The Mutual Influence of Oleoresin Between Rootstock and Scion in Grafted Pine

Abstract

Grafting constitutes a crucial approach for the preservation of pine clones. Slash pine is commonly used as the rootstock for grafting Masson pine scions in Guangxi. In this context, the fresh oleoresin samples of Masson pine, slash pine, and grafted pine (with Masson pine as scion and slash pine as rootstock) were analyzed by gas chromatography–mass spectrometry and gas chromatography, and the key chemical components (α-pinene, β-pinene, longifolene, and isopimaric acid) that can quickly and accurately distinguish the oleoresin of Masson pine and slash pine were found and identified. According to the changes in the relative content of key compounds of oleoresin in scion and rootstock, it was found that the oleoresin of rootstock and scion could interact. Further research showed that the mutual influence of oleoresin between rootstock and scion was persistent, and the influence of rootstock on oleoresin at the scion was affected by height. However, the height effect included a large individual differences, which were not significantly related to the grafting height, tree height, diameter at breast height, etc., but may have been related to the differences in synthesis speed of oleoresin between rootstocks and scions. This work reveals the possible mechanism of mutual influence and secretion of oleoresin in grafted pine trees, laying a foundation for the study of the characteristics of oleoresin from pines grafted by different types, with great significance for the breeding of pine with high yield of oleoresin, and the production and application of special compounds containing oleoresin.

1. Introduction

Masson pine (Pinus massoniana Lamb.), a member of the Pinaceae family, is a widely distributed pioneer tree species for afforestation in southern China [1,2,3]. It plays an important role in global ecosystems due to its fast growth rate, strong adaptability, and tolerance to barren environments [4,5]. In addition, it is also an important economic tree species that can provide raw materials such as wood and oleoresin [6,7]. Therefore, the collection and preservation of Masson pine germplasm resources are of great significance.

Slash pine (Pinus elliottii Engelm.) is a native tree in the southeastern United States, which occurs from South Carolina’s Southern Coastal Plain to Central Florida and Southwestern Louisiana [8]. Since the introduction of slash pine to China in the 1940s, its planting area has gradually increased in southern China [9]. Slash pine is an economic tree species that provides both wood and oleoresin [10]. Slash pine is now one of the main resin-tapping tree species in China, due to its strong adaptability, high oleoresin content, low resin crystallization rate, and high turpentine content [11].

Although grafting is plagued by issues such as scion–rootstock incompatibility and premature tree senescence [12], its advantages in preserving and propagating superior plant traits still render it an important technical measure [13]. It is usually adopted in the construction of seed orchards and gene banks of Masson pine. The impact of different rootstocks on the nutrient supply and growth status of scions varies [14]. Slash pine is commonly used as the rootstock for grafting Masson pine scions in Guangxi. The largest collection bank of Masson pine germplasm resources with slash pine as rootstock is in Guangxi, China.

Oleoresin is a metabolite secreted by pine trees after wounds mechanically inflicted to the trunk. It is a natural, renewable, and important chemical raw material [15], which is widely used in essences, spices, food, medicine, pesticide, ink, paint, and other products [16,17,18,19]. In addition, oleoresin also plays an important role in the defense systems of pine trees themselves. The main compounds in oleoresin are terpenoids (monoterpenes, sesquiterpenes, and diterpenes). The chemical compositions of oleoresins from different sources are different, so their processing processes and specific applications are also different. In order to make better use of oleoresin resources, the chemical compositions of oleoresin from different tree species has been reported [20,21,22,23]. In addition, there have been reports on the synthesis pathway of oleoresin in trees and the key role of oleoresin-related substances in pine tree resistance [24,25].

