Introduction 

As one of the countermeasures for pine wilt disease caused by the pine wood nematode [PWN, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle], which provoked serious damage in Japanese black pine (Pinus thunbergii Parl.), resistant trees have been widely planted in Japan. Seedlings originating from seed production orchards of the resistant mother trees are inoculated with PWN, and the healthy seedlings are available for commercial sale. However, an intensive summer work is required for producing these seedlings, and the mortality rate of approximately 50% after inoculation makes them expensive. Further, the percentages of symptomless seedlings in inoculation tests is not stable, fluctuating over the years because of varying environmental conditions ([24]).

To avoid these problems, resistant trees are propagated by cuttings obtained from healthy young seedlings that have been inoculated with PWN. Although rooting rates are generally low in cuttings of Japanese black pine ([15]), the cuttings obtained from certain stock plants have exhibited high rooting rates ([12]). Moreover, rooted cuttings taken from resistant seedlings have shown a high survival percentage after inoculation with PWN ([14]), suggesting that re-inoculation to confirm the resistance of the rooted cuttings is not required. The production of resistant rooted cuttings has been estimated by Ohira et al. ([19]) to be less expensive than other methods of propagation, and its application for Japanese black pine propagation is expected to increase.

External conditions, such as the bed media type, treatment with a rooting-promoting agent, temperature, humidity, and lighting intensity, are associated with rooting responses in cuttings of black pine. Physiological features of cuttings are also important. The phenomenon wherein differences in the physiological status of cuttings taken from the same tree influence the rooting ability is called “topophysis” ([3]). As an example of topophysis, the rooting ability of cuttings from the lower crown of stock plants is higher than those taken from the upper crown in some coniferous trees ([4], [10], [8], [20]). Furthermore, the rooting percentage varies in different morphological cuttings from the same tree ([6]). In black pine, it has been reported that the length and diameter of cuttings influence the rooting ability ([17], [26]) and that the removal of winter buds from cuttings improves rooting ([22]). However, Miyazaki ([9]) reported that the removal of winter buds was not related with the rooting percentage in black pine. Thus, the effect of the removal of winter buds remains poorly elucidated. Other cutting factors, such as the original number of winter buds, the fresh weight, and the position on the crown of stock plants from which cuttings are obtained, have not been fully explored. Clarifying the association of the rooting ability with crown positions and morphological characteristics of cuttings can increase the practical success rate of the cuttings.

Most previous studies on cutting propagation in black pine have focused on rooting percentages; however, this parameter is insufficient for the assessment of rooting success. The root volume is also important because it affects the growth of rooted cuttings ([23]).

Mori & Miyahara ([11]) suggested a practical though effective method to assess rooting in Japanese black pine: cuttings not easily uprooted from the bed medium have to be considered as rooted. However, this practice has the disadvantage of being stressful for rooted cuttings. In pine tree cuttings, needles from new shoots do not grow before rooting, even if the shoots grow ([15]). The relation between rooting and growth of needles remains unclear. However, this could represent a quick method to infer rooting without excavating cuttings from the bed media. Indeed, the assessment of the root volume of cuttings based on the observation of needles during the propagation period would allow growers to transplant rooted cuttings at the appropriate time.

In the present study, the impact of the crown position of stock plants on the success of cuttings was analyzed in order to improve the propagation of cuttings from Japanese black pine. In addition, two morphological characteristics of cuttings, i.e., the original number of winter buds and the fresh weight, were included in the analysis. Finally, the relation between the root volume and growth of needles was investigated in rooted cuttings.

  Material and methods 

Plant materials

Half-sib seeds were collected from mother trees in seed production orchards of Japanese black pine of the following six pine wilt-resistant clones: Namikata-37, Namikata-73, Shima-64, Tanabe-54, Tosashimizu-63, and Tsuyasaki-50 (Tab. 1). These open-pollinated seeds were sown at the end of March 2010, and the seedlings were transplanted by the middle of March 2011 in a nursery field at the Shizuoka Prefectural Research Institute of Agriculture and Forestry, Forestry and Forest Products Research Center (Hamamatsu, Shizuoka prefecture, Japan). Some 17-19 seedlings per half-sib family were used as stock plants.

