bachou, 2002) and could permit the production of relatively uniform plants on a massive scale in a shorter period of time (Mutandwa, 2008).In-vitro propagation of clonally propagated crops offers promise for rapid multiplication of quality planting materials and sustained optimal agricultural productivity.The production of plants in vitro is independent of season and can continue throughout the year.Sweet potato plants propagated by tissue culture mature earlier are more robust; leading to accelerated growth than the plants propagated through conventional methods (ASARECA, 2008).
Nodal explants are occasionally cultured on media supplemented with GA 3 to increase the length of shoots during multiplication or prior to rooting (Moshkov et al., 2008).
The most characteristic effects of GA 3 on shoot growth are increased inter-node extension, increased leaf growth and enhance apical dominance.The elongated shoots are then subdivided to serve as starting mother stock culture for another multiplication cycle.
The concentration of sucrose is one of the factors controlling the induction and growth of in vitro shoots (Gibson, 2000;Gurel and Gulsen, 1998).The optimum sucrose level for shoot development may vary among species and genotypes (Nowak et al., 2004).The concentration at which sugar is used has a great impact on the photosynthetic abilities of plantlets (Desjardins et al., 1995) and the relative success of subsequent acclimatization process.The optimum sucrose concentration as an efficient carbon source has been examined in tissue cultures systems of some plant species, such as Coffea Arabica cultivar Ruiru 11 (Kahia, 1999), Paederiafoetida (Amin et al., 2003) and Elaeocarpus robustus (Rahman et al., 2004).The current study aimed at evaluating the effect of different concentrations of gibberellic acid (GA 3 ) and sucrose on microshoots proliferation in two elite sweet potato cultivars.

Plant materials
The study was carried out at the plant tissue culture laboratory of Rwanda Agriculture Board (RAB) located in Rubona, Southern Province of Rwanda (Altitude: 1630 m 2°29'07''S, 29°47'49''E).The two sweet potato cultivars were initially propagated in vitro by nodal culture at the Kenya Agricultural Research Institute, Muguga Plant Quarantine Services in Kenya and distributed to Rwanda through the Rwanda Agriculture Board.

Preparation of media
In the first experiment, nodal explants were cultured in Murashige and Skoog (MS) (1962) media supplemented with GA3 evaluated at 2.5, 5, 10, 20 and 40 μM, ascorbic acid (100 µM), calcium nitrate (50 µM ), L-arginine (50 µM), 100 mg/l myo-inositol and 90 mM sucrose.In the second experiment, MS media was supplemented with 30,60,90,120,150,180 and 210 mM sucrose.The medium pH was adjusted to 5.8 before gelrite was added and media heated to dissolve it.Ten (10) ml MS medium with supplements was dispensed into 25 x125 mm test tubes and steam sterilized in an autoclave at 1.06 kg cm 2 and 121ºC for 15 min.

Inoculation and incubation
Inoculation was carried out under aseptic conditions in a laminar airflow hood in the laboratory in Rubona.Explants with two nodes were dissected from in vitro growing cultures using sterile blade and forceps and the leaves were cut off.They were cultured into test tubes containing 10 ml medium under evaluation.Twenty (20) test tubes were used per treatment and these were sealed with parafilm before incubating them in a growth room maintained at 25 ± 2°C under the cool white fluorescent lights and 16 h photoperiod with a photon flux density of about 60 μ mol m -2 s -1 and 70-80% relative humidity.

Transplanting
The in vitro regenerated sweet potato plantlets were carefully removed from the test tubes and the roots gently cleaned with running tap water to remove the gelrite.The plantlets were then taken to the green house where they were soaked with 2% fungicide (Redomil) for 20 min.A weaning pot was filled with sterile potting mixture consisting of top soil, sand and manure mixed in the ratio of 3:2:1(w/w).The vessel was placed in a basin containing water to allow the potting mixture to take up water until the top became moist.The pot was then removed from the basin and the plantlets carefully planted using sharp wooden sticks.The plantlets were irrigated once a week with tap water for the first two weeks and twice a week thereafter.

