Genotype and Grafting Techniques Effects on Survival and Growth of Camu Camu Plants

Camu camu is a fruit native to the Amazon, that stands out for high content of vitamin C (3,133mg / 100 g of pulp). Is in process of domestication and therefore it is investigating a method of vegetative propagation to enable progress in the process of genetic improvement. The objective of this work was to determine the influence of genotype and grafting technique on the success and graft quality in camu camu plants. The trial was conducted using an experimental Randomized Complete Block design (RCB) with factorial scheme 9Ax3B, three blocks and 10 grafted plants per experimental unit. The factor A represented 9 clones of camu camu and factor B, three grafting techniques: Chip budding, b) Splice graft and c) Cleft graft. The experiment was conducted for 110 days to determine the sprouting time (beginning of the expansion of the first pair of leaves), the survival rate, sprout length and number of leaves. Significant interactions between the factors for the variables survival rate, sprout length and number of leaves were observed. The results show that the survival rate was influenced by the intrinsic effects of clone’s genotypic variability, presenting a high degree of dispersion, which was between 96.66% and 26.67%. With respect to grafting technique, the chip budding had higher response of survival rate, sprout length and number of leaves. Based on these results it is possible to conclude that the effect of genotypic variability and grafting method have influenced significantly on quantity and quality of camu camu grafting. The higher values of survival rate and vegetative vigour obtained allow recommending the use of chip budding technique for vegetative propagation of camu camu genotypes evaluated in this study.


Introduction
Twice winner of the Nobel Prize, Linus Pauling, revealed the existence of antioxidants and neutralizing action against destructive free radicals.These radicals are highly aggressive.Causing a body of degenerative diseases such as cancer, diabetes and arteriosclerosis (Pinedo et al., 2010).In this context, camu camu, a native fruit species of the Amazon, comes as a natural source of vitamin C, containing up to 3571 mg/100 gm of pulp, possessing antioxidants and minerals, considered a fruit with nutraceutical value.Currently, it represents an Amazonian agrobiodiversity resource with the greatest prospects in domestic and international markets (Almeida et al., 2014;Abanto et al., 2014).
Camu camu is normally propagated by seed leading to large variation in fruit production, low and late fruiting, diminished fruit quality and varying vitamin C content between one plant and another (Salazar et al., 2012).It also represents high genetic variability due to quantitative and qualitative hermaphrodite flower pollination.This creates a 91% alogamy and 9% autogamy, producing a wide phenotypic variability, which determines a high heterogeneity in yield (kg ha -1 ) and ascorbic acid content, which becomes an unsustainable production (Vasquez, 2000).Therefore it becomes necessary to use duly verified camu camu genotypes of high genetic quality (good fruit yield and vitamin C content) (Salazar et al., 2012).Therefore, asexual multiplication of selected clones may significantly increase the productivity of this species.This will allow the anticipation of the production period and ensure the maintenance of desirable characteristics of the parent plants to the progenies, ensuring the formation of homogeneous plantations to facilitate their management (Pinedo et al., 2010;Giraldo et al., 2009;Chagas et al., 2012).
The IIAP Pucallpa currently has 09 camu camu genotypes (clones) with high yield potential and good agronomic characteristics, which has been propagated through cuttings with sub-irrigation mini-greenhouse.However, previous works verified that the phenotypic variability influenced significantly the rooting process, affecting the propagation of some camu camu clones.In that sense another method of vegetative propagation is needed to allow the propagation for this genotypes.
The grafting methods with different techniques is presented as a viable alternative for propagation.Grafting is a vegetative technique of propagation which is used in timber and fruit species, primarily species with propagation difficulties.The grafting method consists of two parts placed in contact, joining tissues and subsequently originating in a new plant (Hartmann et al., 2002).This process is basically composed of two parts: a scion and a rootstock.This method increases fruit production, decreases plant habit, reduces the period of flowering and fruiting, and guarantees the maintenance of the characteristics of one elite material (Fachinello et al., 2005).The development of this method was a large breakthrough for the domestication and cultivation of plants (Enciso, 1993).
According to Suguino (2002), the grafting techniques that are most used in camu camu are cleft graft, chip budding and splice graft.The camu camu experts cite that grafting should be performed at 30 cm from the ground when the rootstock is between 6 and 9 mm in diameter and about 0.70 to 1.00 mm in length (Suguino, 2002).In Pucallpa's INIA experimental station, several years of research has resulted in camu camu grafting by the chip budding method.Alternatively, Salazar (2012) working with different grafting techniques and three genotypes of camu camu concluded that the MD-017 and MD-015 genotypes using cleft graft, obtained the best results with sprouting percentages of 90% and 87.50%, respectively, and concluded that this graft was an innovation to the camu camu propagation.This gives the plant bushy characteristics typical of simultaneous growth of several branches.In this context the present study was to determine the effect of genotype and grafting techniques in the survival rate and initial growth of camu camu plants under nursery conditions.

