Performance of different Annona species on the grafting success and growth of Soursop (Annona smuricata L.)

Introduction

Soursop (Annona muricata L.) is a tropical fruit tree extensively cultivated in tropical regions due to its aromatic fruits, health benefits, and emerging use in medicine. Despite the significance of soursop, large-scale cultivation is typically challenging because of its susceptibility to soil-borne diseases, inconsistent yields, and sensitivity to environmental fluctuations. Soursop (Annona muricata L.) is commonly propagated from seeds. Propagation by seeds will produce variation among plants, leading to inconsistent orchard performance. To overcome this problem, propagation through vegetative must be done i.e., by grafting. Vegetative propagation by grafting requires the availability of rootstocks and scions [1] . Annonaceous fruits are often propagated via seeds, resulting in significant variety in growth, productivity, and fruit quality among the trees in the orchard. Due to the high variability and heterozygosity among the seedlings, asexual techniques are the easy ways to overcome the problems of seed propagation. The research work on the development of superior-performing as well as known varieties are meager. However, quite often we do stumble upon trees exhibiting prolific yield with superior quality. If such promising trees are further perpetuated through vegetative means, the desirable types can be multiplied and establish the orchard with uniform fruit quality [2] . Among different methods of propagating Annona vegetatively, layering and cuttings are not typically employed for commercial multiplication; nonetheless, budding and grafting are considered advantageous. Rootstocks significantly contribute to the propagation of plants. The compatibility of rootstock and scion is crucial for budding and grafting. Furthermore, the cultivation environment significantly influences the establishment of favorable conditions for optimal growth and development of propagated plants [3] . Annona has shown a higher percentage of success when propagated by grafting in comparison to other methods [10] . Availability of high-quality planting material would augment the cultivation of Annona muricata. This can be achieved by selecting suitable rootstock species to increase the efficiency of its propagation through grafting. Differential anatomical compatibility, cambial alignment, sap flow, and physiological responses post-grafting significantly influence graft survival and growth rate. Identifying the optimal rootstock that synergizes effectively with various plants and exhibits rapid growth can mitigate common challenges in horticulture, such as poor seed germination, uneven root development, and premature seedling mortality [4,5] . This study seeks to evaluate the influence of particular Annona rootstock species on the grafting compatibility and initial growth performance of soursop. The study aims to identify rootstocks that enhance the survival, vigor, and long-term performance of soursop plants by assessing graft union success, scion establishment, and vegetative development. Results will provide valuable insights for farmers, nursery managers, and researchers.

Materials and Methods

The experiment was conducted in the Fruit Genetic Diversity Block of the College of Horticulture, Department of Fruit Science in Bengaluru, Karnataka. The location is situated at 12° 58' N latitude and 77° 35' E longitude, with an elevation of 930 meters above sea level. The region possesses a temperate tropical climate conducive to fruit cultivation.

Rootstock prepration and nursery management

Rootstocks of six Annona species A. muricata, A. squamosa, A. glabra, A. reticulata, A. montana, and A. mucosa were raised in 200-gauge black polybags measuring 8" × 6". Rootstock of six-month-old seedlings that were healthy, rapidly growing, and uniform in height and girth. During growth of the rootstock, standard nursery practices such as irrigation, weed management, and pest control were employed.

Scion selection and curing

Scion shoots were obtained from healthy, disease-free, and pest-free mature soursop (Annona muricata) trees, which were situated in the Fruit Genetic Diversity Park at the College of Horticulture in Bengaluru. The scions were precured to enhance the likelihood of successful grafting, selected mature branches from the current season. The precured scions were collected from 8:00 to 9:00 AM on the grafting day. Upon severance from the mother plant, the scions were enveloped in damp fabric to prevent desiccation and used it for grafting.

Experimental set up and treatment

The experiment employed a Completely Randomized Design (CRD) with six treatments representing different Annona rootstock species. Four replicas of each treatment were produced, and 10 grafts were implemented for each treatment to ensure an adequate sample size and statistically reliable results. Softwood grafting was employed to disseminate all treatments, ensuring uniformity in the grafting procedure across all experimental units.

