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Agrobacterium tumefaciens-mediated in planta transformation strategy for development of transgenics in cotton (Gossypium hirsutum L.) with GFP as a visual marker.

ABSTRACT: Cotton (Gossypium hirsutum L.), a mercantile crop plant, is grown worldwide for fiber and seed oil. As with other economically important crops, cotton is bogged down with many biotic and abiotic stress factors. Towards this, genetic engineering offers numerous protocols to engineer plants for better resilience. However, recalcitrance of cotton to plant tissue culture has been the major constraint for successful in vitro regeneration. Hence, alternate methods that evade tissue culture regeneration have been envisaged. Non tissue culture-based in planta transformation strategies are in vogue due to amenability and ease in the generation of transgenic plants. In the present study, we demonstrate the utility of an in planta transformation protocol and establishment of a stringent selection agent-based screening for the identification of transgenics. The genotype independent nature of the protocol was validated in cotton cv. Pusa 8-6 using GFP. Preliminary transformation efficiency of 28% was achieved with a screening efficiency of 20% in the presence of hygromycin. The proof of T-DNA integration by various molecular and expression analysis in T1 and T2 generations proved that this technique can be employed to generate transgenic cotton.

SUBMITTER: Kesiraju K

PROVIDER: S-EPMC7688863 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Agrobacterium tumefaciens-mediated in planta transformation strategy for development of transgenics in cotton (Gossypium hirsutum L.) with GFP as a visual marker.

Kesiraju Karthik K Mishra Pragya P Bajpai Akansha A Sharma Manju M Rao Uma U Sreevathsa Rohini R

Physiology and molecular biology of plants : an international journal of functional plant biology 20201019 11

Cotton (Gossypium hirsutum L.), a mercantile crop plant, is grown worldwide for fiber and seed oil. As with other economically important crops, cotton is bogged down with many biotic and abiotic stress factors. Towards this, genetic engineering offers numerous protocols to engineer plants for better resilience. However, recalcitrance of cotton to plant tissue culture has been the major constraint for successful in vitro regeneration. Hence, alternate methods that evade tissue culture rege ...[more]

PMID: 33268932

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Project description:BackgroundGenetically modified cotton accounts for 64% of the world's cotton growing area (22.3 million hectares). The genome sequencing of the diploid cotton progenitors Gossypium raimondii and Gossypium arboreum as well as the cultivated Gossypium hirsutum has provided a wealth of genetic information that could be exploited for crop improvement. Unfortunately, gene functional characterization in cotton is lagging behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods. We present here a simple, fast and efficient method for the transient transformation of G. hirsutum that can be used for gene characterization studies.ResultsWe developed a transient transformation system for gene characterization in upland cotton. Using β-glucuronidase as a reporter for Agrobacterium-mediated transformation assays, we evaluated multiple transformation parameters such as Agrobacterium strain, bacterial density, length of co-cultivation, chemicals and surfactants, which can affect transformation efficiency. After the initial characterization, the Agrobacterium EHA105 strain was selected and a number of binary constructs used to perform gene characterization studies. 7-days-old cotton seedlings were co-cultivated with Agrobacterium and transient gene expression was observed 5 days after infection of the plants. Transcript levels of two different transgenes under the control of the cauliflower mosaic virus (CaMV) 35S promoter were quantified by real-time reverse transcription PCR (qRT-PCR) showing a 3-10 times increase over the levels observed in non-infected controls. The expression patterns driven by the promoters of two G. hirsutum genes as well as the subcellular localization of their corresponding proteins were studied using the new transient expression system and our observations were consistent with previously published results using Arabidopsis as a heterologous system.ConclusionsThe Agrobacterium-mediated transient transformation method is a fast and easy transient expression system enabling high transient expression and transformation efficiency in upland cotton seedlings. Our method can be used for gene functional studies such as promoter characterization and protein subcellular localization in cotton, obviating the need to perform such studies in a heterologous system such as Arabidopsis.

