Bulbs of ornamental plants have considerable economic value; however, seed degeneration is a major concern in several bulbous flower varieties due to its adverse effects on reproduction. Radiation mutagenesis bree?ding can help increase variation frequencies, enlarge variation ranges, and induce morphology?associated mutations, which may produce novel varieties within short time. However, plant survival is severely reduced due to physiological and genetic damage caused by 60Co?γ radiation treatments, and the frequency of beneficial mutations is low and insufficient. We used salicylic acid (SA), melatonin (MT) and gibberellin (GA) in order to reduce radiation?induced damage and death rates and to increase survival and flowering rates in freesia bulbs after 60Co?γ radiation treatments. The results may provide important technical information and reference values for cultivation and improvement of flower varieties.
Freesia （Freesia hybrida） bulbs were treated using 60Co?γ radiation at dosages of 55, 65 or 75 Gy after soaking treatments with SA at concentrations of 150, 300 or 450 mg/L and MT and GA at 5, 10 or 15 mg/L. Irradiated bulbs not treated with protective agents and bulbs treated with water only were used as controls. Effects of radiation protection on growth and development were assessed by measuring growth indicators and content of malondialdehyde (MDA) and superoxide dismutase (SOD).
At a radiation dose of 55 Gy, germination and survival rates in freesia bulbs treated with 150 mg/L SA were 86.87% and 90.00%, respectively, and were significantly higher than those of the radiation control group; plant height of freesia was 144.63% larger than that of the radiation control, and the number of leaves and total leaf area were 5.96? and 1.78?fold higher, respectively, compared to the radiation control. Flowering rates showed a maximum at 33.33%. At a radiation dose of 65 Gy, the three tested agents produced protective effects in a dose?dependent manner. In the 450 mg/L SA treatment, the survival rate was 79.67%, which was 91.68% higher than that in the radiation control; the number of small flowers in plants treated with 15 mg/L MT was 187.77% higher than that in the radiation control. In the high?dose irradiation treatment (75 Gy), high concentrations of protective agents showed better effects than low concentrations. Survival rate, plant growth and flowering rate in freesia bulbs treated with 450 mg/L SA were 122.23%, 268.17% and 500.60% higher, respectively, compared to the radiation control, and the number of leaves and total leaf area were 2.22? and 2.57?fold higher, respectively, than in the radiation control. Treatments with 15 mg/L MT and GA produced higher numbers of small flowers in freesia bulbs irradiated at 65 and 75 Gy with 7.83 and 5.68 more flowers, respectively, compared to the radiation control. Moreover, radiation protectants at different concentrations can significantly reduce the levels of MDA in irradiated freesia plants and delay the increase in SOD membrane?protective enzyme activity after radiation, suggesting that radiation protection agents can effectively promote the repair of radiation damage and alleviate physiological damage.
Treatments with SA, MT and GA after irradiation can effectively mitigate radiation damage and may thus be important for radiation protection. Treatments with 150 mg/L SA and 15 mg/L MT showed better protective effects than that of other groups of protective agents after radiation at 55 Gy, whereas under radiation doses of 65 and 75 Gy, 450 mg/L SA, 15 mg/L MT and 15 mg/L GA showed most effective radiation protection.
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has become the most common method for studying gene expression. The appropriate application of qRT-PCR in such studies requires the use of reference gene(s) as an internal control in order to normalize the mRNA levels between different samples for an exact comparison of gene expression levels. The aim of the present study was to screen optimized reference genes for qRT-PCR in Freesiahybrida for future studies on the gene expression of Freesia, especially the genes relative to corm development.
Freesia hybrida cultivar ‘Shangnong Golden Queen’ was used as the plant material in the study. Six different tissues or organs including tepal, pistil and stamen (mixed samples), flower scape, leaf, corm and root, corms at five developmental stages and corms under treatments with three exogenous hormones, were collected, respectively. qRT-PCR was used to analyze the gene expression level of selected eight commonly used housekeeping genes, including Actin, Glyceraldehyde-3-phosphate (GAPDH), β-tubulin (TUB), 18S rRNA, elongation factor 1 beta (EF-1β), ubiquinol-cytochrome C reductase (QCR), cyclophilin (CYP) and S-adenosylmethionine decarboxylase (SAMDC). Three widely-used software, including geNorm, NormFinder and BestKeeper, were used to make a comprehensive analysis on the stability of gene expression.
