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Highlights:
Interspecific grafting of P. rzedowskii with P. pinceana, P. maximartinezii, P. ayacahuite and P. pseudostrobus.
Survival was evaluated with the non-parametric Kaplan-Meier method.
P. pinceana, a species related to P. rzedowskii, had the highest survival (80 ± 0.3 %).
P. pseudostrobus had the lowest survival function (m = 0.66; 20 ± 0.8 %).
Phylogenetic relationships should be considered for the success of interspecific grafting of Pinus.
Introduction
Grafting is one of the most widely used asexual propagation methods for pine species (Aparicio-Rentería, Viveros-Viveros, & Rebolledo-Camacho 2013; Muñoz-Flores, Prieto-Ruiz, Flores-García, Pineda-Ojeda, & Morales-González, 2013; Pérez-Luna et al., 2019). This method accelerates the reproductive cycle to have higher plant production with better genetic quality in less time; the plant is used for reforestation and conservation activities, establishment of clonal seed orchards or development of commercial plantations (Aparicio-Rentería et al., 2013; Guadaño & Mutke, 2016; Muñoz-Flores et al., 2013; Pérez-Luna et al., 2019, 2020a). However, most studies have been conducted with rootstocks of the same species and few studies have been reported with different species (Climent, Prada, Gil, & Pardos, 1997; Darikova, Vaganov, Kuznetsova, & Grachev, 2013; Villaseñor & Carrera, 1980; Pérez-Luna et al., 2020b).
Some of the most important advantages of interspecific grafting are the ability to adapt to environments different from that of the original species and the production of a greater number of reproductive structures with an increase in the availability of seeds (Climent et al., 1997). These advantages have been observed in species of wide distribution and economic importance; in contrast, there are no studies for pines with restricted distribution and endangered species. Grafting in this type of species can be an invaluable tool for conservation, because it mitigates degradation of their natural areas through propagation to help repopulation and reduces the loss of genetic variants that are of great adaptive and evolutionary significance for the genus.
Pinus rzedowskii Madrigal & Caball. Del. is endemic to Mexico and in danger of extinction (Secretaría de Medio Ambiente y Recursos Naturales [SEMARNAT], 2010). The species is distributed in the Sierra de Coalcomán, Michoacán, with fragmented populations (15) and represented by few individuals (9 to 3 500 per population; Castilleja, Delgado, Sáenz-Romero, & Herrerías, 2016; Delgado, Piñero, Chaos, Pérez‐Nasser, & Alvarez‐Buylla, 1999). Its habitat is characteristic of nutrient-poor limestone soils (Cambisol), with populations comprised mostly of senescent adult individuals, which reduces regeneration capacity (Delgado et al., 1999). Nevertheless, genetic variation is relatively high (>0.275), although with significant levels of inbreeding (F = 0.270; Delgado et al., 1999). This condition affects reproductive success with low production of viable seeds (17 % with embryo) and low germination (3 %; Castilleja et al., 2016).
One of the alternatives for increasing the reproduction of P. rzedowskii is grafting, which unfortunately is difficult to perform between individuals of the same species, due to the scarce availability of viable seeds (Castilleja et al., 2016). Therefore, it is necessary to explore the possibility of using other pine rootstock-species, under the assumption that the selection of phylogenetically close species, where anatomical characteristics have probably evolved in a similar way, will allow better tissue coupling and, consequently, greater grafting success. This phylogenetic assumption has not been documented for pines, but other factors have been explored, although also scarcely, such as: the phenological state and viability of the scion and rootstock, time of grafting, the skill of the grafter, control of environmental conditions, and taxonomic and anatomical compatibility of species (Climent et al., 1997; Hartmann, Kester, Davies, & Geneve, 2002; Pérez-Luna et al., 2019, 2020a, 2020 b).
Phylogenetic reconstructions with molecular data from chloroplast and nucleus DNA regions (Gernandt, Geada, Ortiz, & Liston, 2005; Montes et al., 2019) place P. rzedowskii within the Parrya section, Cembroides subsection, whose phylogenetic relationships with P. pinceana Gordon & Glend. and P. maximartinezii Rzed. form a monophyletic clade. Therefore, the use of rootstock plants with these pine species supports the possibility of having grafts compatible with P. rzedowskii.
