Plant material

Scions (terminal bud and basal segment) from nursery stock plants (NSSP) and field-selected trees (FSSP) from a sexual seed orchard in Pueblo Nuevo, Puebla, Mexico (19° 57´ 35.73´´ N; 98° 06´ 22.77´´ W) were used.

Nursery stock plants originated from grafting made in 2016 using scions from field-selected trees from a 14-year-old sexual seed orchard located at the Insurgentes nursery, Zacatlán, Puebla, Mexico (19° 57´ 35.73´´ N; 98° 06´ 22.77´´ W). Plants were kept in 12 L pots (21, 27 and 31 cm in base, height and upper diameter, respectively) with substrate consisting of bark, vermicompost and tezontle (60:20:20; v: v: v) at Montecillo, Texcoco, Estado de México, Mexico (19° 27´37.74” N; 98° 54´15.27” W), under 60 % shade netting.

From tree 16 in the seed orchard, 220 scions with a length of 8 to 12 cm and a diameter of 3.5 mm were collected. This activity was carried out one day before grafting; the scions were transported in Ziploc® bags in a cooler containing frozen water in plastic bottles. On the same day of collection, in the in vitro culture laboratory, the needles were removed from the base of the scions (4 cm), washed with soap and water to remove dust and scrubbed with a fine bristle toothbrush. They were then disinfested with Benomyl (methyl 1-[butylcarbamoyl] benzimidazole 2-yl carbamate; 2 g∙L^-1) for 15 min, left on laid paper to remove excess water, and stored in the same manner as transported. Nursery scions were collected (120 scions) at the time of grafting, but were not disinfested; the average length was 15 to 20 cm. At the time of grafting, scions averaged 8 to 10 cm in length for both origins (Figure 1).

Figure 1 Pinus greggii var. australis scions with terminal bud (A) and basal segment (B) used for grafting. 

Rootstocks

In May 2018, five months seedlings from seed of Pinus greggii var. australis, P. patula Schiede ex Schltdl. et Cham., P. teocote Schiede ex Schltdl. et Cham. and P. leiophylla Schiede ex Schltdl. et Cham, produced in rigid plastic container (140 cm³) were transplanted into 1 L square pots (8.5, 12.5 and 10.7 cm at base, height and top, respectively) with bark, vermicompost and perlite substrate (60:20:20 v/v/v). Plant sizes ranged from 1.74 to 2.98 mm in diameter and from 14.05 to 20.55 cm in height. Fertilizations were made with 2 g∙L^-1 diammonium phosphate solution (DAP®) every two weeks for two months. The rootstocks were kept in a space protected with 60 % shade netting.

In April 2019, the plants of the four species were grafted (15 months, 3 to 4 mm in diameter and 36 to 52 cm in height). Pinus patula and P. greggii had the greatest heights, 52 and 49 cm, respectively, and P. patula and P. teocote had the greatest diameters, both with 4.3 mm. Shoots developed at the base of the rootstocks were removed one month after grafting and, subsequently, according to emergence.

Grafting and post-grafting management

A total of 60 grafts were made per species of rootstock: 15 for each origin (NSSP and FSSP) and 15 of each type of scion (terminal shoot and basal segment); a total of 240. It should be mentioning that, for basal segments of the field scions, the main apex was removed because they had insufficient length to section them, contrary to the nursery scions, where in some cases up to two basal segments were obtained. The type of grafting was cleft; the scion was grafted where the rootstock stem was tender green (12 cm), it was tied with plastic tape and a transparent plastic bag was placed under the union to avoid excess transpiration (Figure 2).

Plants were fertilized with DAP® (2 g∙L^-1), one week after grafting and, subsequently, every three weeks for six months, interspersed with DAP®, Yaramila complex® (N-12 %, P-11 %, K-18 %, S-11 %, Mg-2.7 %, B-0.0015 %, and Fe, Mn and Zn-0.2 %; 2 g∙L^-1) and humic substances (1 mL∙L^-1), adding 100 mL of solution to each plant and Ultrasol micromix® foliarly (1 g∙L^-1).

The plastic bag was removed as follows: one week after grafting one corner of the bag was cut off (about 2 cm), in the second week the next end of the bag was cut off (Figure 2B), in the third week the top of the bag was removed (Figure 2C), and in the fourth week it was completely removed (Figure 2D). The straps to tie the graft were removed three months later.

Figure 2 Post-grafting management of Pinus greggii var. australis on four rootstock species: saturation of the bag with mist to reduce graft transpiration (A), cut at the corners of the bag (B), cut at the top of the bag (C) and bag removal and graft growth (D). 

