Transformation technology as a research or breeding tool to improve maize is routinely used in most industrial and some specialized public laboratories. However, transformation of many inbred lines remains a challenging task, especially when using Agrobacterium tumefaciens as the delivery method. Here we report success in generating transgenic plants and progeny from three maize inbred lines using an Agrobacterium-mediated standard binary vector system to target maize immature embryos. Eleven maize inbred lines were pre-screened for transformation frequency using N6 salts. A subset of three maize inbred lines was then systematically evaluated for frequency of post-infection embryogenic callus induction and transformation on four media regimes: N6 or MS salts in each of two distinct media backgrounds. Transgenic plants recovered from inbred lines B104, B114, and Ky21 were analyzed for transgene integration, expression, and transmission. Average transformation frequencies of 6.4% (for B104), 2.8% (for B114), and 8% (for Ky21) were achieved using MS salts. Availability of Agrobacterium-mediated maize inbred line transformation will improve future opportunities for maize genetic and functional genomic studies.

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Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts.

The objective of this study was to determine if the growth of sweet orange (Citrus sinensis (L.) Osbeck cv. ‘Valencia’) nonembryogenic callus could be regulated and controlled via the mineral nutrient components of the medium. The 14 salts comprising Murashige and Skoog (MS) basal medium were subdivided into five component groups. These five groups constituted the independent factors in the design. A five-dimensional hypervolume constituted the experimental design space. Design points were selected algorithmically by D-optimality criteria to sample of the design space. Growth of the callus at each design point was measured as % increase of fresh weight at 14 d. An analysis of variance was conducted and a response surface polynomial model generated. Model validation was conducted by mining the polynomial for design points to two regions—“MS-like” growth and MS + 25% growth and comparing callus growth to predicted growth. Five of the eight selected MS-like points and three of the six MS + 25% growth points validated, indicating regions within the design space where growth was equivalent to MS, but the salt combinations were substantially different from MS, and a smaller region where growth exceeded MS by greater than 25%. NH4NO3 and Fe were identified as important factors affecting callus growth. A second experiment was conducted where NH4NO3 and Fe were varied, thus creating a two-dimensional slice through the region of greatest callus growth and provided increased resolution of the response.

https://bioone.org/journals/in-vitro-cellular-and-developmental-biology---plant/volume-43/issue-4/s11627-007-9062-5/Regulating-plant-tissue-growth-by-mineral-nutrition/10.1007/s11627-007-9062-5.pdf

Regulating plant tissue growth by mineral nutrition

A rapid and reproducible Agrobacterium-mediated transformation protocol for sorghum has been developed. The protocol uses the nptII selectable marker gene with either of the aminoglycosides geneticin or paromomycin. A screen of various A. tumefaciens strains revealed that a novel C58 nopaline chromosomal background carrying the chrysanthopine disarmed Ti plasmid pTiKPSF(2), designated NTL(4)/Chry5, was most efficient for gene transfer to sorghum immature embryos. A NTL(4)/Chry5 transconjugant harboring the pPTN290 binary plasmid, which carries nptII and GUSPlus expression cassettes, was used in a series of stable transformation experiments with Tx430 and C2-97 sorghum genotypes and approximately 80% of these transformation experiments resulted in the recovery of at least one transgenic event. The transformation frequencies among the successful experiments ranged from 0.3 to 4.5%, with the average transformation frequency being approximately 1% for both genotypes. Over 97% of the transgenic events were successfully established in the greenhouse and were fully fertile. Co-expression of GUSPlus occurred in 89% of the transgenic T(0) events. Seed set for the primary transgenic plants ranged from 145 to 1400 seed/plant. Analysis of T(1) progeny demonstrated transmission of the transgenes in a simple Mendelian fashion in the majority of events.

Rapid and reproducible Agrobacterium-mediated transformation of sorghum

A highly efficient microprojectile transformation system for sorghum (Sorghum bicolor L.) has been developed by using immature embryos (IEs) of inbred line Tx430. Co-bombardment was performed with the neomycin phosphotransferase II (nptII) gene and the green fluorescent protein (gfp) gene, both under the control of the maize ubiquitin1 (ubi1) promoter. After optimization of both tissue culture media and parameters of microprojectile transformation, 25 independent transgenic events were obtained from 121 bombarded IEs. The average transformation frequency (the total number of independent transgenic events divided by the total number of bombarded IEs) was 20.7% in three independent experiments. Transgenic events were confirmed by both PCR screening and Southern hybridization of genomic DNA from primary transgenics (T0). More than 90% of transformants were fertile and displayed normal morphology in a containment glasshouse. Co-transformation rate of the nptII and gfp genes was 72% in these experiments. The segregation of nptII and gfp in T1 progenies was observed utilizing fluorescence microscopy and geneticin selection of seedlings indicating both were inherited in the T1 generation. The transformation procedure, from initiating IEs to planting putative transgenic plantlets in the glasshouse, was completed within 11–16 weeks, and was approximately threefold more efficient than the previously reported best sorghum transformation system.