Data collection
Collection of data on the number of microshoots, roots and their lengths was carried out one week after inoculation and on a weekly basis for four weeks during incubation.While in the glasshouse the number of surviving plantlets was observed.

Experimental design and data analysis
The trials were laid out in a Completely Randomized Design.The number of shoots and roots was counted while the length was  measured in centimeter.The collected parameters were analyzed using SAS software (SAS Institute, 2001).Analyses of variance (ANOVA) were computed for each parameter and the data were summarized as mean ± SE of each parameter and presented in tables.Means were separated using least significant difference test (LSD) with 5% of level of significance

RESULTS
The effects of GA 3 on shoot proliferation and root growth from Ukerewe nodal explants is presented in Table 1.GA 3 at all the concentrations evaluated did not have a significant difference in the number of microshoots per explant.However, GA 3 evaluated at a concentration of 10 µM yielded the highest mean shoot length (2.78 ± 0.36) which was significantly different (P<.0001) from all other concentrations evaluated.The highest mean number (7.43 ± 0.69) of roots was observed on medium supplemented with 20 µM GA 3 .
The effects of GA 3 on shoot proliferation and root growth from Gihingamukungu nodal explants is presented in Table 2.The media supplemented with 20 µM GA 3 produced the highest (5.57± 0.69) mean microshoot which was not significantly different from all other GA 3 concentrations evaluated.Explants cultured on medium supplemented with 2.5 µM GA 3 produced microshoots with the highest (3.23 ± 0.40) mean length.On the other hand, the highest (4.36 ± 0.39) mean number of roots was achieved on medium supplemented with 40 µM GA 3 which was not significantly different from the numbers obtained with 2.5, 10 and 20 µM.Plate 1A shows the regenerated plantlets from explants cultured on medium supplemented with different GA 3 concentrations.
The effects of different concentrations of sucrose on shoot proliferation and root growth from Ukerewe sweet potato nodal explant is presented in Table 3. Increasing the concentration of sucrose from 30 to 150 mM increased the number of microshoots fivefold.The media supplemented with 180 mM sucrose produced the highest (5.93 ± 0.62) mean number of microshoots per explant which was not significantly different from the numbers produced on medium supplemented with sucrose 120, 150 and 210 mM.Nodal explants cultured on media supplemented with 150 mM sucrose produced the highest (2.51 ± 0.26) mean shoot length which was significantly higher at P <.0001 than the length obtained using the lower sucrose concentrations (30, 50 and 90 mM).The highest (7.47 ± 0.74) mean number of roots was produced on the medium supplemented with 210 mM sucrose.
The effects of sucrose concentration on shoot  proliferation and root growth from Gihingamukungu nodal explants is presented in Table 4.The media supplemented with 210 mM sucrose produced the highest (4.93 ± 0.36) mean number of microshoot per explant.On the other hand, nodal explant cultured on media supplemented with 150 mM sucrose produced the highest (2.34 ± 0.24) mean shoot length and highest (5.30 ± 0.42) mean number of roots.Rooting was observed in all the cultures (Plate 1B).The effect of GA 3 and sucrose on regeneration efficiency in the two cultivars is presented in Table 5.There were no significant difference in the responses of two cultivars to the gibberellic acid concentrations evaluated in terms of the number and length of microshoots.The effect of sucrose was significantly higher in cultivar Ukerewe compared to Gihingamukungu for all the parameters evaluated.The plantlets were successful weaned in the greenhouse with an average 60% survival rate (data not shown)