Interspecific grafting of P. rzedowskii with rootstocks of P. pinceana, P. maximartinezii, P. ayacahuite var. veitchii (Roezl) Shaw and P. pseudostrobus Lindl. were carried out to evaluate survival in this study. The hypothesis is that species phylogenetically closer to P. rzedowskii show higher survival compared to species with a more distant phylogenetic relationship.
Materials and methods
Biological material and grafting
Rootstock plants were produced by seed germination of the following five species: P. rzedowskii; P. maximartinezii, P. pinceana and P. ayacahuite var. veitchii, three phylogenetically close species of the Strobus subgenus; and P. pseudostrobus of the Pinus subgenus (Table 1).
Table 1 Source of seeds used to obtain rootstock plants of the Pinus species under study.
| Species | Source | Latitude (N) | Length (W) | Altitude (m) |
|---|---|---|---|---|
| P. pinceana | Guadalcázar, San Luis Potosí | 22° 40’ 10’’ | 100° 29’ 30’’ | 1 600 |
| P. maximartinezii | Pueblo Viejo, Jalisco | 21° 21’ 15’’ | 103° 21’ 22’’ | 2 380 |
| P. ayacahuite var. veitchii | La Palma, Michoacán | 19° 36’ 39’’ | 102° 20’ 24’’ | 2 150 |
| P. pseudostrobus | San Juan Nuevo Parangaricutiro, Michoacán | 19° 27’ 06’’ | 102° 12’ 57’’ | 2 685 |
| P. rzedowskii | El Varaloso, Michoacán | 18° 40’ 59’’ | 102° 59’ 34’’ | 2 800 |
Seeds were treated with 0.2 % sodium hypochlorite for 5 min; subsequently, seeds were rinsed with tap water and soaked in a solution of distilled water with 2 % hydrogen peroxide for 24 h, selecting seeds with embryo by the flotation technique (Castilleja et al., 2016). Finally, seeds were treated with 3 g∙L-1 of Captan® fungicide for 5 min and planted in transparent plastic tray (30 x 30 x 30 x 5 cm) with 100 % vermiculite substrate. After 40 days, seedlings were changed to a substrate composed of 50 % moss peat, 25 % agrolite, 25 % vermiculite and 5.0 g∙plant-1 of controlled release fertilizer (Multicote®), in black plastic containers of 5 L capacity (30 x 20 cm); irrigations were applied every third day. Rootstock plants were selected from three years old according to the recommended characteristics; age between 2 to 4 years, as straight as possible and with a diameter between 1.5 to 2.5 cm (Muñoz-Flores et al., 2013). Due to reproductive problems of P. rzedowskii (Castilleja et al., 2016), only seven plants were obtained, therefore, the number of plants for the rest of the species was approximated to 10 with the intention of avoiding bias in the results, due to differences in the sample size.
The P. rzedowskii scions were collected in the locality of Chiqueritos, Dos Aguas, municipality of Aguililla, Michoacán (18° 04' 17” N and 102° 59' 36” W). Seventy scions with terminal buds of similar size (5 to 8 cm in length) were collected from an adult reproductive tree (140 years old; diameter of 30 cm and height of 25 m). The collection was made from the lower middle part of the tree, which contained the largest number of buds. Collecting and grafting was carried out in a 24 h period (February 18 and 19, 2017) to keep the tissues alive and have grafting success (Aparicio-Rentería et al., 2013; Muñoz-Flores et al., 2013).
Grafting was carried out in a rustic nursery or common garden with the following dimensions 4 x 8 x 3 m and 50 % shade mesh at the “Presidente Juárez” Faculty of Agrobiology of the Universidad Michoacana de San Nicolás de Hidalgo. Grafting stages are shown in Figure 1. The grafting method was side-veneer grafting, recommended for pine species (Muñoz-Flores et al., 2013; Pérez-Luna et al., 2019; Villaseñor & Carrera 1980;). Temperature conditions were constant between 23 to 24 °C and relative humidity ranged from 60 to 80 % in the study. Grafts were kept covered with a brown paper bag to reduce light incidence and a plastic bag with water to keep them hydrated (Muñoz-Flores et al., 2013); the paper bag was removed after 30 days, at this moment grafting success was observed due to bud growth, and the plastic bag was removed after 60 days. To avoid fungal problems, Ridomil Gold® fungicide (3 g∙L-1) was applied to the foliage and graft area once a week.
