Optimisation of methods for Agrobacterium rhizogenes mediated generation of composite plants in Eucalyptus camaldulensis

Balasubramanian, A.; Rathinavel, Venkatachalam; Selvakesavan, Rajendran K.; Mary, Abraham Sanu; Gherbi, Hassen; Svistoonoff, Sergio; Franche, Claudine; Bogusz, Didier; Kumar, N. K. Krishna; Nambiar-Veetil, Mathish

doi:10.1186/1753-6561-5-s7-o45

Cited by 4 publications

(8 citation statements)

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“…We first tested the hypervirulent A. rhizogenes strain A4RS previously shown to be the most effective in infecting other trees (Alpizar et al ., ; Bosselut et al ., ; Diouf et al ., ; Gherbi et al ., ) as well as in another Eucalyptus species, E. camaldulensis (Balasubramanian et al ., ). We compared four protocols combining three types of E. grandis explants and three infection methods: (i) stabbing the hypocotyl or (ii) cutting and inoculating the base of the hypocotyl of 14‐day‐old seedlings, (iii) cutting and inoculating the radicle apex of 3‐day‐old germinating seeds and (iv) stabbing the stem of an in vitro clonal line.…”

Section: Resultsmentioning

confidence: 97%

“…With the aim of improving the rooting capacities of some Eucalyptus clones, the early work of MacRae and Van Staden () had evaluated their susceptibility to several A. rhizogenes strains. Later on, a protocol for the generation of composite plants of E. camaldulensis using a GFP‐based screening was briefly described (Balasubramanian et al ., ), but no functional gene characterization was achieved. In that study, 68% of plants showed root induction, but only 36% of those were co‐transformed, resulting in a co‐transformation efficiency of 25%.…”

Section: Discussionmentioning

confidence: 99%

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Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation

Plasencia

Soler

Dupas

et al. 2015

Plant Biotechnology Journal

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SummaryEucalyptus are of tremendous economic importance being the most planted hardwoods worldwide for pulp and paper, timber and bioenergy. The recent release of the Eucalyptus grandis genome sequence pointed out many new candidate genes potentially involved in secondary growth, wood formation or lineage-specific biosynthetic pathways. Their functional characterization is, however, hindered by the tedious, time-consuming and inefficient transformation systems available hitherto for eucalypts. To overcome this limitation, we developed a fast, reliable and efficient protocol to obtain and easily detect co-transformed E. grandis hairy roots using fluorescent markers, with an average efficiency of 62%. We set up conditions both to cultivate excised roots in vitro and to harden composite plants and verified that hairy root morphology and vascular system anatomy were similar to wild-type ones. We further demonstrated that co-transformed hairy roots are suitable for medium-throughput functional studies enabling, for instance, protein subcellular localization, gene expression patterns through RT-qPCR and promoter expression, as well as the modulation of endogenous gene expression. Down-regulation of the Eucalyptus cinnamoyl-CoA reductase1 (EgCCR1) gene, encoding a key enzyme in lignin biosynthesis, led to transgenic roots with reduced lignin levels and thinner cell walls. This gene was used as a proof of concept to demonstrate that the function of genes involved in secondary cell wall biosynthesis and wood formation can be elucidated in transgenic hairy roots using histochemical, transcriptomic and biochemical approaches. The method described here is timely because it will accelerate gene mining of the genome for both basic research and industry purposes.

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Section: Resultsmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation

Plasencia

Soler

Dupas

et al. 2015

Plant Biotechnology Journal

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“…A . rhizogenes has been successfully employed in the composite plant generation of a number of woody plants such as poplar, coffee, grapevine and Eucalyptus camadulensis 31 – 34 ]; and has been the only available option in the transformation of recalcitrant trees such as black locust and larch [ 35 , 36 ]. Composite plant generation through A .…”

Section: Discussionmentioning

confidence: 99%

“…camadulensis ) and normal roots (eg. potato) have been observed in different plant systems [ 34 , 48 , 49 ]. The rol genes are known to induce and regulate root formation by interfering with the plant hormonal physiology [ 17 , 50 ].…”

Section: Discussionmentioning

confidence: 99%

“…Coffee embryos showed transformation efficiencies of 70% with 35% roots being transgenic [ 32 ]. In eucalyptus, the transformation efficiencies of 36% and 4.1% with ~1 and ~0.5 roots per plant were recorded with seedling explants and in vitro grown plantlets, respectively [ 34 ]. The lower transformation efficiencies in the present study compared to the above studies could be due to the differences in age and nature of the explants in addition to the bacterial strains and the infection conditions.…”

Section: Discussionmentioning

confidence: 99%

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Generation of composite Persea americana (Mill.) (avocado) plants: A proof-of-concept-study

et al. 2017

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Avocado (Persea americana (Mill.)), an important commercial fruit, is severely affected by Phytophthora Root Rot in areas where the pathogen is prevalent. However, advances in molecular research are hindered by the lack of a high-throughput transient transformation system in this non-model plant. In this study, a proof-of-concept is demonstrated by the successful application of Agrobacterium rhizogenes-mediated plant transformation to produce composite avocado plants. Two ex vitro strategies were assessed on two avocado genotypes (Itzamna and A0.74): In the first approach, 8-week-old etiolated seedlings were scarred with a sterile hacksaw blade at the base of the shoot, and in the second, inch-long incisions were made at the base of the shoot (20-week-old non-etiolated plants) with a sterile blade to remove the cortical tissue. The scarred/wounded shoot surfaces were treated with A. rhizogenes strains (K599 or ARqua1) transformed with or without binary plant transformation vectors pRedRootII (DsRed1 marker), pBYR2e1-GFP (GFP- green fluorescence protein marker) or pBINUbiGUSint (GUS- beta-glucuronidase marker) with and without rooting hormone (Dip 'N' Grow) application. The treated shoot regions were air-layered with sterile moist cocopeat to induce root formation. Results showed that hormone application significantly increased root induction, while Agrobacterium-only treatments resulted in very few roots. Combination treatments of hormone+Agrobacterium (-/+ plasmids) showed no significant difference. Only the ARqua1(+plasmid):A0.74 combination resulted in root transformants, with hormone+ARqua1(+pBINUbiGUSint) being the most effective treatment with ~17 and 25% composite plants resulting from strategy-1 and strategy-2, respectively. GUS- and GFP-expressing roots accounted for less than 4 and ~11%, respectively, of the total roots/treatment/avocado genotype. The average number of transgenic roots on the composite plants was less than one per plant in all treatments. PCR and Southern analysis further confirmed the transgenic nature of the roots expressing the screenable marker genes. Transgenic roots showed hyper-branching compared to the wild-type roots but this had no impact on Phytophthora cinnamomi infection. There was no difference in pathogen load 7-days-post inoculation between transformed and control roots. Strategy-2 involving A0.74:ARqua1 combination was the best ex vitro approach in producing composite avocado plants. The approach followed in this proof-of-concept study needs further optimisation involving multiple avocado genotypes and A. rhizogenes strains to achieve enhanced root transformation efficiencies, which would then serve as an effective high-throughput tool in the functional screening of host and pathogen genes to improve our understanding of the avocado-P. cinnamomi interaction.

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Hairy Roots as a Tool for the Functional Analysis of Plant Genes

et al. 2018

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Optimisation of methods for Agrobacterium rhizogenes mediated generation of composite plants in Eucalyptus camaldulensis

Cited by 4 publications

References 4 publications

Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation

Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation

Generation of composite Persea americana (Mill.) (avocado) plants: A proof-of-concept-study

Hairy Roots as a Tool for the Functional Analysis of Plant Genes

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