DISCUSSION
The production of plants from nodal explants has proven to be the most generally applicable and reliable method of regenerating true-to-type in vitro plantlets (George et al., 2008).The elongation of microshoot is one of the crucial aspects in developing protocols for regeneration of sweet potato.In order to promote elongation and accelerate microshoot development, and gibberellic acid (GA3) is sometimes added to the culture medium.In previous studies on micropropagation of sweet potato, the effect of GA 3 on elongation of microshoot has been evaluated and it was used at various concentrations e.g.40 mg/L (Dagnino et al., 1991) and 0.02 g/L (Luo et al., 2006).During the current study, GA 3 promoted induction and elongation of microshoots in both the cultivars evaluated.These results were interesting as Dagnino et al. (1991) reported that GA 3 had no influence on culture of sweet potato meristem of cultivar curacao Alado A B  whereas meristem of cultivar Mae de Familai cultured on medium supplemented with GA 3 formed multiple shoots.
In cassava, Villaluz (2005) reported that when used singly, GA 3 did not elicit any growth response from nodal explants however, Mushiyimana et al. (2011) reported that nodal explants cultured on MS medium supplemented with 40 µM GA 3 produced the highest mean number of microshoots and length.A possible explanation for such disparity seems to be genotypic difference.During the current study, prolific rooting was achieved in all the cultures.The reason for this could be the fact that GA 3 and sucrose are known to induce roots (Kochba et al., 1973;Calamar and De klerk, 2002).The presence of roots is of paramount importance because a good root system is essential for successful acclimatization of the plantlets and subsequent growth in the field since roots facilitate the absorption of nutrients and water from the soil (Xiansong, 2010).
In the current study, increasing the concentration of sucrose from the 90 mM (the concentration that is routinely used) resulted in increased mean number of microshoots per explant for both sweet potato cultivars.
The results of the current work agree with earlier reports that indicate that there are cases where higher concentrations > 3% of sucrose may be beneficial (Boggetti, 1997;Kahia, 1999;Van Huylenbroeck and Debergh, 1996).However, it contradicts other reports which have shown that high sucrose concentration was detrimental to shoot formation.These authors have suggested that the osmotic level in the medium with high sucrose concentration may be inhibitory to shoot growth and development (Karimet al., 2007;Nowak et al., 2004).
A genotype dependent one step protocol for the micropropagation of elite sweet potato cultivar Ukerewe, and Gihingamukungu, was developed.This protocol will provide the basis for the mass production of studied cultivars through in vitro techniques.This is the first report on evaluating the effect of different concentrations of sucrose to regenerate sweet potato plantlets.
Regenerated sweet potato plantlets from nodal explants cultured on 2.5, 5, 10, 20 and 40 µM gibberellic acid B: In vitro regenerated plantlets showing profuse rooting on media supplemented with sucrose 120, 150 and 210 mM (from left to right).

Table 1 .
Effect of different GA 3 concentrations (µM) on in vitro shoots proliferation and roots regeneration of Ukerewe sweet potato.

Table 2 .
Effect of different GA 3 concentrations (µM) on in vitro shoots proliferation and roots regeneration of Gihingamukungu sweet potato.
Values represent means ± SE.Means within a column followed by different letters are significantly different at P = 0.05.LSD: least significant difference test (LSD).

Table 3 .
Effect of different sucrose concentrations (mM) on in vitro shoots proliferation and roots regeneration of Ukerewe sweet potato.

Number of microshoots Length of shoots (cm) Number of roots Length of roots (cm
Values represent means ± SE.Means within a column followed by different letters are significantly different at P = 0.05.LSD: least significant difference test (LSD).

Table 4 .
Effect of different sucrose concentrations (mM) on in vitro shoots proliferation and roots regeneration of Gihingamukungu sweet potato

Table 5 .
Comparing the effect of GA 3 and sucrose on shoot proliferation and root Regeneration of Ukerewe and Gihingamukungu sweet potato cultivars

and sucrose Number of shoots Length of shoots (cm) Number of roots Length of roots (cm)
3 : gibberellic acid, LSD: least significant difference test (LSD).