Experimental design and study variables

The experimental design was generalized randomized blocks with factorial arrangement: the four rootstocks species as blocks, the factors were origins (NSSP and FSSP) and scion types (terminal bud and basal segment); there were 16 treatments in total.

The variables evaluated were grafting percentage (with the total number of plants per treatment), one month after grafting; increase in height (cm) and diameter (mm) six and 12 months after grafting (diameters were measured 0.5 cm above and below the graft union); scion/rootstock ratio (scion diameter/rootstock diameter); and number of shoots emitted on the scions of the combinations, three months after grafting. The grafting percentage was calculated considering the 15 plants per combination; for the rest of the variables, the sample was 10 plants per combination of rootstock and scion (origin and type). Data were analyzed with the statistical packages R 3.6.1 (^{The R Foundation, 2018}) and SAS 9.4 (^{Statistical Analysis System Institute, 2013}).

Height and diameter increment data were subjected to an ANOVA and subsequent Tukey's mean comparison test (P ≤ 0.05); for the first variable only terminal shoots scions were used, and diameter was analyzed in both scion types. The number of shoots generated in the scions of the combinations was analyzed with Poisson regression.

Grafting success

Using 15-month-old rootstocks and scions from nursery or field-collected stock plants, but with active growth, more than 93 % grafting was reported (Figure 3). Grafting success was 100 % when P. teocote and P. patula were used, while with P. greggii and P. leiophylla it was 96.5 % (Figure 3); ^{Baron, Esteves, Amaro, Pina, and Ferreira (2019}) mention that it is necessary to use the same species as rootstock to obtain high grafting percentages; however, with the results of this study it is shown that such assumption may vary and that the pine species used have similar behavior to Citrus (^{Uribe-Bustamante, Curti-Díaz, Hernández-Guerra, & Ticante-Montero, 2013}); Persea (^{Salazar-García, Medina-Torres, Ibarra-Estrada, & González-Valdivia, 2016}) and Prunus (^{Gullo et al. , 2014}) that can be grafted onto other species.

Figure 3 Grafting (n = 15) using terminal budding of Pinus greggii var. australis on four rootstock species. NSSP: nursery scion stock plant; FSSP: field scion stock plant. Evaluation one month after grafting, May 2019. 

Pine rootstocks in slow-growing species or cespitose state are regularly three to seven years old (^{Pérez-Luna et al., 2019}) and fast-growing subtropical pine species are used from two years (Aparicio-Rentería et al., 2013). Plants of the mentioned ages are usually grafted in areas of higher lignification because this is mentioned in the methodologies, which can probably affect grafting. These ages are used because there is a lack of methodology to produce plants for grafting purposes and for these to have the minimum diameter and height measurements; however, with the results shown here, for fast-growing species, 15-month-old plants can be used.

In relation to the origin, the average grafting was 98 %, so either NSSP or FSSP with active growth can be used. The advantages of using NSSP are easy and fast harvesting and selection, since due to the height of the plants (1.5 m) it is not necessary to use persons, tree climbing equipment or transportation costs, as is the case with FSSP.

The NSSP scheme is used in fruit trees such as Citrus (^{Zamora-Rodríguez et al., 2016}) and to obtain cuttings in pine species (^{Martínez-Alonso et al., 2012}). In the present study, the management provided to NSSP such as pruning, fertilization and application of products against pathogens generated scions suitable for grafting.

Regarding the type of scion, the proposal proposed showed that the basal segment scions were suitable for grafting, having 100 % grafting success, which allowed the efficient use of the vegetative material of the selected trees. The average grafting success of both types of scions was 99 % (two grafts failed), possibly because the field scion was harvested one day before grafting and the NSSP scion may have been too tender. Grafting is regularly done with terminal bud scions, because those collected in the field are approximately 10 cm long, which makes the use of basal segments impossible; however, with the implementation of nursery stock plants, the scions reached lengths of up to 30 cm, so that up to three fractions of approximately 10 cm can be obtained: one with terminal bud and two basal segments, which means an advantage to obtain more scions.

In the studies carried out in Mexico, grafting success is less than 50 % three months after grafting (Aparicio-Rentería et al., 2013; ^{Pérez-Luna et al., 2019}), due to the lack of early preparation of rootstocks and scions; in addition, post-grafting management is deficient. Aparicio-Rentería et al. (2013) remove plastic bags three months after grafting, enough time for the temperature and humidity conditions inside the bag to favor the proliferation of fungi, a possible cause of graft death. In the present study, the elimination of plastic bags begins the week after grafting and ends one month later. It should be mentioned that the percentage of grafting one year later remains the same; therefore, proper post-grafting management is important for survival regardless of the rootstock and scion (type and origin) used.