However, at present, few studies have been conducted on the chemical composition of oleoresin after pine grafting treatment. Previously, the effects of sand pine scion on monoterpene composition of slash pine rootstock were reported [26], but only the mutual influence of monoterpenoids was studied in that research. The chemical composition of monoterpenoids in the oleoresin of the selected rootstocks and scions was similar; therefore, the conclusions drawn from that research were not well explained. The chemical composition of oleoresin at the scion of several kinds of grafted pine trees using slash pine as rootstock was reported in prior studies [27]. However, analysis of the chemical composition of oleoresin at the rootstock of the grafted pine was lacking in that research. In current work, Masson pine, slash pine and grafted pine (with masson pine as scion and slash pine as rootstock) at the same location (excluding the influence of geographical factors on the chemical composition of oleoresin) were selected as the research subjects. The interaction between oleoresin from scions and rootstock of the grafted pine can be easily identified, attributed to the high degree of chemical differentiation in the oleoresin components of Masson pine and slash pine. Through studying the interaction between rootstocks and scions, the possible mechanism of mutual influence and secretion of oleoresin in grafted pine trees is proposed, providing a new idea for the breeding of high-resistance pine trees with high oleoresin content.

2. Materials and Methods

2.1. Materials

The Masson pine, slash pine, and grafted pine (with Masson pine as scion and slash pine as rootstock) used in this work were all planted at Nanning Institute of Forestry Science (23°10′ N and108°00′ E). The area is in the monsoon climate of the southern edge of the south subtropical zone, with an annual average temperature of 21.5 °C, an annual average frost-free period of 358 days, an annual average precipitation of 1246 mm, and an annual average evaporation of 1613.8 mm. The microclimate of the dry-hot valley of the Youjiang River has an annual average relative humidity of 79%. The experimental forest land is a broad gentle hill platform between limestone peak forests and peak clusters, with an altitude of about 120 m. The soil is lateritic red soil, with a pH value of 5.5–6.5 and a deep soil layer [28]. The grafted pines were grafted between late April and early May 2011. The grafting method adopted was side grafting. Specifically, 2-year-old slash pine seedlings grown from seeds were used as rootstocks, and the scions were fresh branches taken from superior individual plants in the progeny test plantation of the primary seed orchard of Masson pine. The nitrogen used in the gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS) was purchased from Guangxi Ruida Chemical Technology Co., Ltd., (Nanning, China) with a purity of 99.999%. Ethanol, tetramethyl ammonium hydroxide solution, phenolphthalein, and other chemicals and solvents were commercially acquired as standard laboratory grade.

2.2. Methods for Collecting the Oleoresin

The fresh oleoresin samples were collected by borehole oleoresin tapping at the corresponding positions on the trees. A 1 cm deep circular hole was drilled on the tree trunk using an electric drill, and a 15 mL centrifuge tube (with the same diameter as the circular hole) was inserted at a 45° angle into the circular hole. The centrifuge tube was removed and sealed after a sufficient amount of oleoresin flowed into it, which was then brought back to the laboratory for future use.

2.3. Preparation of Methylated Oleoresin Sample

A small amount of oleoresin was placed into a 5 mL test tube, an appropriate amount of anhydrous ethanol was added to the test tube to dissolve the oleoresin, and, then 1–2 drops of 0.5% phenolphthalein solution were added to the solution. The tetramethylammonium hydroxide solution was added dropwise to the test tube until the solution turned red and did not fade after 30 s. Anhydrous magnesium sulfate was used to dry the methylated oleoresin sample, and then the sample was filtered with 0.22 μm organic filter head, the filtrate was used for GC analysis and GC-MS analysis.

2.4. GC Analysis and GC-MS Analysis

The oleoresin samples were analyzed by GC-MS (Bruker SCIONSQ-TQ, Karlsruhe, Germany) and GC (Nexis GC-2030, Kyoto, Japan). The chemical components of the oleoresin were analyzed by the retrieval system in GC-MS, and the relative content of each chemical component was calculated by the area normalization method in GC. The GC-MS chromatographic column was a DB-5 capillary column (30 m × 0.32 mm × 0.25 μm, Agilent, Hong Kong, China). The detector was a hydrogen flame ionization detector. The temperature of the column was held at 60 °C and maintained for 2 min, then increased at 4 °C/min up to 200 °C and maintained for 2 min, and finally increased at 2 °C/min up to 250 °C and maintained for 10 min. The split ratio was 1:50, and the carrier gas was nitrogen (99.999%). The temperature of the vaporization chamber and detector were 260 °C and 280 °C, respectively. The injection volume was 0.50 μL. The electron bombardment source was EI, and the electron energy was 70 eV. The temperature of the ion source and transmission line were 230 °C and 270 °C, respectively.

