Agrobacterium-mediated genetic transformation of Chinese chestnut (Castanea mollissima Blume)

Abstract

Chinese chestnut (Castanea mollissima) is an important germplasm resource for the breeding of Castanea species worldwide with vital ecological and economic value. Biotechnology overcomes the limitations of traditional breeding and accelerates germplasm improvement. However, a genetic transformation system for Chinese chestnut has not yet been established. In this study, a stable and efficient Agrobacterium-mediated genetic transformation method for Chinese chestnut is described. Embryogenic calli of C. mollissima cv. 'Yanshanhongli' were used as the target material. The sensitivity of embryogenic calli to kanamycin was determined, whereby the proliferation of non-transformed calli was completely inhibited at 180 mg/L. Antibiotic inhibition results for Chinese chestnut embryogenic calli showed that 50 mg/L cefotaxime and 500 μM timentin completely inhibited the growth of Agrobacterium tumefaciens but did not affect the normal growth of Chinese chestnut embryogenic calli. When embryogenic calli were co-cultured for 2 days with Agrobacterium tumefaciens strain AGL1 harboring the PBI121-EGFP plasmid, an embryogenic callus transformation efficiency of 4.55% was obtained, and two transgenic chimera were acquired. This Agrobacterium-mediated transformation system for Chinese chestnut provides a fundamental platform for genetic improvement of core germplasm and for further verification of gene function. Key messageA method of Agrobacterium-mediated transformation was established for Chinese chestnut, providing a basis for genetic improvement of core germplasm and an important platform for further verification of gene function in Chinese chestnut.

Recent Advances in Adventitious Root Formation in Chestnut

“…The availability of complete genomes for this genus should pave the way for a deeper understanding of the reasons that underlie the genotype differences that are found to be the reason behind the differential responses to the rooting treatments in the works analyzed here. Chinese chestnut is on the verge of a significant biotechnological burst due to the completion of the sequencing of its genome and the development of a transformation protocol [ 153 , 154 ]. Together with the work developed by the Hardwood Genomics Initiative in chestnut (hardwoodgenomics.org), available information might be crucial for the development of biotechnological advances that will improve our knowledge concerning this genus, as well as the development of protocols that might help overcome the difficulties in its improvement.…”

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

“…The transformation protocols rely on the availability of regenerative explant sources, and is a time-consuming process to produce transgenic individuals ( Song et al, 2019 ). Embryogenic calli have been demonstrated to be the best target explants for Agrobacterium -mediated transformation in some plant species ( Dutt and Grosser, 2010 ; Yang et al, 2010 ; Sun et al, 2020 ). Somatic embryogenesis is an ideal tool to be used in the study of genetic transformation.…”

“…Compared with microprojectile bombardment and electroporation, Agrobacterium -mediated transformation is a common tool for plant transformation with many advantages, such as easy operation, low cost, low-copy transgene integration, the transfer of large DNA fragments ( Xia et al, 2020 ), and insertion priority into transcriptionally active regions ( Cha et al, 2012 ). Agrobacterium -mediated transformation has been widely used in research and applied to many woody species to acquire transgenic plants ( Shou et al, 2004 ; Prasad et al, 2014 ), such as Populus ( Fillatti et al, 1987 ), Eucalyptus globulus ( Matsunaga et al, 2012 ), Quercus robur ( Vidal et al, 2010 ), elm ( Bolyard et al, 1991 ), Chinese chestnut ( Castanea mollissima ; Sun et al, 2020 ), and European larch ( Shin et al, 1994 ).…”

Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with a Gene (CsTL1) Encoding a Chestnut Thaumatin-Like Protein

“…Cytotoxic effects in plant cells caused by GFP expression have been mentioned in previous studies [ 57 ], but the data obtained in the proliferation of embryos and plant regeneration, as well as the morphological appearance of the transgenic material, suggest that GFP gene expression is not toxic in cork oak. Similarly, deleterious effects were also not observed in the GFP -mediated genetic transformation of somatic embryos of several citrus cultivars [ 58 ], avocado [ 59 ] or Chinese chestnut [ 60 ].…”