Abstract
The aim of this study was to establish a regeneration system of watermelon. Watermelon W1 was selected as the experimental material using seedling shoots as a receptor. The effects of different concentrations of 6-Benzylaminopurine (6-BA) on the shoot-tip of watermelon seedlings were studied. Number of shoots at the stem tip were counted every other day until the new buds reached 2 cm. The new stem tip was cut, and the effect of different concentrations of Murashige and Skoog (MS) medium on the number of regenerated roots and root length of shoots were studied. The results showed that the differentiation rate was highest when the 6-BA concentration was 0.7 mg/mL to 0.8 mg/mL. The optimum concentration for root regeneration was 1/8 MS. At this concentration, the number rooted was the highest, and root length was also promoted.
Watermelon is in the family Cucurbitaceae. Watermelon has its origin in Africa. It is a high-yielding fruit, widely distributed in the world; however, in the cultivation of watermelon production process, often a lot of diseases and pests, such as fusarium wilt and aphids (Chen et al. 1998, 2008), influence the growth and development of watermelon, thereby affecting watermelon yield and quality. Although the common breeding methods have cultivated many new varieties, some of the improvement of watermelon is still quite difficult to achieve. This is particularly important in watermelon transgenic research, to create new and high-quality germplasm resources. Watermelon production and variety improvement are inevitable. However, the regeneration ability of watermelon is weaker than other plants, and the frequency of regeneration is low, which is the limiting factor of genetic engineering improvement. With the rapid development of genetic engineering, so that breeders can use transgenic technology to quickly obtain high quality and high resistance to pests and diseases of new varieties of watermelon, a highly efficient regeneration system is needed (Chiang et al. 2011; Lin et al. 2012; Liu et al. 2016). Conventional breeding methods have cultivated many excellent varieties, but the improvement of some traits is more difficult. Genetic engineering has become an effective way for additional improvement of watermelon. Watermelon stem tip regeneration can be used to improve in vitro regeneration success to improve watermelon characters by transgenic methods. In recent years, many people have studied and explored the establishment of a watermelon regeneration system, looking for an efficient and fast regeneration system. But the methods studied are not the same. The results are also different.
The establishment of a generation system of watermelon is crucial for transgenic technology (Ren et al. 2021). Although in recent years, there have been many research methods that explored many hormone formulations, but in the course of these experiments the conditions should be strictly aseptic, which makes them complicated.
Adventitious bud differentiation induction is the tissue culture method that is the choice of most of the researchers at present (Jin et al. 2024). These are mainly divided into two categories, direct induction and callus re-differentiation (Pizarro and Díaz-Sala 2022). In the in vitro regeneration of watermelon, the probability of budding directly induced by hypocotyls is relatively high. The callus is first formed and then differentiated into buds (Chaturvedi and Bhatnagar 2001; Krug et al. 2005; Venkatachalam et al. 2018; Zhang et al. 2023). Because it is difficult to induce callus to form regenerated plants, watermelon regenerants require a long period and thus are prone to vitrified seedlings. In the establishment of this system, adventitious buds were usually directly induced to form adventitious buds to regenerate intact plants (Hamdeni et al. 2022; Niu et al. 2015). To this end, the characteristics of adventitious buds in the watermelon stem tip can be used to establish the watermelon stem tip regeneration system, laying the foundation for watermelon genetic engineering.
Materials and Methods
Watermelon strain W1 seed
Seed treatment.
A sample of 180 watermelon strain W1 seeds was placed in water at 55 °C to soak. The solution was continuously mixed until the water temperature dropped to room temperature and then soaked for an additional 10 h. The seeds were then wrapped with gauze and watered once a day until germination. After the seeds of watermelon germinated, the germinated seeds were sown to the nursery tray. Each seedling plate held 20 seeds and each hole held a seed. After 2 d, the seedlings were unearthed as the cotyledons had not yet started, with a small blade to remove the stem tip. The stem tip was then removed as a receptor.
Adventitious bud induction.
After removal of the terminal buds, the concentration of 6-BA was 0.0 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, and 0.8 mg/mL. The same amount of 6-BA was added to the stem tip wound for 2 consecutive days. The change of the growth point of the stem tip was observed at the later stage, and the change was observed every day. The moisturizing culture was observed and the leaves remained moist. When the growth point of the expansion, and can be divided into many adventitious buds.
Adventitious buds.
When the adventitious buds reached 2 cm, the adventitious buds were cut into different concentrations of MS medium. The MS concentrations were water (without MS), MS, 1/2 MS, 1/4 MS, 1/8 MS, 1/16 MS, each of four adventitious buds were treated and observed every 24 h. The rooting situation, number of roots, and root length were recorded. Photographs were taken to observe changes. Every 2 d for MS culture medium to prevent contamination of the culture medium, affecting the rooting. In 2 to 3 d adventitious buds will grow roots.
Statistical analysis
The data are presented as the means of replications (plants), data were analyzed with IBM SPSS 25.0 and Excel 2010. All statistically significant differences were tested at the P ≤ 5% level.