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Highly efficient sorghum transformation.

Maize is an important food and feed crop in many countries. It is also one of the most important target crops for the application of biotechnology. Currently, there are more biotech traits available on the market in maize than in any other crop. Generation of transgenic events is a crucial step in the development of biotech traits. For commercial applications, a high throughput transformation system producing a large number of high quality events in an elite genetic background is highly desirable. There has been tremendous progress in Agrobacterium-mediated maize transformation since the publication of the Ishida et al. (1996) paper and the technology has been widely adopted for transgenic event production by many labs around the world. We will review general efforts in establishing efficient maize transformation technologies useful for transgenic event production in trait research and development. The review will also discuss transformation systems used for generating commercial maize trait events currently on the market. As the number of traits is increasing steadily and two or more modes of action are used to control key pests, new tools are needed to efficiently transform vectors containing multiple trait genes. We will review general guidelines for assembling binary vectors for commercial transformation. Approaches to increase transformation efficiency and gene expression of large gene stack vectors will be discussed. Finally, recent studies of targeted genome modification and transgene insertion using different site-directed nuclease technologies will be reviewed.

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Maize transformation technology development for commercial event generation.

Fragments of young panicles and immature embryos of different cultivars of grain sorghum were cultured on modified MS and N6 media supplemented with L-asparagine (6.7 mM), L-proline (17.4 mM) and different concentrations of NO
 3
 -
 , NH
 4
 +
 and PO
 4
 3-
 . The panicle-derived cultures were used in experiments with different nitrogen sources; the influence of PO
 4
 3-
 level was studied in embryo-derived and panicle-derived cultures. An increase of the NO
 3
 -
 and NH
 4
 +
 levels in the media with amino acids significantly increased induction and growth of embryogenic callus (EC) and its regeneration ability. A new M2 medium, which contained 62.5 mM NH
 4
 +
 and 72.4 mM NO
 3
 -
 , exceeded other media which were previously effective for obtaining EC of sorghum. The level of NO
 3
 -
 and NO
 3
 -
 /NH
 4
 +
 ratio in the media supplemented with L-asparagine and L-proline were established to be the critical factors of friable EC formation in sorghum. High level of NH
 4
 +
 with low level of NO
 3
 -
 resulted in formation of compact EC, while increase of NO
 3
 -
 concentration (39.9–82.4 mM) in MS or N6 media favoured proliferation of friable EC in some genotypes. Formation of compact EC in the NH
 4
 +
 -rich media was accompanied by strong pigmentation of the medium, which was never observed in cultures with friable EC. An increase of the PO
 4
 3-
 level (up to 8.8–14.2 mM) was shown to increase the frequency of somatic embryogenesis by 3–4 times, the EC mass by 1.5–2.0 times and its regeneration ability. Media with increased nitrogen or phosphorus could be used for maintenance of sorghum EC for 4–5 passages; for a more prolonged maintenance, their level in the medium should be decreased.

Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum

Methodological Techniques for Developing Cadets’ Pedagogical Skills in Preparation for Military-Political Work in the Armed Forces of the Russian Federation

Relevance. The inflammatory process and endogenous intoxication of the patient’s body has a negative impact on the course of many malignant neoplasms, including salivary gland cancer.The objective of the study was to determine the influence of some factors of systemic inflammation and endogenous intoxication in salivary gland cancer.Methods and materials. A prospective study included the data of 59 patients with salivary gland cancer. The influence of peripheral blood parameters and relative indicators characterizing systemic inflammation on overall and disease-free survival was assessed.Results. As a result of the multivariate analysis, it was revealed that an increase in the level of the relative number of peripheral blood neutrophils by more than 60.08 % increases the risk of death in patients from salivary gland cancer by 3.90 times (p=0.0456; HR 3.90: 95 % CI 1.03–14.79). The level of the absolute number of peripheral blood lymphocytes, not exceeding 1.49x109 /l, increases the risk of disease progression by 8.72 times (p=0.0002, R 8.72: 95 % CI 2.78–27.28).Conclusion. Individual factors of systemic inflammation and endogenous intoxication, it is advisable to evaluate at the stage of planning the primary treatment of patients with salivary gland cancer, in order to determine the prognosis of the disease and optimize the choice of tactics for the primary treatment of patients. 

Natalia V. Pakhomova

Papers

Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum

Methodological Techniques for Developing Cadets’ Pedagogical Skills in Preparation for Military-Political Work in the Armed Forces of the Russian Federation

Systemic inflammation in salivary gland cancer