Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism

Article · November 1998with87 Reads
DOI: 10.1016/S0168-9452(98)00162-9
During selection for transformed tissue, in vitro-cultured barley material rapidly loses regenerability or gives rise to albino plants, and this has caused difficulty in developing successful transformation technologies for important North American barley cultivars. Callus from three spring cultivars, Golden Promise (GP), Galena (GL), and Harrington (HT), was initiated from immature scutellar tissue and grown on callus-induction medium containing 2.5 mg/l of the auxin, 2,4-dichlorophenoxyacetic acid (2,4-d), 0.01 or 0.1 mg/l of the cytokinin, 6-benzylaminopurine (BAP), and 5.0 μM cupric sulfate. The addition of BAP and copper, compared to auxin alone, resulted in shinier, more compact and slightly brown-colored callus, which was more regenerable. When the highly regenerable structures were exposed to dim light and maintained on 0.1 mg/l BAP, they could be cultured for more than a year without a marked loss in regenerability or evidence of albinism. When GP tissues were initiated on auxin alone (2,4-d or dicamba) and transferred to 2,4-d, BAP and copper, as an intermediate step before regeneration, green shoot production increased 2.4 to 11.4 times for both transgenic and nontransgenic calli. Similar increases were found for nontransgenic GL and HT. This increase in regenerability, likely due to a change in the developmental state of the cultures, along with other changes in the transformation protocol, resulted in successful transformation of the previously recalcitrant GL and HT cultivars.
    • We hypothesize that this low recovery of transgenic lines is due to a decrease in plant regeneration potential after a long culture period. Longterm cultures are often reported to lead to decreased plant regeneration efficiency and increases in the number of albino plant regenerants (Cho et al. 1998;Gondo et al. 2005;Lambé et al. 1998). Similar phenomenon has occurred within a 3-month culture term in napier grass (Chandler and Vasil 1984) and our data showed that we obtained a transgenic plant which was recovered from callus with the shortest culture term of 140 days (Table 4).
    [Show abstract] [Hide abstract] ABSTRACT: Napier grass (Pennisetum purpureum Schumach.) is a highly productive C4 tropical forage grass that has been targeted as a potential bioenergy crop. To further increase the efficiency of bioethanol production by molecular breeding, a reliable protocol for genetically transforming napier grass is essential. In this study, we report the creation of transgenic napier grass plants derived from embryogenic callus cultures of shoot apices. Embryogenic callus was initiated in three accessions of napier grass and a napier grass×pearl millet hybrid using Murashige and Skoog (MS) medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L⁻¹ 6-benzylaminopurine (BAP) and 50 µM copper sulfate (CuSO4). Of the accessions tested, a dwarf type with late-heading (DL line) had the best response for embryogenic callus formation. Highly regenerative calli that formed dense polyembryogenic clusters were selected as target tissues for transformation. A plasmid vector, pAHC25, containing an herbicide-resistance gene (bar) and the β-glucuronidase (GUS) reporter gene was used in particle bombardment experiments. Target tissues treated with 0.6 M osmoticum were bombarded, and transgenic plants were selected under 5.0 mg L⁻¹ bialaphos selection. Although a total of 1400 target tissues yielded nine GUS-positive bialaphos-resistant calli, only one transgenic line that was derived from target tissue with the shortest culture term produced four transgenic plants. Thus, the length of time that the target tissue is in callus culture was one of the most important factors for acquiring transgenic plants in napier grass. This is the first report of successfully producing transgenic napier grass plants.
    Article · Sep 2017
    • Fertile plants were regenerated[137]Callus induction and regeneration at Czech cultivars The callus formation frequency and number of green regenerants were influenced significantly both by genotype and auxin[123]Callus induction and regeneration at Nordic cultivars Regeneration of many plants from the same callus over long periods of time and makes available highly efficient regeneration protocols[138]Mature embryos Tissue culture establishment and plantlet regeneration Plantlets regenerated both via organogenesis and somatic embryogenesis[139]Tissue culture and plant regeneration at Indian cultivars Multiple shoot induction and plantlet regeneration in Indian cultivar of barley[140]Anther culture Possible effect of copper during anther culture in barley The positive influence of copper sulphate was characterized by an increase of microspore survival during anther culture[141]Barley (Hordeum vulgare L.) Improvement Past, Present and Future transformation[156]Immature embryos and microsporederived cultures Successful transformation[157]Transformation of recalcitrant species Successful transformation[158]Pre cultured immature embryos Molecular analysis of T1 generation plantlets revealed the amplification of selectable marker hptII gene in the progeny[159]Agrobacterium-mediated transformation Immature embryos Successful transformation[142]Shoot apices Successful transformation[160]Optimization of gene transfer immature embryos Transformation efficiencies 2.6–6.7%[145]Young ovules Successful transformation[161]Microspores Successful transformation[147]Optimization of gene transfer immature embryos 25 % transformation efficiency[148]Mature scutellum Successful transformation[162]Immature embryo-derived callus cultures Improve T-DNA transfer in monocotyledon transformation procedures[163]Mature embryos Successful transformation[164]Tissue electroporation DNA transfer into mature embryos of barley via electroporation Successful transformation[147]first reported Agrobacterium-mediated gene transfer protocol to barley using immature embryos (IEs).
    Full-text · Chapter · Jul 2017 · Plant Cell Reports
    • The supplement of BA (in combination with IAA) had no positive effect on plant regeneration from embryogenic callus of both T. timopheevii and T. kihare, that is in conflict with several reports on wheat and barley embryo cultures (Cho et al. 1998; Ganeshan et al. 2003; Sharma et al. 2005; Yu et al. 2008; Fahmy et al. 2012). Moreover, cultivation of T. timopheevii on the regeneration medium containing BA induced a greater number of albino plants, while the earlier attempts reduced the incidence of albinism in barley when BA was used (Cho et al. 1998). Though the application of Zeatin was not beneficial for regeneration from embryogenic callus of T. timopheevii, it could apparently be used to achieve the acceptable plant production in T. kiharae.
