Starch is the main form of storage carbohydrate in plants and is important in the carbon economy of many organs, tissues, and cell types of plants [27
]. Soluble sugars play an important role in plant growth and developmental processes. They provide energy and mid-metabolites, and also act as signals, regulating the vital movements of plants [28
]. The BA was reported to help in accumulating starch in Lemna minor
] and ETH in increasing starch content in apple [30
], which are in agreement with results of this study. Medium drenching or foliar spray of BA or ETH led to higher starch content in both cultivars of strawberry plants, while BA injection resulted in the lowest starch content for both cultivars. Root drenching or foliar spray of BA or ETH led to increased starch contents in both cultivars of strawberry plants, while BA injection decreased starch content to the lowest level for both cultivars. On the other hand, BA injection induced an increased level of soluble sugar content in both cultivars. Presumably, some functions of BA help in transforming starch into soluble sugars, and the intensity of this transformation is related to different plant tissues. It was observed that injection of any of three PGRs resulted in shortened runner length for both cultivars, while drenching or spraying PGRs did not. This indicates that PGRs have different mobile characteristics and induced effects depending on the location on the strawberry plants where they are applied, just as hypothesized. The actual mechanisms behind this need to be investigated further.
It is reported that CCC is highly mobile in both xylem and phloem tissues, and is rapidly absorbed and translocated [31
]. However, CCC shows different mobility characteristics depending on the species. It was observed that CCC had high mobility in wheat but slow uptake/movement in barley, making CCC more effective in wheat than in barley [32
]. In this study, strawberry plants with CCC treatments displayed a similar number of new leaves and runners, length of the first node, percentage of plants with new runners, and runner dry weights, while no significant differences were observed among the plants with respect to the application method. Therefore, it can be concluded that CCC is highly mobile in strawberries too. However, CCC injection into crowns resulted in a more pronounced shortening of the runner length compared to foliar spray or medium drench. This is probably because CCC injection works directly on the crown where runners are generated, while foliar spray and medium drench result in reduced CCC dosage during the transportation process, making CCC relatively less effective in shortening the runner length.
Truernit et al. (2006) found in Arabidopsis thaliana
that cytokinin regulates the expression of invertase and transports hexose [33
]. As a kind of cytokinin, BA has the effect of inhibiting chlorophyll, nucleic acid, and protein decomposition in the leaves, and various functions such as transporting amino acids, auxin, and mineral salts to plant parts exposed to it [34
]. Another important characteristic of BA is its poor mobility in plants; furthermore, its physiological effects are limited to the treatment site and its vicinity. That is the reason why BA injection into crowns induced many more new runners and leaves but medium drench and foliar spray with BA did not. Due to BA helping in accumulating nutrients, runners of plants injected with BA were much stronger. The competition for nutrients among these strong runners made them have a shorter average length compared to runners under other treatments. As a benefit, growers have more options to get healthy and strong runners, while also saving the nutrients and energy to support the mother plant.
The ETH is quite different from the other two PGRs mentioned above, as it eventually decomposes into ethylene, so temperature (high temperatures accelerate ETH movement inside the plant), pH, period of usage, and the plant growth stage can easily influence the effect of ETH [35
]. In plant production settings, ETH is applied by spraying, dipping, smearing, or air fumigation, among which spraying is the most commonly used. The ETH can be absorbed by leaves, stems, fruits, and other organs but is mainly taken up through leaf surface absorption. When ETH enters the vascular bundle, it is transported to other tissues and organs as the organic matter moves, so ETH has some mobility within the plant [36
]. The ETH that enters the cell is broken down gradually to release ethylene, and then produces its effect on the plant. The ETH is similar to ethylene in that it enhances the synthesis of RNA in cells and promotes the synthesis of proteins. It has also been shown by studies that plants treated with ETH had high protein contents (Figure 2
Both crown injection and medium drench of ETH induced dead leaves, albeit to different degrees, and inhibited formation of new leaves and runner in strawberries. Foliar ETH spray did not induce any leaf deaths. The other two PGRs did not have the same leaf-killing and development-inhibiting effects. These results indicate that ETH has differing effects that depend on the application site, or that different plant tissues or organs have different tolerances to ETH, as they do to certain natural phytohormones. Another possible explanation for the differing effects of ETH by application method is the concentration of the solution. In this study, a concentration at 100 mg·L−1 was used. The higher the applied concentration, the more likely the occurrence of phytotoxicity. Phytohormones are characterized by low doses but high efficiencies. Some of them, such as IAA, have effects that depend on the concentration, where they promote plant growth at a low concentration but inhibit growth at a high concentration. As an analogue of plant hormone, ETH may have similar characteristics. To clarify the reason and mechanism by which dead leaves were induced by crown injection and medium drench of ETH in this experiment, further research is needed.
Stressful environments can cause the accumulation of reactive oxygen species (ROS) or free radicals in plants. Harsh environments, normal oxygen metabolism, certain chemical reactions, or toxic agents in the environment could force plants to produce such substances that continuously threaten the cells and tissues. The ROS and free radicals are able to disrupt the metabolic activity and cell structure. When this occurs, additional free radicals are produced in a chain reaction that leads to more extensive damage to plants, particularly the oxidation of DNA, proteins, and membrane lipids. Fortunately, plants can defend themselves against such damages via synthesizing antioxidant enzymes such as SOD, POD, and CAT to eliminate stresses [37
]. It is true that the use of PGRs could push plants into stressed states. One example is the dead leaves and a low number of newly-grown runners in plants treated with ETH as discussed above. The experimental results also confirmed that the treatments did cause some biological stresses on the strawberry plants, because the activities of antioxidant enzymes in most of the treated plants were higher than that in the control group. Generally, ETH treatments resulted in higher activities of antioxidant enzymes, and BA treatments, especially by injection, resulted in lower activities of antioxidant enzymes in strawberry. To some extent, BA injection had little toxic and side effects on strawberry mother plants.
Although the PGRs selected were reported to have low toxicity, according to a research in mice, ETH could be harmful for the kidney and liver even in small doses [38
]. The CCC was also reported to be toxic on the fertility of mammals such as pigs and mice [39
]. The BA toxicity is seldom reported, and thus can be considered as no concern for human and animal safety. For plant safety, improper application or excessively high concentrations of CCC result in severe marginal leaf chlorosis or chlorotic spotting [40
Typical application methods for PGRs are foliar spray or medium drench, but substrate spray, bulb spray, seed soak, cutting, and liner dips are also used. Each method has advantages and disadvantages, so appropriate methods should be chosen for a particular situation [41
]. The injection method used in the experiment was actually similar with cutting and liner dips in that it damages the surface of plant tissues and lets the PGRs into the plant body. It is not easy to inject PGR solutions into strawberry crowns because strawberry do not have blood vessels like human or animals. If we obey the definition of ‘injection’ strictly, namely, it involves pricking the surface tissues of plants with an injector and then sending the solution inside, as tested, one person can finish one or two plants per minute, and this was the method tried prior to this experiment.
In the second turn of the experiment, a new method for raising efficiency was found and used. An injector was used to make small pores on the crowns, pushed in to let its contents out, without the need for pricking the entire needle inside. This allowed solutions to enter the plant through small pores, and the effectiveness was not compromised at all, compared with the first method in prior experiment discussed above. This allowed for three-four additional plants to be treated per minute. This means that, for example, if a grower wished to control the number and length of 5000 strawberry plants, it would take 1000 man/min (16.7 man/h) under ideal conditions, making this improved injection method practically applicable to real production environments.