Standardized Genetic Transformation Protocol for Chrysanthemum cv. ‘Jinba’ with TERMINAL FLOWER 1 Homolog CmTFL1a

Abstract

Chrysanthemum (Chrysanthemum x morifolium Ramat.) cultivar Jinba is a distinctive short-day chrysanthemum that can be exploited as a model organism for studying the molecular mechanism of flowering. The commercial value of Jinba can be increased in global flower markets by developing its proper regeneration and genetic transformation system. By addressing typical problems associated with Agrobacterium-mediated transformation in chrysanthemum, that is, low transformation efficiency and high cultivar specificity, we designed an efficient, stable transformation system. Here, we identify the features that significantly affect the genetic transformation of Jinba and standardize its transformation protocol by using CmTFL1a as a transgene. The appropriate concentrations of various antibiotics (kanamycin, meropenem and carbenicillin) and growth regulators (6-BA, 2,4-D and NAA) for the genetic transformation were determined to check their effects on in vitro plant regeneration from leaf segments of Jinba; thus, the transformation protocol was standardized through Agrobacterium tumefaciens (EHA105). In addition, the presence of the transgene and its stable expression in CmTFL1a transgenic plants were confirmed by polymerase chain reaction (PCR) analysis. The CmTFL1a transgene constitutively expressed in the transgenic plants was highly expressed in shoot apices as compared to stem and leaves. Overexpression of CmTFL1a led to a delay in transition to the reproductive phase and significantly affected plant morphology. This study will help to understand the biological phenomenon of TFL1 homolog in chrysanthemum. Moreover, our findings can explore innovative possibilities for genetic engineering and breeding of other chrysanthemum cultivars.

Development of a Transformation System and Locus Identification Pipeline for T-DNA in Chrysanthemum seticuspe, A Model Species for Hexaploid Cultivated Chrysanthemum

“…An increased amount of protein leads to the stagnation of metabolic processes and the death of explants [ 30 ]. Compared with other research results, it was reported that 15–25 mg·L −1 kanamycin completely inhibited callus induction and led to explant browning [ 28 ]. In this study, 10 mg·L −1 kanamycin was the optimal concentration for screening.…”

“…As previously shown in some studies, Li et al reported that a concentration ratio of 2:1 of cytokinin to auxin was conducive to the formation and differentiation of chrysanthemum leaf explants, while a high concentration of 6-BA (5 mg·L −1 ) and a low concentration of NAA (0.1 mg·L −1 ) were conducive to the induction and differentiation of shoot tip thin-cell-layer explants [ 28 ]. Similarly, Wang et al reported the highest germination rate of ground-cover chrysanthemum on MS medium containing 2.0 mg·L −1 6-BA and 0.5 mg·L −1 NAA [ 29 ].…”

“…After disinfection and sterilization with 75% ( v / v ) ethyl alcohol, the sterilized seeds were germinated in Murashige and Skoog (MS) medium [ 35 ]. When the seedlings grew, their shoots were removed and cultured in MS medium containing 3.0% sucrose and 0.32% plant gel that was adjusted to pH 5.8 before autoclaving at 116 °C for 30 min; samples were then incubated under a cold white fluorescent lamp (80–100 μmol·m −2 ·s −1 ) with a 16 h light cycle at 23 °C [ 3 , 28 ]. The bacterial strain Agrobacterium tumefaciens EHA105 harboring the pG10 plasmid was obtained from the State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University [ 36 ].…”

Towards the Improvement of Ornamental Attributes in Chrysanthemum: Recent Progress in Biotechnological Advances

“…Constitutive overexpression of CmTFL1a resulted in the delaying of the transition to the reproductive phase and significantly affected plant morphology in chrysanthemum cv. Jinba [ 114 ]. Nakano et al (2020) demonstrated that photoperiodic variations in heat sensitivity fluctuations affected flowering in chrysanthemum, supporting an earlier study wherein heat-induced flowering delay was caused by the suppression of FTL3 expression, suggesting that daily fluctuations in heat sensitivity may correlate with the rhythm of FTL3 regulation [ 115 , 116 ].…”

Genotype Screening of Recipient Resources with High Regeneration and Transformation Efficiency in Chrysanthemum

“…It was reported that the callus of chrysanthemum cultivar 'White ND' could be induced on MS medium supplemented with 1 mg L −1 6-BA and 2 mg L −1 NAA, but it needed to be changed onto medium containing 2 mg L −1 6-BA and 0.5 mg L −1 NAA to continue to differentiate [10]. Furthermore, most reports currently use cultivar 'Jinba' as the material for gene function verification [5,11], but it is a white, double-petaled cut chrysanthemum cultivar, which is not suitable for flower color and flower development research. Therefore, it is important to screen more chrysanthemum cultivars with high regeneration rates and diverse genotypes, and establish an efficient regeneration system for subsequent research.…”