Growth and height increase

One year after grafting, the height of grafts from NSSP increased 253.0, 300.0, 289.3 and 243.1 % and in FSSP increased 227.5, 216.0, 222.1 and 179.0 % in P. greggii, P. teocote, P. patula and P. leiophylla rootstocks, respectively (Figure 4). Pinus leiophylla produced lower growths, possibly because the species produced around 15 epicormic shoots, which involved competition for nutrients and carbohydrates.

Figure 4 Average growth dynamics in grafting (n = 10) of Pinus greggii var. australis made in April 2019, using terminal shoot scions. NSSP: nursery scion stock plant, FSSP: field scion stock plant, HAS: height after six months (October 2019), ADM: height after 12 months (April 2020). 

Scions from nursery stock plants showed higher growth than those in the field, which indicates that the nursery scheme is an excellent alternative to have scions with suitable characteristics for grafting, obtained with management practices through pruning, fertilization, protection against pests and diseases. The results also indicate that it does not depend on the phenology of the species, because the pruning carried out forces the plants to sprout, as occurs in Citrus (^{Zamora-Rodríguez et al., 2016}).

ANOVA showed significance for scion origin and rootstock (P < 0.0001) in the six-month data; however, after 12 months only the scion origin factor (P = 0.0009) was significant. The height increment for NSSP grafts six months later ranged from 13.2 to 19.0 cm, and for FSSP grafts from 6.8 to 10.4 cm. At 12 months, the increases were 32.0 to 35.7 cm and 22.2 to 30.2 cm in NSSP and FSSP grafting, respectively.

Regarding the mean tests of height increment, after six-month evaluation, NSSP grafting had the greatest increase; after 12 months, the averages for scion origin were 33.98 and 27.74 cm in NSSP and FSSP, respectively (Table 1).

On both evaluation dates, rootstocks with P. leiophylla had the lowest increments, regardless of scion origin, probably because the number of epicormic shoots emitted was a competition factor. In contrast, P. patula and P. greggii generated the greatest increases after 12 months of evaluation.

Rootstocks are a key element for grafting growth because they provide water and nutrients that can be used. In fruit, horticultural and ornamental species, the production of rootstocks is an activity that is carried out so that the plant has sufficient reserves to provide nutrients and carbohydrates to the grafted scion. In the case of pines, the production of rootstocks has not been a priority to date, since plants produced for different purposes are used.

For pine species, scions from field trees are generally lignified and sometimes collected and grafted during periods when plants are dormant (Aparicio-Rentería et al., 2013; ^{Pérez-Luna et al., 2019}), which delays the grafting and growth processes and even causes the death of grafts. The results show that growth is greater with NSSP, which can shorten the time to planting, as long as climatic conditions such as the presence of rain and absence of frost allow it.

^{Martínez-Ballesta, Alcaraz-López, Muries, Mot-Cadenas, and Carvajal (2010}) mentioned that vascular connection is fundamental for greater grafting growth. According to this, the results with NSSP could be due to the correct vascular connection, in addition to the nutrition provided with the objective of having scions with adequate characteristics for the activity. It should be noted that the practice of fertilization is little used in plants established under open-field conditions. On the other hand, ^{Castro-Garibay, Villegas-Monter, and López-Upton (2017}) mention that P. teocote has circular vascular cambium, making it a viable option to serve as rootstock, just like P. leiophylla; however, with the latter the lowest increases in height were reported, so, in addition to the anatomical characteristics of the species, there should be another factor affecting the growth of the combinations made.

Diameter and scion-rootstock ratio

The diameters of the scions generated by the stock plants in the nursery were homogeneous (2.8 to 3.1 cm) and none were discarded for use, in contrast to the 220 collected in the open field, of which about 80 were discarded because they did not meet the minimum required for grafting. This indicates that selection and experience of the person performing this activity are also important.

According to the ANOVA, the factors with a significant effect on scion diameter were rootstock (P < 0.0001) and scion type (P < 0.0001), while for rootstock diameter they were rootstock (P < 0.0001), origin (P = 0.0009) and the interaction origin*scion (P = 0.0105) after six months of evaluation. After 12 months, the factors rootstock, scion type and origin were significant for both diameters, and only the rootstock*origin interaction (P = 0.0206) was significant for scion diameter. The highest values of scion-rootstock ratio occurred in basal segment scions, regardless of origin.

After six months of evaluation, the largest scion and rootstock diameter was recorded for the combinations of P. teocote with basal segment scion from the open field (5.05 mm) and NSSP (5.40 mm), respectively. After 12 months, the combination of P. patula with basal segment scion from NSSP had the largest diameters for graft (6.41 mm) and rootstock (6.68) (Table 2). In fruit trees it is mentioned that rootstock vigor is positively related to plant diameter (^{Girardi, Mourão Filho, & Kluge, 2007}), which suggests that P. patula can be classified as vigorous because it has the largest diameters 12 months after grafting.