Data Collection and Observation

Observations about the growth and development of the graft were recorded at 30-, 60-, and 90-days post-grafting (DAG). Root characteristics were assessed destructively at 90 days after grafting (DAG). Three grafts were randomly selected for each replication, and the identical plants were employed across all observation periods to maintain consistency. The duration from grafting to the emergence of the initial sprout on the scion was documented. At 30 days after grafting (DAG), the success of the graft was assessed by calculating the proportion of grafts that had developed, utilizing the formula: At 30, 60, and 90 days after grafting (DAG), we enumerated the leaves on each graft and measured the height from the graft union to the terminal shoot. A digital Vernier caliper was employed to measure the graft girth at the graft union. The girth measurements were subsequently computed utilizing the formula:

Graft success (%) = Number of grafts sprouted × 100 Total number of grafts prepared Every 30 days, the quantity of sprouts per graft was documented, and a digital Vernier caliper was employed to measure the diameter at the graft union. At 90 days after grafting, the grafts were carefully extracted to assess the root parameters. The taproot length was assessed from the collar region to the root apex, and the quantity of primary roots emerging from the main taproot was enumerated. Weigh the freshly uprooted roots using an electronic balance, and subsequently weigh the dried roots after subjecting them to an oven at 70°C for 48 hours. The equation for determining graft survival at 90 days after grafting (DAG) was: The DMSO extraction method to quantify the chlorophyll content in the sample. Measured absorbance at 645 nm and 663 nm. The subsequent equations was employed to get the chlorophyll fractions:

Chlorophyll "a" = {12.7 (A 663) -2.69 (A 645)} × V/(1000 × W×a)

Chlorophyll "b" = {22.9 (A 645) -4.68 (A 663)} × V/(1000 × W×a)

Total chlorophyll = Chlorophyll "a" + Chlorophyll "b"

Where, A= Absorbance at specific wavelengths (OD values) V= Volume of chlorophyll extracted (ml) W= Fresh weight of the sample (g) a= Path of the light (1cm)

The total chlorophyll content was obtained by summing chlorophyll a and chlorophyll b. These metrics provided extensive insights into the physiological and morphological responses of soursop grafts to several Annona rootstocks.

Statistical Analysis

The obtained data was analyzed using Analysis of Variance (ANOVA) with the Web Agri Statistical Package (WASP 2) developed by the ICAR Research Complex, Goa. Prior to analysis, percentage statistics such as graft success and survivorship were modified by arcsine square-root transformation. A significance level of 1% (p = 0.01) was employed for testing. Critical Difference (CD) values were computed wherever the F-test indicated significance, following the statistical procedures of [6] . 1). These findings align with previous reports indicating that A. montana can be a suitable rootstock for soursop grafting. Grafting of A. muricata scions onto A. montana rootstocks yielded graft-success rates comparable to use of A. muricata rootstocks [7] . The superior performance of A. montana in the present study suggests better anatomical or physiological compatibility with the scion than some of the other rootstocks tested.

At 90 DAG, survivability also varied markedly across treatments (S.Em ±4.60, CD₀.₀₅ =13.67). T₅ again led with 67.50% survivors, followed by T₄ with 55.00%. T₁ achieved 37.50%, much lower than the top two. Importantly, T₂ (A. squamosa) and T₃ (A. glabra) recorded zero survival, and T₆ achieved only 12.50% survival (Table 1). This pattern suggests that although initial graft union may form, long-term compatibility and physiological continuity vary strongly by rootstock. The complete failure of A. squamosa and A. glabra as rootstocks in this experiment indicates strong incompatibility under these conditions. Literature on Annona grafting supports the notion that rootstock selection is critical: while A. glabra is tolerant to flooding and used for rootstock under specific conditions, compatibility issues have been reported [8] . The marked difference in survivability between A. montana and the poorest-performing rootstocks underscores the importance of assessing both short-term graft take and longer-term survival. Graft mortality presented an inverse trend to survivability. T₂ and T₃ had 100% mortality, T₆ had 87.50%, T₁ had 62.50%, T₄ had 45.00%, and T₅ had the lowest mortality at 32.50% (Table 1). The fact that A. montana (T₅) showed both the highest success and lowest mortality suggests superior compatibility and vigor of the resulting graft union.

Number of Days to First Sprouting

Variations in the number of days to first sprouting among treatments were significant (Table 2). T₅ sprouted earliest at 12.33 days, followed by T₄ at 15.75 days, and T₁ at 18.00 days. Treatments T₂ and T₃ took much longer (23.92 and 24.83 days respectively). Early sprouting may indicate good physiological connectivity and vitality of the graft union; therefore, the superior performance of A. montana and A. reticulata affirm their suitability as rootstocks.

