Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis

Abstract

The recurring growth of bacterium in newly developed resistant cells and a minimal level of bacterial infection rate are the main limiting factors of Agrobacterium-mediated transformation experiments in Hevea brasiliensis. The current study aimed to optimize crucial factors of the transformation protocol in order to obtain an efficient transformation experimental model for Hevea using cotyledonary somatic embryos as explants. Transformation conditions such as antibiotic concentration, preculture duration, Agrobacterium concentration, sonication and cocultivation conditions were analyzed using the binary vector pCAMBIA2301. Transient transformation was confirmed by GUS histochemical staining. The best transformation efficiency was observed when the explants were not cultured on a preculture medium that contained acetosyringone at a level of 100 μM. The best results were obtained using a bacterial density of 0.45 at OD 600 nm, 50 s of sonication of explants in a bacterial liquid culture and a total incubation time of 18 min in the same bacterial suspension. Transmission electron microscopical analysis confirmed the impacts of sonication on bacterial infection efficiency. Cocultivation conditions of 22 °C and 84 h of darkness were optimal for the transfer of T-DNA. Agrobacterium was eliminated with 500 mg/L of timentin, and the selection of transformants was performed using 100 mg/L of kanamycin in the selection medium. The presence of transgene was confirmed in the resistant embryos by polymerase chain reaction (PCR). The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest into the Hevea genome for the breeding of this economically important plant species in the future.

An optimized somatic embryo transformation system assisted homozygous edited rubber tree generation method mediated by CRISPR/Cas9

“…In addition, the induction of somatic embryo from anther callus is somewhat difficult, which also impedes the genetic transformation efficiency using anther as explants. Alternatively, SEs can be used as explants to continuously provide plentiful receptor materials by highly efficient cyclic proliferation, and with relatively high transformation efficiency (Hua et al ., 2010; Udayabhanu et al ., 2022). Appropriate screening markers are crucial for genetic transformation efficiency, kanamycin is usually used as a suitable selection agent to screen transgenic cell lines in dicotyledonous species, including Hevea (Jayashree et al ., 2003; Kala et al ., 2014; Huang et al ., 2015), however, the impact of different screening markers on somatic embryo transformation has not been systematically studied in Hevea somatic embryo transformation.…”

“…Unexpectedly, this is harmful to embryonic induction and development, and successive constant 10 mg/L hygromycin in the medium is the best favourable for embryo transformation. By combining optimized transformation methods (Udayabhanu et al ., 2022) and suitable hygromycin screening strategy, the embryo transformation efficiency is improved from previous 4% (Huang et al ., 2015) to 27% in present study.…”

“…In the past decades, Agrobacterium -mediated transformation is the most wildly used method in Hevea , and although transgenic Hevea seedlings were regenerated by using embryogenic calli as receptor sources (Jayashree et al ., 2003; Blanc et al ., 2006; Huang et al ., 2010; Lestari et al ., 2018), the transformation efficiency was not satisfactory due to the low level of calli-to-embryogenesis and seedling regeneration, and the explants are not available throughout the year. Alternatively, somatic embryos (SEs) have been proved to be amenable explants for Agrobacterium infection in Hevea , and can achieve a good transformation efficiency (Huang et al ., 2015; Udayabhanu et al ., 2022), but a major drawback is that the regenerated cells are always not the transformed cells (Niazian et al ., 2017; Wang et al ., 2017), thus we inferred that the transgenic seedlings are largely chimeric although there is no powerful molecular evidence. In present study, the efficiency of embryo transformation was improved by antibiotic sensitivity analysis and adopting optimum selection strategy.…”

Induction of Axillary Bud Swelling of Hevea brasiliensis to Regenerate Plants through Somatic Embryogenesis and Analysis of Genetic Stability

“…From the results obtained, we confirmed that the phenological impacts were very high on the induction of ABs. Explant age is a crucial factor in the success of plant micropropagation [32,33]. The most suitable phenological stage was the mature period (70.83 ± 7.22), which exhibited the best AB swelling rates compared with the bronze period, color-change period and pale-green period.…”

Tissue culture and Agrobacterium-mediated genetic transformation of the oil crop sunflower

“…Genetic transformation is usually carried by Agrobacterium -mediated method, its transformation efficiency is often influenced by different factors, such as Agrobacterium strains, bacterial density, duration of infection, co-culture time, acetosyringone concentration, and the type of antibiotics [ 20 ], and so on. The gene was successfully transformed into Hevea brasiliensis using cotyledonary somatic embryos as explants [ 21 ], similarly, immature leaf segments of Switchgrass ( Panicum virgatum ) [ 22 ] and leaves of dandelion ( Taraxacum officinale ) were successfully transformed to obtain positive seedlings by optimizing various conditions [ 23 ]. Genetic transformation in sunflower is less reported.…”