MATERIALS AND METHODS

Location of research site: The study was conducted at the Maseno University, nurseries in Maseno, Kenya. The nurseries are located at an altitude of 1515 m above sea level and at a longitude of 34 and 36° East and a latitude of 0°. The soils comprise a complex of somewhat excessively drained, shallow, stony and rocky soils of varying colour, consistence and texture (dystric regosols with ferralic cambisols, lithic phase and rock outcrops. The soils are acidic with high extractable Ca and K contents. Soil organic carbon and phosphorus content are 1.8% and 4 mg kg^-1, respectively. The pH of the soil ranges between 4.5 and 5.4 (Netondo, 1999). The soils have a water holding capacity of 40%. The area receives a fairly well distributed annual rainfall of 1853 mm. The studies were conducted in a plastic green house measuring 20x10 m in length and width and 30 m in height. The maximum and minimum temperatures in the structure were 26±4 and 35±5°C, respectively with a relative humidity of 60±5%.

Preparation of experimental materials and methods: Overripe avocado (Persea americana) var Fuerte fruits were collected from a market in Kisumu City, 20 km away on January 20th, 2003 and transported to Maseno University and stored overnight in a refrigerator at 5°C. To eliminate infection from avocado rot (Phytophthora cinamomi), the seeds were extracted on January 21st, 2003 and then immersed in hot water at 49 to 50°C for 30 min before planting.

The seeds were then sown in well prepared nursery beds dug to fine tilth to which was added Farm Yard manure and Diammonium Phosphate fertilizer (i.e., inorganic fertilizer). The bed was watered daily at 08:00 and 18:00 h using watering cans. After germination, the seedlings were left in the nursery beds for three months then transplanted into four different container sizes of (V₁) 1.7 L, (V₂) 2. 7 L, (V₃) 3.9 L, (V₄) 4.7 L according to the treatments. After transplanting, standard practices of weeding, irrigation, fertilization and pest and disease control were followed (Rice et al., 1987).

The experiment was set up in a Completely Randomized Design (CRD) comprising 4 treatments of container size namely, V1(1.7 L), V2 (2.7 L), V3 (3.9 L), V4 (4.7 L) and 3 irrigation frequencies: W₁ (irrigating everyday, W₂ (irrigating every 2 days) and W₃ (irrigating every 3 days). The treatments were replicated four times.

Measurements of parameters: This was carried out for a period of 9 months, after which the seedlings were ready for grafting. The studies were conducted between July 2003 and May 2004. Both morphological and physiological parameters were determined. The morphological parameters determined were number of leaves, plant height, stem diameter, shoot and root dry weights and whole plant dry weights. The physiological parameters determined were CO₂ assimilation rate, transpiration rate, stomatal conductance, substomatal CO₂ concentration and soil respiration.

Morphological parameters: Plant height was measured from the base of the stem to the shoot apex using a metre ruler every 4 weeks. All the fully expanded leaves, on each of the mango rootstock seedlings were counted and recorded every four weeks to determine the number of leaves. The diameter of each seedling was measured by a veneer caliper at a distance of 10 cm from the base of the stem every four weeks at a resolution of 1 to 100 cm.

Determination of the plant dry weight involved destructive measurements. The plants were carefully uprooted after loosening the soil and rinsed under tap water. Care was taken to ensure that all the root masses sticking to the soil were removed by soaking the roots in water and sieving out all the root segments. The plants were sorted out into shoots, roots and leaves, dried in an oven at 70°C for 48 h and then weighed. The weight was obtained by using an electronic weighing balance (Denver Instrument Model XL-3100). The measurements were carried out at the expiry of the experiment.

Physiological parameters: Soil respiration was determined on the plastic pots after removing the plants at a soil depth of 5 cm using a portable respiration system type SRS (PP Systems, Hitchin, UK) with no soil temperature probe. It reflected the degree of microbial activity.

Gas exchange measurements were taken on the most recent, fully expanded and well-exposed healthy leaves under bright sunlight. Leaves measured were in position 2 to 5, leaf position being the most recently emerged leaf. An open Infrared gas analyzer (IRGA) Porometer model (CIRAS) (PP systems, Stortfied, Hitchin, Herts, UK) was used. The stem was connected to a cuvette with a Parkinson leaf chamber whose area was 2 cm². The intact leaf lamina was sealed in the leaf chamber and all the major veins were avoided. The boundary layer resistance was 0.2 m^-2sec^-1 while the flow rate was maintained at 200 mL min^-1. The IRGA equipment determined simultaneously net CO₂ assimilation rate, substomatal CO₂ concentration, stomatal conductance and transpiration rate. The data was stored in the data logger in the equipment and analyzed statistically. Measurements were taken on attached leaves and three readings were taken from each leaf for all the four replications and two leaves were taken per plant. Data obtained was subjected to Analysis of variance (ANOVA) and means separation done by Least significant Differences (LSD) using the Statistical Analysis (SAS) Package.

