In Vitro Seed Germination and Seedling Development of Two Avocado Varieties

Abstract

Avocado (Persea americana Mill.) is a tree native to central and eastern Mexico. A basal angiosperm of the Lauraceae family, it produces an oil-rich fruit that is appreciated worldwide for its nutritional value. Mexico is the world’s leading avocado producer. Production is based mainly on the use of rootstocks of Persea americana var. drymifolia, a “Mexican native”. The agronomic characteristics of the rootstock are key to avocado production. This work reports a germination method to obtain seedlings in vitro from two avocado varieties, P. americana var. drymifolia and West Indian P. americana var. americana. With this system, germination success rates of 100% were obtained in a maximum of five days, with homogeneous seedling development. This system could provide rootstock that improves the characteristics of agronomic programs and the selection of genetic material for avocado production.

1. Introduction

The formation, dispersal, and germination of seeds are very important events in the life-cycle of gymnosperm and angiosperm plants [1]. As a member of the basal Angiosperms, the avocado tree, of the Lauraceae family, is considered by numerous botanists to be one of the most primitive dicotyledonous plants [2]. They are species of economic importance that are cultivated in tropical and subtropical areas worldwide [3]. Three botanical varieties (races) have been reported, known as Mexican, Guatemalan, and West Indian [4, 5, 6].

Latin America is the area with the highest production in the world, with Mexico being the largest producer [7]. The “Hass” cultivar has the highest consumer acceptance, production, and commercialization indices. This cultivar results from the hybridization of the Mexican and Guatemalan varieties [8], but cultivation requires rootstock from one of the botanical varieties, usually obtained from seeds [9, 10].

However, seedlings grown in nursery systems may require 40–60 days to germinate after sowing [11] and may lack uniformity due to the high genetic variation characteristic of seeds. Thus, various authors have reported the importance of shortening germination times and favoring this process using in vitro systems. Previous studies have reported 94−99% success rates in the germination of whole seeds and embryos barely covered by 1.5 cm² of cotyledons in in vitro systems, with germination emerging at 3−10 days [12]. Various culture media compositions have also been evaluated with Persea americana var. drymifolia and Persea americana cv. Hass, reported average germination emergences of 20−27 days [13]. The advantages of in vitro germination may provide a solution to cases of the total inhibition of germination, foster increased germination rates, reduce the time required, and improve the homogenization of seedlings that can serve as rootstocks [14]. In this work, we report data on the germination and development of in vitro seedlings obtained from P. americana var. drymifolia and Persea americana var. americana.

2. Materials and methods

2.1 Biological material

Avocado seeds were obtained from two varieties of fruit. P. americana var. drymifolia were obtained from crops in Tingambato and local markets in Morelia, Michoacán, Mexico. P. americana var. americana seeds were obtained from local markets in Merida, Yucatán, Mexico. All fruits were in the ripe stage (Figure 1).

Figure 1.

Characteristics of the avocado fruits in the mature consumption stage used in this work, Persea americana var. americana (A-C) and Persea americana var. drymifolia (D-F). The fruits were washed and separated into pericarp (A, D), mesocarp (B, E), and seeds (C, F). Scale line equivalent to 1 cm.

2.2 Seed disinfection treatment

The surface of the avocados was washed with soap and water, then the fruit was cut to obtain the seeds, which were rinsed with sterile purified water to eliminate remaining parts of the mesocarp. For P. americana var. drymifolia, the clean seeds were placed in 70% alcohol for 15 minutes and then planted in a laminar flow hood with superficial flaming. After that, part of the cotyledons was removed leaving a maximum of 1.5 cm² flanking the embryo. For P. americana var. americana, the rinsed seeds were immersed in a 10% sodium hypochlorite solution for 10 min, then placed in the 70% alcohol solution for later sowing in a laminar flow hood under the conditions just described.

2.3 Culture medium

MS culture medium was used [15], adding 30 g/L of sucrose (Bioxon: Cat. No. 217000), cytokinin (BAP) at 0.05 mg/L (Sigma: Cat. No. B3408), and 15 g/L of bacteriological agar (Bioxon: Cat. No. 215000). pH was adjusted to 5.7–5.8, and the medium was sterilized in an autoclave at 120°C at 20 kgf/cm² for 15 minutes.

2.4 In vitro germination conditions

Sowing was carried out in 105 × 55 mm glass flasks with 25 ml of the medium, in a laminar flow hood with 10 individuals of P. americana var. drymifolia and 10 of P. americana var. americana. Once sown, the seeds were maintained in a culture chamber at a temperature of 25 ± 1°C with a photoperiod of 16 light hours at an intensity of 80 E·m-2·s-1 against 8 hours of darkness.

