**2. Morphology of seeds, embryos, and polyembryony**

The study of polyembryony is receiving increased attention from both industrial and scientific sectors as it provides a form of cloning through seed that avoids the typical complications of sexual reproduction (for example, incompatibility barriers) and vegetative propagation (replication of viruses and other diseases) [20]. Another possible benefit of asexual reproduction by seed implies fixing the hybrid vigor and allowing the propagation of hybrids through many generations of seeds [21]. However, even in 2021, in [22] indicated that fixed hybrid vigor has not been possible because hybrid seeds cannot produce offspring with the same qualities.

Over the years, attempts to link the morphological characteristics of fruits, seeds, and embryos with polyembryony or with the characteristics of nucellar or zygotic embryos to predict the sexual or asexual origin of seedlings have taken place. Some researchers have studied the relationship between fruit weight and polyembryony in Swingle citrumelo, Volkamerian lemon, Cleopatra mandarin, and Amblicarpa [11, 23]. Other studies have tried to select characteristics of polyembryonic seeds as a possible method to anticipate the origin, zygotic or nucellar, of seedlings. For example, the size and shape classification of seeds has been associated with the production of zygotic seedlings on rootstocks [24, 25]. Also, logistic regression models that predict sexual seedlings have been developed [13].

Another variable that has been considered in the possible prediction of predominant sexual or asexual reproduction is the relationship between the percentage of polyembryony in citrus genotypes and the production of nucellar seedlings. Different studies have shown that species with a higher percentage of polyembryony are less likely to develop zygotic seedlings [21, 26, 27] because only one zygotic embryo

possesses competitive disadvantage against all nucellar embryos: zygotic embryos tend to be small and do not survive in field conditions. In contrast, more numerous apomictic embryos tend to be large and produce more vigorous seedlings. One case is *Citrus reshni*: apomictic reproduction occurs based on the degree of polyembryony of the seeds (70–90%) and the small size (≤3 mm) of the sexual embryos [28]. However, this characteristic varies between genotypes; for example, in [12] reported six genotypes with a high percentage of polyembryonic seeds (65–98%), where only Valencia orange and Amblicarpa mandarin have the possibility of apomictic reproduction. Therefore, the presence and degree of polyembryony in the seed is a genetic characteristic dependent on the interaction with the environment, particularly with the weather, the stage of development of the plant and its organs, or the physiological conditions [29, 30]. Therefore, using the phenotypic characteristics of seeds, embryos, and polyembryony to identify the seedlings' sexual or clonal origin often leads to the omission of zygotic seedlings in segregating populations, which is unfavorable in breeding programs. This trait will be discussed later in the topic of molecular markers.

Other features of the embryos, such as size and location, have been studied to define the position and origin of nucellar and zygotic embryos. Zygotes often appear as more diminutive in seed size, with slower growth than adventitious embryos and growth prevailing at the micropylar end [31, 32]. However, during nucellar embryogenesis associated with the fertilized embryo sacs, there seems to be an inhibitory effect in the chalaza region [10, 33]. Consequently, both embryos preferentially grow towards the micropyle. These arguments are helpful to generate reproduction models in polyembryonic citrus; however, it should be noted that these studies focused on the initial stages of embryo and seed formation. Therefore, it is essential to develop innovative methods of identifying nucellar and zygotic embryos in mature seeds and differentiating the seedlings that originate in the early stages of development. We will cite the work of Andrade-Rodríguez et al. [28], who, using RAPD (Random

#### **Figure 1.**

*Embryos classified by size in a seed of Amblicarpa mandarin (Citrus amblycarpa (Hassk) Ochse). E1 corresponds to the largest embryo in length and weight. E7 corresponds to the smallest. For each embryo, its location towards the micropyle or the chalaza and radicle orientation were recorded.*

#### *Citrus Polyembryony DOI: http://dx.doi.org/10.5772/intechopen.105994*

Amplification of Polymorphic DNA) markers, in vitro culture, and embryo morphology, concluded that not all small embryos located in the micropyle produce zygotic seedlings in Volkamerian lemon seeds. This contribution generated various questions about the reproduction models proposed for citrus and motivated us to pursue our current research focus.

Our laboratory has studied polyembryony in citrus from 2000 to date (as well as in other fruit trees not mentioned in this chapter). We have used molecular markers, in vitro culture of embryos separated by size, and grafting of the plants obtained in the greenhouse. These techniques have produced plants from embryos one, two, three, four, five, six, and seven (Example: E1, E2, E3, E4, E5, E6 and E7 in **Figure 1**) and propose new approaches to study the relationship between size and sexual, asexual, or different origin of the plants obtained from each of the embryos. Among the works published in citrus are references [7, 11, 12, 28, 34].
