In this study, meristem culture and chemotherapy has been used to produce virus free planting materials of four sweet potato varieties. The essence of meristem culture is the excision of the organized apex of the shoot from a selected donor plant for subsequent in vitro culture. The culture conditions are regulated to allow only for organized outgrowth of the apex directly into a shoot, without the intervention of any adventitious organs [16].
The excised meristem is typically small (often less than 1.0 mm) and is removed by sterile dissection under microscope. A major advantage of working with such a small explant is the potential that it holds for excluding pathogenic organisms that may have been present in the donor plants from the in vitro culture. The second advantage is the genetic stability inherent in the technique, since plantlet production is from an actively dividing apical meristem [17]. Shoot development directly from the meristem avoids adventitious organogenesis, ensuring that genetic instability and somaclonal variations are minimized.
Chemotherapy techniques drew much attention to get virus free varieties of sweet potato. The technique employs the application of different antiretroviral analogs in tissue cultures to retard or hinder viral replications in explants used.
Shoot initiation from meristem explants depends on many factors. The main factors that affected shoot initiation in our study were the genotype and the concentrations of growth regulators. Thirteen different growth regulators concentrations and combinations on MS medium were evaluated to get shoots from meristem. The first two combinations (1.0 mg/l BAP, 1.0 mg/l GA3, 0.01 mg/l NAA and 1.0 mg/l BAP, 2.0 mg/l GA3, 0.01 mg/l NAA) resulted in relatively better number of shoots for the three varieties, ‘Beletech’, ‘Ogensegen’ and ‘Koka -12’. However, in both combinations, large number of callus was induced by ‘TIS-8250’ variety. It could be suggested that sensitivity to growth regulators is affected by the endogenous levels of hormones and it is reasonable to assume that differences in response among different sweet potato varieties resulted from the genetic differences among genotypes as it was also stated by Otani and Shimada [18]. Different reports show similar results and indicated genotypes and growth regulator concentrations highly affect meristem initiation, callus induction and shoot regeneration from callus [19, 20]. According to these authors, genotypes which form callus from meristem explants are appropriate for callus induction. In the present study, ‘TIS-8250’ variety induced callus in all of the treatments indicating that this variety is the most responsive variety for callus induction among all the four varieties. On the other hand, these authors reported that the responses of meristem initiation to plant growth regulators have varied with the type, concentration and combination of growth regulators, age and type of explants and genotypes.
Results obtained in the present study showed that sensitivity to exogenous growth regulators is probably affected by the endogenous hormonal level, which controls many circumstances expressed by the genetic makeup of the cells. The type of auxin used in initiation medium was found significant for meristem initiation and specific to the individual cultivars [21]. In the light of the results obtained in this study, each of the growth regulators used had different effects on shoot initiation from meristems of sweet potato varieties. As it was reported by Gong et al. [22], shoot initiation from meristem requires specific concentration and combination of growth regulators. Kakuei and Salehi [23] reported that with increasing concentration of growth regulators, most of the shoots of Dracaena sanderiana showed deformity.
Interaction effects among ‘Beletech’, ‘Koka-12’ and ‘Ogensegen’ varieties and growth regulators (BAP, NAA and GA3) were significant at 5% probability level for meristem initiation. MS medium containing NAA, BAP and GA3 are critical for shoot initiation from meristem of sweet potato varieties [21]. On the other hand, Oggema et al. [24] pointed out that lower concentrations of NAA and BAP in initiation medium resulted in higher shoot initiation percentage from meristem. When NAA concentration was increased from 0.01 to 0.05 mg/l, the number of shoot production sharply decreased from 27 shoots to 5 shoots in ‘Beletech’, 21 shoots to 0.0 in ‘Koka-12’ and 17 shoots to 0.0 in ‘Ogensegen’ and enhanced callus formation. Similarly, Rafi and Salehi [25] reported increasing the concentration of NAA to 3.0 μM reduced the number and length of proliferated shoots of Himalayan cedar. Compact callus formation with increase in concentration of NAA was reported by Robbins [26]. Increased concentrations of BAP resulted in more callus than shoots in all of the varieties. Similar trend was also observed with increase in the concentration of GA3.
