Agrobacterium-Mediated Genetic Transformation of Embryogenic Callus in a Liriodendron Hybrid (L. Chinense × L. Tulipifera)

Abstract

A highly efficient genetic transformation system of Liriodendron hybrid embryogenic calli through Agrobacterium-mediated genetic transformation was established and optimized. The Agrobacterium tumefaciens strain EHA105, harboring the plasmid pBI121, which contained the ß-glucuronidase (GUS) gene and neomycin phosphotransferase II (npt II) gene under the control of the CaMV35S promoter, was used for transformation. Embryogenic calli were used as the starting explant to study several factors affecting the Agrobacterium-mediated genetic transformation of the Liriodendron hybrid, including the effects of various media, selection by different Geneticin (G418) concentrations, pre-culture period, Agrobacterium optical density, infection duration, co-cultivation period, and delayed selection. Transformed embryogenic calli were obtained through selection on medium containing 90 mg L−1 G418. Plant regeneration was achieved and selected via somatic embryogenesis on medium containing 15 mg L−1 G418. The optimal conditions included a pre-culture time of 2 days, a co-culture time of 3 days, an optimal infection time of 10 min, and a delayed selection time of 7 days. These conditions, combined with an OD600 value of 0.6, remarkably enhanced the transformation rate. The results of GUS chemical tissue staining, polymerase chain reaction (PCR), and southern blot analysis demonstrated that the GUS gene was successfully expressed and integrated into the Liriodendron hybrid genome. A transformation efficiency of 60.7% was achieved for the regenerated callus clumps. Transgenic plantlets were obtained in 5 months, and the PCR analysis showed that 97.5% of plants from the tested G418-resistant lines were PCR positive. The study of the Liriodendron hybrid reported here will facilitate the insertion of functional genes into the Liriodendron hybrid via Agrobacterium-mediated transformation.

The Identification and Expression Analysis of the Liriodendron chinense F-Box Gene Family

“…Liriodendron is a basal genus of angiosperms within the Magnoliaceae family, consisting of two species: L. tulipifera , which is primarily distributed in North America, and L. chinense , mainly found in East Asia. Additionally, L. sino-americanum is a hybrid species formed by crossing these two species [ 27 ]. Liriodendron is an extensively cultivated ornamental tree species due to its beautiful tree shape and exceptional ability to withstand environmental adversity [ 28 ].…”

Overexpression of Liriodenron WOX5 in Arabidopsis Leads to Ectopic Flower Formation and Altered Root Morphology

“…There are many reports on WOX5 function in various crops, as mentioned above, but limited research has been performed on the WOX5 ortholog in woody plants, especially in non-model organisms. Liriodendron is widely distributed in Eastern North America [ 20 ] and has a high commercial value in China [ 21 ]. Liriodendron belongs to the Magnoliidae family, which takes up an evolutionarily intermediate position in between basal angiosperms and eudicots [ 20 ].…”

“…The study of Liriodendron may help us better understand the functional diversification of genes during plant evolution. Moreover, Liriodendron has a mature somatic embryogenesis and transgenesis system [ 21 ], making it an ideal woody plant. Although transgenic Liriodendron can be obtained through gene transformation, it requires significantly more time than the quintessential model plant, Arabidopsis .…”

AT-Hook Transcription Factors Show Functions in Liriodendron chinense under Drought Stress and Somatic Embryogenesis

“…Under drought stress, its low natural reproduction efficiency and seed germination rate limit regeneration of the natural populations [ 18 ]. The embryogenic callus of L. Hybrid possessed a mature genetic transformation system [ 19 ]. Therefore, finding an alternative gene that improves drought resistance and somatic embryogenesis efficiency can help to cultivate and promote new transgenic varieties with excellent resistance.…”