2.5 Determination of physio-morphological parameters

The seeds were evaluated at 0, 1, 3, 5, 10, 15, 30, and 60 days, using the first evaluations (0, 1, 3, 5, 10, and 15 days) to determine the days of rupture and emergence of the seedlings, while 30 and 60 days were selected to determine four key parameters of the two varieties: percentage of germination, contamination, oxidation, and death or necrosis. Growth parameters such as seedling size, root length, stem and root diameter, and dry weight.

2.6 Statistical analysis

All parametric tests were carried out using the Statistical Package for Social Sciences (IBM SPSS V.20). Mean differences were determined by a Tukey test with a significance value of p < 0.05 in 10 independent measurements.

3. Results

3.1 Characteristics of the fruits of P. americana var. drymifolia and P. americana var. americana

The fruits were weighed, measured, and separated into their tissues (pericarp, mesocarp, seeds) to determine proportions. The results for P. americana var. drymifolia were weights of 83.3−154.5 g with averages for the pericarp of 7.37 ± 1.43 g (7%), mesocarp of 73.09 ± 17.28 g (65%), and seeds of 31.32 ± 7.67 g (28%) (Figure 2). Seed size was 4.87 ± 0.49 cm. For P. americana var. americana, the fruits had weights of 332.4−481.5 g, with averages for the pericarp of 32.51 ± 6.16 g (8%), mesocarp of 316.46 ± 38.27 g (79%), and seeds of 50.06 ± 12.48 g (13%) (Figure 2). Seed size was 7.63 ± 0.15 cm.

Figure 2.

Proportion of the avocado fruit tissues used in this study: A) Persea americana var. drymifolia; B) Persea americana var. americana.

3.2 In vitro establishment

The medium and conditions used to ensure asepsis of the seeds in this study allowed the germination of all the seeds sown. For P. americana var. drymifolia, the surface cleaning with 70% alcohol and superficial flaming were sufficient to lower the percentages of contamination, so that on the initial days of observation. and at the maximum time, homogeneity in seedling size and development of the leaves, stems, and roots was observed (Figure 3). For P. americana var. americana, high contamination percentages occurred due to the presence of endogenous bacteria, so a first cleaning step was carried out with 10% sodium hypochlorite to reduce contamination. Observations showed that homogeneity was achieved in the size and development of the seedlings (Figure 3).

Figure 3.

In vitro establishment of seeds of two avocado varieties, Persea americana var. drymifolia (A-E) and Persea americana var. americana (F-J). Embryos covered with 1.5 cm² of the cotyledon were planted in MS media (A, F). At 3 ± 1 days post-planting, the radicle emerged from the embryo (C, H). At 5 days, the emergence of the seedling is visible (B, G). At 10 and 30 days, development of the seedling and root is evident (D, E, I, J).

3.3 Germination, oxidation, and contamination

The seeds of both varieties showed germination and seed breakage 3 ± 1 days after sowing. Determination of germination was made from the appearance of the radicle [16]. Emergence of the seedling was generally seen at five days, so the observations at 10, 15, and 30 days only served as controls for development and the possibility of loss due to contamination under the culture conditions used. The elimination of part of the cotyledon allowed an adequate exchange of water and nutrients in the germinative phase. The levels of this tissue allowed low oxidation and/or loss of the embryo that favored the development of the seedlings. The study obtained 100% germination and survival for both varieties, null oxidation levels, and only 5% contamination in the drymifolia variety (Table 1).

Avocado variety	GD	G	O	C	S
drymifolia	3 ± 1	100%	100%	5%	100%
americana	3 ± 1	100%	100%	0%	100%

Table 1.

Percentages of germination, oxidation, contamination, and seedling survival for seeds of two varieties of avocado, Persea americana var. drymifolia (Mexican native avocado) and Persea americana var. americana (west Indian avocado).

GD.- Germination Day; G.- Germination; O.- Oxidation; C.- Contamination; S =.

3.4 Survival and development of the avocado seedlings

The seedlings obtained from the in vitro germination process using the two varieties presented a homogeneity in size and development of their organs 60 days after sowing. Variation in the length of the aerial part and roots was not statistically significant between or within the varieties. Similarities in their dry weight were observed and quantified. However, statistically-significant differences were observed in the stem and root diameters, as these were slightly higher in the americana variety (Table 2). Regarding the proportion of the developed organs, observations showed that the root data presented the greatest inter- and intra-variety variation (Figure 4).

Avocado variety	SS (cm)	RL (cm)	SD (cm)	RD (cm)	DW (g)
drymifolia	56.94 ± 9.28	37.41 ± 8.9	0.29 ± 0.08*	0.23 ± 0.1*	0.52 ± 0.14
americana	54.28 ± 9.2	39.21 ± 7.67	0.31 ± 0.06*	0.25 ± 0.2*	0.54 ± 0.12

Table 2.

Statistical significance in the comparison of the means of the growth variables for seedlings between the varieties Persea americana var. drymifolia (Mexican native avocado) and Persea americana var. americana (west Indian avocado) after 60 days.