On May 20, 2017, the first pruning of the total foliage and branches below the graft area was carried out. On June 3, 2017, the second pruning of the foliage and branches was carried out in the continuous area at the top of the graft, without pruning the terminal end of the rootstock. This pruning was carried out so that the sap would not feed its main bud, giving priority to the graft (Goldschmidt, 2014; Muñoz-Flores et al., 2013). On June 17, 2017, advanced growth of graft buds was observed and total pruning of rootstock plants and their release was chosen.
Figure 1 Sequence of grafting stages. a) Pinus rzedowskii scion, b) cutting the rootstock stem, c) size matching, d) side-veneer grafting, e) tying the graft with plastic tape (MD brand), f) plastic bag with water to avoid dehydration, g) brown paper cover to reduce light incidence, h) grafting with rootstock plants of P. ayacahuite var. veitchii, with developing male strobiles, i) second pruning of P. pinceana at the central-top part of the graft with the terminal end unpruned and male strobiles, j) graft of P. rzedowskii with larger male strobiles at the pollen release stage, k) third pruning and graft release, and l) grafting with new needles and twigs of P. rzedowskii.
Data collection and analysis
Live and dead grafts were quantified every seven days until six months after grafting (March-August, 2017). A value of 0 was assigned to plants with loss of bud turgor and yellowing in the graft area, and 1 to plants with live buds for each evaluation (Barchuk & Díaz, 2000; Sigala, González, & Prieto-Ruíz, 2015). Each species was considered as a treatment (five treatments) with 10 replicates, except for P. rzedowskii (control treatment), since only seven plants were obtained.
Survival curves were performed using the non-parametric Kaplan-Meier model (Sigala et al., 2015), which allows statistical significance studies on the survival of each treatment over time; that is, it analyzes the time elapsed for an event to occur (in this case the death of plants) also regarding the censored data, which correspond to the individuals that, until the last moment of measurement, had shown no event (live plants) (García, 2012). A total of 12 measurements were taken with an interval of 15 days for six months. Differences between treatments were evaluated using the Log-Rank test, based on the comparison of observed versus expected events through the survival function (Le, 1997) defined as: S (t) = P (T > t); where, S (t) is the probability that at least one death occurs at a time T, as large as time t12. Furthermore, with this non-parametric approximation, the mean value of the survival function (m) of the samples of each treatment was collected. When following up each sample that did or did not present the event (plant death), the mean value of survival over time was estimated, obtained from the number of days (md) elapsed from the observations made in six months (180 days). Finally, paired Chi-square (X2) comparisons were made to determine the contribution of treatments. Data was analyzed in the R platform (R Development Core Team, 2016) with the Survival (2.41.3) and Survminer (0.4.0) packages.
Results and Discussion
The highest final survival value was for rootstock-plant grafts of the same species: P. rzedowskii with six out of seven grafts (85.7 ± 0.21 %), followed by P. pinceana with eight out of 10 (80 ± 0. 316 %), both with the highest mean values of expected survival function (m = 0.97 and m = 0.95) and mean estimated survival values in number of days (md = 163.4 and md = 162.2) (Table 2; Figure 2).
A recent genomic study of the Cembroides subsection indicates that P. pinceana is one of the two species phylogenetically closest to P. rzedowskii (Montes et al., 2019); their anatomical characteristics and modifications over time have probably been similar, allowing better coupling and functionality of cambium tissues (Castro-Garibay, Villegas-Monter, & López-Upton, 2017; Pérez-Luna et al., 2020a). It has been reported that xylem characteristics and the shape of vascular bundles should also be similar for the contact to be as homogeneous as possible (Castro-Garibay et al., 2017; Daricova et al., 2013; Pérez-Luna et al., 2019). In this sense, and according to the results, P. pinceana is recommended as a rootstock for successful grafting with P. rzedowskii.
