**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,

#### **Figure 2.**

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

In Vitro *Seed Germination and Seedling Development of Two Avocado Varieties DOI: http://dx.doi.org/10.5772/intechopen.107005*

#### **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 cm<sup>2</sup> 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).*

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**).

#### **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**).


#### **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).*


#### **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.*

#### **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).*

#### **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**).

### **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 [24–26].

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 [27–39] 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 [40–42]. 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.
