Although the plant materials are disinfected and all vessels and media are sterilized properly, microbial contamination in tissue culture, especially bacteria are reported (Leifert and Cassells 2001; Mona et al. 2019). Such contaminants resistant to surface disinfestations are likely to be endophytic (Reed et al. 1995), and they can be controlled by antibiotics (Gubišová and Gubiš 2019). The role of cefotaxime antibiotics in controlling bacterial contamination of enset shoot tip culture was investigated on the two landraces of enset, guarye and kibnar. The result showed that, the inclusion of two concentrations (250 mg l− 1 and 500 mg l− 1) cefotaxime on MS medium resulted in less bacterial contamination than the control (the medium devoid of cefotaxime). Similarly, Kumari et al. (2015), who compared the effect of different antibiotics on micropropagation of sugarcane, reported that cefotaxime at a concentration of 250 mg l− 1 and 500 mg l− 1 was the most effective in reducing systemic bacterial contamination as compared to other antibiotics.
Cefotaxime is usually considered to be non-toxic to plant cells (Ogawa and Mii 2004). No toxicity effect was also visible in the current study. The average level of contamination during culture initiation of the current study (13.17) was much lower than the loss of explants (50–60%) reported due to internal contamination of enset shoot tip culture without the use of antibiotics (Birmeta and Welander 2004), which indicates that the use of cefotaxime in the growth media is advantageous for efficient micropropagation of enset.
The amount or plant growth regulators required for micropropagation usually depends on the genotype used. In the current study, however, the interaction effect of PGRs and genotype was not significant. Similarly, the highest shoot proliferation rate of 7 enset landraces was recorded from one type of formulated medium (Birmeta and Welander 2004). The highest mean number of shoots (8.8) regenerated in the current study was much higher than the shoot number (3.6) reported by Diro (2003) from splitted shoot tips of oniya enset landrace on MS medium containing different concentration of PGRs. Negash et al. (2000) also reported less values (2.5, 2.6 and 2.3) of shoot number from three enset landraces (choro, nobo and ketano) respectively.
The mean shoot number recorded in the current study was much lower than the values (23 and 16) from shoot tip explants of mazia and digomerza enset landraces respectively, but comparable to the shoot number (7) from arkiya landrace on MS medium containing 4.5 mg l− 1 BAP and 1.5 mg l− 1 NAA (Gezahegn and Mekbib 2016). Regeneration of very high number of shoots from shoot tip culture on formulated macronutrients medium with double-standard concentration of MS micronutrients, with a wide range of variation among the landraces (14.7 to 74.7 shoots) was also reported (Birmeta and Welander 2004). Since the authors used different basal media composition, and data recording was made lately, 9 to 10 weeks after initiation, making direct comparison is difficult. However, the wider variations even in the same experiments reported indicate much of the variation among the results seems to be due to the differences in the types of enset landraces used. The variation in multiplication response among enset landraces was also observed in the current study; from the two enset landraces, significantly higher number of shoot was regenerated from guarye.
The increase in concentration of BAP from 0.0 to 5.0 mg l− 1 significantly increased the number of shoots regenerated per shoot. However, the increase in concentration of BAP from 5 mg l− 1 to 7.5 mg l− 1 didn't result in significant increase in shoot numbers. Diro (2003) also reported that, higher concentration of other forms of auxins, benzyl adenine, didn't show a significant increase in the number of shoots per shoot both with intact and halved shoot tips. From the successive culturing of shoots on the best performed media (MS medium supplemented with mg l− 1 BAP and 1.0 mg l− 1 IBA), an average 8.0 new shoots per shoot tip could be regenerated in one month.
Rooting of micro shoots was primarily initiated on full and half-strength MS medium containing different concentrations of IBA after 12 days of culturing. Similarly, Negash et al. (2000) reported the development of primary root in approximately ten days and the occurrence of secondary roots within the next four days. However, some of the explants showed the tendency of rooting while they were in multiplication and elongation medium, with or without PGRs. Birmeta and Welander (2004) also reported the development of roots in the elongation medium without a need of rooting stage. The highest mean number of roots per shoot (3.0) regenerated on both full and half-strength MS medium supplemented with 3.0 and 2.0 mg l− 1 IBA, respectively, was slightly lower than the root number (3.77) reported by Gezahegn and Mekbib (2016) on MS medium supplemented with 2.0 mg l− 1 IBA, but higher than the values (0.0 to 1.5) reported by Diro and Staden (2004) on MS medium supplemented with 12 different concentrations of auxins and cytokinins. The highest mean root lengths recorded in the current study was also by far lower than the value (5.96 cm) reported by Gezahegn and Mekbib (2016). The variation of the results could be due to the difference in the types of landraces used.
The results also indicated that there was an increasing trend of root number per shoot and mean root length with increased concentration of IBA up to 3.0 mg l− 1 on full strength MS and, up to 2.0 mg l− 1 and 3.0 mg l− 1 on half-strength MS medium respectively. On the other hand, the highest root length was recorded from lowest concentration (0.5 mg l− 1) of IBA (Gezahegn and Mekbib 2016), the variation could be due to the difference in the types of landraces used.
Some level of shoot-tip necrosis was observed during rooting of shoots, of which, few (11%) of the shoots were died. Mathew and Philip (1996) reported that, shoot- tip necrosis is one of the constraints during in vitro regeneration of Ensete superbum. According to the authors, the cause for that was explained to be shortage of calcium in the growth medium. Birmeta and Welander (2004) could minimize enset shoot-tip necrosis by replacing calcium chloride with calcium gluconate monohydrate in the growth medium. On the other hand, Martin et al. (2007) reported that, shoot-tip necrosis in micropropagation of banana was influenced by cultivar type, and from several methods tested to reduce the necrosis, only addition of calcium chloride was effective.
During acclimatization of plantlets in a simple greenhouse, which is not automated 75% survival rate was recorded for both guarye and kibnar after 21 days. A comparable survival rate (80 to 90%) of in vitro regenerated enset after 15 days of acclimatization was reported (Gezahegn and Mekbib 2016). Whereas, Birmeta and Welander (2004) also reported (100%) success in acclimatization of in vitro regenerated enset plantlets in a glass house. The variation of the results could be probably due to the difference in type of landrace, the condition of the greenhouse, or the season of acclimatization.