The results of the present study showed that 1.0 mg/l BAP, 2.0 mg/l GA3 and 0.01 mg/l NAA were found to be optimum in shoot initiation from meristem in ‘Beletech’, ‘Ogensegen’ and ‘Koka-12’. This combination was repeated once and similar results were recorded. In all of the growth regulators combinations, shoot initiation was not possible for ‘TIS-8250’ but callus formation was highly evident.
Shoots initiated from meristem culture were cultured on different concentrations of BAP. Shoot multiplication response per explant was increased tremendously when 2.0 mg/l and 1.5 mg/l BAP was used for ‘Beletech’ and ‘Koka-12’ respectively. However, further increase in concentration of BAP resulted in sharp decrease in shoot multiplication response for both varieties. This is possibly due to the reason that normal development of somatic tissue requires a fine temporal and spatial regulation of cell division, enlargement and differentiation that could be achieved by optimum concentration of cytokinins as also reported by Ammirato [27]. Similarly, Shahcheraghi and Shekafandeh [28] reported increasing kinetin from 6 to 8 mg/l significantly reduced the number of shoots in Ficus carica indicating supra-optimal concentration of cytokinin reduces rate of shoot multiplication.
The results indicated that IBA is important factor for root length, root number and abundance of root hair formation. However, there was no significant difference in percentage of rooting between medium containing IBA and growth regulators free medium that was used as a control. Since sweet potato is vegetatively propagated plant, it roots easily. Wondimu et al. [9] transferred microshoots directly to greenhouse for acclimatization by bypassing the in vitro rooting stage and obtained 100% rooting. Significant differences (P < 0.05) were shown between the two sweet potato varieties in number of roots and root length. The highest mean number of roots was exhibited by variety ‘Beletech’ in growth regulator free medium, which was used as a control. This was different from results obtained by Lity and Conooer [29] who reported that IBA significantly increased the number of roots produced in sweet potato varieties. On the other hand, the highest mean number of roots in ‘Koka-12’ was obtained on medium containing 0.1 mg/l IBA. These differences may be due to differences in genotype, which may have different concentrations of endogenous auxin. The number of roots per shoot increased when IBA was used in low concentration for ‘Beletech’ and relatively high concentration for ‘Koka -12’. Growth regulator free medium resulted in longest root with root hairs in both genotypes. Similarly, Sepehrtaj and Shahsavar [30] reported increasing the concentration of IBA resulted in decrease in the number of roots in citrus. Spontaneous rooting was also common in initiation medium as also reported by Oggema et al. [24] probably due to high endogenous concentration of auxin.
Plantlets are ready to be established in the soil when they have two or three leaves and at least one strong root. Soil: sand: compost mixture in a ratio of 1:2:1 resulted in 91.4% survival of plants after acclimatization. Kuo et al. [31] reported soil mixtures with high peat content often lead to slower growth and a higher death rate.
Since sweet potato is vegetatively propagated by the use of vine cuttings and storage roots, almost all germplasms are known to be infected with viruses [32]. Viral infections can have a dramatic effect on yield and marketable quality of a crop and also can adversely affect international distribution of sweet potato germplasm [6]. Some viruses like sweet potato feathery mottle virus can alone decrease the yield by 40% and in combination with other viruses, the loss reaches 90% [33]. SPFMV is the most common sweet potato virus which occurs world-wide. This was also confirmed in our study that all of the stock plants tested positive for SPFMV. The best control method for potato viruses is production of healthy plants from meristem culture. To produce healthy plants, small meristems (0.11- 0.25 mm diameter) are more responsive than big meristems. In the present study, the two main virus elimination methods, meristem culture and chemotherapy, were used for virus elimination. Serological method, NCM-ELISA, was used to test for the presence of viruses in the stock plants and meristem derived and chemical treated plantlets. Meristem culture was found to be very effective in eliminating viruses. Its virus elimination efficiency reached 100% in our study and 75-80% elimination efficiency was reported by Kuo et al. [31]. In another study, Mellor and Stace-Smith [34] cultured apical meristems of four potato cultivars in modified MS solid medium with BAP, NAA and GA3 and they obtained 68.1-86.6% virus free plantlets. In the stock plants tested, ‘Ogensegen’ showed very deep purple color showing that it is severely attacked by viruses. Symptoms in the leaves of this plant are also indicative of infection by viruses. NCM-ELISA confirmed the synergistic effect of the three viruses (SPFMV, SPCSV and SPVG) in this variety. ‘Beletech’ and ‘Koka-12’ showed light purple color showing that they are infected only by single virus, SPFMV.