2.5. Sampling Design

Thirty Masson pine trees, thirty slash pine trees, and eighty-four grafted pine trees were select randomly for resin tapping (the oleoresin from rootstock and scion of grafted pine was collected separately), and the chemical composition of collected oleoresins were analyzed and compared. The detailed data of the growth indicators of grafted pines are provided in Table S1 of the Supplementary Materials.

An individual plant with the oleoresin of scion affected by the rootstock and an individual plant with the oleoresin of rootstock affected by the scion were selected, and a cutter was then used to cut a 45° wound at the scion and rootstock, respectively. This method was followed for 20 consecutive days (once a day and the wounds were downward in turn). The oleoresin was collected with a straw after it flowed out. The fresh oleoresin samples were collected at different specific times (0 h, 1 h, 2 h, 4 h, 8 h, 2 d, 3 d, 5 d, 10 d, and 20 d).

Thirty-two grafted pines whose rootstock had influence on scions were selected as the test trees. Fresh oleoresin was collected from the scions at the distance of 30 cm, 80 cm and 130 cm from the grafting interface, respectively, and the chemical composition was analyzed.

An individual plant with the oleoresin of the scion affected by the rootstock was selected as the research object. A lateral branch of the scion was cut off to collect the oleoresin at the fracture. The cut lateral branch was cut into small segments after removing the epidermis, and these were placed in the anhydrous ethanol solution to dissolve the oleoresin. The chemical compositions of the two oleoresin samples were analyzed.

2.6. Statistical Analysis and Graph Drawing

One-way analysis of variance (ANOVA) and multiple comparisons (LSD) were used in SPSS 25.0 to analyze the differences in the relative content of major compounds in oleoresin. Correlation between the mutual influence of oleoresin (represented by the relative content of isopimaric acid) and growth indicators was analyzed by Pearson correlation analysis, and the graphs were drawn in Origin 9.0.

3. Results and Discussion

3.1. Characteristics of Oleoresin from Masson Pine, Slash Pine and the Grafted Pine

3.1.1. Differences in Chemical Composition and Relative Content of Oleoresin from Masson Pine and Slash Pine

The oleoresin samples from Masson pine and slash pine were analyzed by GC-MS and GC for their chemical composition and relative content, and the results are shown in

Table 1. Chemical composition and content of oleoresin from Masson pine and slash pine.

No.	Compound		Retention Time (min)	Relative Content (%)
				Masson Pine	Slash Pine
1	Monoterpene	α-Pinene	5.210	42.76 ± 3.07	25.18 ± 4.72
2		Camphene	5.571	0.53 ± 0.11	0.38 ± 0.07
3		β-Pinene	6.272	1.15 ± 0.76	14.90 ± 6.22
4		Myrcene	6.612	0.42 ± 0.11	0.53 ± 0.06
5		Limonene	7.702	0.70 ± 0.20	-
6		β-Phellandrene	7.710	-	3.58 ± 2.69
7		Terpinolene	9.537	0.08 ± 0.09	-
8		Estragol	13.167	-	0.43 ± 0.27
9	Sesquiterpene	Longipinene	18.134	0.46 ± 0.12	-
10		Longicyclene	18.785	0.55 ± 0.15	-
11		Sativene	19.414	0.28 ± 0.06	-
12		Longifolene	19.900	9.07 ± 2.18	-
13		Caryophyllene	20.345	1.72 ± 0.58	-
14		Farnesene	21.403	0.23 ± 0.15	-
15		Humulene	21.488	0.33 ± 0.12	-
16	Diterpenoid (resin acid)	Pimarinal	41.106	-	0.47 ± 0.49
17		Pimaric acid	42.592	3.19 ± 0.59	0.93 ± 0.27
18		Elliotinoic acid	42.961	0.03 ± 0.05	2.52 ± 0.27
19		Sandaracopimaric acid	43.142	0.60 ± 0.13	2.70 ± 0.54
20		Isopimaric acid	44.457	0.12 ± 0.25	9.68 ± 1.36
21		Palustric acid/levopimaric acid	44.808	25.76 ± 2.67	22.23 ± 1.20
22		Dehydroabietic acid	45.904	2.50 ± 0.82	1.27 ± 0.25
23		Abietic acid	47.279	3.74 ± 0.75	3.53 ± 0.57
24		Neoabietic acid	49.103	4.04 ± 0.75	8.30 ± 0.92

