This study was aimed at assessing the influence of rootstock/scion compatibility on grafting union success in avocado. The trial was laid out in a simple randomized experiment made up of 3 x 6 x 20 factors including 3 avocado varieties (Anaheim, Taylor and Hass), 6 treatments correspondingly as rootstock + scion, notably (T1=Anaheim + Anaheim, T2= Anaheim + Taylor, T3= Anaheim + Hass; T4= Taylor + Taylor, T5= Taylor + Anaheim, T6= Taylor + Hass); and 20 repetitions. T1 and T4 were described as homografts since rootstock and scions are from same avocado varieties, while T2, T3, T5 and T6 were considered heterografts given that rootsocks and scions are from different avocado varieties. Days to first bud initiation, Number of buds, number of leaves, height of buds, % failed graft were used to score success in grafting union. Data collected was analyzed using SPSS ver. 22 and means were separated by the LSD using student t test at CI of 95%.

Results showed that homografts (T1 and T4), recorded earlier and robust bud development, followed by   T2 and T5 and the least results record in T3 and T6. A mean of 23 days after grafting were required for homografts (T1 and T4), to break first bud dormancy and get a first bud developed against 35 days in heterograft treatments (T2, T3, T5 and T6). Considering, % graft union failure, homografts recorded 1 and 2% for T1 and T4 respectively, against a failure rate of 9, 13, 58 and 68 % correspondingly for heterograft T5, T2, T6 and T3.  Rootstock and scions from the same variety showed higher compatibility expressed via least rate of grafting unions failures as compared to uniting rootstock and scions from different cultivars. Based on these results, rootstock and scion from same avocado variety are recommended for sustainable graft unions in grafting projects.

Keywords: avocado, chimeras, compatibility, grafting, heterograft, homograft

Avocado (Persea americana), a tropical crop of the Luraceae family is considered to be one of the most commercialized and profitable fruits at the international scale.¹ This might probably be due to the fact that avocado fruit has been recognized for its benefits in enhancing cardiovascular health given the richness of its pulp in monounsaturated fatty acids, phytosterols and phytostanols reported to reduce cholesterol levels.² Avocado trees are partially self-pollinating so to obtain true-to-type planting materials as well as shorten the juvenile stage, the crop is artificially propagated through vegetative methods like cutting, layering, budding and grafting to maintain a predictable quality and quantity of the fruit.³ Among these, grafting, a vegetative propagation technique that connects two severed plant segments together, is the main method of avocado seedling production in commercial nurseries. A grafted plant has two parts, notably the rootstock, which provides the root and the scion, the piece with desirable fruit traits that is attached to the rootsock. For a successful and sustainable graft union between the severed plant segments, the cambium of the scion must line up as closely as possible with that of the rootstock.⁴ The chimera, consisting of the rootstock and scion survives as a new individual after wound healing.  Studies have indicated that grafts between plants from different genera of the same family are rarely compatible, whereas grafts of different species within the same genus can survive by forming an effective graft union.⁵ A common hitch faced by avocado graft men is low take off rate of graft union characterized by premature death or failure of the graft combination, plant die back and incapacity to form a solid and lasting functional scion/rootstock union, and complications in plant growth and development in the field like unhealthy trees, breakage at the graft union etc. Several potential causes could account for graft failure amongst which are contamination, anatomical mismatch at the union interface, weak tightening during banding and incompatibility between rootstock and scion.⁶ Graft incompatibility is generally referred to as inability of the stock and scion to bind together to form a successful graft union. Among several factors like microbial contaminations during manipulation, anatomical deformities at graft interface,⁷ lack of compatibility between the rootstock and scion is the major limiting factor in propagation of fruit trees, particularly stone fruits.⁶ Though failure to take off is just an immediate consequence, incompatibility issues have been reported to be evident long after planting the tree in the field, which affects the yield and fruit quality in the advanced years of the crop.⁸ Graft incompatibility is therefore a critical issue for breeding rootstocks of fruit trees and longevity of an orchard.⁹ Thus, to ensure a successful and sustainable graft union the selection of a mutually compatible rootstock/scion combination is important.⁵ Following the recent increase in demand than supply of avocado in Cameroon,¹⁰ a lot of farmers are now embarking on establishing avocado plantations. In a recent survey carried out in the North West region of Cameroon, this research team noticed that the pursuit to plant varieties with high market value like Hass, Anaheim, Fuerte and Taylor has also mounted pressure on graft men to try to catch up with supply. Unfortunately among avocado nurseries visited, graft men report graft failures as high as 41%. Given that in Cameroon, there is no clonal rootstock currently used for grafting avocado, rootstocks are raised from seeds picked up haphazardly. It was within this scope that it was hypothesized in this study, that genetic incompatibility between rootstock and scion used by most graft men accounts for 35 % of graft union failures noticed during the survey.

