Fronds Out! Establishing a Novel Transformation System in Lemna Minor
Fronds Out! Establishing a Novel Transformation System in Lemna Minor
Isabelle Deng Thomas Jefferson High School for Science and Technology
This article was originally included in the 2020 print publication of the Teknos Science Journal.
I poke at the clump of duckweed with the end of my pencil. In the span of just three hours, my healthy green fronds have shrunken into blistered white masses. After taking observations, they join my pile of failed duckweed cultures. Some are moldy, some are shriveled, some are stored in agar plates and some in liquid medium -- but each failure is still a step closer to establishing a novel genetic transformation protocol for the TJHSST Biotechnology Lab.
Lemna minor, or the common duckweed, is an attractive model system due to its rapid growth, ease of metabolic labelling, and small genome. Duckweed shows high potential for biofuel production, due to its efficient photosynthesis output and high starch accumulation. Approximately 28.9% of duckweed dry weight is protein, and it contains 40 different types of vitamins and minerals. According to the FAO Amino Acid Reference Pattern (a measure of nutritive value centered around amino acid abundance), duckweed has a favorable abundance of the amino acids critical to a nutritious diet [1]. Because of this high protein, starch, and vitamin content, duckweed could be a potential inexpensive dietary supplement for livestock and poultry [3]. Despite this potential for industrial applications, however, current duckweed research is constrained by a lack of efficient genetic transformation tools. Until researchers can rapidly introduce or knockout genes of interest within the duckweed genome to enhance desired features, duckweed will not be commercially viable.
The most common method of genetic manipulation for plants is Agrobacterium-mediated transformation. Agrobacterium tumefaciens and related Agrobacterium species are gram-negative bacterial “plant pathogens” known for using horizontal gene transfer to infect plants with crown-gall disease. Agrobacterium induces tumor-like growths, called galls, by conjugatively transferring a DNA segment (T-DNA) from the bacterial tumor-inducing (Ti) plasmid. Since the 1990s, Agrobacterium has been used to transform plants and fungi, using a modified Ti plasmid. The three main genetic components necessary for transformation are: the T-DNA, the virulence (vir) genes, and three chromosomal virulence loci [2].
The T-DNA, which acts like a mobile genetic element, is the DNA segment transferred from the Agrobacterium and integrated into the plant genome. This T-DNA is called the T-region, and is located on the tumor-inducing (Ti) or rhizogenic (Ri) plasmid inside Agrobacterium. Virulence (vir) genes regulate the processing and transfer of T-DNA from Agrobacterium to plant cells. The VirD1/VirD2 T-DNA border-specific endonuclease processes the T-DNA from the Ti plasmid. These vir proteins cleave the T-DNA borders, resulting in a single-stranded T-DNA molecule called the T-strand. It is this T-strand, and not a double-stranded T-DNA molecule, that is transferred to plant cells. The VirD4 and the 11 VirB proteins make up the type IV secretion system (T4SS) that transfers the T-DNA from the Agrobacterium to the plant cells. The three chromosomal virulence loci (chvA, chvB, and exoC) aid the T4SS in transforming plant cells, as they are responsible for exopolysaccharide production, modification, secretion, and other roles in bacterial attachment to plant cells [4].
My research compares the transformation efficiency of a traditional callus induction system (CIS) to the newer frond transformation system (FTS) when introducing new genetic material to duckweed. The CIS was introduced in the 1990s and has remained the most popular duckweed transformation method since then. Callus tissue is a mass of undifferentiated plant cells, formed to cover plant wounds, with the potential to become any type of specialized cell. When induced via selective injury of the duckweed meristem and incubation with Murashige and Skoog (MS) basal salts, calluses has a high frequency of successfully retaining novel genetic information after incubation with recombinant Agrobacterium. While the CIS has a high transformation effciency and produces stable recombinant fronds, it is genotypically restricted, resource intensive, and time consuming. Not every strain of duckweed, as derived from geographic isolates, can successfully induce calluses using the established CIS protocol. The entire procedure, from callus induction to frond regeneration, takes anywhere between 7-9 months, thus making it impossible to replicate in a school laboratory [2]. These constraints limit commercial-scale production of genetically-modified duckweed.