Methods
The study was conducted from April to August 2014, in the Research Institute of the Peruvian Amazon nursery, located at km 12.4 of Highway Federico Basadre Yarinacocha district province of Coronel Portillo Region Ucayali, located 8º22′31″ south latitude and 74º34′35″ west longitude and at an altitude of 154 meters (Figure 1).

UCAYALI
The climate is tropical, with warm temperatures throughout the year, classified as equatorial climate according to the Köppen system.The average annual temperature is 26 °C, with an average of 25 °C in July and 26.5 ºC September to January.The average maximum temperature is 33 °C and the minimum temperature is 21.5 ºC.Annual rainfall precipitation is approximately 1,570 mm with higher precipitation between October and April.In the study period the rainfall precipitation, relative humidity, minimum and maximum temperature were 452.9 mm, 85.5%; 22.7 ºC and 31.1 ºC respectively (IIAP, 2014).Plants used as rootstocks were obtained from seeds of mother plants from the Research Institute IIAP Ucayali, and were germinated in September 2013.Seedlings were planted in substrate composed of soil and chicken manure at a 1:1 ratio.After 8 months, when the plants reached an average basal diameter of 0.95 mm and a height of 1 m, the experiment was conducted with the three grafting technique: Chip budding, cleft and splices grafting.The cuts were made 30 cm from ground using plastic tape, parafilm, razor blades and pruning shears (Figure 2a).Nine genotypes were used as a graft, from camu camu collection of IIAP-Ucayali, noted for having a high content of vitamin C (1800 mg/100 g pulp) and high fruit yield (20 t/year).They are: ca39, CA242, ca253, CA48, ca294, CA51, CA81, ca306 (Figure 2b).For chip budding technique, a transverse cut was made in the rootstock.Then about 2 cm above the same direction a second cut down was performed until it connects with the first cut.The cut was made to remove the bud to be used for grafting, and was performed exactly the same as was performed in the rootstock (Rosati & Arana, 2003).Subsequently, the wrapping is performed with polyethylene tape, being removed after 25 to 30 days, the time required for the scion/rootstock tissue formation (Figure 3).For cleft grafting, rootstock and scion should be the same diameter.In the center of rootstock, a longitudinal cut with approximately 2 cm was performed.The scions are packaged or prepared with a blade.Then its two sides in the form of, trying to preserve the cambium, as it is the key of the graft.Subsequently subjecting them both sides are joined with polyethylene tape firmly, ensuring 100% match crusts.Finally the graph is protected with Parafilm tape to create a humid chamber protection and prevent ingress of water (Figure 4).To run the splice graft technique, a bevel cut was made of approximately 2 cm at a height of 30 cm from the ground in the rootstock.The same method was done in the scion, and then the rootstock and scion are wrapped with tape so they are securely attached.It is also necessary that the rootstock and the graft be about the same diameter.Finally the graft is protected with Parafilm tape in order to create a wet chamber and protection against outside humidity (Figure 5).The study was conducted using a complete randomized block design with factorial arrangement with three replicates 9A × 3B and 10 grafted plants per experimental unit.Factor A consisted of nine clones of camu camu and factor B by three techniques of grafts: Chip budding, b) Splice grafting and c) Cleft grafting.The experiment was assessed weekly determining the time of sprouting, after of 110 days was obtained the percentage of survival, sprout lenght (cm) and the number of leaves.Data were transfomed and analyzed using analysis of variance and treatment means were compared statistically by the Tukey test at 5% probability (P < 0.05) using the statistical analysis program SISVAR (Ferreira, 2010).

Results
In Figure 6, it is observed that the Splice grafting and Cleft grafting were the first to sprout in an average time of The summary of the analysis of variance (Table 1) shows interaction between genotype and graft techniques factors for the variables:% of survival, sprout lenght and number of leaves.The results show that camu camu presents variability and clones with marked influence on the process of survival rate and initial growth of plants by grafting techniques used.Note.**Significant at 1%; ns not significant by ANOVA.
Regarding the survival rate clones ca306 and ca84 obtained the best results with 96.66% each, when used chip budding.With the cleft grafting they obtained 76.66 and 86.67% respectively.On the other hand, when splice graft was used it had lower results.The same behavior was obtained when ca39 and ca51 clones were splice grafted, obtained 46.67, 26 and 67% respectively.However it is observed that, although there were no statistically significant differences in most genotypes, the chip budding had a superior performance (Table 2).Note.In each genotype (line), averages followed by at least one capital letter do not differ by Tukey test (p < 0.05).
In each technique of graft (column) averages with the at least one lower case letter do not differ by Tukey test (p < 0.05).
Also shown in Table 2, the survival rate was influenced by the genotypic variability of the clones, which was expressed at a high degree of dispersion ranged between 96.66 and 26.67%.This result is not suitable because, on a commercial level, the vegetative propagation below 70% is not considered suitable for any species (Leakey et al., 1990).
In relation to the shoot length, shown in Table 2, the genotypes with chip budding presented an increased growth and number of leaves.The clone ca294 reached 35.46 cm long and 30.87 leaves, showing a more vigorous vegetative growth in all variables.The splice grafting technique presented the lowest results with an overall average of 4.38 cm (Figure 6).