This research was carried out at the teaching and research farm of the department of crop production technology, College of technology of the University of Bamenda, Cameroon located at 5^o59’0’’ North, 10^o15’0’’ East, with an altitude of 1,558m above sea level. This area has a mean annual temperature ranges from 13-18 ⁰C, characterized by annual rainfall of 2230 mm and average humidity of 70% and 52% in the rainy season and dry season respectively

Experimental design and field layout

A simple complete randomized design made up of 3 x 6 x 10 factors including 3 avocado varieties (Anaheim, Taylor and Hass), 6 treatments correspondingly as rootstock + scion, repeated 10 times was used. The choice of these three varieties was based on their high market value in Cameroon. The treatments (Table 1) comprised of each avocado variety serving as a rootstock and a scion within and with two other varieties, except in the case of Hass where the research team couldn’t find seeds to develop rootstock and so Hass only served as scions in this study (Table 1). Treatments in which, rootstocks and scions used are from the same avocado variety were designated as homografts (T1 and T4) while in cases where rootstocks and scions are from different avocado varieties were denoted heterografts (T2, T3, T5 and T6).

Standard method of grafting and follow up

Preparation of rootstocks

For each variety, trees for seeds were identified and mature fruits harvest from a local orchards in which only a given variety is cultivated. The seeds were removed from the mature fruits after ripening. They were sun dried for 4hrs and then disinfected by soaking in fungicide and insecticide solution prepared by respectively dissolving 50g of Ridomil gold 75 + 20 ml of Cigone in 15 L of water for 15 mins. The disinfected seeds were later potted in poly bags of 20 x 25 cm containing sterilized garden soil and allowed in open air. Six months later healthy mature avocado rootstocks with an average height of 30cm and a girth of 5cm were considered to have attained maturity for grafting. Allowing the seedlings to develop for up to 6 months was to allow the cambium layer to fully develop for grafting operation.

Scions with 5 well-developed buds were harvested from same plant from where fruits for rootstocks were collected with the use of secateurs. The leaves were pruned and the scions placed in a plastic bag to minimize rate of transpiration and dehydration from the bud zones. The scions were then disinfected by soaking in a solution of Ridomil and Cigone for 15 mins before air drying.

The Cinchona veneer grafting method developed by J.M. Benitez (cit. 11) in which the rootstock is not completely cut off was used. The stock was trimmed off its lower leaves and cleaned of any soil or other foreign matter on the stem. A tangential cut of 5-7 cm long was made through the bark and just into the wood (cambium) in an area where the stem is straight, approximately 7-10 cm above the soil using a grafting knife. A short second cut was made at the base of the first one, forming a notch. A scions with five buds and of relatively equal diameter with the stock was prepared by making a slanting cut on one side equal in length to that made on the rootstock. A small cut was made at the base of the scion on the opposite side so that the scion could fit into the notch on the stock. Finally the scion was carefully placed into position on the stock such that it lined up the juncture between bark and wood on at least one side. The entire graft was secured with wrapping material in a spiral beginning at the bottom, taking care to maintain alignment of the cut edges of the rootstock and scion. 

The graft was then protected by inserting a white polythen bag over the graft to form a healing chamber with microclimate different from ambient. The primary purpose of healing chamber is to minimize transpiration and provides a microclimate around the graft point that prevents dehydration, contamination, and general favorable conditions for fast wound healing and effective graft take-up. The healing chamber was maintained and only eliminated once graft takes off was observed with outgrowth of buds by the scions.

Data collection and analysis

In this study, a graft union take off was considered successful if at least one scion bud break domancy followed by continuous increase in growth and development in terms of number of new leaves and height. On the other hand three criteria were used to score graft union failure; notably, (1) complete drying off of the scion, (2) scion remained fresh but without developing any bud 9 weeks after grafting and (3) scions that developed a bud(s) but later died within the study period or bud development was arrested. Parameters assessed were days to first bud outgrowth, number of developed buds over time, number of leaves, height of first bud and graft union failure level. Data collected were subjected to statistical analysis using analysis of Variance (ANOVA) to determine the significant effect on parameters measured. Data collected was analyzed using the statistical pack for social science (SPSS) version 22. Least Significant Difference at P<0.05 was carryout using Turkey’s HSD mean comparison test to detect the significant differences between the means of the various treatments.

