Avocado seed physiology aspects.

Abstract

The avocado seeds/seedling is needed as rootstock for other economic trees and loss of tress stand in orchids after establishment is of great commercial loss in avocado orchids around the worldwide and South Africa. The aim of this study was to evaluate and compare avocado seeds development of various seeds ages by investigating seeds germination percentage over three generations, as there is little information on avocado seeds growth and development, despite the importance of the seeds in avocado propagation. Seed harvesting was carried out over various developmental stages, from early fruit development to two-year-old seeds (Generation 1, 24 to 29 months after full bloom MAFB). Seed from current season (Generation 2, 12 to 17 MAFB) and newest seeds (Generation 3, 0 to 5 MAFB) of two cultivars (‘Hass’ and ‘Fuerte’) was analysed. Seed of three generations were analysed: ‘Hass’ Generation 1seed (seed from the oldest, commercially over-mature, fruit full bloom in July/ August 2017); Generation 2 (full bloom in July/ August 2018) and Generation 3 (full bloom in July/August 2019). Similarly, ‘Fuerte’ fruit of three generations were compared: from the avocado fruit, (Generation 1, full bloom in June/July 2017), to Generation 2 (full bloom in June/July 2018) to Generation 3 (full bloom in June/July 2019). Seed were extracted from fruit to determine seed parameters, such as germination percentage, seed viability, seed moisture content and seed respiration rate. Further, seed physiological parameters, such as cotyledonal sugars and starch concentrations, seed coat phenolic compound concentrations and polyphenol oxidase (PPO) concentrations were determined. Anatomical features of the seed coat, such as seed coat thickness and seed coat ultrastructure were also observed. In both cultivars, the germination percentage was higher in Generation 2, 12 to 18 MAFB), than in Generation 1, 24 to 29 MAFB) seed from June to September. From October to November Generation 3 (0 to 5 MAFB) had a higher germination percentage than Generation 2. Seed viability was higher in Generation 2 of both cultivars and lower for the Generation 1; similar results were found for the germination percentage, with seed from Generation 2 having a higher germination rate than seed from the Generation 1. Seed viability differed significantly between seed age, and the interaction between generations and months was statistically significant (P ˂ 0.001). The seed collected from fruit of the Generation 2 of both cultivars had a slightly higher moisture content and a higher germination percentage than the Generation 1. Seed moisture content ranged between 54.5 and 62.1 % in ‘Hass’ (Generation 2 seed age 12 to 15 MAFB), harvested in June to September, while the Generation 1 seed age 23 MAFB) seed had a lower moisture percentage (39.2%) in June. ‘Hass’ seed of (Generation 3 seed age 4

MAFB) harvested from October to November had a higher seed moisture than seed from (Generation 2,15 MAFB). ‘Fuerte’ seed showed a similar pattern with the highest moisture percentage (60.5%) in July and the lowest in June (33.2%). (Generation 2’ seed age 13MAFB seed had higher moisture percentages than Generation 1 from June to September, and (Generation 3, 3 MAFB) had higher moisture percentage than Generation 2. Seeds respiration rate, determined following fruit harvest, decreased over the time. Generally, Generation 2 respired more than the Generation 1, from June to September. From October to November 2019 the Generation 3 respired more than Generation 2 seed. The respiration rate of seed extracted from June to September 2018 Generation 2 declined rapidly. The Generation 3 (collected October to November 2019, seed age 4 to 5 MAFB) were characterized by a higher respiration rate than seed of Generation 2, seed age 16 to 17 MAFB; therefore, younger seeds generations respired more than older ones. It is concluded that the contribution of seeds respiration rate to avocado whole fruit respiration decreases with development over the time. The ability of the avocado seed to germinate quickly and produce seedlings is dependent on the carbohydrate reserves in the cotyledons, which make up the bulk of the avocado seed. In seed coats of both cultivars, phenolic concentrations inhibited seed germination of Generation 1, probably due to the higher level of phenolic concentrations in older seed coats. Seed coats generally contained high amounts of phenolics (2.3 mg GAE*g-1 DM for ‘Hass’ and 2.02 mg GAE* g-1 DM for ‘Fuerte’). Seed extracted from Generation 1 fruit in June to September 2018, had a higher amount of seed coat phenolics than those from Generation 2 fruit. In fruit from October to November 2019 the Generation 3 seed coat had lower phenolic concentrations than Generation 2 seed coats, confirming that older seed coats contain more phenolics than younger seed coats. Germination percentages of Generation 3 seed were higher than those of Generation 2 seed. The high phenolic concentration in the seed coats seems to be aligned with the seed turning dark brown upon maturation, probably due to sufficient oxygen present in the fruit to allow phenolic oxidation of the seed coat; the seed coat becoming entirely brown and very thin, could, therefore, be used as an indication that the fruit has reached physiological maturity. Seed at this stage of maturation are, however, characterized by a low germination percentage, possibly due to the seed coat phenolic compounds interfering with germination. This is supported by the positive correlation between lower seed coat phenolic compound concentration and higher seed germination rate for both cultivars (r = 0.11, P ˂ 0.61). Seed coat thickness of Generation 1 (24 to 28 MAFB) and Generation 2 (12 to 16 MAFB) ‘Hass’ seed coats differed, with

