Before reading this article, please pay attention so that you can receive more valuable information in the future!
Grafting is an effective horticultural practice for vegetable crops that enhances soil-borne disease resistance, tolerance to abiotic stresses, nutrient uptake, and improved crop quality. Watermelon is a very popular and widely cultivated fruit crop with high economic value and was the first vegetable crop to be grafted commercially on a large scale.
Double root cutting grafting is a recently commonly used watermelon grafting method that involves cutting off the roots of the scion and rootstock. DRC grafting is an effective technique that has a higher survival rate and shorter healing time compared to other traditional grafting modes, and also enhances the vitality and productivity of crops.
In addition to temperature and humidity, a growing body of research has shown that light is another important environmental factor affecting the survival rate and quality of grafted plants. Compared to traditional light sources, light-emitting diodes (LEDs) have low energy consumption, low heat output, controllable light quality and intensity, and long service life.
Supplementation with a variety of LED lighting could significantly promote the scion-rootstock interaction, enhance the vascular reconnection of grafted wounds, and change the total leaf area, total chlorophyll/carotenoid ratio, soluble protein and sugar content of grafted seedlings.
In this study, the effects of different light quality on DRC grafted watermelon seedlings were studied, and it was found that Fr could significantly promote growth, especially AR regeneration. In order to verify the effect of AR regeneration on graft healing, the acidic magenta coloring method was used to explore the transport activity of the vasculature, and the healing conditions under different red/far-red light treatments were compared.
In addition, transcriptome analysis showed that auxin and photochromin interacting factor related genes were greatly affected by Fr, and the expression profiles of these genes were verified by qRT-PCR. Then, HPLC-MS/MS was used to analyze the concentrations of different forms of auxin in AR under different light treatments. These results may be helpful in identifying effective light patterns that promote root transformation of grafted watermelon seedlings and identify possible regulatory mechanisms.
1. Materials and methods
1.1. Plant material, grafting and light treatment
Watermelons are used as scions and pumpkins are used as rootstocks. All of them belong to commercial cultivars. The seeds of the scion and rootstock are sown in a plastic plug with 72 cells. Scion seeds are sown 2 days after rootstock seeds, so that the scion and rootstock seedlings have approximately the same stem diameter at the time of grafting.
DRC grafting is carried out when both the scion and the rootstock have a true leaf present. Half of the cotyledons of each rootstock are removed to avoid mutual shading caused by adjacent seedlings. Insert the scion into the rootstock with a grafting needle and hold it together with a plastic grafting clip.
Subsequently, the grafted seedlings are planted in 72-well vegetable seedling trays and immediately covered with matching dish lids to maintain humidity. In this study, seedling substrate was used as the object of plant growth. After grafting, in order to prevent leaf dehydration, the relative humidity was above 3% for the first 95 days, and gradually decreased to 93%, 90% and 85% every day, after which the lid was removed.
Different light treatments were applied to the first 3 days after grafting, and then the seedlings were transplanted to 100 μmol·m-2·s-13 days. "Fr" stands for Mixed Light, which adds far-red light to white light, reducing the red/far-red ratio to 0.3. All auxiliary lighting is provided by LED light modules.
In this study, a pattern of gradually increasing the light intensity was used. In the first 3 days, we chose a relatively low light intensity, which was about 50 μmol·m-2·s-1, and in the 4-6 d and 7-10 days, the light intensity increased to 100 and 200 μmol·m-2·s-1, respectively.
1.2. Root morphology and growth index analysis
After 8 days of light treatment, the roots of grafted seedlings were randomly selected from each treatment. After rinsing with distilled water, the morphology of the roots is imaged using a root scanning device. The WinRHIZO 2009 software analyzes the morphological index of the root according to the method described earlier.
The growth index of grafted watermelon seedlings was analyzed under different light treatments, and the seedlings were grown under standard white growth light for 20 days. Use a scale to weigh fresh and dry weight. The height and thickness of the scion and rootstock were measured with a ruler and vernier calipers, respectively.
1.3. Analysis of the transport capacity of the vasculature of grafted seedlings under different light treatments
Grafted seedlings treated with D (dark color), W (white), Fr 3.0 and Fr0.3 were randomly selected for 20 days to determine the transmission capacity. Franc 0.3 and Fr3.0 red/far-red ratio light is 0.3 and 3.0, respectively.
After the root system was removed, the stem base of the grafted seedlings was placed in 1% acidic magenta solution for 3 h to absorb magenta. Then, all the leaves are weighted using an electronic scale. The supernatant was collected and the absorbance was measured using a spectrophotometer with a wavelength of 5200 nm.
2.4. RNA extraction, transcriptome sequencing, and quantitative RT-PCR validation
We selected rootstocks treated with dark light (D), white light (W), and far-red light (Fr) at 4 and 8 days as samples for transcriptome analysis. The roots and shoots of the rootstock were sampled separately. Since there were no obvious adventitious roots in the D and W treatments, we collected 1.5 cm of tissue from the incision of the roots, in which the location of the adventitious roots appeared, as a root sample for RNA sequencing.
