Agrobacterium-mediated transformation of avocado (Persea americana Mill.) somatic embryos with fluorescent marker genes and optimization of transgenic plant recovery

Palomo-Ríos, Elena; Cerezo, Sergio; Mercado, José A.; Pliego‐Alfaro, Fernando

doi:10.1007/s11240-016-1122-2

Cited by 13 publications

(9 citation statements)

References 43 publications

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“…The restricted GUS activity spots to the actively growing root tips and not the whole root could be due to the poor diffusion of the substrate into the root interior. Though DsRed1 expressing vectors have been used successfully in avocado [ 13 ], no transformants were observed here which could have been due to insertion of the T-DNA harbouring dsred1 in a transcriptionally inactive region of the chromatin (few randomly chosen roots from pRedRootII-treated plants however turned negative for dsred1 ) or the Arabidopsis Pubq10 promoter may not be functional in avocado. However, the sunflower polyubiquitin promoter has been successfully used in avocado transformation [ 14 ].…”

Section: Discussionmentioning

confidence: 94%

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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Section: Discussionmentioning

confidence: 94%

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

et al. 2017

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“…Cytotoxic effects in plant cells caused by GFP expression have been mentioned in previous studies [ 57 ], but the data obtained in the proliferation of embryos and plant regeneration, as well as the morphological appearance of the transgenic material, suggest that GFP gene expression is not toxic in cork oak. Similarly, deleterious effects were also not observed in the GFP -mediated genetic transformation of somatic embryos of several citrus cultivars [ 58 ], avocado [ 59 ] or Chinese chestnut [ 60 ].…”

Section: Discussionmentioning

confidence: 99%

“…Plant regeneration from somatic embryos is considered one of the main bottlenecks in the application of somatic embryogenesis [ 65 ]. In the case of transgenic embryos, plant regeneration is often even more difficult [ 59 , 66 , 67 , 68 ], usually due to the continued cultivation in media with antibiotics [ 69 ]. However, in cork oak, regeneration was obtained in all 18-cork oak transgenic lines evaluated, and the plantlets exhibited normal morphology and growth.…”

Section: Discussionmentioning

confidence: 99%

“…The frequency of conversion of cork oak lines transformed with CsTL1 varies considerably depending on the genotype. The effect of genotype on plant regeneration has been reported for non-transgenic embryos [ 70 , 71 , 72 , 73 , 74 ], as well as for transgenic somatic embryos [ 48 , 49 , 59 ]. In addition, during genetic transformation by A. tumefaciens , it is not possible to control either the number of gene copies that are inserted or their position in the plant genome [ 75 ].…”

Section: Discussionmentioning

confidence: 99%

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Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (CsTL1) Encoding a Chestnut Thaumatin-Like Protein

Cano

Martínez

Couselo

et al. 2021

IJMS

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We present a reproducible procedure for transforming somatic embryos of cork oak with the CsTL1 gene that codes for a thaumatin-like protein, in order to confer tolerance to Phytophthora cinnamomi. Different concentrations/combinations of the antibiotics carbenicillin and cefotaxime, as bacteriostatic agents, and kanamycin, as a selective agent, were tested. A lethal dose of 125 mg/L kanamycin was employed to select transgenic somatic embryos, and carbenicillin was used as a bacteriostatic agent at a concentration of 300 mg/L, which does not inhibit somatic embryo proliferation. The transformation efficiency was clearly genotype-dependent and was higher for the TGR3 genotype (17%) than for ALM80 (4.5%) and ALM6 (2%). Insertion of the transgenes in genomic DNA was confirmed by PCR analysis, whereas expression of the CsTL1 gene was evaluated by semi-quantitative real-time PCR (qPCR) analysis. A vitrification treatment successfully cryopreserved the transgenic lines generated. The antifungal activity of the thaumatin-like protein expressed by the gene CsTL1 was evaluated in an in vitro bioassay with the oomycete P. cinnamomi. Of the eight transgenic lines analyzed, seven survived for between one or two times longer than non-transgenic plantlets. Expression of the CsTL1 gene and plantlet survival days were correlated, and survival was generally greater in plantlets that strongly expressed the CsTL1 gene.

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“…Moreover, the same laboratorydeveloped early screening techniques for transgenic tissue and optimized the parameters influencing transformation using fluorescent markers such as DsRed. In addition, by partially removing the cotyledons of the underdeveloped SEs or SEs with partial shooting and culturing them in media supplemented with cytokinins -benzyl adenine (BA) and thidiazuron (TDZ) -the authors saw improvements in transgenic plant recovery of >50% when compared to previous reports (Palomo-Ríos et al, 2017). This was a significant step towards overcoming the long-standing bottleneck in avocado transformation -the recovery of transgenic plants.…”

Section: Agrobacterium Tumefaciens-mediatedmentioning

confidence: 95%

Advances in Understanding Defense Mechanisms in Persea americana Against Phytophthora cinnamomi

et al. 2021

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Avocado (Persea americana) is an economically important fruit crop world-wide, the production of which is challenged by notable root pathogens such as Phytophthora cinnamomi and Rosellinia necatrix. Arguably the most prevalent, P. cinnamomi, is a hemibiotrophic oomycete which causes Phytophthora root rot, leading to reduced yields and eventual tree death. Despite its’ importance, the development of molecular tools and resources have been historically limited, prohibiting significant progress toward understanding this important host-pathogen interaction. The development of a nested qPCR assay capable of quantifying P. cinnamomi during avocado infection has enabled us to distinguish avocado rootstocks as either resistant or tolerant - an important distinction when unraveling the defense response. This review will provide an overview of our current knowledge on the molecular defense pathways utilized in resistant avocado rootstock against P. cinnamomi. Notably, avocado demonstrates a biphasic phytohormone profile in response to P. cinnamomi infection which allows for the timely expression of pathogenesis-related genes via the NPR1 defense response pathway. Cell wall modification via callose deposition and lignification have also been implicated in the resistant response. Recent advances such as composite plant transformation, single nucleotide polymorphism (SNP) analyses as well as genomics and transcriptomics will complement existing molecular, histological, and biochemical assay studies and further elucidate avocado defense mechanisms.

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Agrobacterium-mediated transformation of avocado (Persea americana Mill.) somatic embryos with fluorescent marker genes and optimization of transgenic plant recovery

Cited by 13 publications

References 43 publications

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

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

Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (CsTL1) Encoding a Chestnut Thaumatin-Like Protein

Advances in Understanding Defense Mechanisms in Persea americana Against Phytophthora cinnamomi

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