Project description:The plant immune system is made up of a complex response network that involves several lines of defense to fight invading pathogens. Fungal plant pathogens on the other hand, have evolved a range of ways to infect their host. The interaction between Ascochyta lentis and two lentil genotypes was explored to investigate the progression of ascochyta blight (AB) in lentils. In this study, we developed an Agrobacterium tumefaciens-mediated transformation system for A. lentis by constructing a new binary vector, pATMT-GpdGFP, for the constitutive expression of green fluorescent protein (EGFP). Green fluorescence was used as a highly efficient vital marker to study the developmental changes in A. lentis during AB disease progression on the susceptible and resistant lentil accessions, ILL6002 and ILL7537, respectively. The initial infection stages were similar in both the resistant and susceptible accessions where A. lentis uses infection structures such as germ tubes and appressoria to gain entry into the host while the host uses defense mechanisms to prevent pathogen entry. Penetration was observed at the junctions between neighbouring epidermal cells and occasionally, through the stomata. The pathogen attempted to penetrate and colonize ILL7537, but further fungal advancement appeared to be halted, and A. lentis did not enter the mesophyll. Successful entry and colonization of ILL6002 coincided with structural changes in A. lentis and the onset of necrotic lesions 5-7 days post inoculation. Once inside the leaf, A. lentis continued to grow, colonizing all parts of the leaf followed by plant cell collapse. Pycnidia-bearing spores appeared 14 days post inoculation, which marks the completion of the infection cycle. The use of fluorescent proteins in plant pathogenic fungi together with confocal laser scanning microscopy, provide a valuable tool to study the intracellular dynamics, colonization strategy and infection mechanisms during plant-pathogen interaction.

Project description:Seed germination is considered the beginning of the spermatophyte lifecycle, and it is a crucial stage in determining subsequent plant growth and development. Although many previous studies have found that melatonin can promote seed germination, the role of melatonin in cotton germination remains unexamined. The main objective of this study is the characterization of potential promotional effects of melatonin (at doses of 0, 10, 20, 50, 100 and 200 μM) on cotton seed germination. This experiment demonstrated that low concentrations of melatonin can promote germination, while high concentrations failed to promote germination and even inhibited germination. Together, these results indicate that a 20 μM melatonin treatment optimally promotes cotton seed germination. Compared with the control, germination potential (GP), germination rate (GR), and final fresh weight (FW) increased by 16.67%, 12.30%, and 4.81%, respectively. Although low concentrations of melatonin showed some improvement in vigor index (VI), germination index (GI), and mean germination time (MGT), these effects were not statistically significant. Antioxidant enzyme activity during seed germination was most prominent under the 20 μM melatonin treatment. Superoxide dismutase (SOD) and peroxidase (POD) activities were significantly increased by 10.37-59.73% and 17.79-47.68%, respectively, compared to the melatonin-free control. Malondialdehyde (MDA) content was reduced by 16.73-40.33%. Two important plant hormones in seed germination, abscisic acid (ABA) and gibberellins (GAs), were also studied. As melatonin concentration increased, ABA content in seeds decreased first and then increased, and GA3 content showed a diametrically opposite trend, in which the 20 μM melatonin treatment was optimal. The 20 μM melatonin treatment reduced ABA content in seeds by 42.13-51.68%, while the 20 μM melatonin treatment increased GA3 content in seeds to about 1.7-2.5 times that of seeds germinated without melatonin. This study provides new evidence suggesting that low concentrations of melatonin can promote cotton seed germination by increasing the activity of antioxidant enzymes, thereby reducing the accumulation of MDA and regulating plant hormones. This has clear applications for improving the germination rate of cotton seeds using melatonin.

Dataset Information

Agrobacterium tumefaciens-mediated in planta transformation strategy for development of transgenics in cotton (Gossypium hirsutum L.) with GFP as a visual marker.

Publications

<i>Agrobacterium tumefaciens</i>-mediated <i>in planta</i> transformation strategy for development of transgenics in cotton (<i>Gossypium hirsutum</i> L.) with <i>GFP</i> as a visual marker.

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