Rootstock origin and fertilization

The seed to produce the rootstock was collected from natural stands of P. pseudostrobus var. oaxacana in two localities (Santa Catarina Ixtepeji and Santa María Jaltianguis) in Oaxaca, Mexico. No evidence of natural hybridization was observed at the sites, so rootstock plants were considered as representative trees of that variety. Rootstocks were grown for 10 months in 310 cm³ plastic containers in a substrate formed by a mixture of peat, vermiculite and perlite in a 2:1:1 ratio, respectively; subsequently, they were transplanted into black polyethylene bags with a capacity of 5.2 L (20 cm wide and 35 cm long) in a substrate of mountain soil (Andosol 80 %) and ground pine bark (20 %). Rootstocks received three doses of fertilization: 3, 5 and 8 kg of Multicote® 15-7-15 + 2MgO + micronutrients per cubic meter of substrate. At grafting, rootstocks (1.5 years) with 3 kg of fertilizer·m^-3 of substrate had average height of 136.2 ± 4.7 cm and diameter at stem base (D) of 14.0 ± 1. 5 mm (rootstock case); on the other hand, those with 5 kg fertilizer·m^-3 had average height of 143.5 ± 3.8 cm and D = 15.4 ± 1.7 mm, and those produced with 8 kg fertilizer·m^-3 reached an average height of 148.0 ± 4.1 cm and D = 16.9 ± 1.3 mm.

Budwood collection

Budwood for grafting were collected from trees selected using the neighbor comparison method proposed by ^{Zobel and Talbert (1988}), located in the same communities where seed was collected for rootstock production. An average of 120 buds per location were collected at each grafting season. The average age of donor trees was 43.5 ± 4.7 years with a diameter of 53.5 ± 2.2 cm and average height of 35.0 ± 3.7 m. Healthy wood buds with an average length of 18 to 20 cm and caliber of 10 to 15 mm were cut from the mid-upper part of the canopy of trees to facilitate union with the rootstock during each grafting season. Wood buds were placed in labeled cool boxes, covered with dehydrated sawdust, wet pieces of fabric and ice, and transported to the nursery of the Uruapan experimental field where they were grafted 24 h after harvesting.

Grafting technique and grafting period

Grafting was in three periods: 1) early winter (December 2018), 2) late winter (February 2019), 3) early summer (July 2019). The methodology described by ^{Muñoz et al. (2013}) was used with two types of grafting: side-veneer and terminal tip/insertion; the average grafting height was 19.3 ± 1.7 cm and 74.5 ± 2.4 cm, respectively. In each grafting season, 108 intravarietal grafts were made (54 of each type). A total of 324 grafts were made in the three grafting seasons.

To evaluate the success of grafting, the criteria indicated by ^{Muñoz et al. (2013}) were used, which includes two categories: a) unsuccessful grafting (value of 0), where apparently there was no functional union of the cambium of both structures and b) successful grafting (value of 1) with response from 45 to 90 days after grafting (dag), which include the formation of new needles, bud break or bud burst and shoot growth (Sg). Successful grafting and survival (%) were recorded every third day, from 3 to 90 dag. Sg was measured with a Neiko® digital vernier and a graduated ruler (cm). Similarly, and in a complementary manner to determine its effect on grafting success, length (cm) and bud diameter (mm), total height (cm), diameter at the base of the rootstock stem (mm) and grafted height (cm) were measured.

Experimental Design and Statistical Analysis

A total of four factors with different levels were evaluated: (a) bud origin (Santa Catarina Ixtepeji and Santa María Jaltianguis); (b) rootstock fertilization (3, 5 and 8 kg of Multicote® 15-7-15 + 2MgO + micronutrients per m³ of substrate); (c) grafting season (early winter, late winter, early summer); and (d) grafting technique (side-veneer and terminal tip/insertion). A completely randomized experimental design with a 2 x 3 x 3 x 3 x 2 factorial arrangement was used with nine replicates per treatment; each graft represented one experimental unit.

An analysis of variance (ANOVA) was carried out to determine the effect of factors and their interactions on the values of success in grafting and Sg; the ANOVA for both variables was carried out with the data from the evaluation at 90 dag, with the following statistical model.

where,

y_ijklm = response variable value of repetition l of level i of A, level j of B, level k of C and level l of D

µ = Overall mean

A_i = effect of level i of factor A (origin of the bud)

B_j = effect of level j of factor B (rootstock fertilization)

C_k = effect of level k of factor C (grafting period)

D_l = effect of level l of factor D (grafting technique)

AB_ij = A*B interaction, corresponding to level i of A and level j of B

AC_ik = A*C interaction, corresponding to level i of A and level k of C

AD_{i l}= A*D interaction, corresponding to level i of A and level l of D

CB_jk = B*C interaction, corresponding to level j of B and level k of C

BD_jl = B*D interaction, corresponding to level j of B and level l of D

CD_kl = C*D interaction, corresponding to level k of C and level l of D

ABC_ijk = A*B*C interaction, corresponding to level i of A, level j of B and level k of C

ABD_ijl = A*B*D interaction, corresponding to level i of A, level j of B and level l of D

ACD_ikl = A*C*D interaction, corresponding to level i of A, level k of C and level l of D

BCD_jkl = B*C*D interaction, corresponding to level j of B, level k of C and l of D

ABCD_ijkl = A*B*C*D interaction, corresponding to level i of A, level j of B, level k of C and level l of D.

