Citrus is rated as a high-value fruit crop known for its refreshing juice, vitamin C content, secondary metabolites, and high antioxidant properties. So immense are the nutritional values that it was recommended as an immunity booster during the pandemic (WHO, 2021). These desirable attributes signify the glory of this fruit in world commerce by an export quantum of 1.7 million tonnes of fruits per annum. Globally, citrus is grown in an area of 10.07 million hectares (ha) with a production of 143.75 million tonnes. Although sweet orange dominates more than 50% of global citrus production, mandarin and its groups dominate India’s citrus industry by accounting for more than 43% of production (FAO, 2021). Among mandarins, Kinnow, an interspecific hybrid introduced from California, USA, made a breakthrough in the citrus industry of India and has established itself as the most preferred fresh fruit among consumers. There is a great demand for Kinnow fruits for processing into juice. However, it could not get a boost due to its high seediness leading to delayed bitterness (Mallick et al., 2016; Kumar et al., 2021). The development of low-seeded scion varieties is a major target for citrus breeders worldwide, which, however, is difficult to achieve through conventional breeding.

Modern breeding approaches such as in vitro mutagenesis have opened new vistas for achieving the traits of interest over ex vitro mutagenesis (Penna et al., 2012). In this direction, solid mutant induction can be considered a powerful speed breeding tool in perennial fruit crops. The totipotency of single cells can be utilized to induce solid mutants through in vitro techniques. Since indirect somatic embryogenesis (ISE) and direct somatic embryogenesis (DSE) play an essential role in single-cell regeneration, these techniques have immense value in citrus species (Omar et al., 2016). Standard ISE protocols in citrus using female reproductive organs such as stigma, style, ovary, and ovule are available (Gmitter and Moore, 1986; Cardoso et al., 2012; Omar et al., 2016). Earlier workers have suggested ISE from undeveloped ovules as the most effective protocol for citrus improvement (Grosser et al., 2000; Dutt et al., 2010; Ge et al., 2015; Omar et al., 2016). Contrary to the recommendations given by earlier workers, few researchers have also reported the frequent occurrence of somaclonal variations from ISE. Culture-induced somaclonal variation is undesirable when efforts are underway to produce genetically identical regenerants (Hao and Deng, 2002; Grosser et al., 2007; Dutt and Grosser, 2010; Meziane et al., 2017). Similarly, the ISE system, although has been reported to induce high-frequency somatic embryogenesis, ex vitro characterization of obtained plantlets is difficult due to the low recovery of hardened plantlets (Grosser and Gmitter, 1990; Fleming et al., 2000). Recent studies emphasize the importance of DSE for overcoming the somaclonal variation as well as rapid mass multiplication and high plantlet recovery in different fruit crops such as banana, citrus, date palm, mango, and passion fruit (Kochba et al., 1972; Navarro and Juarez, 1977; Panis et al., 1993; Wu et al., 2007; Silva et al., 2021).

The DSE technique holds promise particularly in polyembryonic fruit crops. In some citrus species, the sporophytic mixed apomixis type of polyembryony has been reported and contains both zygotic and nucellar embryos within the same seed (Bewley et al., 2012). Polyembryony in citrus is referred to as nucellar embryony because of its nucellus origin (Kepiro and Roose, 2007). For achieving in vitro DSE, nucellus tissue can be utilized by taking advantage of predetermined embryogenic and primordial cells in nucellar embryonic varieties. It leads to embryo development from a single cell to fulfill the need of its usage in solid mutant induction (Bitters et al., 1972; Kochba et al., 1972; Esen and Soost, 1977; Navarro and Juarez, 1977; Kobayashi et al., 1979). From a mutagenesis perspective, removing the embryos before explant inoculation is a prerequisite to avoiding zygotic variation and chimera formation. Migration of embryos toward the micropylar end after pollination is the characteristic feature of nucellar embryony, and the duration of its complete migration varies among citrus species (Wakana and Uemoto, 1987). Therefore, histological identification of explant developmental stages with probable complete migration of embryos toward the micropylar end must be standardized to confirm the absence of embryos from explants before inoculation.