Total RNA was extracted from each sample by RNAprep Pure Extration Kit and biology was replicated 260 times. After verifying RNA purity by spectrophotometry, first-strand cDNA synthesis was performed using DNA-free total RNA from different samples at a ratio of 280/1 nM >9.5.
Total RNA was extracted from samples treated with D, W, and Fr for 4 and 8 days. The rootstock was sampled by dividing the buds and roots. Primer sequences for all genes were set using Primer Premier 6 software.
2. Data presentation and statistical analysis
Statistical analysis was performed using the SPSS statistical software package. One-way ANOVA combined with Duncan's multi-range test was used to analyze the differences between treatments, and P<0.05 was the threshold.
3. Results
3.1 Effects of different light treatments on root regeneration and survival rate of rootstocks
Compared with other light treatments, the far-red light accelerated the rooting of rootstock after 4 days of grafting. Therefore, far-red light can induce the formation of AR in DRC-grafted watermelon seedlings. In order to further verify the effect of far-red light on AR formation, a root scanning device was used to detect the root morphology of AR.
After 8 days, there were significant differences in root growth under different light treatments. Far red light significantly improves AR regeneration. In contrast, dark and blue light is not conducive to the formation of AR. White light, red light, red light + blue light had no significant effect on AR formation. Root scans showed the same results.
Far red light improved the AR regeneration of DRC-grafted watermelon seedlings. With the exception of the blue treatment, all light treatments increased the total length of the roots and the number of root tips. Among them, the total root length under far-red light was increased by more than 4 times compared with the dark light treatment.
Similarly, the number of root tips was increased by more than 1.9 times in far-red light compared to the dark treatment. Blue light had a negative effect on the root growth of grafted seedlings. Red + blue light mixed light increased root growth, but was lower than far red light.
3.2. Far red light treatment had a significant effect on auxin concentration
Our transcriptome data and qRT-PCR results showed that the transcription levels of auxin-associated DEGs were significantly altered by Fr. As a result, we detected different forms of auxin in AR. During the 4 d and 8 d, the IPyA concentrations of D and Fr treatments were higher than those of W treatments.
Fr induced IAM and TAM concentrations at 4 days, but there was no significant difference between D and W. At 8 d, the concentrations of IAM and TAM reached the highest and lowest concentrations in Fr and W, respectively. Fr significantly increased the IAN concentration at 4 and 8 days.
In addition, at 4 and 8 d, the concentration of IAA in Fr was significantly induced, the concentration of IAA in W decreased, and the inactivated auxin and auxin catabolite IAA-Glu were significantly decreased in D, reaching a peak at W. Similarly, IAA-Asp concentrations were lower than W in both D and Fr, but the difference was not significant.
In summary, Fr increases the concentration of activated auxin and decreases the concentration of inactivated auxin. In other words, the mutual signal transduction process of auxin is affected by Fr, indicating that Fr can activate auxin-related pathways.
4. Discussion
Nowadays, there is an increasing demand for grafted seedlings with higher yield, quality, and resistance to abiotic and biotic stresses around the world. Many studies have shown that the healing process of graft binding is influenced by supplemental lighting and illustrates its mechanism.
However, the mechanism of light-induced rooting of watermelon seedlings grafted by DRC has not been reported. In addition, many studies have shown that the first 3 days after grafting are the most critical processes for the growth of grafted seedlings, which include the exchange of signals between scion and rootstock cells, auxin-related vascular reconnection, and the resumption of vasculature transport activities.
Therefore, in this study, we determined the effect of supplemental light on enhancing rootstock root regeneration during healing. We believe that root regeneration is induced by Fr and that it may be related to auxin-related pathways.
IAA-induced root formation with intracellular and extracellular calcium (Ca2+) concentrations. Our transcriptome data show that DEGs involved in calcium signaling are significantly affected by Fr. In Fr treatment, all DEGs involved in calcium signaling were detected by RNA-seq.
In contrast, some DEGs were not detectable in the D and W treatments. In addition, the number of DEGs at 4 days was greater than that at 8 days, indicating that early light had a stronger effect on the transcription level of DEGs related to calcium signal transduction.
The qRT-PCR results also showed that the expression level of calcium-binding protein 27 in Fr was 3 times higher than that in group D, and the expression level in group W was lower than that in group D. The results showed that Fr light could promote calcium signaling during AR rooting, which was beneficial to auxin-induced AR regeneration.
- conclusion
The results of this study provide a new idea for the regulation mechanism of far-red light regulating the root regeneration of rootstocks of watermelon seedlings grafted by DRC. Fr induces the expression of CmPIFs. At the same time, CmPIF7 and CmPIF1 activated the transcription of auxin-related genes such as CmIAA3, CmIAA11 and CmAUX17, thereby activating the auxin pathway and promoting root regeneration.
In addition, the increase of auxin accumulation further induced the transcription levels of CmSAUR28 and CmSAUR3 involved in cell elongation and callus formation, which was conducive to the regeneration of AR in DRC-grafted watermelon seedlings.
This study preliminarily elucidated the mechanism of Fr influencing the growth of grafted seedlings and the formation and development of AR in rootstocks. The results of this study provide new ideas for speeding up grafting recovery and improving the quality of grafted watermelon seedlings.