Use of DoE Methodology to Optimize the Regeneration of High-quality Transgenic Maize Plants

Authors of the manuscript, from left: Jeff Adelberg, Keith Lowe, Uyen Cao Chu and Todd Jones

The basic tissue culture process of maize transformation has changed little since the first reports in 1990. The maize Agrobacterium-mediated transformation process normally takes about 10 to 15 weeks from embryo infection and co-cultivation to when “T0” plants can be sent to the greenhouse (GH).  At Corteva AgriscienceTM, a research team recently developed a new transformation method that utilizes the maize transcription factors Babyboom (BBM) and Wuschel2 (WUS2) to stimulate direct transgenic embryo formation and plant regeneration. This method by-passes the need for prolonged tissue culture and regeneration from embryogenic callus. While the new method was rapid and convenient, the conversion of somatic embryos into strong plantlets was not optimal and there had been no systematic attempts to improve the quality and survivability of the somatic embryos produced by the process.  We chose to use a Design of Experiment (DoE) method to improve the conversion of somatic embryos to plants and define parameters that enhanced the quality of regenerated plants. This method tested 10 factors to optimize the quality of somatic embryo maturation, root formation and subsequent plantlet survival, without compromising the molecular event quality. Testing 10 factors in traditional one-factor-at-a-time (OFAT) experiments would have been protracted and impractical.

In addition, OFAT experiments would not have revealed key interactions between the factors. The use of DoE methodology enabled the testing to be done in one single, large experiment. The results of the DoE showed that the concentration of NO3 and the ratio of NH4+ to K+ had significant effects on the morphology of plantlets from directly germinated transgenic somatic embryos. During early development, optimal tissue morphology required a ratio of 1 NH4+/K+ and 20 mM [NO3] with the highest concentration of BAP tested (14.2 µM) and at low light intensity (50 µMolm-2s-1). Later development of rooted shoots required more macronutrients with less NH4NO3 (15 mM  NH4NO3 and 25 mM KNO3), less BAP (7.4 µM) and more ABA (1 µM) and at high light intensity (140 µMolm-2s-1). Using the optimized parameters, we improved the frequency of plants sent to the GH by 2-fold compared with the initial process and doubled the number of single copy T-DNA events. This experiment illustrated the utility of DoE methodology for optimizing plant tissue culture conditions where multiple paramaters need to be tested with limited resources and time.

Uyen Cao Chu, Jeffrey Adelberg, Keith Lowe, and Todd J. Jones. Use of DoE methodology to optimize the regeneration of high quality, single copy transgenic Zea mays L (maize) plants. In Vitro Cellular & Developmental Biology – Plant, 55:678-694, 2019.

The Proposed APHIS Regulation Modernization Could Enhance Agriculture Biotechnology Research & Development in the U.S.

Top row (left to right): Wayne A. Parrott, John Harbell, Heidi Kaeppler, and Todd Jones.
Bottom row (left to right): Dwight Tomes, Joyce Van Eck, Kan Wang, and Allan Wenck.

Recently the USDA’s Animal and Plant Health Inspection Service (APHIS) – one of three agencies that govern the importation, interstate movement, or environmental release of certain genetically engineered (GE) organisms – issued a call for comment on proposed changes to 7 CFR part 340.  This law has major impacts on researchers working in agricultural biotechnology in academic, government and private laboratories.  The law was originally issued in 1987.  Significant changes have not occurred since, despite over 3 decades of experience with biotechnology not only as a research tool, but as a widely adopted technology fully incorporated into agricultural practices.  During this time, over 5 billion acres of biotech crops have been planted globally.  The recent call, therefore, presented a rare opportunity to provide input on how to improve science-based regulation.

As a professional scientific society devoted to fostering the exchange of knowledge of in vitro biology of cells, tissues and organs from both plant and animals (including humans), the SIVB is uniquely positioned to comment on the proposed changes. SIVB members have played leading roles in the development, safety evaluation, and deployment of Genetically Engineered Organisms, and their efforts have directly and indirectly contributed to plant biology and agricultural biotechnology advancements. Members of the Public Policy Committee took this opportunity to focus on the science behind genetically engineered organisms and the proper assessment that should be considered when regulating their importation, interstate movement and potential environmental release. We authored a science-based document specifically addressing the questions raised by APHIS and submitted the response.  This was one of over 6,000 submissions that APHIS received.

The need for SIVB members to be involved in such initiatives – as part of the committee and/or as individuals – is clear. We decided to publish the response in our journal to raise awareness of that need and to share the science-based rationale behind our response. While regulatory policy is seldom a high priority of some researchers, it is vitally important to our long-term survival. We hope for a positive outcome.  If implemented appropriately, the final rule will remove many current barriers and will likely stimulate university and business “startup” innovation. Innovations are critical as we develop agriculture with better sustainability. Rule changes that rely on scientific knowledge will help get us there. We all need to be aware and involved.