Mandarins are parents for many citrus species of polyembryonic origin. It is well-established that mandarin varieties are recalcitrant and give poor in vitro regeneration response (Gmitter and Moore, 1986; Tomaz et al., 2001; Ricci et al., 2002; Germana, 2003; Benelli et al., 2010). The wild and primitive nature may be the reason for a few of its varieties to respond poorly under in vitro culture conditions. Furthermore, the interspecific hybridity of Kinnow (Citrus nobilis × Citrus deliciosa) makes embryogenesis complex (author’s unpublished data). Although nucellus cultures can induce DSE in citrus, the injury caused during tissue excision gives a lower success (Bitters et al., 1972; Kochba et al., 1972; Navarro and Juarez, 1977). Since the technique has an immense advantage for obtaining high recovery of solid mutants, the present study is an attempt toward modification of protocol through in ovulo nucellus culture for getting genetically stable regenerants with high plantlet recovery from nucellus tissue in Kinnow mandarin. The study also includes histological examination and analysis of clonal fidelity employing the ISSR marker system for seeking histological and molecular evidence for the proposed protocol’s robustness.

The possibility of DSE competence of the in ovulo nucellus culture in Kinnow mandarin was assessed in the present study to explore the regeneration potential of embryogenic cells present within the nucellus tissues. Several reports show that degeneration of the nucellus tissue coincides with the enlargement of the embryo sac; hence, the explant collection stage is important in citrus (Bitters et al., 1972; Koltunow et al., 1995; Xu et al., 2021). A weekly collection of fruits has been advocated in citrus for somatic embryogenesis (Bitters et al., 1972). However, since flowering and fruiting are region-specific and weather-dependent, identifying optimum-size fruits with a well-developed nucellus and liquid endosperm is a prerequisite for efficient in ovulo culture. The identification and classification of immature fruit growth stages in the present study revealed that for the stage II fruits (>14–21 mm in diameter), although provide a chance for true-to-type regeneration, complete removal of the preexisting embryos from the ovule is not feasible due to the scattered distribution of proglobular nucellar embryos. Attempts to remove the preexisting embryos at this stage may deviate from the objective of solid mutant induction because there are ample chances for chimera formation. Stage III fruits having a size between >21 and 25 mm had ovules with a well-developed intact nucellus and liquid endosperm; the proglobular embryos migrated completely to the micropylar end. It facilitated easy removal of the preexisting embryos and was convenient to handle in vitro. Earlier reports on nucellus culture in citrus species had also emphasized that at the time of explant collection, the ovule must have a liquid endosperm with an intact nucellus for better somatic embryogenesis (Bitters et al., 1972). There is a dearth of literature pertaining to the specific recommendation on fruit size in Kinnow mandarin for efficient ovule culture, which was accomplished in this study. Standardization of fruit size will enable researchers to directly collect the fruits at the recommended growth stage and use them for in vitro culture initiation.

Several factors, such as the developmental stages of the explant, culture medium, hormonal concentration, and combinations of other factors, influenced the DSE of cultured explants. In the present investigation, although preexisting embryos were eliminated entirely from the inoculated explants, somatic embryogenesis was witnessed possibly due to the stimulative effect of culture media composition. The findings correlate well with the histological observations and are in consonance with the results of the earlier workers in the nucellus culture of different citrus species (Bitters et al., 1972; Kochba et al., 1972; Navarro and Juarez, 1977) as well as mango (Wu et al., 2007). Although the use of MT medium has been widely reported in citrus somatic embryogenesis (Carimi, 2005; Dutt et al., 2010), in the present study, DKW medium stimulated a higher degree of DSE and maintained continuous proliferation of somatic embryos than the MT medium. The rapid induction and maturation of somatic embryos on DKW medium in contrast to MT medium indicate enhanced physiological activity as a consequence of reduced amounts of ammonium and chloride and increased calcium and sulfate ions as reported in citrus (Tallon et al., 2012; Tallon et al., 2013; Navarro-Garcia et al., 2016). In our study, kinetin and its combination with ME induced somatic embryos. The requirement of high concentration of cytokinin and low concentration of auxin in the medium for the induction of somatic embryos in citrus has also been suggested by Dutt et al. (2010) and Agisimanto et al. (2016). The use of 2,4-D in the culture medium of citrus species has been reported to suppress embryogenesis by inducing non-embryogenic cells due to induced osmotic stress (Pan et al., 2010; Ge et al., 2012). However, in the present study, 2,4-D in combination with CW induced somatic embryo formation in low frequency. It clearly explains the need for high cytokinin and low auxin for induction of morphogenesis, i.e., somatic embryos on nucellus tissues via rapid cell division and differentiation. Histological examination of the ovules in our study confirms the polyembryony nature and induction of somatic embryos through direct embryogenesis from nucellus cells in Kinnow mandarin.

