**3. Identification of nucellar embryos and possible zygotes**

Successfully harnessing apomixis for citrus propagation requires understanding its facultative nature; the formation of nucellar and zygotic embryos varies among environments and times of the year. Additionally, each species genetic makeup acts as an activation "switch" that relies on environmental changes. All these variables lead to variations in the degree of polyembryony, number of embryos per seed, and even the type of reproduction that will prevail, asexual or sexual [3, 30, 35]. Understanding the apomictic phenomenon in citrus is central to various studies to benefit from its dual characteristics: clonal propagation from seed and hybrids production. We seek to generate models that relate the characteristics of polyembryony, size, and location of the embryos in the seed, with their sexual or asexual origin. However, the selection of the hybrids that result from the cross with polyembryonic parents is complex if they do not express a reproducible dominant trait under several environmental conditions. In addition, the percentage of zygotic progenies in various citrus hybrids has been found to vary based on the seed parent used [36], the pollen origin [37], and environmental factors [38]. For this reason, researchers used various morphological, biochemical, and molecular markers to identify seedlings in the early stages of development.

Plant morphology is the first visible marker for hybrid selection in plant breeding. The first morphological trait used to differentiate nucellar plants from zygotic ones relied on linking vigor with asexual origin. For example, in 1949 in Ref. [39] published that nucellar plants are those that develop juvenile characteristics, such as vigorous growth, presence of thorns, or slow fruiting. Another morphological marker, which is also a dominant phenotypic trait, is leaf morphology [26]. In **Figure 2**, Amblicarpa nucellar plant (**Figure 2a**) and the hybrid that exhibits more than one leaflet (**Figure 2b**) are compared; both plants have thorns, which according to Cameron and Johnston [39] are juvenile characteristics. It has been used in nurseries since the first decades of the 20th century to identify hybrids by crossing trifoliate genotypes. The method is still valid and is used to obtain trifoliate rootstocks such as citrandarins (Cleopatra mandarin× P. trifoliata). However, both the presence of juvenile traits and multifoliate leaves are markers that vary depending on environmental conditions and plant development, so they are unreliable [40].

Not all morphological markers can reliably distinguish between zygotic and nucellar seedlings [40]. The expression of "trifoliate leaf" is a dominant trait over

#### **Figure 2.**

*Mandarin Amblicarpa seedling (Citrus amblycarpa (Hassk) Ochse), obtained from embryos in vitro cultured, grafted on Volkamerian lemon (50 days after grafting). 2a nucellar plant and 2b sexual plant, both plants show "vigor" in height, leaf development, and thorn size; however, the sexual plant develops leaflets.*

the recessive unifoliate leaf trait makes it easy to identify the first-generation hybrid seedlings in crosses between unifoliate citrus and trifoliate orange male parents [41]. Thus, hybrid seedlings with multifoliate leaves would be expected to appear when crosses of trifoliate male parents (*P. trifoliata*) are made. However, when Poncirus hybrids (such as citranges or citrumelos) are backcrossed to Citrus, seedlings with bior trifoliate leaves may vary considerably [37]. Furthermore, it is particularly difficult to remove off-type seedlings based on seedling morphology when pummelo has been used as a seed parent because the morphological characteristics associated with pummelo dominate the seedling phenotype [29]. An alternative to morphological markers is molecular markers, which can unequivocally discriminate the zygotic seedlings [28–30, 42, 43].

Biochemical tests have also been used as genetic markers in discriminating zygotic seedlings. Such is the case of colorimetry [44], chromatography [45], polyphenol darkening [46], spectroscopy [47], and isozyme analysis techniques [48]; however, these techniques fail to identify all clones and ignore some hybrids. For example, although they are codominant markers with simple methods, isoenzymes express few polymorphic loci to differentiate F1 seedlings from the female parent. Additionally, their expression level is qualitatively and quantitatively affected by environmental factors, plant development stage, or physiological conditions [29, 30].

