Visual Starch Stain Procedure Assists Cycad Propagation Decisions

Abstract

Stem cuttings are often exploited for asexual propagation of cycad plants, and impaired health of the source plants may reduce success. A reliable procedure that screens potential source plants for predicted success would benefit conservationists. The cut surfaces of Cycas revoluta stem cuttings obtained from source plants that had endured 0–39 weeks of Aulacaspis yasumatsui herbivory were stained with potassium triiodide to determine if this visual test could predict asexual propagation success. The stem cuttings were sorted into three groups based on stain intensity, then specific gravity and starch concentration of pith and cortex tissue were measured. The cuttings were maintained in a propagation nursery for 27 weeks, then root growth was quantified as dry weight. Specific gravity ranged from 1.01 to 1.11 and scaled linearly with starch concentration, which ranged from 16 to 195 mg·g⁻¹. The group of cuttings with the darkest stain intensity exhibited 100% propagation success with 23 g of roots per cutting, and the group with the least stain intensity exhibited 30% success with 2 g of roots per cutting. The group of stems with intermediate stain intensity exhibited intermediate values of every metric. These results indicated that cycad cuttings may be stained with iodine to estimate starch content in the field to provide an accurate instantaneous visual test for selection of the source plants that reliably generate the greatest level of asexual propagation success.

1. Introduction

Asexual propagation of new plants through adventitious root formation on stem cuttings is widely used in cycad horticulture and conservation [1,2]. The procedure is successful if source plants are healthy, and a prophylactic sealant of some type is used to protect the open wound of the pachycaulous cycad stem. Conservationists are often forced to manage threatened forest populations that exhibit compromised health of tree individuals for many years before ultimate mortality occurs. The reduced health of the source plants in cases such as this may thwart attempts to asexually propagate the plants.

These issues have been investigated in the case study from Guam, where the native Cycas micronesica K.D. Hill has become endangered by the invasive armored scale Aulacaspis yasumatsui Takagi [3,4,5]. The highly successful adventitious root formation on stem cuttings from healthy C. micronesica plants is compromised when cuttings are derived from unhealthy plants [4]. Therefore, a reliable procedure that accurately screens potential source plants for predicted propagation success would benefit conservation practitioners who are expected to select which C. micronesica trees to propagate during conservation protocols.

Cycad stems are a rich source of starch [1]. Chronic herbivory of a Cycas plant by A. yasumatsui leads to depletion of stem starch, which then leads to the reduction in asexual propagation success [4]. Starch reacts with iodine to create a blue-black color that is more intense as starch concentration increases in the plant tissues [6]. The iodine reacts with the helical amylose portion of starch. These facts have been exploited to stain the cut surface of apple (Malus domestica Borkh.) fruits with potassium triiodide to provide a visual protocol to identify when starch content of developing apple fruits has declined sufficiently to signify appropriate harvest date [7]. A similar procedure conducted on the cut surface of cycad stem cuttings may prove to be effective in diagnosing which source plants contain the greatest levels of nonstructural carbohydrates and therefore the greatest likelihood of successful development of adventitious roots.

The aim of this study was to exploit the potassium triiodide starch stain procedure on stem cuttings of a representative Cycas species to determine if the visual test could accurately predict which cuttings would most successfully generate success in the vegetative propagation procedures. Stem cuttings exhibiting the darkest stain on the cut surfaces were predicted to contain the most starch and be the most successful in root formation during the subsequent propagation phase.

2. Materials and Methods

Cycas revoluta is endemic to several islands in the Ryukyu Archipelago but is cultivated in more countries than any other cycad species. The distinctive tree is often found in public landscapes of temples, churches, government buildings, and schools (Figure 1a).

Thirty healthy C. revoluta plants were obtained from a commercial nursery in Angeles City, Philippines on 2 December 2021. The plants were originally derived from asexual propagation of stem suckers, which is the most common form of propagation of this species in the Philippines. Maximum stem diameter ranged from 6 to 9 cm. The container medium was inconsistent among the plants, so all plants were carefully bare-rooted and replanted in 2.6 L containers in a medium of 50% washed river sand and 50% dried rice hulls. The plants were grown inside of 2 × 2 × 2 m cages that were constructed with polyester screening (130 holes per centimeter), which excluded all insects. Each container received irrigation as needed, and plant nutrition was provided by weekly drenches of 100 mL per container of a complete nutrient solution (Excel, Everris, North Charleston, SC, USA) with a concentration to provide 7.5 mM nitrogen.