Note: The relative content data of each compound is the average of 30 samples. The “-“ in the table indicates that the compound was not detected.

. The oleoresin from Masson pine mainly consisted of monoterpenes, sesquiterpenoids, and resin acids, while the oleoresin from slash pine mainly consisted of monoterpenes and resin acids (the presence of sesquiterpenes in oleoresin is the most intuitive and obvious difference between Masson pine and slash pine). In fact, except for the difference in sesquiterpenoids, most of the compounds of oleoresin from Masson pine and slash pine were common. However, the relative contents of these compounds in oleoresin differed between Masson pine and slash pine. For example, the relative contents of α-pinene in oleoresin from Masson pine and slash pine were 42.76% and 25.18% respectively. The relative contents of β-pinene of oleoresin from Masson pine and slash pine were 1.15% and 14.90%, respectively. The relative contents of isopimaric acid in oleoresin from Masson pine and slash pine were 0.12% and 9.68%, respectively. The independent sample t-test results showed that their differences were significant. Previous reports by Song [29] show that the relative contents of α-pinene, β-pinene, and isopimaric acid in oleoresin from Masson pine were 31.7%, 1.2%, and 0.2%, respectively, and the relative contents of α-pinene, β-pinene and isopimaric acid in oleoresin from slash pine were 15.5%, 12.6%, and 11.4%, respectively. Our current research results are relatively close to those results. In addition, Song utilized the relative content of isopimaric acid as a diagnostic marker for detecting the presence of slash pine rosin in Masson pine rosin. So, the difference in the relative content of these specific compounds in oleoresin is also an important means to distinguish Masson pine and slash pine.

3.1.2. Chemical Composition and Relative Content of Oleoresin from the Grafted Pine

Previous reports [27] had shown that the oleoresin from scions still retained the characteristics of the original tree species (with slash pine as rootstock and Pinus glabra, Pinus patula, Pinus echinate as scion). However, in this work, by analyzing the oleoresin from the scion part of some grafted pines, it was found that the relative contents of α-pinene and longifolene (two representative compounds of oleoresin from Masson pine) were much lower than in oleoresin from Masson pine, the relative contents of β-pinene and isopimaric acid (two representative compounds of oleoresin from slash pine) were much higher than in oleoresin from Masson pine, and these changes were synchronous, as shown in Figure 1. We believe that this phenomenon was caused by the mixing of two kinds of oleoresin, that is, the oleoresin sample of the rootstock permeated the grafting interface and flowed out from the scion. By analyzing 84 samples of oleoresin from the scion, it was found that 50% of the samples were affected by the oleoresin from the rootstock, and the remaining 50% of the samples still retained the characteristics of oleoresin from Masson pine. Similarly, we tested the oleoresin samples from the rootstock and found that sesquiterpene components were detected in some grafted pines, as shown in Figure 1 (these compounds were not present in the oleoresin from slash pine), which indicated that the oleoresin at the scion migrated downward to the rootstock. It is further explained that the resin channels of scion and rootstock were interconnected.