Vegetative Growth, Root Traits and Physiological Parameters

The number of sprouts per graft, number of leaves, graft height and graft girth, diameter at graft union, and root traits (tap-root length, primary root number, fresh & dry root weights) all followed patterns consistent with the earlier results (Table 3; Fig 3): rootstocks T₅ and T₄ generally outperformed the rest. For example, at 90 DAG, T₄ had the highest number of sprouts (11.58) followed by T₅ (7.41) and T₁ (7.25), while T₂ and T₃ produced none. Leaf numbers at 90 DAG were highest in T₅ (18.66 leaves) followed by T₄ (16.91) and T₁ (14.66). In root parameters, T₁, T₄ and T₅ produced measurable tap-root lengths (23-24 cm) whereas T₂ and T₃ recorded none. Specific Leaf Weight (SLW, Fig 4) and chlorophyll content further supported these growth differences. T₄ and T₅ registered the highest SLW (0.03 g/cm²), and for chlorophyll, T₅ recorded the highest total chlorophyll (3.42 mg/g FW), followed by T₄ (2.50) and T₁ (2.38). Treatments T₂ and T₃ recorded essentially zero values (Fig 5). These results reaffirm that rootstock choice not only influences graft take and survival, but has downstream effects on vegetative development, root system establishment, leaf physiological properties and likely the future fruiting capacity. Previous studies have noted similar trends: in sugar-apple (Annonaceae) grafted combinations, root systems with better development translated to higher leaf chlorophyll content and improved growth [9] . T1: Annona muricata, T2: Annona squamosa, T3: Annona glabra, T4: Annona reticulata, T5: Annona montana, T6: Annona mucosa The overall pattern from these results clearly highlights that among the six Annona species, Annona montana (T₅) and Annona reticulata (T₄) exhibited superior compatibility with soursop scion in the experimental conditions. They both delivered high graft success, higher survival, early sprouting, vigorous vegetative growth and strong root development. In contrast, A. squamosa and A. glabra performed poorly, indicating likely graft incompatibility or sub-optimal physiological interactions. The compatibility between scion and rootstock is a critical determinant of graft success and subsequent growth. Successful grafting depends on anatomical alignment, vascular connectivity, and physiological integration including nutrient and water translocation [14,10] . The findings here suggest that A. montana and A. reticulata rootstocks may facilitate more effective graft-union integration for soursop scions. However, the complete failure of A. squamosa and A. glabra under these conditions raises questions about their viability as rootstocks for soursop under similar nursery regimes. Previous literature indicates that although A. glabra can perform as a rootstock under certain stress conditions (e.g., flooding tolerance), it may be incompatible with some scions [11][12][13] . Incompatibility may manifest as poor graft union formation, weak vascular connectivity, or physiological stress leading to death over time. The physiological measurements (SLW and chlorophyll content) further reflect the nutritional and functional status of the grafted plants. Higher chlorophyll content in grafts on A. montana and A. reticulata indicates better leaf health and photosynthetic potential, likely translating into improved growth and future yield.

Conclusion

This study showed that the choice of rootstock significantly influences the grafting success and growth rate of soursop (Annona muricata L.). A. montana and A. reticulata were the most suitable matches among the six species examined. They exhibited the highest rates of graft success and survivorship, accelerated sprouting, enhanced vegetative growth, superior root system development, and increased chlorophyll content. The results indicate that the soursop scion and the other plants exhibited effective physiological compatibility. A. squamosa and A. glabra exhibited complete graft failure, whereas A. mucosa displayed very restricted compatibility. A. montana and A. reticulata are the optimal rootstocks for enhancing the growth of soursop.

Acknowledgement

AcknowledgementWe acknowledge the support of the Department of Fruit Science, College of Horticulture, Bengaluru and University of Horticultural Sciences, Bagalkot, Karnataka, India for providing us with the field and laboratory facilities to accomplish the research. This work was carried out in collaboration of all authors. Author Kavana S C conducted the study, collected the data, arranged and analyzed them and wrote the original draft. Authors Manu Kumar H. R., G S K Swamy, Babu A. G., and Shivakumar K. M. edited and revised the manuscript. All authors read and approved the final manuscript.

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