The goal was to create a wide range in duration of A. yasumatsui infestation among the individual plants to ultimately generate a group of plants with a wide range in health. One of the cages was purposefully infested with A. yasumatsui, such that all biological control organisms were excluded by the screening. One plant was transferred to the cages containing the living population of A. yasumatsui on 6 March 2022, and one more plant was transferred every 10 days until the end of the infestation period on 14 December 2022. This protocol provided plants that had endured A. yasumatsui herbivory for 0–39 weeks.

To end the herbivory stress phase and begin the propagation phase, the leaves were pruned from each plant at the base of the petioles, then stem cuttings were harvested from the plants by excising the apical stem section. Each of the 30 stem cuttings exhibited the same gestalt appearance at the end of this propagation procedure, so the propagator could not determine the health status or duration of A. yasumatsui herbivory for each cutting by looking at the freshly prepared plant material. The basal 1 cm of stem was cut from each cutting for specific gravity and starch analysis of the pith and cortex tissue. Specific gravity was calculated by measuring the fresh weight of a cutting in air, followed by measuring of the weight after submerging in water [8]. A metric with no units results from dividing the submersion weight into the air weight. Starch was quantified by drying the tissue at 75 °C in a forced draft oven for 48 h, milling the tissue, using amyloglucosidase to hydrolyze the starch, then quantifying the resulting glucose pool [9].

The final cut surface of each cutting was stained with potassium triiodide solution (1.7 mg iodine per 100 μL water). The 30 cuttings were sorted visually by observing iodine stain intensity from the least intense to the most intense stained cutting. The cuttings were then assembled to provide one group of the 10 least stained cuttings, one group of the 10 most stained cuttings, and one group of the 10 intermediately stained cuttings (Figure 2).

Adventitious root formation was fostered using standard cycad stem cutting protocols with horticultural perlite as the substrate. Indole-3-butyric acid in powder form (3 mg·g⁻¹) was applied to the cut surfaces then petroleum-based commercial pruning sealant was used to cover the wounds. The cuttings were inserted into the perlite in 2.6 L containers on 14 December 2022 and maintained under 50% shade for 27 weeks. The perlite was irrigated three times per week. All 30 cuttings were harvested on 22 June 2023.

Cuttings with no new roots or leaves were examined to determine viability. Cycad stem cuttings that remain alive in a nursery facility were turgid, and the parenchyma exhibited an off-white color. Cuttings that died were pliable or brittle, their tissues were dry, and their colors became dark. Therefore, diagnoses of living versus dead cuttings were unambiguous. A binary variable was created to determine propagation success, with living cuttings assigned a 1, and dead cuttings assigned a 0. The adventitious roots were cut from the base of each cutting, dried in a forced draft oven at 75 °C, and then dry weight was measured.

The specific gravity and starch concentration data were subjected to a 2 × 3 factorial ANOVA with two tissue types and three levels of stain intensity. Means separation among levels of significant factors was conducted with Tukey’s HSD test. The binary survival variable and root dry weight data did not meet prerequisites for parametric tests, so the non-parametric Kruskal–Wallis H test was used.

3. Results

3.1. Specific Gravity and Starch

Specific gravity of C. revoluta stem tissue exhibited significant differences between pith and cortex tissues and among the three levels of potassium triiodide stain intensity (

Table 1. Analysis of variance results for specific gravities and starch concentrations of Cycas revoluta stem cuttings. Sources of variation included two tissue types (pith and cortex), three levels of potassium triiodide stain intensity, and the interactions of tissue and intensity. n = 10.

Trait	Source	df	f	p
Specific gravity	Tissue	1	34.335	<0.001
	Intensity	2	162.172	<0.001
	T × I	2	7.2717	0.001
Starch	Tissue	1	30.314	<0.001
	Intensity	2	164.144	<0.001
	T × I	2	8.430	<0.001

). The interaction between these two sources of variation was also highly significant.