3.2. Persistence of Mutual Influence Between Oleoresin at Rootstock and Scion

After Masson pine is grafted onto slash pine, the chemical composition of oleoresin at the scion and rootstock interacts. Whether this influence is temporary or continuous is a special concern. The relative content of main chemical composition of oleoresin collected at different times is shown in Figure 2. In the grafted pine where the oleoresin of scion was affected by the rootstock, with the extension of sampling time, the relative content of the main compounds of oleoresin from the scion fluctuated, as shown in Figure 2a, but the relative content of isopimaric acid and β-pinene still remained significantly higher than in the oleoresin from Masson pine, which may confirm that the effect of rootstock on scion (about oleoresin) is a continuous process. During the continuous trauma process, the oleoresin at the rootstock flowed continuously to the scion and then flowed out from the wound; the relative content of α-pinene tended to decrease, and the relative content of β-pinene and isopimaric acid tended to increase, indicating that the proportion of oleoresin from the rootstock gradually increased in the outflow of oleoresin from the wound. Similarly, the same experimental treatment was carried out on the rootstock with oleoresin affected by the scion, and the results are shown in Figure 2b. In the process of continuous trauma, the sesquiterpene components (mainly longifolene) unique to oleoresin of Masson pine were still detected in the oleoresin samples from the rootstock, indicating that the influence of scion on rootstock was also not temporary.

3.3. Changes in Main Compounds of Oleoresin Under Different Sampling Heights in the Grafted Pine

The oleoresin samples at different heights of the scion with oleoresin affected by the rootstock were collected and analyzed, and the results were shown in Figure 3. With the increase of sampling height, the relative contents of α-pinene and longifolene gradually increased, while the relative contents of β-pinene and isopimaric acid gradually decreased. The relative contents of the four main compounds in oleoresin collected at 30 cm and 130 cm were significantly different. This indicates that the influence of rootstock on scion has a height position effect. The influence of rootstock on scion became smaller and smaller with the increase of sampling height. When the sampling height exceeded a certain value, the oleoresin of scion part were not affected by the rootstock. However, this effect varied greatly among individuals. Part of the grafted pine basically returned to the characteristic of oleoresin from Masson pine at a distance of 80 cm from the grafting interface, while part of the grafted pine showed no signs of reducing the relative content of typical compounds (β-pinene and isopimaric acid) at a distance of 130 cm from the grafting interface.

3.4. Correlation Between Mutual Influence of Oleoresin and Growth Indicators of the Grafted Pine

The oleoresin at the rootstock and scion influenced each other in the grafted pine, and the occurrence or extent of this influence could easily be identified by the relative content of specific compounds (such as longifolene, isopimaric acid, etc.). The correlation between the relative content of isopimaric acid (RCI) and growth indicators such as the height of the rootstock (HR), the height of the tree (H), diameter at breast height (DBH), clear bole height (CBH), and the proportion of rootstock height to tree height (HR/H) was studied. The results showed that HR, DBH, CBH, and HR/H were not significantly related to RCI. There was a negative correlation between H and RCI, but the correlation value was small (as shown in Figure 4). Therefore, the growth indicators were not the main reasons for the mutual influence of oleoresin between rootstock and scion.

3.5. Possible Mechanism of Mutual Influence of Pine Resin at Rootstock and Scion

The chemical composition of oleoresin flowing out from the lateral branch fracture and oleoresin extracted from the lateral branch with ethanol were analyzed separately, and the results showed that the chemical composition of the two oleoresins was similar, both having the characteristics of mixed oleoresin from Masson pine and slash pine. This indicates that the oleoresin of scion and rootstock had been transported and mixed within the tree body before resin tapping.

After a pine tree is mechanically damaged, oleoresin flows out from the wound. The outflow of oleoresin is controlled by pressure. When a pine tree (4-year-old slash pine) was cut down, it was observed that there was oleoresin outflow at both cutting interfaces. The length of the stump in the lower part was much smaller than that in the upper part, but the oleoresin flowing out of the lower part was much higher than that in the upper part (as shown in Figure 5), indicating that the upward pressure at the root was the main driving force for the outward secretion of oleoresin.