Preparation, planting and culture condition

Cutting experiments were conducted in the middle of February 2013, which is an appropriate season for obtaining cuttings of Japanese black pine ([18]).

Fifty cuttings were taken from each half-sib family for a total of 300 cuttings. Twenty-five cuttings were randomly obtained from branches in the upper crown of 17 to 19 stock plants without stumping, while the remaining half of the cuttings were taken from branches in the lower crown (Tab. 1). The height of stock plants was approximately 50-100 cm.

The cuttings were 5-cm long, and the needles were removed from all but the most upper part (2 cm) of the cuttings. A standard two-way slanting cut was made across the base. The original number of winter buds and the fresh weight of each cutting were recorded. The average fresh weight and number of winter buds of cuttings are shown in Tab. 1. Because of the better growth of branches in the upper crown than in the lower crown, both the average fresh weight and number of winter buds of cuttings from the upper crown were higher than those from the lower crown.

The cuttings were then dipped in the rooting hormone solution (indole butyric acid, 4000 ppm) for 5 s ([22]). They were then vertically inserted into the bed container (47×23×9 cm - L×W×H) filled with moist Kanuma soil medium (which contains low levels of organic nutrients suitable for cuttings) and planted within 1 day after their removal from stock plants.

The bed containers were placed on shelves in a greenhouse and misted for 25 to 30 s every 25-30 min all day long. During the experiment period, the temperature in the greenhouse ranged from -2.0 °C to 40.7°C. Bed containers were rotated once a week in order to avoid differences in irrigation and light conditions.

Measur length, rooting and root volume

Winter buds of the cuttings revealed a gradual growth into new shoots after being planted. The length of the longest new needle from a new shoot in each cutting was measured at the beginning of October 2013 (approximately 8 months after planting).

Cuttings were excavated from the bed containers and their rooting was examined. Roots were washed with water to rinse off soil residuals, and then gently wiped with a towel. The root volume was estimated by measuring the increase in the water volume after the roots were dipped into a 100-mL graduated cylinder filled with 80 mL water, according to Kathiravan et al. ([7]).

Data analysis

To identify the factors associated with rooting, a logistic regression analysis was performed using the R statistical software (version 2.15.1) on data from 300 cuttings of six half-sib families. The rooting was designated as the response variable, and the family of the stock plant, the crown position of the cutting (location on the stock plant), the fresh weight of cuttings, and their original number of winter buds were considered as explanatory variables. The additive models, including all four explanatory variables and an intercept, were constructed using the logit link function as rooting is binomial. The model used is represented by the following equation (eqn. 1):

where β₀ is the intercept, and β₁, β₂, β₃ and β₄ are the effects of the family, crown position, fresh weight, and number of winter buds, respectively.

The analysis of the root volume was conducted on data obtained from 164 rooted cuttings. For the additive model of the root volume designated as the response variable, a generalized linear model (GLM) was used with a log function because the root volume data exhibited a gamma distribution. The family, crown position, fresh weight, number of winter buds, and length of the longest needle were designated as explanatory variables, such that (eqn. 2):

where β₀,is the intercept, while β₁, β₂, β₃, β₄ and β₅ are the effects of family, crown position, fresh weight, number of winter buds, and length of the longest needle, respectively.

After conducting the logistic regression and GLM analysis, only explanatory variables which markedly affected response variables were considered for further analysis. Comparison of the rooting percentages between cuttings obtained from the lower and the upper part of the crown (crown position) was carried out by χ² test. In addition, the correlation between root volume and the length of the longest needle of cuttings (both from upper and lower crowns) was calculated using a permutational Spearman’s rank test. Furthermore, all cuttings were divided into three groups based on their fresh weight (light, medium and heavy) with a nearly equal number of cuttings (55, 55, and 54, respectively), and the correlation coefficients between the root volume and length of the longest needle were also calculated for each group.

  Results 

Factors influencing rooting

The results of the logistic regression analysis indicated that family (p < 0.001) and crown position (p < 0.001) significantly associated with the rooting of cuttings (Tab. 2). The crown position did not interact with any other variable; however, family interacted with the fresh weight and winter bud number of the cuttings.