In vitro somatic embryogenesis and polyembryony (sporophytic apomixis) can be considered artificial and natural paths of clonal multiplication mechanisms, respectively. In polyembryonic citrus species, nucellar embryony resembles somatic embryogenesis, and it is regulated by complex molecular mechanisms such as upregulation of specific genes, transposable element insertion, and gene regulation (Xu et al., 2021). Exogenous hormonal supplementation is among the other factors responsible for the expression of the above molecular signals (Mendez-Hernandez et al., 2019). Waki et al. (2011), in 2011, for the first time, demonstrated the role of the RKD gene in regulating the early developmental stages of embryogenesis in Arabidopsis. Subsequently, in 2018, Shimada et al. (2018) demonstrated a positive correlation between the higher expression of citRKD1 and somatic embryogenesis in nucellus tissue of polyembryonic Satsuma mandarin. The dominant allele CitRKD1-mg2 that bears the miniature inverted-repeat transposable element (MITE)-like insertion in the upstream region is predominantly transcribed in manifesting the upregulation of citRKD1 and is associated with polyembryony in citrus. Similarly, the repression of csSPL transcription factors (CsSPL3 and CsSPL14) for initiating somatic embryogenesis in different citrus species is primarily mediated by the upregulation of miR156a (Long et al., 2018). Very recently, Feng et al. (2022) demonstrated that the binding of Fus3 and AGL15 complexes at the promoter region of miR156a is essential for its activation. Since most of the molecular regulatory pathways are conserved across the higher plants, induction of somatic embryogenesis in Kinnow possibly occurred via similar molecular cascade activation by hormonal stimuli. The enhanced somatic embryogenic response from in ovulo nucellus culture can be considered an improvement over the nucellus culture technique in mandarin, where a low embryogenic response (10%) has been reported by Bitters et al. (1972). Conversely, 22%–84% of embryo initiation has been reported from nucellus culture in sweet oranges (Kochba et al., 1972). This evidence and our experimental results suggest that mandarins have inherited in vitro recalcitrance. Thus, embryogenic response in citrus depends on genotype and not on the presence or absence of integuments in the developing ovules.

The practical utility of any somatic embryogenesis protocol depends on the conversion of induced embryos into healthy and quality germinated seedlings with a balanced shoot:root system (Gmitter and Moore, 1986; Corredoira et al., 2019). In our study, somatic embryos transferred from SE₂ combination of ICM medium onto GM₃ treatment of GCC medium gave better germination and bipolar conversion. It was possible due to the better starch and protein accumulation and post-maturation regime attained by somatic embryos from the SE₂ combination of ICM medium. The physiological role of starch and protein accumulation for the maturation of somatic embryogenesis was demonstrated by Peng et al. (2020) in Korean pine. The molecular evidence of miR156-mediated regulation of starch accumulation during somatic embryogenesis has also been recently reported in citrus by Feng et al. (2022). Furthermore, Joshi et al. (2022) reviewed the involvement of MCM1, AGAMOUS, DEFICINS and SRF, serum response factor (MADS)-domain transcription factor AGAMOUS-Like 15 (AGL15) in somatic embryo development by influencing functions such as transcription factors, hormone signaling, and epigenetic regulation. Our result on better germination of SE₂ embryos correlates well with the above findings and confirms the need for the above factors for the somatic embryogenesis pathway in plant species.