The eight candidate housekeeping genes could be expressed in diffe-rent tissues and organs of Freesia hybrida ‘Shangnong Golden Queen’ with individual expression richness. Among those, two genes, 18S rRNA and Actin were the most stable, while EF???1β was the most unstable gene based on the analysis with the three software. In the corms at five developmental stages, three candidate genes, QCR, Actin and TUB presented a stable expression level, which can be selected as reference genes in future studies on the gene expression involving corm development. While CYP and 18S rRNAwere not stable, these two genes were not introduced to analyze gene expression in corms at different developmental stages. In corms under treatments with exogenous hormones, the expression of CYP and 18S rRNA was quite stable, while EF?1β was the most unstable gene in this experimental condition.
This is the first time reference genes were screened for a given set of experimental conditions in Freesia hybrida. Taken together, 18S rRNAand Actin were the preferred internal reference genes for gene expression analysis in different tissues of Freesia. QCR，Actin and TUB were the optimized genes in corm development, while for the gene expression analysis under hormone induction, the preferred reference genes were 18S rRNA and CYP. Our findings provide a guideline for any future work on gene expression in Freesia by using qRT-PCR.
We investigated the effects of gibberellin acid (GA3) (0, 100, 200, 300 and 1 000 mg/L) on dormancy release to replace low-temperature treatments; furthermore, physiological changes in endogenous hormones and carbohydrate metabolism were examined. The results may provide theoretical and technical support for applied use of GA3 to terminate dormancy and regulate flowering time in Paeonia lactiflora.
We investigated effects of GA3 at different concentrations on plant growth and flowering using open-field overwintered potted P. lactiflora ‘Da Fugui’ as a control. We measured fructose, glucose and sucrose concentrations using high performance liquid chromatography (HPLC). Activities of enzymes associated with the metabolism of five sugar compounds and content of five endogenous hormones were determined using enzyme-linked immunoassays (ELISA) and liquid chromatography-mass spectrometry(LC-MS), respectively.
The results showed that 100, 200, 300 and 1 000 mg/L GA3 affected dormancy termination in P. lactiflora ‘Da Fugui’, and substantial differences in plant growth and development were observed in treated seedlings. Stem lodging and wilting rates of seedlings treated with 100, 200, 300 and 1 000 mg/L GA3 were 0.88%, 18.1%, 65.7% and 100%, respectively. Treatments with 100-300 mg/L GA3 led to substantially decreased height (variation rate 17.9%-23.5%), number of flowers (variation rate 46.6%-65.9%), and flower diameter (variation rate 11.8%-16.7%); however, the flowering process was normal. During dormancy and during the bud scale bursting period, fructose and glucose were the predominant soluble sugars in ‘Da Fugui’ plants, and the proportion of sucrose increased du?ring bud swelling. During dormancy, bud swelling, and bud scale bursting, sucrose synthase, cell wall invertase, and vacuolar invertase activity increased, whereas sucrose phosphate synthase and alpha-amylase decreased slightly initially and then increased. Gibberellin, auxin, abscisic acid and salicylic acid content initially decreased and then increased, whereas jasmonic acid content showed the opposite pattern.
Treatments with 100-400 mg/L GA3 affected dormancy release in ‘Da Fugui’ plants, and the strongest effect was observed at 100 mg/L GA3 which produced the best growth and flowering results. Fructose and glucose are the main soluble sugars during bud dormancy and bud scale burs?ting, and nutrients produced in the roots are transferred to buds in form of sucrose during bud swelling. GA3 treatments induced considerable changes in the levels of five endogenous hormones, and subsequent synergistic effects elicited the transition from dormancy to seedling growth.