Molecular biology expanded the tools available for identifying seedling sexual or asexual origin through molecular markers based on PCR (polymerase chain reaction). Among the most used markers are RAPD (Random Amplification of Polymorphic DNA), ISSR (Inter Simple Sequence Repeat), SSR (Simple Sequence

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

Repeat), and SNP (single-nucleotide polymorphism) [30, 34, 42, 43]. Various studies compare the efficiency of genetic markers in selecting hybrids, in Ref. [29] evaluated RAPD and EST-SSR (Expressed Sequence Tag-SSR), in [43] worked with morphological markers and SSR, and in [45] compared morphological markers and SNPs. RAPDs (dominant marker) and EST-SSRs (codominant marker) efficiently and accurately identified nucellar plants of mandarin (*C. reticulata* Blanco) and pumelo (*C. maxima* Meer.); both markers were highly related (p = 0.001) [29]. The expression of trifoliate leaves with molecular markers was complemented in Ref.s [43, 49]; their research showed the viability of early selection of hybrid seedlings based on the morphological marker and subsequent analysis with molecular markers in the seedlings that did not express multifoliate leaves. This procedure reduces the cost and time of the analysis.

Not only can genetic markers complement each other, but in vitro germination can also be used in embryo identification to increase the development of small embryos (which do not germinate under in vivo conditions). Similarly, faster acclimatization and growth of plants resulting from these embryos result from grafting on a rootstock. Three studies show the advantages of combining in vitro culture and identifying hybrids with SSR markers. It is worth mentioning that SSRs have been widely used to discriminate embryos according to their origin because they favor the selection of plants obtained by self-pollination and cross-pollination. Embryos from F1 seeds from a cross between 'Shiranuhi' mandarin and 'Shiranuhi' orange were cultivated *in vitro* to determine the percentage of zygotic embryos detected with SSRs depending on the days after pollination (90, 105, 125, 145 and 180 days, DAP) [50]. Growth in an artificial medium allowed them to maintain constant humidity and nutrient supplementation and achieved 36–75% germination depending on DAP, allowing identification of zygote embryos: 12% at 90 days, 8% at 105 days, 7% at 125 days, 1% at 145 days and 4% at 180 days after pollination. On the other hand, *in vitro* culture, SSR markers, and morphological markers have been used [49]; they first selected using a trifoliate leaf morphological marker and then, with SSRs, selected 41% of hybrid seedlings of rough lemon and citrandarin X-639c, and 46% of rough lemon and Swingle citrumelo hybrids. Likewise, these authors state that not all zygotic embryos survived until fruit maturation as the ratio of hybrid seedlings decreases with advancing stages of embryonic development after 95 days after pollination. Finally, *in vitro* culture, seedling grafting, and SSR markers have been used to identify nucellar embryos in citrange C-35, Amblicarpa mandarin, and Volkamerian lemon rootstocks, and Valencia orange and Minneola tangelo cultivars [12]. Also, this chapter includes the identification of nucellar embryos in Parson Brown orange, not published in the 2021 paper. This study differs in its approach as the authors only studied the largest embryo per seed, finding that the weight of the seed correlates with the weight of the largest embryo (0.76–0.96, p ≥ 0.05). They used in vitro germination until they achieved 3–4 mm long seedlings and grafted them on Volkamerian lemon to promote their growth for 5 months until foliar collection. This procedure allowed for enough leaf material for DNA extraction (1.8–2.0 at 260/280 nm absorbance) without damage to the grafted plant, which developed under simulated nursery management. The SSR markers identified 70% nucellar seedlings in C-35, 65% in Volkamerian and Minneola, 85% in Amblicarpa and Valencia, and 100% in Parson Brown (**Figure 3**). Focus on the embryos with the most significant capacity to germinate in each seed allowed linking size (8–10 mm in length, 6–19 mg in weight) and the probability of generating nucellar plants; only Amblicarpa, Valencia, and Parson Brown showed a tendency to clonal propagation.

#### **Figure 3.**

*Dendrograms showing the Genetic Similarity Indices (GSI) between nucellar seedlings and the female parent, obtained from the largest embryo per seed in six polyembryonic citrus cultivars (A, Citrange C-35; B, Amblicarpa Mandarin; C, Volkamerian lemon; D, Parson Brown orange; E, Tangelo Minneola; F, Valencia orange). The GSI was calculated with 30 SSR markers.*