Although somatic embryo maturation plays a significant role in germination, without the stimulation of meristematic activity, bipolar conversion into a complete seedling is not possible. In the present investigation, GM₃ treatment stimulated germination and bipolar conversion in somatic embryos. A high level of three B vitamins (20.95%), i.e., thiamine, nicotinic acid, and pyridoxine, in the MT medium as compared to the DKW medium is responsible for the higher meristematic stimuli. The reports on the effect of vitamins on somatic embryo germination are scanty. However, the involvement of the same in germination as well as meristem formation has been reported by few researchers (Chen and Xiong, 2005; North et al., 2011; Ashihara et al., 2015). Moreover, the requirement of hormonal combinations for meristematic activity acquisition through enhanced cell division, cell elongation, and meristem formation in somatic embryos cannot be ignored. The positive effect of GA₃ on shoot elongation and root initiation in citrus somatic embryos were suggested by earlier researchers (Kochba et al., 1972; Gmitter and Moore, 1986; Grosser and Gmitter, 1990; Germana, 2005). Similarly, adding CW to the germination medium on the shoot apical meristem formation and regulation of endogenous auxin levels due to substitutive cytokinin-like activity in somatic embryos was reviewed by Corredoira et al. (2019). However, the effect of cytokinin needs to be counteracted by a slight increase in auxin for balanced root:shoot growth (Corredoira et al., 2019). Hence, the enhanced bipolar conversion ratio in the present study was possible due to the addition of a lower concentration of NAA. The support of spermidine on somatic embryogenesis and meristem conversion in somatic embryos is well explained by Kaszler et al. (2021) in Arabidopsis. Thus, the precise combinations of the above supplements in GM₃ medium are essential for high-frequency germination in Kinnow mandarin. Earlier studies emphasized the high vulnerability of somatic embryos toward poor germination in mandarin cultivars due to abnormal morphological defects (Benelli et al., 2010). However, in the present study, even the abnormal embryos were converted to the bipolar structure, which suggests the positive effect of GM₃ treatment of GCC medium in Kinnow mandarin.

Efficient establishment and acclimatization of in vitro-raised emblings enhance survival and induce stress tolerance. In the present investigation, the germinated seedlings of SE₂ × GM₃ were established well in a PBR-free liquid MT medium. The phenomenal influence of GCC treatment on plantlet establishment is unclear. However, Corredoira et al. (2019), in their review on the establishment of emblings, speculated the involvement of germination medium. Preconditioning of somatic embryo germinated emblings on a hormone-free medium before transferring to a hardening medium has recently been emphasized by Asadi-Aghbolaghi et al. (2021). Our results also revealed a positive effect on growth and development of a liquid culture medium that suggests its superiority for better nutrient utilization, ease of plant removal, lesser damage to the roots, and solubilization of synthesized inhibitors near the root zone, thus resulting in the better establishment of the emblings. Spiegel-Roy and Vardi (1984) have also reported the positive effect of the liquid medium with various growth regulator combinations on the establishment of citrus species. Our protocol is an alternative wherein the hormone-free liquid medium can induce better in vitro establishment particularly in DSE-obtained plantlets. During primary hardening, potting medium containing cocopeat:vermiculite:perlite (2:1:1) exhibited cent percent survival of liquid medium-established plantlets due to the improved water use efficiency.

From the histological studies, it could be inferred that there is a gradual depletion of nucellus tissue during the ascending stages of fruit growth. Hence, the ideal stage of explant collection for efficient in vitro success is crucial (Koltunow et al., 1995; Xu et al., 2021). Our observations while studying histology revealed exciting insights. Although mandarin cultivars are reported to be facultative sporophytic apomixis polyembryony type except a few, the formation of multiple embryos through a non-apomictic pathway via different mechanisms such as a supernumerary bud off from the zygotic embryo, fission of a zygotic embryo, and a single ovule with two or more embryo sacs is a common phenomenon in citrus (Kepiro and Roose, 2007; Aleza et al., 2010). Hence, it is practically challenging to predict the occurrence of polyembryony based on the number of embryos. In the present study, histological observations on the existence of independent embryos other than zygotic embryo at the micropylar end, along with the migration of a few embryos at the micropylar half, clearly suggest the occurrence of sporophytic apomixis with nucellar adventive embryony in Kinnow mandarin. Suppression of zygotic embryo by nucellar embryo is the usual hypothesis in citrus, but in the present study, embryo connected with endosperm (zygotic embryo) was larger than the other embryos. Since the connection between the embryo and the endosperm is the unique distinguishable character for identifying zygotic embryos (Wakana and Uemoto, 1987), it can be concluded that Kinnow mandarin has zygotic dominance capability and hence can be used as a female parent in the breeding program.