In 2018, Yang et al. introduced the FTS, a novel duckweed transformation protocol that successfully transformed and regenerated recombinant duckweed in half the time needed for the CIS. Rather than using the callus induction method, researchers used vacuum infiltration to forcefully introduce the recombinant vector into adult fronds [4]. The processing time is significantly shorter because the FTS involves manipulating fully differentiated plant cells, rather than cultivating tissue cultures in vitro. Furthermore, the FTS is not reagent intensive, as the brute force method of vacuum infiltration replaces traditional induction. Although its transformation efficiency is slightly lower than that of the CIS, its accessible nature makes it ideal for commercial use [1].
Prior researchers have successfully transformed and regenerated recombinant duckweed using Agrobacterium. Liu and his team (2019) refined Agrobacterium-mediated transformation protocols for two species of duckweed, Lemna minor (8627 and 8744) and Lemna gibba. Partially differentiated duckweed fronds were co-cultivated with Agrobacterium tumefaciens containing the nptII expression cassette, which confers kanamycin resistance. The study transformed the duckweed using an Agrobacterium-mediated method, in which they put the duckweed in a broth culture with Agrobacterium containing the gene of interest. Their results showed the gene was successfully transformed into the duckweed without the help of traditional genome manipulation devices [2]. However, as Dr. Yamamoto acknowledges, “the genotypic restrictions caused by callus propagation indicates that my lab’s protocol will not necessarily create stable recombinants for some geographic isolations of L. minor” (Yamamoto, personal communication, Feb. 1, 2020).
By selectively incorporating components of various transformation systems, I hope to establish a new genetic manipulation protocol for the Biotechnology Lab. Selectively injuring both the meristem and parenchyma of duckweed fronds increases the rate of success in the callus induction protocol [1]. By incorporating this finding into the traditional CIS, I will observe if a higher rate of calluses are successfully generated, and if the selective injury mitigates genotypic restrictions arising from varying geographic isolates of duckweed. Yang et al. (2018) used green fluorescent protein (GFP) as the reporter gene for duckweed transformation, and used a flow-cytometer and quantitative polymerase chain reaction (qPCR) to quantify and evaluate the gene expression of the GFP gene in the transformed duckweed [4]. By introducing the GFP gene into the Agrobacterium vector and incubating this vector into duckweed calluses, I hope to evaluate the transformation efficiencies of the CIS and the FTS.
The FTS has opened new doors to the commercialization of duckweed as industrial livestock feed and a biofuel source. With a quick turnaround between initial transformation and frond regeneration, future students in my lab will be able to transform duckweed and replicate their trials multiple times within a single school year. Although researchers are encouraged to develop novel techniques, verifying previously published protocols are vital to a successful academic environment. By testing and establishing the FTS protocol within the Biotechnology Lab, we have created a new path for future students to explore.
References
[1] Kozlov, O. N., Mitiouchkina, T. Y., Tarasenko, I. V., Shaloiko, L. A., & Firsov, A. P. (2019). Agrobacterium-Mediated Transformation of Lemna minor L. with Hirudin and alpha-Glucuronidase Genes. Applied Biochemistry and Microbiology, 55, 808-815. https://doi.org/10.1134/S0003683819080076
[2] Liu, Y., Wang, Y., Xu, S., Tang, X., Zhao, J., Yu, C., ... Zhou, G. (2019). Efficient genetic transformation and CRISPR/Cas9-mediated genome editing in Lemna aequinoctialis. Plant Biotechnology Journal, 17(11). https://doi.org/10.1111/pbi.13128
[3] Yamamoto, Y. T., Rajbhandari, N., Lin, X., Bergmann, B. A., Nishimura, Y., & Stomp, A.-M. (2017). Genetic Transformation of Duckweed Lemna gibba and Lemna minor. In Vitro Cellular & Developmental Biology - Plant, 37(3). https://doi.org/10.1007/s11627-001-0062-6
[4] Yang, G.-L., Fang, Y., Xu, Y.-L., Tan, L., Li, Q., Liu, Y., ...Zhao, H. (2018). Frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor. Plant Molecular Biology, 98(4-5), 319-331. doi.org/10.1007/s11103-018-0778-x
[5] Yang, J., Hu, S., Li, G., Khan, S., Kumar, S., Yao, L., ... Hou, H. (2020). Transformation Development in Duckweeds. In X. H. Cao, P. Fourounjian, & W. Wang (Eds.), Compendium of Plant Genomes: The Duckweed Genomes (pp. 143-155). https://doi.org/10.1007/978-3-030-11045-1_15.