Days to first bud outgrowth

Globally, variations were noticed as concerns the mean number of days various grafts associations took to break bud dormancy and initiate first bud development. The fastest bud outbreaks were recorded in homografts i.e. T1 (23 DAG) and T4 (25 DAG) where bud development started at 23 and 25 days respectively after grafting. Among the heterografts, T3 and T6 in which Hass was used as scion recorded the least speed for first bud outbreak requiring at least 38 days for the first bud to begin developing. Though T1 began bud outgrowth 5 days earlier than T3, the difference in number of days wasn’t significant given a p value of 0.07510231 (Figure 1), In this regards, homografts appeared to be highly compatible as they took a shorter time to initiate bud development.

Figure 1 Effect of rootstock vs scion source on time to first bud outgrowth.

*Same letters on histogram means no significant difference at p<0.05

*Bars represent mean and SD for time to first bud growth obtained from 10 repetitions of the graft union

T= (Rootstock + Scion): T1= Anaheim + Anaheim, T2= Anaheim + Taylor, T3= Anaheim +Hass, T4= Taylor +Taylor, T5= Taylor + Anaheim, T6= Taylor + Hass

Effect of rootstock vs scion origin on number of bud outgrowth on scion per week

Variations were observed in the number of bud outgrowth in the various treatments over time (Table 2). Bud development started at the 4^th WAG in T1, 2, 4 and T5 while in T3 and T6, bud development only commenced 21 days later. Throughout the period of observations, T1 scored highest mean number of buds compared to the other treatments. Significant differences were obtained between T1 and T2 as the obtained p value 0.043421 is <0.05. No significant differences were noticed between T1 and T4 where rootstock and scions were obtained from same variety for example the p value obtained when T1 and T4 were compared was 0.056152 at 7^th week. No significant differences were observed between T2 and T4. Significant differences were also obtained between T1 and T3.

Effect of rootstock + scion source interaction on number of leaves

The common factor in T1, T2 and T3 was the rootstock source specifically Anaheim. Statistically significant differences were obtained among these three with T1 scoring the highest cumulative number of leaves per week throughout the observed period. At each instance, the mean number of leaves in T1 was at least twice the number in T2 while T3 showed the least number of leaves. Statistically, P value of 0.000532067 was obtained at the 9^th week between T1 and T2, T1 and T3 gave 0.000002486. T1 and T4 also showed significant differences with a p value of 0.001972. Unlike in T1 where the number of leaves was on a relative constant increase of at least 4 new leaves per week, in T2 the increase as averagely 2 leaves per week (Table 3). As concerns T4, T5 and T6 where the common factor was Taylor as the rootstock, a similar trend was observed with T4 recording the highest mean cumulative number of leaves per scion of 4 per week against two and one leaf per week correspondingly for T5 and T6 respectively. Interaction T4 vs T5 gave a p value of 0.005874498 indicating there was no significant difference up to week 7. However, by week 8, the number of leaves in T4 increased significantly while in T5 the number was maintained. Significant differences were obtained between T4 and T6 as p obtained was 0.000017911 which is far < 0.05.

Table shows cumulative mean number of leaves produced by all developing buds on the scions of 10 graft repetitions for each treatment from the 4^th to 9 weeks after grafting.

Effect of rootstock scion source compatibility on height of first bud

Observations showed significant differences in mean height of first bud between treatments in which both rootstock and scion are from same variety (T1 and T4). Least first bud growth was obtained in T3 and T6 where variety Hass was used as the scion. Statistically significant differences were obtained in the grafting unions where variety Anaheim was the rootstock when the mean scores of T1 were compared with T2 and T3 at p < 0.05. Comparing T1 and T2 gave a p obtained value of 0.002553 while T1 and T3 gave 0.001703 which were all < 0.05. Comparing T1 and T4 also gave a p value of 0.001523.A similar trend was also obtained for T4, T5 and T6 where variety Tylor was used as the rootstock at p < 0.05 (Figure 2). For instance, p values obtained between T4 and T5 was 0.009836 while between T4 and T6, p obtained was 0.002475.

Figure 2 Effect of rootstock/scion compatibility on mean length of first bud 9 weeks later.