The pith tissue exhibited greater specific gravity than the cortex tissue, and the specific gravity increased in the order of lightest to darkest potassium triiodide stain intensity within each tissue type (

Table 2. Specific gravities and starch concentrations of Cycas revoluta stem cuttings as influenced by stem tissue type and potassium triiodide stain intensity. Mean ± SE, n = 10.

Tissue Type	Stain Intensity	Specific Gravity	Starch (mg·g−1)
Pith	Light	1.026 ± 0.003 d *	43.4 ± 6.1 d
	Intermediate	1.066 ± 0.003 b	117.8 ± 6.9 b
	Dark	1.095 ± 0.002 a	179.2 ± 7.8 a
Cortex	Light	1.024 ± 0.003 d	42.8 ± 5.1 d
	Intermediate	1.048 ± 0.004 c	86.2 ± 6.8 c
	Dark	1.069 ± 0.004 b	128.8 ± 6.1 b

* Means within columns with same letters were not different according to Tukey’s HSD test.

). The greatest specific gravity occurred in the pith tissue of the darkest stained stems. The interaction was significant because the specific gravity of the lightest stained stems did not differ between pith and cortex tissue, but the specific gravity of the intermediate and darkest stained stems was greater for pith than for cortex tissue.

The factorial analysis for starch concentration of C. revoluta stem tissue exhibited patterns that corroborated the specific gravity results, with both sources of variation and their interactions all being highly significant (Table 1). Starch was greatest in pith tissue from the darkest stained stems and decreased in the order dark pith > dark cortex = intermediate pith > intermediate cortex > light cortex = light pith (Table 2). Again, the interaction was significant because pith contained more starch for the dark and intermediate stain intensities, but there were no differences in starch between pith and cortex for the light stain intensity.

Specific gravity and starch concentration scaled linearly for pith and cortex tissues (Figure 3). The slope of the line for pith tissue was 5% greater than the slope of the line for cortex tissue, indicating that the increase in starch for each unit increase in specific gravity was greater for pith tissue. The upper limits of the starch concentration for pith tissue were 21% greater than for cortex tissue.

3.2. Survival and Root Growth

Cutting survival differed among the three levels of visual stain intensity (Figure 4a). The cuttings with the darkest stain exhibited 100% success, but more than 40% of the cuttings in the intermediately stained group and more than 60% of the cuttings in the lightest stained group died during the propagation phase. The adventitious root dry weight for the intermediately stained cuttings was 38%, and the lightest stained cuttings were 11% of that for the darkest stained cuttings (Figure 4b).

4. Discussion

Starch and other nonstructural carbohydrates decline in host Cycas stems as the duration of A. yasumatusi herbivory increases, and these declines are correlated with the limited success of asexual propagation after long-term A. yasumatsui herbivory [4]. The results herein revealed for the first time that a potassium triiodide stain is effective for detecting which scale-infested plants contain the greatest contents of stem starch, illuminating a new protocol to add to the cycad conservation toolbox. The results also revealed that the plants exhibiting the darkest stained stem surfaces may be selected from the general population of scale-infested plants to obtain the greatest level of successful adventitious root formation on cuttings. Allocation of resources under conditions of data uncertainty often occurs during conservation of endangered plant species. In this context, this new visual stain procedure may reduce that uncertainty and thereby increase efficiency of cycad conservation propagation expenditures by filtering out the source plants that would most likely end in propagation failure.

4.1. Stem Starch Estimation

This study has revealed that starch concentration, specific gravity, and potassium triiodide staining may each be employed to predict which Cycas stem cuttings may foster the greatest level of asexual propagation success. A comparison of these three procedures is warranted. The measurement of specific gravity is cumbersome but may be quantified in the field with the appropriate hardware. This includes a battery-operated balance with high precision and wind-exclusion capabilities. Light-weight vessels to contain water for the submersion procedure are also needed to obtain the denominator to calculate specific gravity. The measurement of starch requires extraction of stem tissue, which must be then dried, milled, and subjected to laboratory chemical analysis. These steps cannot be accomplished in the field, the cost may become prohibitive depending on the number of samples, and the starch data may require days or weeks to obtain. Moreover, the direct measurement of starch and specific gravity requires destructive harvesting of tissue from the stems, and some situations may occur where the cuttings are not large enough to sacrifice the tissue. Therefore, some benefits to the use of the potassium triiodide visual stain procedure instead of starch concentration or specific gravity procedures include the instantaneous result, the ease of use in the field, the lack of a need to destructively harvest tissue, and the minimal cost. Moreover, no special skills are required of practitioners to visually examine the cut stem to see how dark the stain develops. The approach only needs to generate a range in stain intensity for the procedure to be exploited to cull the cuttings with the least amounts of starch. The stain intensity range may be altered if the stain intensity appears dark for every cutting by diluting the potassium triiodide with water or washing the stained surface with ethanol. Although other solutions are available to deliver iodine to stain starch in plant tissues, a new conservation protocol is most efficacious if easily available products are used so practitioners can readily adopt the new protocol. Potassium triiodide may be purchased over the counter as Lugol’s Solution from major retail pharmacy outlets, so access to the stain is achievable for most conservation practitioners.