Although P. pinceana is under the special protection status (NOM-59-SEMARNAT-2010), in contrast with P. rzedowskii, it shows no extreme reproductive problems with 54 to 55 % seed efficiency (Quiroz-Vázquez, López-Upton, Cetina-Alcalá, & Ángeles-Pérez, 2017) and some of the populations show adequate regeneration, represented by 22 to 59 % of young plants (1 to 25 years old) with a recruitment rate higher than one individual per tree (Martínez-Ávalos et al., 2015; Molina-Freaner, Delgado, Pérez, Piñero, & Alvarez-Buylla, 2001). Based on the above, P. pinceana can be a safe seed source for the production of rootstock plants for P. rzedowskii grafting, without affecting the demographic and genetic balance of the species.
Table 2 Kaplan-Meier survival function for grafting with Pinus rzedowskii with five treatments (rootstock species) and 12 evaluations at 15-day intervals for six months.
| Species | m | SD | CI (95 %) | md | SD | CI (95 %) | ||
|---|---|---|---|---|---|---|---|---|
| Lower limit | Upper limit | Lower limit | Upper limit | |||||
| P. pinceana | 0.951 | 0.035 | 0.886 | 1 | 162.203 | 2.962 | 156.291 | 167.616 |
| P. maximartinezii | 0.758 | 0.085 | 0.609 | 0.944 | 130.993 | 6.15 | 116.975 | 145.01 |
| P. ayacahuite var. veitchii | 0.852 | 0.065 | 0.834 | 0.988 | 145.796 | 4.69 | 145.211 | 164.381 |
| P. pseudostrobus | 0.666 | 0.106 | 0.558 | 0.873 | 121.509 | 8.523 | 102.844 | 140.174 |
| P. rzedowskii | 0.973 | 0.027 | 0.922 | 1 | 163.421 | 0 | - | - |
m = mean values of the expected survival function, md = mean values of survival in relation to the number of days, CI = confidence interval (95 %), SD = standard deviation of the mean.
P. maximartinezii, the second species closely related to P. rzedowskii, had a low final survival with three out of10 grafts (30 ± 0.91 %) and lower than expected according to the survival function (m = 0.758; md = 130.9). This result was possibly due to the fact that rootstock-plants were larger in size (diameter and stem height) compared to the other species used, although they were of the same age, as recommended (Aparicio-Rentería et al., 2013). Probably, due to differences in natural growth with anatomical structures with different size (v. g., vascular bundles with larger diameter), an adequate coupling of tissues was not achieved. To corroborate this assertion, it is necessary to study grafts with rootstock-plants younger than three years and see if tissue adjustment is possible to increase the survival rate. It is worth mentioning that P. maximartinezii is considered microendemic and under special protection status (NOM-59-SEMARNAT-2010). Although it shows no problems of demographic stability (López-Mata, 2013), its seed is used for production and marketing of ornamental plants without any regulation. Due to the above, natural regeneration of P. maximartinezii could be affected if plants produced in nurseries are not reintroduced into their own stocks, therefore, the development of a repopulation program for protection could be required.
Figure 2 Distribution of the Kaplan-Meier survival function for Pinus rzedowskii grafts using five rootstock species. Colors in light shades indicate confidence intervals (95 %) for each species.
One of the promising species for grafting success is P. ayacahuite var. veitchii, which is also part of the Strobus subgenus with a vascular bundle as P. rzedowskii; although it is grouped in another section (Quinquefoliae) and subsection (Strobus) (Gernandt et al., 2005) it has been observed that trees of both species have very similar physiognomic characteristics when they are young (2 to 15 years), so it is very probable to obtain good tissue attachment. Being the second species with the highest percentage of final survival (50 ± 0.628) and with an acceptable mean expected survival time (m = 0.85, md = 154.7), is proposed as a rootstock species suitable for grafting with P. rzedowskii. In addition, obtaining seeds from P. ayacahuite, for plant production, has no effect on the demographic balance of its natural populations, since it has no reproductive problems with 54.9 % seed efficiency and 71.5 % germination (Castilleja et al., 2016).
The least indicated species was P. pseudostrobus with lower final survival (20 ± 0.89 %) and mean expected survival time (m = 0.66; md = 121.5). One of the main factors that probably influenced this result was its distant phylogenetic relationship with P. rzedowskii, because it is part of another subgenus (Pinus) with different morphological and anatomical characteristics that possibly prevented the complete union of the tissues (Darikova et al., 2013; Goldschmidt, 2014).
