Optimization of Tissue Culturing and Genetic Transformation Protocol for Casuarina equisetifolia

Abstract

Casuarina equisetifolia is widely used in agroforestry plantations for soil stabilization, ecosystem rehabilitation, reclamation, and coastal protection. Moreover, C. equisetifolia has remarkable resistance to typhoons, desert, low soil fertility, drought, and salinity, but not cold. Therefore, it is significant to breed high-quality Casuarina varieties to improve the tolerance and adaptability to cold weather by molecular techniques. The establishment of a rapid and efficient callus induction and regeneration system via tissue culture is pre-requisite for the genetic transformation of C. equisetifolia, which is so far lacking. In this study, we reported an efficient and rapid regeneration system using stem segment explants, in which callus induction was found to be optimal in a basal medium supplemented with 0.1 mg⋅L–1 TDZ and 0.1 mg⋅L–1 NAA, and proliferation in a basal medium containing 0.1 mg⋅L–1 TDZ and 0.5 mg⋅L–1 6-BA. For bud regeneration and rooting, the preferred plant growth regulator (PGR) in basal medium was 0.5 mg⋅L–1 6-BA, and a combination of 0.02 mg⋅L–1 IBA and 0.4 mg⋅L–1 IAA, respectively. We also optimized genetic a transformation protocol using Agrobacterium tumefaciens harboring the binary vector pCAMBIA1301 with β-glucuronidase (GUS) as a reporter gene. Consequently, 5 mg L–1 hygromycin, 20 mg L–1 acetosyringone (As), and 2 days of co-cultivation duration were optimized to improve the transformation efficiency. With these optimized parameters, transgenic plants were obtained in about 4 months. Besides that, Agrobacterium rhizogenes-mediated transformation involving adventitious root induction was also optimized. Our findings will not only increase the transformation efficiency but also shorten the time for developing transgenic C. equisetifolia plants. Taken together, this pioneer study on tissue culturing and genetic transformation of C. equisetifolia will pave the way for further genetic manipulation and functional genomics of C. equisetifolia.

Genome-wide identification of WRKY transcription factors in Casuarina equisetifolia and the function analysis of CeqWRKY11 in response to NaCl/NaHCO3 stresses

“…The C. equisetifolia seedlings were selected from the plant tissue culture room at 25 ℃ with 16 h/8 h light/dark cycle in Zhejiang Agriculture and Forestry University. The stem segments were cut from the selected seedlings and grew in the rooting medium for about two months [ 30 ]. The regenerated seedlings with similar size (about 15 cm in height) were transferred into hydroponic solution for different treatments, including 1/2 Hoagland nutrient solution (CK), 1/2 Hoagland nutrient solution with 300 mM NaCl, 300 mM NaHCO 3 , and pH = 8.5, respectively.…”

“…For phenotype analysis, the seedlings of CeqWRKY11 overexpression lines ( CeqWRKY11OE-1 and CeqWRKY11OE-2 ) generated by using hairy root transgenic system [ 30 ] and control plants (transformed with empty vector, named as CK) were transplanted in 1/2 Hoagland nutrient solution, and cultivated in plant greenhouse at 25 ℃ with 16 h/8 h light/dark cycle for about two months. During the process, the nutrient solution were replaced each week.…”

“…In addition, C. equisetifolia plays significant roles in wind prevention, sand fixation, and soil improvement [ 27 , 28 ]. With the publication of the C. equisetifolia genome data and establishment of the regeneration and genetic transformation systems, it is possible to study the molecular mechanisms of stress tolerance in C. equisetifolia [ 29 , 30 ]. In recent years, there has been increasing research on the mechanism of C. equisetifolia response to stress, especially in response to salt stress.…”

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

“…Regeneration conditions are determined by optimizing various factors, such as different explants, combination and concentration of plant hormones. Mullein ( Casuarina equisetifolia ) stem segments [ 6 ] and garland chrysanthemum leaf disks as explants [ 7 ] can induce callus, adventitious buds, and adventitious roots with different hormone combinations. Two regeneration pathways in sunflower were reported in the late 20 th century, including organogenesis (immature embryos [ 8 , 9 ], cotyledons [ 10 – 12 ], the thin cell layer of the hypocotyl [ 13 ], stem tips [ 14 ], dicotyledons [ 15 ], protoplasts [ 9 ]) and somatic cell embryogenesis (embryogenesis immature embryos [ 16 , 17 ] and pollen sacs [ 18 ]), but these process were unsystematic and superficial.…”

Micropropagation, encapsulation, physiological, and genetic homogeneity assessment in Casuarina equisetifolia

“…In vitro propagation of tree plants is a difficult process for several reasons, viz ., excessive secretions of phenolic, high rate of contamination, callus formation at the basal end of the explant, and difficulty in the rooting process (Harada and Murai, 1996 ; Giri et al, 2004 ). A very limited number of studies are available on the micropropagation of C. equisetifolia (Lin et al, 2012 ; Satheeshkumar and Gupta, 2012 ; Ren et al, 2022 ). However, the available methods are not suitable for mass production because of the limited rate of shoot multiplication and poor rooting of the microshoots of C. equisetifolia .…”