The upper range in starch concentrations of the cuttings with the darkest stains was greater for the C. revoluta pith tissue than for cortex tissue. Moreover, the starch concentration of pith tissue increased more rapidly per unit of increase in specific gravity than cortex tissue. These radial differences were likely due to the dilution of parenchyma tissue by vascular strands that permeated the cortex, as these vascular cells with high lignin and cellulose contents were absent from the pith tissues of cycad stems [1]. These observations indicated that future studies or conservation protocols that employ cycad stem starch or specific gravity measurements may be most effective by focusing exclusively on pith tissue.

4.2. Conservation Applications

Cycads comprise the most threatened group of plants worldwide [10], and the armored scale insect A. yasumatsui has emerged as one of the greatest threats to contemporary cycad survival [11]. As more cycad species become threatened by this invasive insect herbivore, the lessons learned from the recent history of C. micronesica and C. revoluta research may allow conservationists to avoid some of the costly and ineffective protocols that have occurred on the island of Guam [3,4,12]. The mistakes in Guam combined with results from the current study indicate that conservation projects designed to rescue cycad trees within habitats that are newly invaded by A. yasumatsui should transpire immediately after the invasion occurs to ensure that extensive plant mortality did not occur due to apathy of the conservation decision-makers. The early attempts at plant rescue would also enable greater success because stem nonstructural carbohydrates would remain elevated when compared to rescue attempts carried out after years of neglect. Moreover, most conservation decisions are made within a zero-sum game reality, whereby funds for conserving threatened plant species are limited. When A. yasumatsui is the primary threat to a cycad population, spending limited conservation resources on the rescue of a few individuals from the general population does nothing to address this primary threat, and plant mortality among the remaining population will continue [12]. Even the rescued and transplanted cycad plants will remain vulnerable to the ubiquitous A. yasumatsui herbivory after the appropriated funds to maintain the recipient restoration site have been expended and maintenance is abandoned. This occurred on Guam after only one year of maintenance of the transplanted trees. Alternatively, if all available conservation resources were initially dedicated to the rapid establishment of a multi-species biological control program to relegate the A. yasumatsui population to non-lethal status, the host cycad population would no longer be threatened, and species recovery would ensue without the need for expensive propagation and translocation rescue projects.

Global changes during the Anthropocene are causing more case studies where unexpected speed and magnitude of localized tree mortality occurs [13]. The observations highlight the uncertainty that defines attempts to predict sudden mass tree mortality events into the future. Unfortunately, habitats that suffer from unusually high tree mortality events may experience irreversible alterations of ecosystem function, so improving the conservation community’s ability to predict when and where these events may occur is crucial. The case study on the island of Guam illuminates an example where mass mortality of the host tree species of a specialist invasive alien insect species was predicted before the invasion of that insect occurred, and the predictions were accompanied by recommendations from cycad specialists to immediately establish classical biological control to mitigate the new biotic threat [12]. The decades of mass tree mortality that ensued did not result from a failure to accurately predict mass tree mortality but resulted from the lack of response by the conservation decision-makers to act on the initial recommendations from the scientific community.

Although the newly determined protocol for using potassium triiodide staining to help select which surviving trees contain the most stem starch may reduce wasteful spending on propagation projects in the future, the primary need continues to be the establishment of effective biological control of A. yasumatsui. Moreover, in novel insular settings suffering from invasive alien species, the irruption of a fortuitous biological control organism may occur at any time, so careful monitoring of the endangered plant population may enable early detection of these fortuitous events. The C. micronesica populations on the Guam and Rota islands are threatened by the A. yasumatsui invasions, but there is no reason to believe that the long-term response will be similar for the two island populations. Early detection of a fortuitous irruption of a new biological control organism on one of these islands may enable purposeful introduction of this organism to the other island. This can only occur if uninterrupted funding of knowledgeable cycad biologists enables the routine monitoring that would generate success in these endeavors.