the younger seed generation displaying thicker seed coats than the older ones (0.51 versus 0.11 mm, respectively). In ‘Fuerte’, in June and July older seed coats Generation 1, 24 to 25 MAFB, respectively) were thicker than Generation 2 (12 to 13 MAFB) (0.46 and 0.15 mm, respectively. There was, however, negative relationship between seed coat thickness and germination percentage (r = -0.11). Polyphenol oxidase (PPO) and phenolic concentrations of avocado seed coats were also investigated in the seed coat of Generation 1and Generation 2 ‘Hass’ and ‘Fuerte’ seed. Polyphenol oxidase (PPO) and phenolic concentrations of the avocado seed coats of the two avocado cultivars of Generation 1, 27 to 26 MAFB) and Generation 2, 15 to 14 MAFB) respectively, seed coats were investigated. During the colder (winter) season (June-August), Generation 1, fully mature ‘Hass’ seed coats showed higher polyphenol oxidase (PPO) concentrations than seed coats from the Generation 2. From October to November the Generation 3 seed coat also had a lower PPO concentration than those of Generation 2. Generation 2 ‘Hass’ seed coats had relatively low PPO concentrations in June /July, when fruit were 12 to 14 MAFB, but PPO concentrations increased thereafter and remained at a higher-level until October/ November. Generation 1 ‘Fuerte’ seed coat had a similar PPO concentration during all investigated months. Phenolic compounds were present in seed coats of both avocado cultivars, with seed coats of older seeds containing a much higher phenolic concentrations than the seed coats of the newer generation. The seed (cotyledons plus embryo) sugar profile was dominated by the C7 sugar perseitol, followed by the C6 sugar, sucrose, while mannoheptulose and glucose were present in very small amounts. Perseitol was present in in both cultivars with 14 months-old ‘Hass’ (September) cotyledons containing 9.8 mg*g-1 DM and 15-months-old ‘Fuerte (September) containing 10.3 mg*g-1 DM. Avocado cotyledons were found to also be a large starch source, probably providing carbohydrates for seed development and germination. The Generation 2, 14 to 15 MAFB) of ‘Fuerte’ and ‘Hass’ had a higher starch concentration than the Generation 1, 26 to 27 MAFB) and similarly, Generation 3, 4 to 5 MAFB, respectively, had higher starch concentration than Generation 2 for both cultivars, indicating the use of this carbohydrate reserve to sustain embryo development. The highest concentration of starch in ‘Hass’ seeds was detected in August as 88.8% of seed DM (Generation 2, seed age 13 MAFB), while for ‘Fuerte’ seed the highest starch concentration was in August at 90.5% of seed DM (Generation 2, 14 MAFB). Starch seems, therefore, more related to avocado seed development than to avocado fruit growth and development. Delaying fruit harvest to October (seed age 16 to 18 MAFB) allows seed to fully mature and to continue accumulating sugars and starch. To improve

percentage and velocity of germination, seeds were soaked in various concentrations of aqueous moringa leaf extract (MLE, 0, 2.5, 5.0 and 7.5 % w/v) over different periods (0, 10, 30 or 120 minutes). Younger seed were stronger affected by the increasing MLE concentration. Soaking in 2.5% MLE tended to enhance the germination percentage more so than the other MLE concentrations. The lowest germination percentage was determined for seeds soaked in 7.5% MLE for 120 minutes, indicating that younger seed (from10 to 12 months after fruit set ‘Fuerte’ fruit harvested April to June) should be used as ‘nurse seed’. Overall, this study revealed that avocado seed germination and development do not coincide with the commercial fruit harvesting period, the avocado fruit needs 15 to 18 months to change from its flowering blooming period to a full harvest, and seed age12 MAFB can germinate for both cultivars. The study further confirmed perseitol as the dominant free storage sugar that assists in seed development, while starch is also an important energy provider for the developing embryo.