Needles converted the carbon dioxide and water absorbed into carbohydrates and oxygen through photosynthesis under light conditions in the grafted pine, and carbohydrates (or their derivatives) were transported from top to bottom to the scion trunk and rootstock. After a series of biochemical reactions, oleoresin of Masson pine and oleoresin of slash pine were formed at the scion and rootstock, respectively (as shown in Figure 6).

Due to the difference in the synthesis speed of oleoresin at the rootstock and scion, there is a concentration difference between the oleoresin at the rootstock and scion within the tree. In order to ensure the balance of oleoresin concentration inside the tree, oleoresin is transported from high concentration areas through resin channels to low concentration areas, forming a mixed type of oleoresin. During the resin tapping of the grafted pine, the mixed oleoresin flows out under the drive of root pressure.

The difference in synthesis speed of oleoresin between rootstock and scion determines the mixed form of oleoresin in grafted pine. If the synthesis speed at the rootstock is greater than that at the scion, the oleoresin at the scion is affected by the oleoresin at the rootstock (i.e., significant increases in the relative contents of β-pinene and isobaric acid are detected in the oleoresin from the scion). If the synthesis speed at the scion is greater than that at the rootstock, the oleoresin at the rootstock is affected by the oleoresin at the scion (i.e., sesquiterpenoid compounds are detected in the resin at the rootstock).

The sampling tests at different heights at the scion showed that the higher the sampling height, the lesser was the impact of the rootstock on the oleoresin outflowing from the scion. This is because the resistance of the oleoresin at the rootstock during upward transportation gradually increased, and the oleoresin flowing from the scion was no longer affected by the rootstock until the sampling height reached a certain height. There are many possible sources of differences in the synthesis speed of oleoresin between rootstocks and scions, which may be genetic, physiological, or the result of multiple factors. Therefore, further research is needed at a deeper level.

3.6. Potential Application Areas

The secretion of oleoresin and volatiles is the most direct means to defense against diseases and insect pests of pine trees. The chemical components of oleoresin from Masson pine and slash pine are quite different; so, their resistances to different diseases and insect pests are different. Selecting the appropriate rootstock (slash pine) can realize the mutual circulation of two types of oleoresins in the tree, which not only enriches the diversity of the chemical properties of oleoresin but also provides the possibility to improve the resistance of pine trees to disease.

In terms of physical properties, the oleoresins from Masson pine and slash pine are also quite different. Oleoresin from Masson pine volatilizes quickly and easily coagulates in the air. By comparison, oleoresin from slash pine does not easily coagulate, and the oleoresin flows for a longer time. In grafted pine, the mixing of two kinds of oleoresin can prolong the time of oleoresin outflow, and the continuous secretion of mixed oleoresin can reduce the frequency of manual tapping, which is a cost-reducing approach to increasing the yield of oleoresin. Appropriate selection of rootstocks and scions can combine pine trees with characteristics of high oleoresin yield, high timber yield, and high resistance, thereby improving economic benefits.

4. Conclusions

The differences in chemical composition and relative content of oleoresin between Masson pine and slash pine were studied, and the key compounds distinguishing them were found and identified. The chemical compositions of oleoresin in the scion and rootstock of the grafted pine were analyzed and compared with those in Masson pine and slash pine. It was found that the oleoresin of rootstock and scion affected each other, indicating that the resin channels of rootstock and scion were interconnected, and the oleoresin produced at rootstock and scion could be transported up and down. Furthermore, this kind of interaction was a continuous process. In addition, the rootstock’s effect on the oleoresin of the scion varied with height. The higher the sampling height, the lesser was the influence of the rootstock on the oleoresin at the scion. This work lays a foundation for the study of the characteristics of oleoresin from pines grafted by different types, but the deeper mechanism of the interaction of oleoresin after grafting still needs further exploration.