RESULTS

Irrigation frequency significantly (p≤0.05) increased canopy heights of avocado seedlings. There were taller canopies at more frequent irrigation intervals (W₁) than less frequent ones (W₂) and (W₃) (Table 1). There were also taller canopies in larger containers (V₄, V₃) than smaller ones (V₂ and V₁). In contrast, stem diameter was only increased by irrigation frequency but not container size. The interaction between container size and irrigation frequency was significant for canopy height but not stem diameter. There were heavier shoot and root fresh weights at more frequent irrigation (W₁) intervals than less frequent ones (W₂, W₃). Similar trends were obtained with container sizes where larger container sizes (V₄, V₃) had lower fresh weights than smaller ones (V₂, V₁) (Table 2). The interaction between container size and irrigation frequency was significant (p≤0.05) and there were far heavier fresh weights under more frequent irrigation (W₁) and in larger containers (V₄, V₃) than smaller containers (V₂, V₁). Shoot dry weights had similar trends and both container size and irrigation frequency increased them (Table 3). Root dry weight and whole plant dry weight were similarly increased by container size and irrigation frequency. There were heavier root and whole plant dry weights as container size and irrigation frequency increased. The magnitude of these increases were more at higher irrigation frequencies and larger containers showing an interaction between container size and irrigation frequency (Table 3).

Plant height was only significantly (p≤0.05) increased by container size from the first second and fourth and seventh month after transplanting. In contrast irrigation frequency significantly (p≤0.05) increased plant heights at all sampling dates. Plant heights were taller in larger containers than smaller ones from the second to fourth month after transplanting. Similarly, more frequent irrigation intervals (W₁) had taller plants than less frequent ones (W₂ and W₃), at all the sampling dates (Table 4).

Table 1:	Effect of container size and irrigation frequency on the canopy height and stem diameter of avocado (P. americana) rootstock seedlings grown at Maseno, Kenya
Statistical parameter

Table 2:	Effect of different container sizes and irrigation frequency on the shoot fresh weight, root fresh weight and whole plant fresh weight of avocado (P. americana) rootstock seedlings grown at Maseno Kenya
Statistical parameter

Table 3:	Effect of different container sizes and irrigation frequency on the shoot dry weight, root dry weight and whole plant dry weight of avocado (P. americana) rootstock seedlings grown at Maseno, Kenya
Statistical parameter

Container size did not significantly (p≤0.05) affect the number of leaves from months 3 to month 6 after transplanting but increased it month 1 and month 2 after transplanting (Table 5) Conversely, irrigation frequency increased it at all sampling dates (Table 5).

The physiological parameters were generally significantly (p≤0.05) affected by container size and irrigation frequency. Container size significantly (p≤0.05) increased the CO₂ assimilation rate in months 2 and 3 after transplanting. Stomatal conductance was similarly increased by container size month 2 and 3 after transplanting (Table 6). In larger container sizes there were higher CO₂ assimilation rates and stomatal conductance and vice versa. However, transpiration rate was only increased in month 2 after transplanting. In contrast, substomatal CO₂ concentration was unaffected by container size and irrigation frequency. There were significantly (p≤0.05) higher values of stomatal conductance, net CO₂ assimilation rate and transpiration rates in larger containers than smaller ones during the months in which it affected them. In contrast, irrigation frequency increased CO₂ assimilation rate, stomatal conductance, transpiration rate at all the sampling dates.

Table 4:	Effect of different container sizes and irrigation frequency on plant height (cm) of avocado (P. americana) rootstock seedlings grown at Maseno, Kenya
Statistical parameter

Table 5:	Effect of different container sizes and irrigation frequency on the number of leaves of avocado (P. americana) rootstock seedlings grown at Maseno, Kenya
Statistical parameter

Table 6:	Effect of different container sizes and irrigation frequency on the physiological parameters of avocado (P. americana) rootstock seedlings grown at Maseno, Kenya
Statistical parameter

The interaction between the two factors was also significant for these gas exchange parameters at all the sampling dates (Table 6) and the values were far smaller in smaller containers and in less frequent irrigation (W₂, W₃) than larger ones under more frequent irrigation intervals. Soil respiration was significantly (p≤0.05) increased by container size and irrigation frequency and as the two factors increased soil respiration also increased and vice versa (Table 6).

DISCUSSION

Height of canopy, fresh and dry weights of shoot, root and whole plant were increased by container size. Plant height was not consistently increased by container size and this explains why canopy height was not significantly affected by container size in the present study i.e., the latter was not determined monthly while plant height was measured at the expiry of the experiment in previous studies These increases show that larger containers have a more favourable growth environment for the avocado rootstock seedlings caused by less root restriction which resulted in increased growth rates (Vizzotto et al., 1993; Peterson and Krizeki, 1992). Furthermore, larger containers apparently provided for more nutrient uptake, increased hormone synthesis and root metabolism (Peterson and Krizeki, 1992). Further studies are recommended to measure these parameters to confirm these statements. Under such favourable environments existing in large containers there was increased development of primary shoots and total length of the shoots producing taller plants (Alvarez and Caula, 1993). Although container size increased final canopy height it did not consistently increase plant height. Therefore in a follow up study canopy heights should also be determined monthly and not only at the end of the experiment because it is affected by plant heights. In an earlier study on citrus, (C. sinensis) rootstock seedlings (Ouma, 2005) container size increased both plant height and height of canopy but in that study the experimental conditions were very different. The number of leaves, of avocado seedlings in the present study was not also consistently increased by container size. This also differs with the results of my previous study on another plant species, (Citrus sinensis) (Ouma, 2005). But in that study the final leaf count was determined but not monthly leaf counts as in the present study.