Antiviral chemicals can be used as additives in the culture medium [35], and one of the most widely used is ribavirin (1-β-ribofuranosyl-1, 2, 4-triazole-3-carboxamide) also known as virazole. This compound is a guanosine analog with broad-spectrum activity against animal viruses and appears also to be active against plant virus replication in whole plants [22]. Increasing concentrations of ribavirin and increasing length of culture incubation in the presence of the compound typically increases the effectiveness of virus elimination [34], but slow growth and phytotoxicity may be evident at high concentrations. The efficiency of ribavirin in the elimination of plant viruses is subject to its concentrations, type of host plant genetic material, and type of virus. Studies indicate applications of antivirus agents inhibit synthesis of virus RNA [36]. Mangal et al. [37] reported that by in vitro chemotherapy, with application of 20 mg/l ribavirin, 55.5% virus-free sweet potato plants were obtained but in our study, ‘Koka-12’ and ‘Ogensegen’ varieties, 100% of the shoots treated with 20 mg/l ribavirin were free from viruses. Nevertheless, lower concentration (10 mg/l) failed to produce similar results in all treated shoots. Al-Mazrooei et al. [21] reported that 30 mg/l ribavirin treated shoots were 100% free from viruses with sever phytotoxicity effect. In our study, shoots cultured on medium containing ribavirin at concentrations ranging from 10-30 mg/1 had both virus elimination and phytotoxicity effects. In 30 mg/l ribavirin treated samples, significant number of shoots of ‘Koka-12’ followed by ‘Beletech’ and all shoots of ‘Ogensegen’ died. All shoots of ‘TIS-8250’ cultured in all concentrations of ribavirin turned to calli. All shoots cultured on the control (ribavirin free medium) tested positive for viruses present in the mother plants.
The development of pale green pigmentation in the early storage of meristems could be an indication of survival of meristem. If the meristem cultures become white and translucent, it could be an indication of degeneration. When the first initiated shoots were transferred to the same fresh medium or 1.0 or 2.0 mg/l BAP, all of them died. This happened frequently during the subsequent cultures. Several authors drew the attention to the effect of initiation medium on stimulation of plantlet survival for sweet potato as Al-Mazrooei et al. [21] pointed out that the carry over effect of the initiation medium on subsequent growth of plantlets especially in recalcitrant varieties. According to these authors, the effect of auxin on initiation medium is critical and specific to the individual cultivars. One of the most important approaches for overcoming in vitro recalcitrance problems is the optimization of plant growth regulators. However, cultures are under the control of both endogenous and exogenous plant growth regulators. Therefore, achieving the optimum exogenous balance of the key growth regulators can be critical and their appropriate application can effectively overcome certain recalcitrance problems [38]. Finally, callusing in the initiation medium was frequently observed in all varieties especially in ‘TIS-8250’ in which no shoot initiation was possible in all of the treatments used.
In this study, initiation of shoots was successful on auxin, cytokinin and gibberellins containing medium. Growth regulator free initiation medium showed significant difference than plant growth regulator containing medium. BAP containing medium showed the highest number of shoots per explant. Shoots cultured on growth regulators free medium and IBA containing medium rooted well. Rooted plantlets were then transferred into pots and successfully acclimatized in the greenhouse.