The heterogeneity observed between the species was highly significant with the Log-Rank test (X2 = 25.24, P < 0.0001), which is due to differences between P. rzedowskii with P. pseudostrobus (X2 = 13.480, P < 0.0001) and P. maximartienzii (X2 = 8.390, P < 0.004), and these last two species in relation to P. pinceana (P. pseudostrobus, X2 = 15.357, P < 0.0001; P. maximartinezii, X2 = 9.105, P < 0.003) both with the lowest survival (Figure 2). Differences were also observed between P. ayacahuite var. veitchii and P. pseudostrobus (X2 = 5.344) with lower but significant statistical support (P < 0.021). The results suggest that phylogenetic relationships are an important factor for grafting success, showing that the most distant species (P. pseudostrobus) was the least successful and had the most significant differentiation.
To date, studies on heterospecific grafting are scarce and no clear pattern of their behavior has been determined. However, some of these studies show trends similar to the results obtained in this study; the study of P. patula Schiede ex Schltdl. et Cham. on P. radiata D. Don. with 96.7 % survival (Dyson, 1975), both belonging to the Australes subsection (Gernandt et al., 2005); P. patula (Australes subsection) on P. pseudostrobus and P. douglasiana Mtz. (both in the Ponderosa subsection) with 80 % and 69 % survival, respectively (Villaseñor & Carrera 1980), which belong to the Trifoliae section (Gernandt et al., 2005), which probably made their survival possible. Another study is that of P. brutia Ten. on P. nigra Arnold. with 56 % survival (Climent et al., 1997), both from Pinus subsection (Eckert & Hall, 2006).
A recent study on P. engelmannii Carr. grafting with the same species and with a hybrid of P. engelmannii x P. arizonica Engelm. var. arizonica indicated higher survival with the hybrid (83 %) than with the pure species (25 %) (Pérez-Luna et al., 2020b). The authors conclude that the low grafting success was due to the high density of resiniferous canals in P. engelmannii scions (Pérez-Luna et al., 2019); while, in the case of grafting success with the hybrid, the authors indicate that the presence of few stomata in the needles of P. arizonica makes the grafts more resistant to environmental stress with an increase in survival. These differences are present despite the fact that both taxa are closely related (Ponderosae subsection; Eckert & Hall, 2006), which can also be considered as another important factor for the results obtained with the grafts of the hybrid plants.
In a study on the anatomy of rootstock and grafting rootstocks of four pine species (P. patula, P. greggii Engelm. ex Parl, P. teocote Schiede ex Schltdl. et Cham. and P. leiophylla Schiede ex Schltdl. et Cham.), Castro-Garibay et al. (2017) demonstrated that a circular and continuous cambium is the most suitable for a complete connection of graft tissues; these authors observed these characteristics for P. teocote and P. leiophylla, considered as candidate rootstock-species for grafting the rest of the species they evaluated. In the same study, the anatomical results suggested the influence of phylogenetic proximity, where the most promising species (P. teocote and P. leiophylla) were included in a phylogenetic study of Pinus (Gernandt et al., 2005), within the Australes subsection and all (the four species) within the Trifoliate section.
The above studies show that phylogenetic proximity is a common factor for grafting success. Therefore, the heterogeneity of the results obtained in this study is associated with the phylogenetic and evolutionary differences of the species. Currently, there is no information on the anatomy of the stems of the species studied; however, some taxonomic studies report anatomical differences in needles (Farjon & Styles, 1997; Madrigal & Caballero, 1969; Martínez, 1948). Therefore, future studies on vascular tissues of rootstock-plants and scions are required to corroborate the compatibility of vascular tissues of species systematically and rigorously.
Conclusions
Side-veneer grafting propagation strategy is viable for P. rzedowskii, because it achieves a survival of more than 80 %. It is also shown that interspecific grafting is possible, and that the species factor affects survival over time, which is associated with the phylogenetic closeness of the species. The highest survival was obtained with rootstock plants of P. pinceana, which forms a monophyletic group with P. rzedowskii. This information is an initiative with a solid scientific and biotechnological basis for the propagation of the species on a larger scale, of great utility for its ex-situ conservation and for future genetic improvement studies.