4.3. Role of Scientists

The contributions of scientists to publicly funded programs are sometimes ignored or actively suppressed [14,15,16]. The Guam C. micronesica conservation case study provides many examples, some of which directly involve asexual propagation protocols [3,4,12]. The construction site for a Live-Fire Training Range Complex (LFTRC) for military use was the first large-scale conservation project designed to rescue 1000s of C. micronesica trees prior to construction [17]. The previously published recommendation to include cycad expertise on the conservation mitigation team was suppressed by the federal funding agency deciders, and the funded practitioners did not include a demonstrable cycad specialist.

Two costly mistakes resulted from these decisions and defined the outcomes. First, the extent of dichotomous branching of a cycad tree is useful for sexing mature trees because of sexual dimorphism of this trait [4]. The practitioners for the large-scale translocation project from the LFTRC selected mostly branched individuals because more female trees had died by this time than male trees, ensuring very few female trees were added to the recipient restoration site. The mistake could have been avoided by including an appropriate cycad expert who would have ensured that most transplanted individuals were monopodial, and this mistake ensured lack of future regeneration within the restoration site due to the propensity of male trees. Second, the pachycaulous, manoxylic stem of cycad trees requires a prophylactic covering of the cut surface when stem cuttings are exploited for asexual propagation. The practitioner funded to propagate more than 1000 stem cuttings to rescue trees within the LFTRC footprint rejected this horticultural knowledge, claiming that the cut surfaces would callus to seal the wounds. A cycad stem does not produce calluses in response to wounding because most of the stem is already parenchyma.

Competent cycad propagators worldwide are aware of these facts and understand that the asexual propagation procedure demands strict hygiene and an artificial prophylactic sealant of the wounds to ensure the tissue is not temporarily exposed to dehydration, insect herbivore ovipositioning, or pathogens. This decision to exclude knowledgeable cycad experts from the mitigation project led to failure, and the source trees had been destroyed at the federal construction site by the time the propagation failures were apparent. The cycad genotypes that taxpayer funds were appropriated to save were instead killed by ill-informed conservation methods. Considering the fact that the use of a phellogen to heal plant wounds evolved more than 400 million years ago [18], and Triassic era cycad stems revealed phellogen and phellem as a prominent feature of stem tissue protection [19], the rejection of this knowledge by the Guam practitioners and the federal agents that approved the methods guaranteed the project’s failure.

The biologists from the federal permitting agency responsible for conserving species listed on the United States Endangered Species Act have also demonstrated sustained apathy toward the future of C. micronesica. For example, this agency compiled a recent list of consequential invasive species in Guam [20]. This list included 11 named animal species, none of which were A. yasumatsui. This unique tree was the most abundant tree in Guam in 2002 [21], serving as a foundation species. It is the only native gymnosperm in the region and the only Cycas species that is native within the United States.

Suppression of science in the medical field may cause maladministration of public funds, and people die [22]. Suppression of science in the global biodiversity crisis also causes maladministration of public funds, but endangered species die.

5. Conclusions

Conservation of plant species may require practitioners to make decisions despite a lack of empirical data and an abundance of uncertainties. In this context, the findings reported herein are useful for removing the uncertainties surrounding which host Cycas trees may enable the greatest levels of asexual propagation success. The cuttings exhibiting the darkest staining when potassium triiodide is employed will consistently lessen propagation failure and increase success. This lethal specialist armored scale known as A. yasumatsui has recently invaded insular populations of C. revoluta within the Ryukyu Islands [12]. The predictions of mass tree mortality and the emergency need to establish a multi-species coalition of specialist biological control organisms are no less important for C. revoluta today than they were for C. micronesica twenty years ago. If asexual propagation of individuals within the newly invaded Japan habitats is exploited as one of the urgent conservation protocols, the potassium triiodide staining technique may allow the Japan conservationists to be more successful in their endeavors than the Guam conservationists were.