The scion-rootstock ratio is an indicator of compatibility (^{Berdeja-Arbeu, Villegas-Monter, Ruiz-Posadas, Sahagún-Castellanos, & Colinas-León, 2010}). In the case of P. leiophylla, although the diameters were smaller, the FSSP combination with basal segment scion had ratio 1, which indicates that growth is homogeneous and the same applies to some combinations with P. teocote and P. patula with values of 0.98 and 0.99, respectively. However, after 12 months, the results with P. greggii and P. patula are more attractive than with the rest of the combinations (Table 2). ^{Baron et al. (2019}) mention that taxonomic affinity is a requirement for grafting compatibility that may explain why all values are higher than 0.90 in the P. greggii/P. greggii combination after 12 months.

Differential diameter growth may be a symptom of incompatibility because physiological mechanisms are modified and trigger changes in height, diameter and crown size (^{Berdeja-Arbeu et al., 2010}).

^{Aloni, Cohen, Karni, Aktas, and Edelstein (2010}) refer to the accumulation of organic compounds at the graft union, which can cause proliferation of parenchymal tissue and increased stem diameter in both parts, which involves anatomical irregularities. ^{Souza, Diniz, Neves, Alves, and Oliveira (2018}) mention that the difference between diameters impairs the flow of photoassimilates and hydraulic conductivity that will be reflected in plant growth.

Number of shoots

Only origin and type of scion had a significant effect on the number of shoots (P < 0.0001). The highest number of shoots emitted was in basal segment scions, due to the loss of apical dominance, since the concentration of cytokinins increases (^{Tanaka, Takei, Kojima, Sakakibara, & Mori, 2006}), and with this the proliferation and growth of axillary buds, the opposite case for terminal shoot scions. NSSP exceeded in number of shoots (4.4) to FSSP (2.40), which may be associated with the accumulation of organic compounds due to the management provided to NSSP (Figure 5).

Figure 5 Lateral shoots produced in grafts of Pinus greggii var. australis on four rootstocks. NSSP: nursery scion stock plant, FSSP: field scion stock plant, BA: apical bud, SB: basal segment. Data collected three months after grafting (n = 10). 

Shoots can be used to shape the plant depending on the objective set. The scions produced by nursery plants showed a higher number of shoots compared to field plants, perhaps due to the handling provided to the stock plant, which could have led to a higher concentration of organic compounds, contributing to sprouting. After evaluating the number, some shoots of the scions were removed, leaving two or three per plant to promote growth and thickening and to form the crown architecture.

P. greggii var. australis is endemic to Mexico; most research on this species has focused on provenance-progeny studies for selection (^{Ruíz-Farfán et al., 2015}) and nursery plant production (^{Castro-Garibay, Aldrete, López-Upton, & Ordaz-Chaparro, 2018}), but in relation to propagation by grafting there is a lack of information. In Mexico, for P. patula and P. arizonica, complicated grafting methodologies are used, in addition to poor pre- and post-grafting practices (^{Aparicio-Rentería et al., 2013}; ^{Pérez-Luna et al., 2019}).

This study shows important contributions for grafting of P. greggii var. australis on other rootstock species because they are regularly used from the same species. In addition, the methodology is simple and can be adapted to other conifers because the implementation of NSSP has the following advantages: they are not exposed to field environmental conditions (dust, drought, pest or disease attack), they do not depend on the phenology of the species and there is greater control of fertilization and pathogens. On the other hand, by means of pruning, it is possible to provide grafting scions at least three times a year with characteristics of interest to the propagator, and those that do not yet have them are left on the stock plant and monitored until they reach the required size; therefore, NSSP are an excellent option that should be promoted to make grafting propagation of pine species more efficient. Likewise, NSSP, being 1.5 m high, do not require qualified people to collect, there are no transportation costs, and it is not necessary to disinfest the rods before using them, since there is control over the incidence of pests and diseases in comparison with those collected from trees in the open field.

There is no established methodology for rootstock production in forest species. With the present proposal of transplanting five-month-old plants to 1 L pots, pruning roots, removing lateral shoots and making individual fertilizations, vigorous rootstocks are achieved with better roots and greater content of reserves that will be used by the scions when they are grafted; in addition, by programming the planting of species, there will be adequate plants and they can be used between March and May. Due to their growth, the species used as rootstocks were transplanted after five months; however, growth is different for cespitose and stone pine species, so it is necessary to determine methodologies for the production of rootstocks of these or similar species. The proposed methodology can be useful for propagating trees selected in the open field or from progeny trials that have been evaluated to generate stock plants in nurseries