Footnote: *Bars represent mean and SD for number of branches/scion obtained from 10 repetitions of the graft union. Same letters on histogram means no significant difference at p<0.05

T= (Rootstock + Scion): T1= Anaheim + anaheim, T2= Anaheim + Taylor, T3= Anaheim +Hass, T4= Taylor +Taylor, T5= Taylor + anaheim, T6= Taylor + Hass

Rate of graft union failure

Homografts (T1 and T4) showed higher compatibility expressed by least graft unions failures of 1 to 2 % while heterografts showed poor companionability expressed via a wide range of graft failure. Among the heterografts, T2 and T5 showed faily poor compartibility with graft failures ranging from 9 to 13 %  rescpectively for T5 and T2 while T3 and T6  showed poorest compatibility recording alarming graft union failure rates as high as 59 % for T6 and  68 % in T3 (Figure 3).

Figure 3 Effect of rootstock/scion source in a graft union on mean number of branches.

*Bars represent mean and SD for graft union failure obtained from 10 repetitions

*Different letters on bars indicate significant difference at p<0.05

T= (Rootstock + Scion): T1= Anaheim + anaheim, T2= Anaheim + Taylor, T3= Anaheim +Hass, T4= Taylor +Taylor, T5= Taylor + anaheim, T6= Taylor + Hass

Generally, a compatible graft is one with high grafting success rate to be economically viable for nurseries as well as sufficient yield and longevity to be economically viable in the field.⁹ Based on the fact that there is no consensus among researchers on definition of graft compatibility/incompatibility within species,¹² in this study compatible and incompatible unions were expressed in terms of degrees i.e. highly or poorly compatible, rather than absolute given that some level of success was noticed in all the rootstock/scion combinations used and there was no case of absolute compatibility i.e 100% success and absolute incompatibility i.e. 0% noticed in this study. However, it was noticed that in treatments T1 and T4 where rootstock and scions were from the same cultivars showed superiority in all parameters evaluated hence were considered as highly compatible compared to experiments were rootstock and scions were from different cultivars. The latter is an indication that the level of compatibility increases with increase in taxonomic closeness. This result tie with the assertion of Mudge et al.⁴ who reported that taxonomic proximity is a prerequisite for graft compatibility. Autografts or homografts are taxonomically quite close and thus are expected to be always compatible. When grafting is performed within the same species it forms compatible combinations, if the grafting partners are from different species but the genus to which they belong is same, grafts are more or less compatible, intra familial grafts are rarely compatible, while inter familial grafts are essentially unsuccessful due to incompatibility. Taxonomic proximity between the grafting partners is essential for the successful re-establishment of both the rootstock and scion fused together at the level of the graft interface.¹³ Previous studies have well established evidence that presence of vascular cambium is a necessity for grafting thus anatomical deformities of vascular tissue at the graft union junction are one among major causes of incompatibility,⁷ leading to graft failure and or less compatible grafts that leads to failure during that later developmental stages of the plants in the field. The higher compatibility observed in union of rootstock and scion from same cultivar in this study, could be associated to smooth molecular dialogue given that arrangement of vascular cambium is maintained at the interface of the rootstock and the scion, hence enhancing higher compatibility expressed as high rate of successful graft unions. In order to achieve maximum success, grafting should be performed between or within the clones.¹⁴ However, the results of the present study revealed successful grafting even between cultivars though take off rate is delayed. The results corroborate with that of Fazio et al.¹⁵ who showed variations in flexural strength and flexibility at the graft interface between 7 rootstock and 6 scions from different apple cultivars. This corroborates the need to test rootstock and scion combinations form sustainable grafts.

The study revealed variations in graft union success among the avocado varieties used. The general trend is that, the more the rootstock is genetically closer to the scion, the higher the compatibility expressed via higher graft union success rate as well as a shorter time for graft take off. Averagely, homografts needed 23 days after grafting to takeoff against a mean of 34 days for heterografts.

Second revelation from this study is that all cultivars do not have the same potential to serve as suitable rootstocks and or scions in avocado grafting companionability. Among the three cultivars used in this study, Hass scion showed poor compatibility while Taylor and Anaheim appeared to be more compatible either as rootstock or scions.

In all, evidence has been shown in this study that the further the taxonomic proxy of the rootstock and the scion, the lower the compatibility, the higher the rate of graft failure and the slower the rate of graft take off.

To minimize cases of graft failures and improve on percentage success rate of graft union take off in avocado, rootstock and scion should be collected from same avocado cultivar.

None.

The authors declare that there is no conflicts of interest.

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