Wayne A. Parrott, John Harbell, Heidi Kaeppler, Todd Jones, Dwight Tomes, Joyce Van Eck, Kan Wang, and Allan Wenck.  The proposed APHIS regulation modernization could enhance agriculture biotechnology research and development in the USA.  In Vitro Cellular & Developmental Biology – Plant, 56: 1–7, 2020. 

In Vitro Propagation of Medicinal and Aromatic Plants: The Case of Selected Greek Species with Conservation Priority

From left: Dr. N. Krigas (corresponding author) and Dr. K. Grigoriadou (first authors contributing equally to this work), together with Dr. G. Tsoktouridis, Dr. E. Maloupa, K. Papanastasi and Dr. V. Sarropoulou (all co-authors)

Worldwide, numerous medicinal and aromatic plants (MAPs) are still collected from the wild (uncontrolled harvesting) and only a small fraction of them are exclusively sourced from cultivation. When performed non-sustainably, this practice (which in the Mediterranean dates to antiquity) threatens the populations of wild-growing species and currently it is considered as a major threat, especially for MAPs. On the other hand, micropropagation is a powerful tool which can facilitate the conservation of rare and/or threatened MAPs, enabling rapid and massive production of high-value plant material for cultivation without seasonal constraints. Focusing on Greek native MAPs that are assigned with conservation priority (threatened, endemic, range-restricted and/or protected), this study reviewed and assessed the in vitro propagation protocols produced to date for 17 range-restricted plants (herbaceous perennials, bulbous, subshrubs or trees), and 5 MAP taxa of Orchidaceae. For the latter, current micropropagation efforts include seed germination, callus induction, and protocorm formation for successful plantlet development; however, these propagation protocols are still fragmentary. For the rest of the cases reviewed (n = 17), a five-stage detailed procedure was outlined (plant material, establishment, proliferation, rooting, and acclimatization), while materials, treatments, and data per stage were presented comparatively and discussed.

Emphasis was given on the selection and preparation of plant material obtained from nature for research, sustainable use, and ex situ conservation actions, as well as on the effectiveness thereof either for conservation purposes or for mass production needs. A protocol effectiveness was calculated using a specific equation to estimate the potential number of acclimatized plants raised from a single explant within a year. After this assessment, all protocols reviewed can facilitate conservation, and almost half of them could be used for commercialization with high cost (five cases of MAPs), intermediate cost (eight), or low cost (four). This assessment may facilitate the possible sustainable use of Greek native MAPs assigned with conservation priority.

Grigoriadou, N. Krigas, V. Sarropoulou, K. Papanastasi, G. Tsoktouridis, E. Maloupa. In vitro propagation of medicinal and aromatic plants: the case of selected Greek species with conservation priority. In Vitro Cellular & Developmental Biology – Plant, 55:635-646, 2019.

New Generation Benzimidazole Based Plasmid Delivery Reagents with High Transfection Efficiencies on the Mammalian Cells

Current members of the Immunotechnology laboratory at Mersin University, Turkey. PI: Dr. Furkan Ayaz (L), Lab manager: MSc Serra Kuzay (R). We would like to thank our master graduates Qadar Ahmed Isse and Rusmeenee Kheeree for their valuable contributions to our other research studies.

Genetic engineering has paved new venues for biotechnology studies. It enables mass production of biological molecules that can either be used in medicine or industry. We utilize from bacterial plasmids and pieces of mammalian DNA incorporated into them during these processes but there is also another essential element in the picture: a transfection agent! From insulin production to gene therapy methods there has to be an efficient gene or plasmid delivery agent that can carry the genetic material into the mammalian cells. Carrying the package does not complete the job, these agents should also be able to release the piece of gene or plasmid that they interact with, inside the cell so that it can be incorporated into the genome and expressed by the cells. In our study, we selected a set of molecules (benzimidazoles) that are well known for their abilities to interact with DNA. In order to increase their efficiencies LogP values of the candidates were kept high and they had some chemical differences in their structures to observe the structure activity relationship while deciphering the most active compounds in terms of gene delivery (transfection) efficiencies to the mammalian cells in vitro.

We used a commercially available transfection agent that is mostly used in molecular and cellular biology studies and compared our products with the optimal protocol that they suggest to prevent any bias. The transfection efficiencies were measured by determining the GFP expression levels after delivering a GFP expressing plasmid in the mammalian cells. Based on our results, we can claim that we created new generation transfection agents that had superior activity than that of the commercially available popular one. The cytotoxicities of our compounds were minimal and they have high potential to be utilized in the field of biotechnology for transfection of the mammalian cells. In our current research, we are increasing the span of our library with similar molecules while further examining their intracellular journey. In our future studies, we are also aiming to test them for gene therapy purposes especially with a focus on cystic fibrosis disease model in mice.   

Furkan Ayaz, Ronak Haj Ersan, Burak Kuzu, Oztekin Algul.  New-generation benzimidazole-based plasmid delivery reagents with high transfection efficiencies on the mammalian cells. In Vitro Cellular & Developmental Biology – Animal, 56:34-41, 2020