Discussion
The cleft and splice grafting techniques took less time to sprout, because the portion of plant tissue used as graft has increased stem length (5 cm), thereby retaining as much nutrient reserves and requiring less time for sprouting.Furthermore the chip budding has less nutritional reserves, because the region used only comprises a small, thin piece of plant tissue, causing more difficulty of sprouting and survival in a short time, therefore it needs a longer time of development (Simões & Carvalho, 2006).
For survival rate similar results (65% success) were obtained by Ferreira and Gentile (1997) working with chip budding on camu camu plants.Enciso (1993) found that chip budding was the best technique achieving greater success in camu camu.Similarly, Suguino (2002) working with camu camu grafting with different rootstocks of Myrtaceae, showed that the best method was the chip budding technique with 78.96% of survival, while the whip and tongue graft had an average of 25.83%, resulting in lower statistical percentage compared to the other treatments.Moreira Filho ( 2009) working with camu camu shrub (Myrciaria dubia) grafting on camu camu tree (Myrciaria floribunda), verified that the best survival rates were achieved with the same species, but found different behaviors in terms of success of grafting technique, with chip budding (89.3%, cleft graft 51.6% and 31.5% in splice graft, and very similar to the results found in this work. The observed differences between genotypes and graft success may be related to various aspects.The genetic factor is important, since different genotypes may respond differently regarding the ability of vegetative propagation, either by grafting or by another method, such as the cleft graft (Franzon et al., 2010).In that sense Salazar et al. (2012) working with different genotypes and different techniques of graft found that MD-017 and MD-015 genotypes with cleft graft.They achieved the highest percentage of sprouting with 90.0 and 87.5%; respectively, while the chip budding technique only obtained 55% of seizure.A very different results was found in this study, probably because it worked with different genetic material.Regard Rodríguez et al. (1995) stated that the formation of plant organs (shoots, leaves, flowers and fruits) is genetically controlled.
Similarly Rojas et al. (2004) express that genotype affects the concentration of the hormones responsible for growth, which is important when the vegetative propagation of any plant species is performed.Moreover this indicates that proper hormonal regulation favors the formation of shoots from the grafts.On the other hand Bezerra et al. (2002) mentioned that potentially lower rates observed in some clones are caused by compatibility issues between the tissues, once it is physiological and anatomical adjusted.The first is with respect to the region of the grafting, which can be selective and can hinder the transport of sap between the parts.Second would be related to the intimate association of the cambial tissue at the formation of the connection, which can be compromised when the scion and rootstock have different cell sizes, shape and consistency.
Moreover, Simão (1998) emphasizes that for successful union between two plants of the same variety or species, a perfect union of the cambial layers and bonded parts should be made smooth and free from contamination.The time must be appropriate for each species or technique of graft, executed in suitable climatic and soil conditions and be proficient in the handling of tools and appropriate cultural practices during and after welding.
Similar results related to sprout lenght and number of leaves were obtained by Leal (2001) to verify that the chip budding technique had the highest shoot growth in Neem (Azadiracha indica A. Juss).According to the results, the sprout lenght and the number of leaves are closely related because the new leaves synthesize gibberellins, which activate the production of cytokinins that are responsible for stimulating the meristematic activity and mobilization of nutrients, creating a good photosynthetic balance is favorable for shoot growth (Vallejo, 2001).
According to the results, chip budding is best suited for camu camu, because in addition to presenting the best rates of graft success and higher growth, this was the most practical to implement.The speed of execution provided less exposure tissues to the environment, reducing the oxidation reaction of the phenolic compounds.These compounds, when in contact with air, results in oxidation of tissues hindering callus formation and the healing process (Fachinello et al., 2005).

Conclusions
These work conditions concluded that genotypic variability and graft technique significantly influence the grafting process of camu camu plants.
The graft success and more vigorous vegetative growth obtained with the chip budding technique allow us to recommend the use of this technique for vegetative propagation of camu camu genotypes studied.

Table 1 .
Summary analysis of variance (ANOVA) the percentage of survival, sprout lenght (cm), number of leaves per shoot, by the effect of the clones and techniques of grafts, after 110 days of grafting process on camu camu plants/Yarinacocha-Ucayali, Peru

Table 2 .
Percentage of survival, sprout lenght (cm) and number of leaves by the effect of the genotypes of camu camu and different techniques of grafts/Yarinacocha-Ucayali, Peru