Є_ijklm = experimental error corresponding to the repetition m of level i of A, j of B, k of C and l of D.

Prior to ANOVA, data on the success of grafting were transformed with the arcsine function of the square root of the original value expressed as a decimal fraction [T = arcsine (√Y)]. After analysis, average values were transformed back to the original units. When significant differences were observed, a Tukey's mean comparison test (P = 0.05) was carried out. To assess survival, dead grafts were coded as zero and live grafts as one. Survival dynamics for the four factors were analyzed by the Log-Rank test, from survival curves constructed by the ^{Kaplan-Meier method (Kaplan & Meier, 1958}) in which the survival function is defined as:

where,

S (t) = probability of a death occurring within a certain time T

P = probability of survival at the time intervals during evaluation

T = total survival time to the end of the assessment, which must be greater than t

t = period at any time from the beginning of evaluation.

Statistical analyses were carried out using STATISTICA 13.0 (^{StatSoft Inc., 2018}).

Grafting success

The formation of needles, floral structures and bud sprouting was manifested from 45 days after grafting (dag), which was indicative of the activity and success of grafting. Table 1 shows that significant differences (P < 0.05) were obtained at grafting success stage and Sg by the effect of bud origin, period, grafting technique and some of their interactions.

According to Table 2, grafts using the side-veneer technique showed on average 36 ± 0.1 % of success and terminal tip/insertion 13 ± 0.1 %. With respect to time, grafting success increased to 41 ± 0.1 % when grafting was in February 2019; differences were up to 27 % with respect to grafting in December 2018 (period 1: 13 ± 0.1 %) and July 2019 (period 2: 20 ± 0.1 %). During the three periods, budwood coming from Santa Catarina Ixtepeji was more successful (26 ± 0.1 %) than that from Santa María Jaltianguis (23 ± 0. 2 %) in both grafting techniques, especially for side grafting (Table 2). The effect of the grafting period is due to differences in the phenological and physiological state of the bud; at the time of harvesting, during period 2 (February 2019), the bud showed initial quiescence favoring greater growth in diameter (8 to 14 ± 1.2 mm) and length (12 to 16 ± 3.3 cm). Furthermore, the diameter of the rootstock (14 to 18 ± 1.4 mm) and bud (8 to 14 ± 0.8 mm) affected the union of both structures; grafting success involves morphological, anatomical, physiological and biochemical aspects of a plant (^{Yin et al., 2012}). Therefore, the faster the union (welding) of the tissues of both structures, the greater the success of grafting because this efficiency decreases the time in which the graft is at risk of dehydration (^{Gaspar, Wendling, Stuepp, & Angelo, 2017}).

The significant effect of interaction of the factors bud origin-grafting season (Table 1; Figure 1a) reinforced the results obtained; buds from Santa Catarina Ixtepeji grafted during period two had 48 ± 0.04 % of success. On the other hand, the bud origin-grafting technique interaction (Table 1) showed higher (40 ± 0.03 %) success when grafting using side-veneer grafting technique with buds from Santa Catarina Ixtepeji (Figure 1b).

Figure 1 Effect of interactions: bud origin-grafting period (a) and bud origin-grafting technique (b) on grafting success in Pinus pseudostrobus var. oaxacana (bars indicate standard error of the mean). 

It is common that grafting success and reduced growth rate are the result of poor bud-rootstock union quality often related to poor callus formation caused by poor contact or intra- or interspecific incompatibility (^{Castro-Garibay, Villegas-Monter, & López-Upton, 2017}; ^{Yin et al., 2012}). A factor that favored grafting was the genetic affinity between scion and rootstock, since intraspecific grafts are more successful for having greater anatomical, morphological and physiological similarity between graft components (^{Goldschmidt, 2014}). These results agree with those reported by ^{Muñoz et al. (2013}) for intraspecific side-veneer grafting on P. pseudostrobus var. pseudostrobus, since they obtained 40 % of grafting success at 90 dag; with those described by ^{Pérez-Luna et al. (2019}) for grafts of the same type, but with P. engelmannii Carr. who recorded 25 % of success at 180 dag.