Rooting percentages of the six families ranged from 34.0% to 76.0%, with a mean of 54.7% for all the 300 cuttings (Tab. 1). The rooting percentage was considerably higher for cuttings taken from the lower crown (74.7%) than from the upper crown (34.7%). These rooting percentages significantly differed according to the χ² test (p < 0.001).

Factors influencing root volume

GLM analysis revealed that the length of the longest needle significantly correlated with the root volume (p < 0.001); however, family, crown position, fresh weight, and number of winter buds did not show any significant effect on the same parameter (Tab. 2). However, given the significant interaction (p < 0.05) of the length of the longest needle with two factors, i.e., crown position and fresh weight of cutting, were significant, further analyses were conducted.

The mean root volume ranged from 2.60 to 3.41 cm³ among families, and the mean volume across all families was 3.17 cm³ (Tab. 1). The correlation between the length of the longest needle just before excavation and the root volume was significant in cuttings taken from both the upper (r_s = 0.723, p < 0.001) and lower (r_s = 0.402, p < 0.001) crowns (Fig. 1a, Fig. 1b). The correlation coefficients between the length of the longest needle and the root volume were 0.317 (p < 0.05), 0.446 (p < 0.01), and 0.700 (p < 0.001) for cuttings in the light, medium, and heavy groups, respectively, and they showed an increasing trend with the fresh weight of the cuttings (Fig. 2a, Fig. 2b, Fig. 2c). Such analyses confirmed the significant correlations between the length of the longest needle and the root volume.

Fig. 1 - Correlation between the length of the longest needle and the root volume of rooted cuttings excised at two different positions on the stock plants: (a) cuttings taken from the upper crown; (b) cuttings taken from the lower crown. (rs): correlation coefficient based on a permutational Spearman’s rank test; (n): sample size; (***): p < 0.001.

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Fig. 2 - Correlation between the length of the longest needle and the root volume among groups of rooted cuttings. All cuttings were divided into three groups with nearly a equal number of cuttings, based on their fresh weight: (a) light, (b) medium, and (c) heavy. (rs): correlation coefficient based on a permutational Spearman’s rank test; (n): sample size; (*): p <0.05, (**): p < 0.01, (***): p < 0.001.

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  Discussion 

The preparation of cuttings from stock plants with a high rooting ability is critical in order to achieve a high propagation rate. It has been universally recognized that the rooting ability differs among families not only in Japanese black pine ([13]) but also in other pine species ([1]). In the present study, the rooting percentage varied among families of Japanese black pine, thus confirming that the genetic potential is an important factor to be considered for the successful establishment of cuttings.

The crown position on the stock plant the cutting are taken from is one of the decisive factors in producing successful cuttings of economically important tree species. Cuttings taken from the lower part of the crown have shown a higher rooting percentage in western hemlock (Tsuga heterophylla (Raf.) Sang. - [4]), dawn redwood (Metasequoia glyptostroboides - [16]), black spruce (Picea mariana (Mill.) B. S. P - [25]), fraser fir (Abies fraseri (Pursh) Poir - [21]), larch (Larix spp. - [20]), and Japanese cypress (Chamaecyparis obtusa - [8]). The 3-year-old stock plants used in this experiment were slightly younger and smaller than those used in the aforementioned examples. However, as already observed in other coniferous tree species, the crown position of cuttings was substantially associated with their rooting in Japanese black pine. Indeed, the rooting percentage of cuttings from the lower crown was significantly higher than that from the upper crown.

In other coniferous species, the different rooting potential of cuttings from different parts of the tree is believed to be related to their varying amounts of growth-promoting and rooting-inhibiting substances. In dawn redwood (M. glyptostroboides), cuttings with high rooting ability taken from the lower crown contained high levels of hormones ([16]). Tannins, which seemed to inhibit rooting, were present at low levels in the lower branches of Japanese cypress ([5]). Browne et al. ([2]) found that the nutrient status of cuttings differed with the cutting position in jack pine (Pinus banksiana Lamb.). Moreover, Tousignant et al. ([25]) pointed out differences in the physiological maturation of plant tissues from different crown positions. The cuttings from the upper crown revealed advanced maturation, while those from the lower crown remained at a juvenile stage. The rooting percentage was higher in cuttings from shade-grown stock plants in Japanese umbrella pine (Sciadopitys verticillata - [27]), suggesting that sunlight may negatively affect the rooting ability of tissues to be excised. Indeed, the cuttings from the lower crown are subject to shading.