Avoidance of nucellar and zygotic embryos is a prerequisite for obtaining solid mutants via single-cell and true-to-the-type regeneration. The histological observation could decipher stage III (>21–25 mm) of fruit growth as the best explant (ovule) collection stage for easy removal of preexisting embryos. The reason is the complete migration of proglobular embryos at the micropylar end. Similar were the observations of Wakana and Uemoto (1987) in a few mandarin cultivars. From the nucellus tissue of inoculated explants (stage III), induced embryogenic cells were witnessed at the micropylar end. The induced embryogenic activity was confirmed by a dense cytoplasm and loose arrangement of cells from surrounding cells with active cell division. The division of cells and formation of embryos further confirmed the single-cell regeneration and fulfilled the requirement for inducing solid mutants through the standardized protocol. In addition to the above, the normal developmental events, such as globular to cotyledonary somatic embryos, showed the efficiency of the induction medium on Kinnow somatic embryogenesis. The results confirm the hypothesis of Kobayashi et al. (1979), who advocated that any nucellus cell of embryogenic capacity can give rise to embryos.

Assessment of genetic stability of in vitro-grown plants is an unavoidable requirement due to the variability enforced on the regenerated plants by culture environments such as epigenetic changes, transposon activation, or chromosomal aberrations. Emphasis on using various molecular markers for genetic fidelity assessment has gained importance during the last few decades due to innumerable advantages of early and nondestructive detection of variation. The ISSR markers did not reveal any variation in the established emblings, thus confirming genetic stability with the current protocol. In citrus, earlier studies also suggest the efficiency of ISSR markers in assessing genetic stability (Siragusa et al., 2007; Meziane et al., 2017; Devi et al., 2021). The current study is the first report on the genetic stability of in ovulo culture-induced plants obtained through DSE in Kinnow mandarin.

The outcome of the detailed investigation suggests that in ovulo nucellus culture can be an alternative to the nucellus culture technique in Kinnow mandarin. The size-specific recommendation, i.e., ovules of Kinnow mandarin obtained from stage III (>21–25 mm in diameter) fruits can induce DSE when inoculated on somatic embryo ICM medium containing DKW supplemented with kinetin 5.0 mg L^-1 and ME 1,000 mg L^-1. Transfer of cotyledonary embryos from the above medium to GCC medium containing MT basal medium supplemented with GA₃ 2.0 mg L^-1 + NAA 0.5 mg L^-1 + spermidine 100 mg L^-1 + CW 10% resulted in higher germination. Preconditioning germinated seedlings in the liquid medium revealed maximum plant establishment and cent percent survival on a potting medium containing cocopeat:vermiculite:perlite (2:1:1). The occurrence of embryogenesis from single nucellus cells confirms its practical utility in in vitro mutation studies for induction of solid mutants. Since the ISSR markers reveal the genetic fidelity of regenerated plants, the developed protocol can be effectively utilized not only for mutagenesis study but also for mass multiplication, virus elimination, synthetic seed technology, in vitro conservation, gene editing, and germplasm exchange.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

OA conceived the idea, TM and OA designed the study, collected data and wrote the manuscript. TM, GJ, and SK performed data analysis; SS reviewed the manuscript. GC and TM conducted histological studies. AS and TM assessed the molecular marker based genetic fidelity. All the authors contributed equally to the revision and accepted the conclusion. All authors contributed to the article and approved the submitted version.