Transformation of Teosinte (Zea mays ssp. parviglumis) via Biolistic Bombardment of Seedling-Derived Callus Tissues

Abstract

Modern maize exhibits a significantly different phenotype than its wild progenitor teosinte despite many genetic similarities. Of the many subspecies of Zea mays identified as teosinte, Zea mays ssp. parviglumis is the most closely related to domesticated maize. Understanding teosinte genes and their regulations can provide great insights into the maize domestication process and facilitate breeding for future crop improvement. However, a protocol of genetic transformation, which is essential for gene functional analyses, is not available in teosinte. In this study, we report the establishment of a robust callus induction and regeneration protocol using whorl segments of seedlings germinated from mature seeds of Zea parviglumis. We also report, for the first time, the production of fertile, transgenic teosinte plants using the particle bombardment. Using herbicide resistance genes such as mutant acetolactate synthase (Als) or bialaphos resistance (bar) as selectable markers, we achieved an average transformation frequency of 4.17% (percentage of independent transgenic events in total bombarded explants that produced callus). Expression of visual marker genes of red fluorescent protein tdTomato and β-glucuronidase (gus) could be detected in bombarded callus culture and in T1 and T2 progeny plants. The protocol established in this work provides a major enabling technology for research toward the understanding of this important plant in crop domestication.

The green strategy for recombinant protein production in maize using mesoporous silica nanoparticles

“…This plant is categorized as a recalcitrant plant and its transformation and regeneration are dependent on genotype, type of explant, and several parameters such as the composition of the culture media and plant growth regulators (Lagrimini 2018). The current transformation protocols require a very long time to con rm the gen introduction and a totally about 8-10 months to generate transgenic seeds (Zobrist, et al 2021). Furthermore, the average transformation frequency is between 4-7% in the best and optimal situation (Abhishek, et al 2014;Cao, et al 2014;Hood and Howard 2009).…”

Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum

“…Various research groups have explored alternative starting explants for the transformation of grass species. Except for japonica rice, readily transformed via Agrobacterium infection of mature seeds 20 , these groups have used either mature seed-derived embryos 21,22 or seedling-derived leaf bases [23][24][25][26] to first establish callus cultures, which are then used as the target for Agrobacterium infection. The use of Wus2 and Bbm expanded the list of alternative explants for transformation, as demonstrated by the regeneration of fertile transgenic plants using mature embryo slices and leaf-base tissue segments in maize 10 .…”

“…The leaf base has been an attractive tissue culture explant for almost four decades, with callus initiation and regeneration being demonstrated in numerous Poaceae species including wheat [41][42][43][44] , oat 45,46 , barley 47,48 , rye 49 , maize 23 , rice 50 and various apomictic grasses 51 . Accordingly, researchers have used leaf bases to generate embryogenic callus as the target explant for Agrobacterium infection and the regeneration of transgenic plants-for example, in maize 23 , indica rice 24,52 , Ma bamboo 25 and teosinte 26 . However, except for a report in orchard grass (Dactylis glomerata) 53 where transgenic plants were produced, studies where leaf-base tissue has been used as the direct transformation target were limited to demonstrating only transient transgene expression in rice 54 , wheat 55 and maize 56 .…”

Application of CRISPR/Cas9 for targeted mutagenesis in teosinte Zea mays ssp. parviglumis

“…We recently established the first genetic transformation method for teosinte ( Zea mays ssp. parviglumis ) using a biolistic delivery method and leaf‐derived callus explants (Zobrist et al ., 2021 ). In this work, we report the first successful use of CRISPR/Cas9 in teosinte to generate targeted gene knockouts via short indel mutations using a single guide RNA (sgRNA) and precise sequence deletion using two sgRNAs.…”