Results and Discussion
As shown in Table 1, adventitious buds were found at concentrations of 0.1 mg/mL, 0.2 mg/mL, 0.7 mg/mL, and 0.8 mg/mL. The highest differentiation rate was 0.8 mg/mL, but the number of adventitious buds decreased. Concentrations of 0.3 mg/mL-0.6 mg/mL did not produce adventitious buds. As can be seen from Table 1, the 6-BA-treated plants at concentrations of 0.7 mg/mL and 0.8 mg/mL produced the most adventitious buds and had a significant effect on the adventitious bud formation, and the 6-BA concentration of 0.7 mg/mL produced the largest number of adventitious buds. In the experiment, it was found that high concentrations of 6-BA (0.7 or 0.8 mg/mL) had an inhibitory effect on plant height growth compared with the control group. Studies have shown that concentrations of 6-BA exceeding a certain level can inhibit the growth of maize hypocotyls (Qi et al. 2024), which is consistent with our research findings.
Effects of different concentrations of 6-BA on adventitious buds in watermelon stem tip.
According to Table 2 and Fig. 1, the treatments of water (without MS), MS, 1/2 MS, 1/4 MS, 1/8 MS, and 1/16 MS, produced different degrees of rooting. When the concentration was 1/8 MS, the number of roots was the largest and the length of the root was also the longest. In Fig. 1, the overall trend of growth showed first a rise and then decline. Root length also showed such a trend. Root length and root number are the highest at 1/8 MS. In Fig. 2, it can be clearly seen that 1/8 MS contributes to root production and growth.
Effects of different concentrations of MS on rooting of stem tip.
Effects of different concentrations of MS on adventitious root.
Citation: HortScience 59, 9; 10.21273/HORTSCI18094-24
Comparison of morphological characteristics of root growth under different MS concentrations.
Citation: HortScience 59, 9; 10.21273/HORTSCI18094-24
From Table 3, after 10 d of observation and data measurement, we can find that when the concentration was 1/8 MS, the rooting speed was the fastest and the number of roots was the most. But when the concentration was MS, the rooting speed was the slowest, the first 3 d without root, after 7 d gradually took root.
The effects of different concentrations of MS on adventitious roots.
From Table 4, it is clear that when the concentration was 1/8 MS, adventitious shoots took root rapidly in the first 4 d, then from days 4 to 7 showed slow growth, and after 7 d growth leveled off to a certain range. Based on the preceding data analysis, it can be concluded that 1/8 MS could promote the rooting of adventitious buds.
1/8 MS 10 d the number of root changes.
In this experiment, the stem tip was removed, and 6-BA was added to the wound before the cotyledon was completely expanded, and the adventitious buds could be directly induced. In the establishment of watermelon stem tip regeneration, most researchers use 6-BA as the main hormone. In this experiment, we wanted to select the 6-BA concentration suitable for inducing adventitious buds from shoots. The concentration of hormone 6-BA was designed to be different. When the adventitious buds were induced to grow to 2 cm to 3 cm, the adventitious buds were cut and the appropriate MS concentration was induced.
The biggest advantage of this experiment is it does not need strict aseptic operation. The whole process can be completed at the seedling stage. Experiments proved 1/8 MS is conducive to root generation. The culture medium only required changing every few days given the low requirements for sterile conditions. Hormones play an important role in the differentiation of adventitious buds, and Song et al. (2023) has found that induction of explant differentiation on hormone-free media requires a long time. He indicates that the plant's regenerative capacity depends on its genetic type but not hormone content. He found in the experiment the explants cultured on the hormone-free medium will not differentiate adventitious buds for an extended period (Song et al. 2023). Induction of differentiation of watermelon explants requires the need for a certain amount of concentration of 6-BA (Vasudevan et al. 2017). In Cucurbitaceae, 6-BA plays an important role in explant tissue culture induction of adventitious bud differentiation. This is also true in cucumber and pumpkin plant tissue regeneration, which was confirmed by experiments (Kintzios et al. 2002). In the establishment of shoot-tip regeneration of watermelon, most people use 6-BA as the main hormone. However, the explants used by Wang et al. (2013) were cotyledons, which were differentiated into adventitious buds by inducing callus. Moreover, the 6-BA concentration they used was higher than that in this experiment.
The optimal conditions for the establishment of adventitious buds of watermelon stem tip, especially in the proportion of plant hormones, are important conditions for the success of the establishment of watermelon stem tip regeneration The appropriate hormone ratio can effectively promote the watermelon stem tip differentiation rate, and make the plant robust, induced rooting quickly. The survival rate of the plant is very high. In this experiment, the concentration of 6-BA was graduated. The results showed that 6 mg of 0.7 mg/mL to 0.8 mg/mL had the greatest effect on adventitious buds. The concentration of 0.7 mg/mL up to 0.8 mg/mL had the highest differentiation rate.
In the course of the experiment, after the addition of hormones, it was critical to keep the foliage wet to prevent injury to the stem tip and prevent desiccation. However, excess water causes rot. The experiment also has some problems worthy of our consideration. In this experiment, we only considered 6-BA on the stem tip of the adventitious buds. We did not consider the interaction between hormones. In the future, experiments will take full account of these factors.
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