In the present study, the reason underlying the differences in the rooting percentage observed between the two crown positions of cuttings was not investigated. Japanese black pine may possess different quantities of growth-promoting and rooting-inhibiting substances or exhibit different levels of physiological maturation in different parts of the tree. Both shade and nutrient effects may contribute to the high rooting ability of cuttings taken from the lower crown of Japanese black pine. The association of these factors with the rooting ability of cuttings calls for future research on this species. In any case, we can recommend that cuttings should be taken from the lower crown, particularly in the case of 3-year-old Japanese black pine stock plants. Nonetheless, Browne et al. ([2]) reported that the lower branches of jack pine did not always exhibit higher rooting percentages than the upper branches among stock plants of various ages. Thus, further experiments are required for supporting our findings in older stock plants of Japanese black pine.

Cuttings of appropriate sizes should be used for effective propagation, as mentioned by Kathiravan et al. ([7]). Ohira et al. ([17]) reported that small cuttings (mean length 3.9 cm; mean diameter 3.8 mm) of Japanese black pine exhibited a higher rooting percentage than both medium (mean length 7.0 cm; mean diameter 4.5 mm) and large (mean length 12.3 cm; mean diameter 7.6 mm) cuttings. In order to further explore the effect of the size of cuttings on rooting ability, we investigated the influence of fresh weight of 5-cm-long cuttings on rooting. However, based on our results, the fresh weight of cuttings did not affect the rooting ability. Because fresh weight of cuttings is not reported by Ohira et al. ([17]), an exact comparison of the fresh weight of cuttings in their study with that observed in this investigation is not feasible. However, based on the estimated volume of cuttings reported by Ohira et al. ([17]), they seems to use cuttings of a wider range of fresh weights than those used in the present study. This difference in the cutting size may likely explain possible differences in their rooting.

Statistical analysis revealed that the number of winter buds did not affect rooting. Although the influence of winter bud removal on rooting was previously reported only twice ([9], [22]), the present study can support the conclusion of Miyazaki ([9]) that the winter bud removal did not influence the rooting percentage in Japanese black pine.

Rosier et al. ([21]) found that the primary needle length of cuttings just before their insertion into the bed correlated with the rooting percentage and root length of cuttings in Virginia pine (Pinus virginiana Mill.). According to Morishita & Ooyama ([15]), the needles of non-rooted pine cuttings do not grow, regardless of the elongation of the new shoots. In addition to these associations, we report a new finding about Japanese black pine - the length of the longest needle just before transplanting correlated with the root volume in the case of young stock plants. This result suggests that the growth of needles may successfully predict the root volume without excavation of cuttings. Based on this prediction, growers can transplant rooted cuttings at the appropriate time, resulting in well-grown rooted cuttings.

  Conclusions 

This study identified the factors that affect the rooting ability of cuttings from young Japanese black pine stock plants, as well as the significant correlation between the root mass and needle growth. The crown position substantially and independently associated with the rooting. Furthermore, the rooting percentage of cuttings taken from the lower crown was markedly higher than that of cuttings excised from the upper crown. The length of the longest needle significantly correlated with the root volume of cuttings and also interacted with other factors. These findings constitute a useful contribution to the practical propagation of Japanese black pine cuttings resistant to pine wilt disease.

  Acknowledgements 

We thank Dr. I. Tamaki (Gifu Academy of Forest Science and Culture, Japan), Dr. T. Mitsunaga (NARO Agricultural Research Center, Japan) and Mr. T. Hoshikawa (Shizuoka Prefectural Research Institute of Agriculture and Forestry, Forestry and Forest Products Research Center, Japan) for providing us with appropriate suggestions.

TH and SY carried out experiments of cuttings. YH performed the statistical analysis. KK conceived the study and helped to draft the manuscript.

  References