In vitro regeneration of disease free enset [Ensete ventricosum (Welw) Cheesman] planting materials from bacterial wilt diseased plants using shoot tip culture
“…Based on these observations, explants were cultured on media supplemented with 5 mg/l BAP (designated as bud induction medium [BIM] for further experiments) for optimal bud induction. Previous studies reported bud induction at lower BAP concentrations (1.5–4.5 mg/l) [18, 19] or in combination with an auxin such as IAA or ABA [19, 20]. Fig.…”
Section: Resultsmentioning
confidence: 83%
“…Our results agree with previous reports of successful shoot regeneration using media containing either BAP alone or a combination of high BAP with low auxin. Genene and Mekbib [18] reported the development of 20–23 shoots per explant per subculture for different enset cultivars cultured on media having 4.5–6 mg/l BAP and 2 mg/l NAA. In this study SEM4 media with 2 mg/l BAP (hereafter referred to as SEM) was identified as the most ideal for shoot regeneration and elongation.…”
Section: Resultsmentioning
confidence: 99%
“…Previous studies have reported the induction of a fewer number of roots within a longer culture duration. Genene and Mekbib [18] reported the production of an average of 3.55 roots per explant after 12–14 days post culture of explants on MS media with 1 mg/l IBA. This differences in root induction response can be explained by the fact that root development is dependent on several factors including media type and plant genotype.…”
Section: Resultsmentioning
confidence: 99%
“…A major drawback to extending these transgenic approaches to other economically important species in the Musacae family (including E. ventricosum ) is the lack of efficient regeneration protocols [16]. To date, the main tissue culture technique applied to enset is micropropagation using either intercalary meristems [17] or shoot-tips [18–21].…”
Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.
“…Based on these observations, explants were cultured on media supplemented with 5 mg/l BAP (designated as bud induction medium [BIM] for further experiments) for optimal bud induction. Previous studies reported bud induction at lower BAP concentrations (1.5–4.5 mg/l) [18, 19] or in combination with an auxin such as IAA or ABA [19, 20]. Fig.…”
Section: Resultsmentioning
confidence: 83%
“…Our results agree with previous reports of successful shoot regeneration using media containing either BAP alone or a combination of high BAP with low auxin. Genene and Mekbib [18] reported the development of 20–23 shoots per explant per subculture for different enset cultivars cultured on media having 4.5–6 mg/l BAP and 2 mg/l NAA. In this study SEM4 media with 2 mg/l BAP (hereafter referred to as SEM) was identified as the most ideal for shoot regeneration and elongation.…”
Section: Resultsmentioning
confidence: 99%
“…Previous studies have reported the induction of a fewer number of roots within a longer culture duration. Genene and Mekbib [18] reported the production of an average of 3.55 roots per explant after 12–14 days post culture of explants on MS media with 1 mg/l IBA. This differences in root induction response can be explained by the fact that root development is dependent on several factors including media type and plant genotype.…”
Section: Resultsmentioning
confidence: 99%
“…A major drawback to extending these transgenic approaches to other economically important species in the Musacae family (including E. ventricosum ) is the lack of efficient regeneration protocols [16]. To date, the main tissue culture technique applied to enset is micropropagation using either intercalary meristems [17] or shoot-tips [18–21].…”
Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.
“…Plants propagated by cuttings also show a lower longevity and possess a lower drought and disease resistance than those propagated by seeds (Sujatha et al, 2005). Again this conventional propagation technique has its own draw back since planting materials are one of sources for disease transmission from place to place (Genene, 2014). Besides, for an effective large scale commercialized production of Jatropha maintaining true to type genotypes, producing disease free planting materials and high number of propagule in vitro culture has a paramount importance.…”
Jatropha curcas L. is among important tree crops in the world with a potential for biofuel production. In Ethiopia, there is a soaring investors' interest to produce Jatropha in the country for biodiesel production. However, insufficient good quality propagation material is a major production constraint. A study was undertaken to establish a protocol for in vitro mass propagation of three Ethiopian Jatropha accessions viz. Metema, Adami Tulu and Shewa Robit through direct organogenesis from nodal explant. The result revealed that the highest percentage of shoot induction (86-90%) was achieved on MS medium with BAP (1 mg/L) and IBA (0.5 mg/L) for all the three accessions. The maximum number of shoots (6) was obtained for Metema when BAP (0.5 mg/L) with Kn (0.5 mg/L) was used. Whereas, the maximum (3.2 cm) shoot length was recorded for Shewa Robit on media with 0.5 mg/L Kn. The highest rooting percentage (84.8-88%) and maximum root number (5.43) were recorded on media supplemented with 0.25 mg/L IBA. Shewa Robit and Metema had longer roots on media with 0.25 mg/L IBA. Finally, the plantlets were successfully established in greenhouse with survival rate of 86.67% for Shewa Robit followed by 73.33 and 66.67% for Metema and Adami Tulu, respectively. This study provided optimal protocol for micro-propagation of Jatropha accessions through direct organogenesis to boost its production.
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