Shoot growth

Table 1 indicates that Sg had significant differences (P ≤ 0.05) only for individual factors period and grafting technique. According to Table 2, grafting made during period 2 (February 2019) had 3.3 cm (14.5 ± 0.1 cm) more than the other periods. Sg was higher for side-veneer grafting on 13.6 ± 0.1 cm, compared to terminal tip/insertion with 11.4 ± 0.1 cm. Differences in Sg are because, terminal tip/insertion, the leader bud was replaced and grafted at a higher height (74.5 ± 2.4 cm); consequently, the rootstock could reduce the flow of water and nutrients required for growth (^{Świerczyński, Kolasiński, Urbaniak, Stachowiak, & Nowaczyk, 2018}). In contrast, for side-veneer grafting, the aerial part of the rootstock was removed at three stages as mentioned by ^{Muñoz et al. (2013}); in addition, the height of the graft was lower (19.3 ± 1.7 cm) and, therefore, the needles of the graft developed at 45 dag carried out all photosynthesis and transpiration activities. These conditions impacted and established differences in Sg.

It has been mentioned that Sg differences are due to the growth habit of the sources; however, no significant differences were observed in this case; rather, Sg was determined by the time of grafting (^{Koepke & Dhingra, 2013}; ^{Martínez-Ballesta, Alcaraz-López, Muries, Mota-Cadenas, & Carvajal, 2010}; ^{Świerczyński et al., 2018}). According to Martínez-Ballesta et al. (2010), improper bud-rootstock callus formation can reduce Sg. The use of buds in initial quiescence or recess stage (period 2) increased the growth of the grafted scion, as Sg was initiating its active phase (^{Viveros-Viveros & Vargas-Hernández, 2007}). On the other hand, the use of scions in post-dormant quiescence (period 3) interrupted Sg at the grafting phase (first 45 days), while the connections at the graft union point were being established.

Grafting survival

Grafting survival was determined in accordance with than mention by several authors, as dead grafts without PI and live grafts with response and Sg were evaluated (^{Cuevas et al., 2015}; ^{Muñoz et al., 2013}; ^{Pérez-Luna et al., 2019}). For the entire experiment (including all treatments), survival decreased from 100 % to 50 ± 5 % at 40 dag and 27.9 ± 4.6 % at 90 dag (Table 3).

The Log-Rank test showed statistical differences (P < 0.001) only for the grafting technique regarding the time of grafting. The average survival of side-veneer grafting at 90 dag was 20.5 % and for terminal tip/insertion 30.3 %. The highest percentage of mortality was recorded during the first 40 days, because more than 50 % had problems at grafting (Table 3; Figure 2).

Figure 2 Survival estimated with the Kaplan-Meier model for side-veneer grafting and terminal tip/insertion in Pinus pseudostrobus var. oaxacana. Values with different letters indicate statistical differences according to Tukey's test (P ≤ 0.05). 

The survival function estimated with the Kaplan-Meier model allowed identifying significant differences at 90 dag; side-veneer grafting had 24.1 %, 19.2 % and 18.4 % of survival for period 1, 2 and 3, respectively, and for the terminal tip/insertion, the estimated survival was 33.3 %, 34.3 % and 23.3 % for period 1, 2 and 3, respectively (Figure 2).

Grafting season has been shown to influence the percentage of grafting success and survival; most studies recommend grafting conifers in winter season (^{Almqvist, 2013}; ^{Cuevas et al., 2015}; ^{Gaspar et al., 2017}; ^{Świerczyński et al., 2018}). In a similar study with side-veneer grafting on P. engelmannii at the end of winter, had a survival rate of 22.5 % six months after grafting (^{Pérez-Luna et al., 2019}). In the case of broadleaf species, terminal intraspecific grafting on Khaya grandifoliola C. DC., increased survival significantly (81.67 %), and height growth during winter (^{Opoku et al., 2019}). Based on the results obtained, it can be said that the best time for grafting on P. pseudostrobus var. oaxacana is at the end of winter, during period 2 (February). Gaspar et al. (2017) mention that successful grafting of woody species from any part of the world is possible at any time of the year under controlled climatic conditions, as long as the maximum temperature is not higher than 24 °C and the minimum temperature is not lower than 3 °C.

The grafting technique plays a determining role in survival, since, if the rootstock and bud have no union in the cambium, there will be no grafting success; in this study, side-veneer grafting had higher Sg and grafting success. Although average percentages of grafting and survival were low (36 ± 0.1 %) and in most of the factors there was no significant interactive effect, there was an additive effect that increased these values (up to 66 %). The above was reflected in the case of side-veneer grafting made on rootstocks produced with 8 kg fertilizer·m^-3 with buds from Santa Catarina Ixtepeji during period 2 (February 2019).

In this regard, the results represent an advance in knowledge of the subject taking into account some of the determining factors for the success of intraspecific grafting on P. pseudostrobus var. oaxacana. It was found that grafting and survival depend mainly on external factors such as the correct execution of the grafting technique and the time of grafting. Some authors indicate that internal factors such as bud/rootstock compatibility and the anatomical characteristics of both structures (factor not studied) are also important (^{Almqvist, 2013}; ^{Castro-Garibay et al., 2017}; ^{Darikova et al., 2013}; ^{Martínez-Ballesta et al., 2010}; ^{Ranjith & Ilango, 2017}; ^{Reig et al., 2018}).