*

: statistically-significant difference; SS.- Seedling size; RL.- Root length; SD.- Stem diameter; RD.- Root diameter and DW = Dry weight.

Figure 4.

Development of avocado seedlings germinated from seed in an in vitro system using two varieties, Persea americana var. drymifolia (A), and Persea americana var. americana (B) 60 days after sowing. The seedlings show full development of their leaf, stem, and root tissues (B, D).

4. Discussion

Seeds transfer an organism’s genetic information from one generation to another, thus allowing variability to develop in the species. Seeds in unfavorable conditions can enter dormancy, a survival mechanism in the presence of certain climatic conditions, such as very low temperatures, alternating dry and wet seasons, or desert climates [17]. On average, germination takes 20–75 days [18]. Elements such as the quality of light, or its absence, are other determining factors in the success of germination [19, 20]. The emergence of seedlings and their establishment are the key stages of development after germination and preconditions for successful establishment at the final site [21]. The controlled conditions of in vitro systems allow the germination and propagation of many plants in shorter periods [22]. In the case of avocado production systems, the commercial and nutritional importance [23] of the fruit and the need to obtain rootstocks suitable for the establishment of important cultivars –such as “Hass”– demand greater propagation of materials with desirable traits in a shorter time but with homogeneous development [24, 25, 26].

Various studies of avocado have sought to achieve the massive propagation of materials with outstanding characteristics, but numerous factors can affect success. Interaction with the environment, the type of plant material, and the developmental stage [27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39] are factors that can cause low spread rates. During traditional propagation, avocadoes may be exposed to attacks that can reduce germination, so a controlled germination system that optimizes plant development would seem to be ideal for producing and selecting plants of agronomic interest as future rootstocks. For avocado production, in vitro germination of the P. americana var. drymifolia and P. americana var. americana varieties are necessary for the medium- and long-term selection of plants with some degree of tolerance for biotic or abiotic stress.

The natural differences in the size and weight of avocado fruits have led producers to search for greater homogeneity in order to satisfy different market needs. The classification systems and taxonomy of the varieties of this fruit have multiple parameters used to identify the members of a specific botanical variety. For P. americana var. drymifolia, fruit weight averages 118−134 g in the ripe stage. Proportions of 82−94 g of the weight correspond to the pericarp-mesocarp, while 35−38 g correspond to the seed [40, 41, 42]. These data do not differ greatly from the figures obtained in this study, considering that the latter fall between the maximum and minimum values reported. For P. americana var. americana, fruit weights of 250−312 g have been reported, with pericarp-mesocarp proportions of 150−280 g and seed weight of 35-60 g [40].

Microbial contamination and darkening of tissues in in vitro systems, however, are two of the greatest challenges for avocado fruit [39]. Therefore, aseptic processes are a determining factor for the establishment and germination of seeds. Previous studies have reported protocols with up to 3% contamination in whole seeds and 5% oxidation in seeds from which part of the cotyledon was removed. As an oxidation inhibitor, researchers have added antioxidant compounds like thiosulfate and silver nitrate to the cultivation media [12]. The main causes of contamination in earlier studies involved bacteria contained in, or associated with, embryos. Under those conditions, low percentages of established embryos (up to just 60%) were achieved [33]. In the present study, in contrast, the percentages of success in the establishment of embryos and the elimination of contaminants (maximum 5% for P. americana var. drymifolia) are clearly superior to those previously reported (Table 1).

The percentages of in vitro germination in the avocado seedlings observed in this work (3 ± 1 day) also contrast with previous reports, which found variations in the composition and concentration of the components of the culture media, with germination times of 10−35 days [33, 40, 43], and 94−99% germination of the seeds, both whole and those that presented only partial sections of the cotyledon of up to 1.5 cm² covering the embryo [44] (Table 1).

Seedling emergence was observed with no differences days after sowing. Once again, this result contrasts with previous reports where emergence occurred after 15 days [12] The seedlings evaluated at 60 days of development showed a marked difference in size, as well, when compared to seedlings obtained from germinating embryonic axes [12]. Although there are significant differences in the stem and root diameters of the two avocado varieties, the production of cell mass is not reflected in the measurements of dry weight, suggesting that the increase in this diameter was due to the absorption of water from the culture media and/or to natural variations in the two varieties (Table 2).

The findings of this study show that the method applied contributed to obtaining avocado seedlings from seeds of the varieties analyzed that were free of pathogens and had clear homogeneity in their development. Therefore, this approach could be useful in avocado genetic improvement programs.

5. Conclusion

The germination of seeds of P. americana var. drymifolia and P. americana var. americana in an in vitro system made it possible to obtain homogeneous seedlings with complete tissue development that were free of pathogens. This system also improved germination time compared to ex vitro germination, so it could be useful in future research on avocado plants. The method may also prove useful for work on topics like genetic selection, environmental stress, and pathogen tolerance.