Apart from plant height and number of leaves, the results of the present study agree with (Ouma, 2005; Vizzotto et al., 1993). However, container size did not affect the stem diameter in this study disagreeing with the findings of (Ouma, 2005; Vizzotto et al., 1993). This is may have been due to the different plant species and different experimental conditions. Irrigation frequency increased plant height, height of canopy, fresh and dry weights of shoots and roots, stem, diameter, number of leaves, fresh and dry weights of whole plants. This is in agreement with Ouma (2005) working on young citrus (C. sinensis) rootstock seedlings which are different from avocado and it is apparently due to the participation of water in the early growth processes of cell division and cell enlargement, metabolic activities and as a medium of nutrient uptake (Ouma, 2005; Luvaha, 2005). Water from frequent irrigation regimes seems to have had a more pronounced effect on the growth parameters in larger containers than small containers less frequent irrigations under small containers was apparently very stressful and seriously limiting to plant growth. Under larger containers more water added may have enhanced nutrient uptake and growth processes many-fold. In Kenya where the nursery industry is increasing in prominence and complexity this study is important because the issue of container size is often neglected resulting in disastrous consequences in reduced nursery productivity particularly with respect to the nursery production of avocado rootstocks. However, container size did not affect the stem diameter in this study and this disagrees with Ouma (2005) and Vizzotto et al. (1993). This may have been due to the different plant species which have different growth requirements, patterns and adaptabilities.

Irrigation frequency increased plant height, height of canopy, fresh and dry weights of shoots and roots, stem diameter, number of leaves and whole plant dry weights. This is in agreement with Ouma (2005) working on citrus (C. sinensis) rootstock seedlings and it is apparently due to participation of water in the early growth processes of cell division and cell enlargement, metabolic activities and as a medium of nutrient uptake, water from frequent irrigation seems to have had a more pronounced effect on the growth parameters in larger containers but less frequent irrigations under small containers was apparently very stressful.

Physiological parameters: Container size neither affected transpiration rate nor substomatal CO₂ concentration. Further, it only significantly affected stomatal conductance and CO₂ assimilation rates during months 2 and 3 after transplanting and transpiration rate during month 2 after transplanting. The effect on CO₂ assimilation rate for the two months is apparently due to its effect on stomatal conductance over the same period. The small effect on stomatal conductance may be attributed to the fact that the stomatal conductance is strongly affected by growth conditions and changes with leaf age characteristically maximum stomatal conductance does not attain a peak value until several days after leaf emergence (Jones, 1992). The plants in the present study were still too young to have noticeable leaf conductance hence, also the small effect on CO₂ assimilation rates.

Another important factor which may have a profound effect on CO₂ assimilation rate in plants is the substomatal CO₂ concentration which, in the present study was not affected by container size. Therefore, the increases in CO₂ assimilation rate during the two months, without a corresponding decline in substomatal CO₂ concentration could be due to non-stomatal effects on the photosynthetic processes, possibly an increase in the mesophyll resistance (Cornic et al., 1989). A reduction in substomatal CO₂ concentration can be detrimental to the photosynthetic process especially in the presence of the rubisco enzyme. For many species substomatal CO₂ concentration tends to remain constant over a range of environmental conditions (Pearcy, 1981). This may explain the lack of effect of container size on substomatal CO₂ concentration in the present study. Other workers have also reported increase of CO₂ assimilation rates, transpiration rates and stomatal conductance from increased irrigation frequency (Luvaha, 2005).

Soil respiration was significantly (p≤0.05) increased by container volume and as the volume increased there was a consistent increase of soil respiration and this was apparently less enhanced in small containers and less frequent irrigations showing a significant container size x irrigation frequency interaction. The conditions in the larger containers such as increased soil volume, nutrient uptake, hormone synthesis as discussed else where in this study all appeared to increase soil microbial activities thus increasing soil respiration. These conditions were more enhanced under frequent irrigations. The increase of soil respiration under more frequent irrigation can be attributed to that fact that water enhances nutrient uptake and metabolic activities such as protein or enzyme synthesis which enhance microbial activities hence soil respiration.

CONCLUSION

It can be concluded that Container size significantly increases plant growth when it is increased from 1.7 to 3.9 L through its effect on morphological parameters. Irrigation frequency of (W₁, irrigating everyday) increases morphological and physiological parameters more than irrigating less frequently (W₂ and W₃). Canopy height, stem diameter, transpiration rate and substomatal CO₂ concentration are not significantly affected by container size and irrigation frequency. Both container size and irrigation frequency affect plant growth through their effects on morphological and physiological parameters.