    [Show abstract] [Hide abstract] ABSTRACT: The efficiency of immature embryo-derived in vitro culture of G genome wheats is significantly influenced by various auxins and sugars which are used for induction of embryogenic response, and by regeneration media composition for promotion of plant development from subcultured embryogenic calli. The embryogenic calli of Triticum timopheevii has demonstrated the highest regeneration ability when the initial explants were cultured on the media supplemented with 4 mg l−1 of Picloram (29.0 %), 4 mg l−1 of Dicamba (28.7 %) or 3 mg l−1 of 2,4-D (29.1 %). The media supplemented with 5–6 mg l−1 of Picloram were considered to be the most effective for promotion of embryogenic/regenerable callus production in Triticum kiharae cultures (73.7–75.0 %). Both T. timopheevii and T. kiharae embryogenic structures were characterized by the formation of green and albino plantlets. Generally the medium that was initially supplemented with Picloram promoted the formation of lower albino plants fraction rather than 2,4-D and Dicamba. As it was measured by the total green plant production per initial explant, the overall efficiency has been reduced when sucrose was substituted by glucose or maltose. The regeneration medium supplemented with 0.25 mg l−1 TDZ significantly enhanced the regeneration capacity of embryogenic callus in T. kiharae. In culture of T. timopheevii the difference between the medium lack of growth regulators and the medium supplemented with TDZ was not prominent, though both of the media have demonstrated the greater efficacy as compared to those supplemented with BA and Zeatin.
    Article · Mar 2016
    • Similar results have been described by Chang et al (2003) for cereals. The elongated shoots of Bermuda grass obtained from callus were rooted on B5 medium with 14.7 mM IBA by Cho et al (1998). The IBA was an efficient auxin to produce the roots was proved in maize by Patel et al (2006).
    [Show abstract] [Hide abstract] ABSTRACT: An efficient regeneration was developed using mature and immature embryos by using Maize (Zea mays L) variety MU 2092. Mature embryos are removed from surface sterilized seeds, slice them into halves and immature embryos are detached from seed endosperm. Both are used as explants to initiate callus on N6 medium supplemented with 2,4 D @ 4.0 mg.L-1. The induction frequency of primary calli i.e embryogenic callus was 90% in maize. The embryogenic calli on N6 medium supplemented with 6-benzylaminopurine (BAP) @ 0.5 mg.L-1 and kinetin @ 0.5 mg.L-1 was more effective in producing shoots. The culture expressed maximum plant regeneration potential with eight shoots per embryo on regeneration. Green shoots thus developed were successfully rooted within 20 days on MS media containing IBA (Indole-3-Butyric acid) 1mg L-1. Over 86 % of rooted plants grew well and produced seeds normally when transferred to green house. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for mature and immature embryos.
    Full-text · Article · Jan 2016
    • However, highly efficient Agrobacterium-mediated transformation in modern elite inbreds is not routine and remains essential for commercial biotechnology product development. Green regenerative tissue (GT: highly regenerative, green tissue) technology with the use of high copper and BAP was developed to reduce a genotypic limitation in transformation via microprojectile bombardment in recalcitrant commercial monocot crop varieties such as barley (Cho et al. 1998), wheat (Cho et al. 1999; Kim et al. 1999; Li et al. 2009) and Kentucky bluegrass (Ha et al. 2001).
    [Show abstract] [Hide abstract] ABSTRACT: The current study describes a robust, high-frequency Agrobacterium-mediated transformation protocol suitable for multiple recalcitrant modern elite commercial maize inbreds employing media modifications with glucose, cupric sulfate and a cytokinin, 6-benzylaminopurine (BAP). An optimal combination of these three key elements in the co-cultivation, resting, and selection media resulted in 4- to 14-fold improvements in transformation frequencies at the T0 plant level of 9.7, 31.9, 9.6 and 10.0 % for PH4CN, PH12BN, PHW0V and PH17R8, respectively. Transformation frequency in PH1CP1 was also improved at the T0 tissue level from 2.5 to 8.3 %. The addition of cupric sulfate and BAP in the co-cultivation medium improved transformation frequency in all inbreds except PH4CN. The use of cupric sulfate and BAP in combination with additional glucose in the selection medium was especially important, significantly improving the transformation frequency in 3 (PH4CN, PHW0V and PH1CP1) out of 5 inbreds by increasing the proliferation of high quality regenerable tissue. It was observed that the amount/ratio of these three components needed to be optimized for each inbred. The results in this study can be applied to optimize the tissue culture response and improve transformation frequency in other recalcitrant elite commercial maize inbreds.
    Full-text · Article · Jun 2015
    • These green regenerative tissues contained multiple lightgreen structures similar to shoot meristems. Similar results by a combination of cytokinin and copper were observed in other monocot crops (Cho et al. 1998Cho et al. , 1999aCho et al. , 1999bCho et al. , 2000Cho et al. , 2003Cho et al. , 2004 Kim et al. 1999; Lemaux et al. 1999; Li et al. 2009; Ha et al. 2001; Wu et al. 2014). More specifically, in barley, these tissues had physiological and developmental similarities with shoot meristematic tissues cultured from the excised shoot apices on medium containing 2,4-D and BAP (Lemaux et al.
    [Show abstract] [Hide abstract] ABSTRACT: Key message An improved Agrobacterium -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds. Abstract This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T0 plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T0 plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.
    Full-text · Article · Jul 2014
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