RESEARCH NEWS STORY
May 1, 2024
Chiba University

Novel Genetic Plant Regeneration Approach
Without the Application of Phytohormones
Researchers develop a novel plant regeneration approach by modulating the expression ofgenes that control plant cell differentiation
Conventional plant regeneration approaches by cell culture require the external applicationof plant growth regulators, including hormones. However, optimizing culture conditions canbe laborious. Now, researchers have developed a novel plant regeneration system thatomits the need for hormone application by genetically regulating the expression of genesthat control plant cell differentiation. Their work holds significant potential in thedevelopment of genetically modified plants in a simpler and cost-effective manner.

Image title: Researchers develop novel gene expression-based plant regeneration approachwithout the application of plant growth regulators
Image caption: Conventional plant regeneration techniques require the application of plant
growth regulators, like hormones, which can be species-specific and laborious. In a new study,
researchers have developed a novel system of plant regeneration by modulating the functionand the expression of genes involved in plant cell dedifferentiation (cell proliferation) andredifferentiation (organogenesis).
Image credit: Associate Professor Tomoko Igawa, Chiba University, Japan
Image license: CC BY
Usage restrictions: Credit must be given to the creator.
For ages now, plants have been the primary source of nutrition for animals and mankind.
Additionally, plants are used for the extraction of various medicinal and therapeuticcompounds. However, their indiscriminate use, along with the rising demand for food,underscores the need for novel plant breeding practices. Advances in plant biotechnology canaddress the problems associated with food scarcity in the future by enabling the productionof genetically modified (GM) plants with higher productivity and resilience to the changing
climate.
Naturally, plants can regenerate an entire new plant from a single ‘totipotent’ cell (a cell thatcan give rise to multiple cell types) through dedifferentiation and redifferentiation into cellswith various structures and functions. Artificial regulation of such totipotent cells throughplant tissue culture is widely used for plant conservation, breeding, generation of GM species,
and scientific research purposes. Conventionally, tissue culture for plant regeneration
requires the application of plant growth regulators (PGRs), such as auxins and cytokinins, tocontrol cell differentiation. However, optimum hormone conditions can vary significantly withplant species, culture conditions, and tissue type. Therefore, establishing optimum PGR
conditions can be time-consuming and laborious.
To overcome this challenge, Associate Professor Tomoko Igawa, along with Associate
Professor Mai F. Minamikawa from Chiba University, Professor Hitoshi Sakakibara from theGraduate School of Bioagricultural Sciences, Nagoya University, and Expert Technician Mikiko
Kojima from RIKEN CSRS, have developed a versatile method of plant regeneration bymodulating the expression of ‘developmental regulator’ (DR) genes which control plant celldifferentiation. Giving further insights into their research work published on 3 April 2024 inVolume 15 of Frontiers in Plant Science, Dr. Igawa says, “Instead of using external PGRs, oursystem uses the DR genes, which are involved in development and morphogenesis, to control
cellular differentiation. The system utilizes transcription factor genes and resembles induced
pluripotent cell generation in mammals.”
The researchers ectopically expressed two DR genes, namely — BABY BOOM (BBM) andWUSCHEL (WUS) from Arabidopsis thaliana (used as the model plant), and examined theireffects on the differentiation of tobacco, lettuce, and petunia tissue cultures. BBM encodes atranscription factor that regulates embryonic development, while WUS encodes atranscription factor that maintains stem cell identity in the shoot apical meristem region.
Their experiments revealed that the expression of Arabidopsis BBM or WUS alone wasinsufficient to induce cell differentiation in tobacco leaf tissue. Conversely, co-expression offunctionally enhanced BBM and functionally modified WUS induced an accelerated andautonomous differentiation phenotype. The transgenic leaf cells differentiated into calli (adisorganized mass of cells), greenish organ-like structures, and adventitious shoots in theabsence of PGR application. Quantitative polymerase chain reaction (qPCR) analysis (atechnique used to quantify gene transcripts) revealed that the expression of Arabidopsis BBMand WUS was associated with the formation of transgenic calli and shoots.
Given the key role of phytohormones in cell division and differentiation, the researchers went
on to quantify the levels of six phytohormones, namely — auxins, cytokinins, abscisic acid(ABA), gibberellins (GAs), jasmonic acid (JA), salicylic acid (SA), and their metabolites in thetransgenic plant cultures. Their findings revealed that the levels of active auxins, cytokinins,
ABA, and inactive GAs increased as cells differentiated to form organs, highlighting their rolein plant cell differentiation and organogenesis.
Furthermore, the researchers used transcriptome by RNA sequencing (a technique used for
qualitative and quantitative analysis of gene expression) to assess the gene expression
patterns in the transgenic cells showing active differentiation. Their results suggested thatgenes related to cell proliferation and auxins were enriched among the differentiallyupregulated genes. Further validation using qPCR revealed that four genes were upregulatedor downregulated in the transgenic cells, including those regulating plant cell differentiation,
metabolism, organogenesis, and auxin response.
Overall, these findings shed light on the novel and versatile approach to plant regeneration
without the need for externally applying PGR. Moreover, the system used in this study has
the potential to advance our understanding of the fundamental processes of plant celldifferentiation and improve the biotechnological breeding of useful plant species.
Highlighting the applications of their work, Dr. Igawa remarks, “The reported system canimprove plant breeding by providing a tool to induce cellular differentiation of GM plant cells
without PGR application. Therefore, in societies where GM plants are accepted as products, itwould accelerate plant breeding and reduce associated production costs.”
About Associate Professor Tomoko Igawa
Dr. Tomoko Igawa is an Associate Professor at the Graduate School of Horticulture, Plant
Molecular Science Center, and Research Center for Space Agriculture and Horticulture atChiba University, Japan. Her research interests include plant sexual reproduction anddevelopment, as well as plant biotechnology. Her work focuses on understanding themolecular mechanisms underlying the sexual reproduction and differentiation of plant cells
using various transgenic systems. She has authored several publications in these domains andis a member of The Japanese Society for Plant Biotechnology, The Botanical Society of Japan,
Japanese Society of Breeding, The Japanese Society of Plant Physiologists, and The
International Association of Sexual Plant Reproduction Research.
Funding: This work was supported by Japan Society for the Promotion of Science KAKENHI
Grants (JP17K07600 to TI, JP19H04852 to TI, JP23H04732 to TI) and JST SPRING grants(JPMJSP2109 to YS).

Reference:
Title of original paper: Autonomous differentiation of transgenic cells requiring no external
hormone application: the endogenous gene expression and phytohormone behaviors
Authors: Yuka Sato1, Mai F. Minamikawa2, Berbudi Bintang Pratama1, Shohei Koyama1, Mikiko
Kojima3, Yumiko Takebayashi3, Hitoshi Sakakibara3,4 and Tomoko Igawa1,5,6
Affiliations: 1Plant Cell Technology Laboratory, Graduate School of Horticulture, Chiba
University, Matsudo, Japan
2Institute for Advanced Academic Research (IAAR), Chiba University, Chiba, Japan,
3RIKEN Center for Sustainable Resource Science, Yokohama, Japan
4Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
5Plant Molecular Science Center, Chiba University, Chiba, Japan
6Research Center for Space Agriculture and Horticulture, Chiba University, Matsudo, Japan
Journal: Frontiers in Plant Science
DOI: 10.3389/fpls.2024.1308417
Contact: Tomoko Igawa
Associate Professor, Chiba University
Email: tigawa@chiba-u.jp
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