Various works that use molecular markers to classify seedlings according to their sexual or asexual origin state that those with total similarity to the female parent are nucellar. In [29] consider seedlings of Swingle citrumelo (*Citrus paradisi* Macf. × *Poncirus trifoliata* (L.) Raf.) and sour orange (*C. aurantium* L.) as "true nucellar seedlings" when they were identical to the female parent at all loci (100%), using EST-SSR and Euclidean distance with UPGMA cluster analysis. However, in [12] seedlings of six citrus genotypes with a Genetic Similarity Index [51] of 0.95 (95%) were considered "nucellar" compared to the female parent (**Figure 3**). Both studies show the discrepancy between the genetic similarity values needed for a plant to be considered "true nucellar" or "possible zygotic" to the female parent. As any marker has the disadvantage of characterizing only a tiny part of the genome, a discussion on the number of molecular markers needed for these studies is required. As an example, in [29] evaluated 12 EST-SSR markers, of which eight primer pairs revealed polymorphism, whereas in [12] examined 30 SSR markers, but only 17 were polymorphic enough to differentiate hybrids (TAA 41 and F4 being the most informative).

One of the objectives of breeding programs is to select plants based on agronomic and economic traits, such as the expression of polyembryony. However, it is relevant to consider the advantage of using molecular markers. These have the possibility of generating QTL (Quantitative Trait Loci) by associating the polymorphism to a phenotypic trait. The QTL Apo1, Apo2, Apo3, Apo4, and Apo6 associated molecular markers (RAPD, SSR, and CAP -Cleaved Amplified Polymorphic Sequence) with apomixis in the progeny of *C. volkameriana*× P. *trifoliata* cv 'Rubidoux.' *Apo2* is also associated with the activation of the type of embryo (mono-embryonic or

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

poly-embryonic) in apomictic genotypes [52]. Further information has been obtained from AFLPs (Amplified Fragment Length Polymorphism) as marker loci have identified a genomic region associated with the percentage of polyembryonic seeds in *Citrus*× Poncirus hybrids [53]. The genetic control of adventitious embryology was also documented in *C. reticulata* [2, 22]; it has been proposed that the CiRKD1 gene regulates the somatic embryogenesis that occurs with two alleles that originate polyembryony and embryogenesis [54, 55]. So, the genetic control of apomictic reproduction and polyembryony is "more than a single switch" to activate it; it involves various genome regions in citrus.

Despite the effectiveness of molecular markers to discriminate between hybrids and nucellar plants, ongoing efforts try to relate the expression of morphological features of the embryo and its location in the seed with sexual origin. The purpose is to separate hybrid plants from clonal plants from seed or in the early stages of development without a laboratory. Various studies have related polyembryonic traits and origin; however, we will only analyze the *Citrus volkameriana* Pasq rootstock. The sexual origin was related to the embryo's size employing the trifoliate leaf marker expressed by the cross between the Volkamerian lemon and *P. trifoliata* [26]. They report large embryos (>5 mm) generate up to 82.7% of hybrid plants, while small embryos (1–2.9 mm) produce only 5.8%. Meanwhile, 25.9% of zygotic plants were identified with RAPDs in mature polyembryonic seeds, of which small embryos produced less than 43% (2–3 mm) and near the micropyle [28]. On the other hand, in Volkameriano seeds, when the largest embryo was separated from each polyembryonic seed and studied its location; all older embryos are in the chalaza and 45% of it produced non-clonal plants [12]. It is relevant to mention that the relationships between embryo morphology and sexual origin vary among genotypes [12, 26], strengthening the idea that embryos with a greater capacity to germinate in vivo in a polyembryonic genotype do not imply clonal reproduction. Thus, the competition caused by the asynchronous development between embryos during seed formation [7, 56] does not always favor the growth of nucellar embryos.

Therefore, molecular markers and complementary techniques (in vitro culture and grafting) are adequate to identify sexual and asexual seedlings with greater certainty than the exclusive use of morphological markers. Nevertheless, the characteristics of these techniques need to be assessed based on the objective and possibilities of the investigation. For example, although RAPDs and ESS-SSRs show a similar capacity to differentiate zygotic and nucellar seedlings [29], RAPDs are simpler and cheaper than EST-SSRs. However, the latter shows greater reproducibility among laboratories and detects all alleles of a locus (codominance). In the case of SNPs, their limitation is the cost and availability of the necessary equipment in the laboratory. Instead, in vitro culture is an excellent technique to be applied in research where germination of embryos is required for subsequent identification, but it is expensive in commercial citrus propagation schemes.
